麻风树的遗传转化 Genetic transformation of Jatropha

[0001] 相关申请的交叉引用 Cross [0001] REFERENCE TO RELATED APPLICATIONS

[0002] 本申请要求享有2008年12月15日提交的美国临时专利申请系列号61/122，454的优先权，该临时专利申请并入本文作为参考。 [0002] This application claims the benefit of US Provisional Patent Application Serial No. 61 December 15, 2008 submitted / 122,454, filed the provisional patent application is incorporated herein by reference.

[0003] 发明背景 [0003] Background of the Invention

[0004] 本发明涉及植物再生和转化领域，尤其涉及用于麻风树属(JatiOpha)的再生和转化的方法。 [0004] The present invention relates to the field of plant regeneration and transformation, jatropha (JatiOpha) regeneration and more particularly to a method of transformation. 更具体地，本发明涉及用于麻风树(Jatropha curcas)植物的再生和转化的方法和培养基组合物。 More particularly, the present invention relates to Jatropha (Jatropha curcas) Regeneration and transformation methods and media compositions plants.

[0005] 本文中用于说明本发明背景或提供有关实施的补充细节的出版物和其他材料并入本文作为参考，并且为了方便起见分别在文献目录中分组。 For explaining the background of the invention or provide additional details on the implementation of the [0005] As used herein the publications and other materials are incorporated herein by reference, and for convenience are respectively grouped in the bibliography.

[0006] 世界面临着化石燃料供给日益减少和温室作用不断恶化的问题。 [0006] The world faces dwindling supply of fossil fuels and the greenhouse effect of worsening the problem. 亟需增加可再生能源的产生和消耗。 Urgent need to increase renewable energy generation and consumption. 对于许多国家寻找替代能源来说，生物燃料已经公认为国家的优先项目以满足它们的能源安全需要，同时有助于减少造成温室作用的CO2排放。 For many countries to find alternative energy sources, the bio-fuel project has been recognized as a national priority in order to meet their energy security needs, while helping to reduce CO2 emissions caused by the greenhouse effect. 对生物燃料的需要导致食物生产的压力增加。 Need biofuels cause pressure increase food production. 例如，为了满足德国政府要求的德国2017年的生物燃料需求，该国的全部农用土地都将用于生长生物能作物，而没有留下土地用于食物生产。 For example, in Germany in 2017 in order to meet the demand for biofuels required by the German government, all of the country's agricultural land will be used to grow bioenergy crops, but did not leave the land for food production. 为了减轻对土地的这种竞争并满足我们对可再生燃料的需要，亟需利用边缘土地来进行生物能产生。 To mitigate this competition for land and meet our need for renewable fuels, need to make use of marginal land to produce bioenergy. [0007] 麻风树是属于大戟科的小木本植物。 [0007] Euphorbiaceae Jatropha belongs to a small woody plants. 麻风树的数种独特性质使其成为用于生物柴油生产的理想植物。 Several unique properties make it ideal for jatropha for biodiesel production plant. 这些性质包括其迅速生长、容易繁殖、种子的低成本、高含油量、短孕育期、广泛的适应性、干旱耐受性和在退化土壤上茁壮成长的能力。 These properties include its rapid growth, easy propagation, the seeds of low cost, high oil content, a short incubation period, a wide range of adaptability, drought tolerance and ability to thrive on degraded soils. 此外，其植株大小使种子的收集非常方便(Jones, 1991 ；Sujatha et al.,2008) „ In addition, the size of the seeds of the plant is very easy to collect (Jones, 1991; Sujatha et al, 2008.) "

[0008] 但是，麻风树具有几个缺点，这限制了它的广泛应用。 [0008] However, jatropha have several disadvantages, which limits its wide application. 植物的产量受到不利的雄花与雌花比的限制，并且其含油量没有通过育种进行优化。 Production plants adversely male and female ratio limits, and its oil content is not optimized through breeding. 该植物对生物应激如病毒(Narayanna et al.，2007)、真菌和细菌病原体，和非生物应激特别是寒冷和干旱也是敏感的(http colon www dot jatropha dot org)。 The plant biological stress such as viruses (Narayanna et al., 2007), fungal and bacterial pathogens, and abiotic stress is particularly sensitive to cold and drought (http colon www dot jatropha dot org). 在植物的种子和叶子中存在数种毒性组分(例如蛋白毒素、麻风树毒蛋白和致癌剂佛波酯)给麻风树产业的农民和生物加工工人带来健康危害。 There are several toxic components (such as protein toxins and carcinogens curcin phorbol ester) in plant seeds and leaves to jatropha farmers and bioprocessing industry worker health hazards.

[0009] 改进植物质量性状的传统方法是通过培育优良的基因型。 [0009] The traditional method for improving plant quality traits is by fostering excellent genotypes. 但是，利用分子标记物对遗传多样性的评价公开了当地麻风树(J.curcas)种质中的低附件间(inter-accessional)变异性(Sujatha et al.,2008)。 However, the use of molecular markers to evaluate the genetic diversity of the local public Jatropha (J.curcas) between germplasm low attachment (inter-accessional) variability (Sujatha et al., 2008). 因此，亟需诸如遗传转化方法的替代性遗传操作工具来为该作物的遗传改良提供另外的策略。 Therefore, an urgent need, such as genetic transformation method of alternative genetic manipulation tools to provide additional strategies for genetic improvement of crops. 土壤杆菌(Agrobacterium)介导的遗传转化已经成为产生转基因植物的主要选择。 Agrobacterium (Agrobacterium) mediated genetic transformation has become the main choice to produce transgenic plants. 但是，很少有报道涉及属于大戟科植物的土壤杆菌介导的转化的应用。 However, few have been reported involving the application of Euphorbiaceae plants belonging to Agrobacterium-mediated transformation. 唯一报道的用于麻风树属的转化方案(Li et al.,2008)在我们手中是无法重复的。 The only reported for Jatropha transformation program (Li et al., 2008) in our hands can not be duplicated.

[0010] 因此，亟需转化麻风树的方法以便为该作物种类的遗传改良提供工具。 [0010] Accordingly, the need for transformation of Jatropha methods provide tools for the genetic improvement of crop species.

[0011] 发明概述 [0011] Summary of the Invention

[0012] 本发明涉及用于麻风树属的植物，更具体的是麻风树的再生和土壤杆菌介导的转化的方法。 [0012] The present invention relates to a plant for Jatropha, more specifically, is a method and regeneration of Jatropha Agrobacterium mediated transformation.

[0013] 因此，在一方面，本发明通过优化组织培养和枝条(shoot)再生条件提供了有效且可重复的用于麻风树的植物再生方案。 [0013] Accordingly, in one aspect, the present invention is to optimize tissue culture and shoot (shoot) regeneration conditions by providing an effective and reproducible for the jatropha plant regeneration scheme. 这种再生方案已经与土壤杆菌介导的转化联用以产生Ttl转基因麻风树属枝条/植物。 This regeneration program has been used to Agrobacterium-mediated transformation associated with the generation of transgenic Jatropha Ttl branches / plant. 本发明还提供了利用Ttl转基因枝条作为接穗和非转基因植株作为根状茎的嫁接步骤的用途。 The present invention also provides the use of transgenic shoots as Ttl scion and non-transgenic plants as Rhizome grafting procedure uses. 这种嫁接步骤不需要再生的植物在组织培养中产生根，并且显著地缩短了转基因枝条开花和产生T1种子的时间。 This step does not require regeneration grafted plants in tissue culture to produce roots, and significantly shorten the flowering branches and produce transgenic seed time T1.

[0014] 在一实施方案中，本发明提供了一种用于再生麻风树植物的方法。 [0014] In one embodiment, the present invention provides a method for the regeneration of Jatropha curcas plants. 根据该实施方案，从5-7天龄幼苗的子叶获得外植体。 According to this embodiment, the 5-7-day-old seedlings of cotyledon explants obtained. 将外植体在愈伤组织形成培养基上培养，所述愈伤组织形成培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-苄氨基嘌呤(6-BA)和1-萘乙酸(NAA)。 The explants were cultured on callus formation in a medium, medium containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as the plant hormone 6- callus formation benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA). 然后将愈伤组织转移到第一枝条再生培养基，其包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、腺嘌呤、蔗糖以及作为植物激素的6-BA和3-吲哚丁酸(IBA)。 The callus is then transferred to the first shoot regeneration medium, containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, adenine, sucrose as well as a plant hormone 6-BA and 3- indole butyric acid (IBA). 将从愈伤组织再生的任何枝条转移到第二枝条再生培养基，其包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA、IBA和赤霉酸(GA3)。 Any branches from the Transfer calli regenerated shoots to a second regeneration medium, containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as a plant hormone 6-BA, IBA and gibberellic acid (GA3). 将不具有再生的枝条的愈伤组织转移到第三枝条再生培养基，其包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA和IBA以用于枝条的进一步再生。 The callus having no regeneration of shoots transferred to the third shoot regeneration medium, containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as a plant hormone 6-BA and IBA branches for further regeneration. 将已经再生的枝条转移到枝条伸长(elongation)培养基,其包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA和GA3以用于伸长和芽增殖。 Will have regenerated shoots are transferred to shoot elongation (elongation) medium, containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as a plant hormone 6-BA and GA3 to use in elongation and shoot proliferation. 将伸长的枝条转移到生根培养基，其包含MS无机盐、维生素B5、蔗糖和IBA。 The elongation of shoots transferred to rooting medium containing MS salts, vitamins B5, sucrose and IBA. 生根后，将幼苗转移到土壤中。 After rooting, the seedlings were transferred to soil. 可选的，可以将伸长的枝条嫁接到麻风树的根状茎。 Alternatively, you can stretch the branches grafted into rhizomes jatropha.

[0015] 在第二实施方案中，本发明提供了一种用于土壤杆菌介导的麻风树植物转化的方法。 [0015] In a second embodiment, the present invention provides a method for Jatropha curcas plant Agrobacterium-mediated transformation. 根据该实施方案，土壤杆菌介导的麻风树转化利用与上述用于麻风树再生的基本方案相同的基本方案。 According to this embodiment, Agrobacterium-mediated transformation and utilization of Jatropha for Jatropha to the basic program of the same basic regeneration scheme. 为了进行转化，首先将外植体与土壤杆菌细胞共培养，然后转移到愈伤组织形成培养基，随后转移到如上所述的枝条再生培养基、枝条伸长培养基和生根培养基。 For transformation, first of all the explants co-cultured with the Agrobacterium cells, then transferred to callus formation medium, then transferred to shoot regeneration medium as described above, shoot elongation medium and rooting medium. 共培养培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖、葡萄糖、乙酰丁香酮以及作为植物激素的6-BA和NAA。 Co-cultivation medium containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, glucose, acetosyringone, and as a plant hormone 6-BA and NAA. 愈伤组织形成培养基与用于再生的培养基相同，除了它还包含选择剂和土壤杆菌铲除剂。 Callus formation medium with the same medium for regeneration, except that it also contains a selection agent and Agrobacterium eradicate agent. 类似地，枝条再生培养基还包含选择剂和土壤杆菌铲除剂。 Similarly, shoot regeneration medium also contains a selection agent and Agrobacterium eradicate agent. 为了转化，在避光条件下的愈伤组织形成培养基上进行培养。 For transformation, under dark conditions, were cultured on callus formation medium. 可以将常规选择剂用于土壤杆菌介导的麻风树植物转化。 Conventional selection agent for Agrobacterium-mediated plant jatropha can be converted. 选择剂的实例包括但不限于除草剂BASTA、潮霉素等。 Select agents include, but are not limited to the herbicide BASTA, hygromycin, etc.

附图说明[0016] 图1示出本发明的土壤杆菌介导的麻风树属转化方法。 BRIEF DESCRIPTION [0016] Figure 1 illustrates the present invention, Agrobacterium-mediated transformation methods Jatropha. 左边所列的时间尺度使用生根方案，而右边使用嫁接方案。 Time left scale use rooting programs listed, and the right to use grafting solution.

[0017] 图2示出用于进行本发明的转化方法的土壤杆菌转化载体。 [0017] Figure 2 illustrates a method of Agrobacterium for transformation of the present invention is the transformation vector.

[0018] 图3A-3K示出麻风树的转化、再生、开花和出苗。 [0018] Figure 3A-3K shows jatropha transformation, regeneration, flowering and emergence. 图3A:麻风树MD5天的幼苗适于转化。 Figure 3A: MD5 day Jatropha seedlings suitable for conversion. 图3B:愈伤组织形成和枝条产生。 Figure 3B: callus formation and shoot production. 左侧，用不携带任何载体的土壤杆菌接种的子叶。 Left side, does not carry any of the carriers with the Agrobacterium inoculated cotyledons. 右侧，用携带含性状基因的载体的土壤杆菌接种的子叶。 To the right, with the carry the trait gene vector containing Agrobacterium inoculation of the cotyledon. 注意来自外植体的枝条再生。 Note shoot regeneration from explants. 图3C:褐色子叶表面上潮霉素抗性愈伤组织和枝条样器官的放大图。 Figure 3C: zoom on the brown surface map hygromycin cotyledon callus and shoot like organs. 图3D:麻风树的潮霉素抗性枝条的再生。 Figure 3D: jatropha hygromycin shoot regeneration. 图3E:枝条伸长。 Figure 3E: shoot elongation. 图3F:转基因枝条的生根。 Figure 3F: Transgenic shoots rooting. 图3G:转基因麻风树的高生根效率。 Figure 3G: transgenic Jatropha high rooting efficiency. 图3H:土壤中生长的转基因麻风树。 Figure 3H: soil growing transgenic Jatropha. 图31和图3J:嫁接于非转基因根状茎上的转基因麻风树枝条。 Figure 31 and Figure 3J: grafting in non-transgenic transgenic shoots of rhizomes on leprosy. 白色箭头指示嫁接部位。 White arrows indicate the graft site. 图3K:转基因麻风树开花和结籽。 Figure 3K: transgenic Jatropha flowering and seed. 比例尺表示10mm。 Scale representation 10mm.

[0019] 图4示出hyg-抗性ub1:GFP麻风树植物的PCR分析。 [0019] Figure 4 shows hyg- resistance ub1: GFP Jatropha plant PCR analysis. 泳道-:野生型麻风树属对照；泳道+:pl300-GFP的质粒DNA ;泳道#1-#10来自潮霉素抗性麻风树属的枝条叶。 Lanes -: Jatropha wild type control; lane +: pl300-GFP plasmid DNA; lane # 1- # 10 from hygromycin Jatropha branches leaves.

[0020] 图5A-5P示出T0植物根部(图5B、图5D)、雄花(图5F、图5H)以及受精后3周T1种子(图J、图K、图L、图N、图O、图P)中GFP的表达。 [0020] Figure 5A-5P shows T0 plant roots (Fig. 5B, 5D), male (FIG. 5F, FIG. 5H), and three weeks after fertilization T1 seeds (Figure J, FIG K, Fig L, FIG N, O FIG. FIG P) in GFP expression. 图A、图C、图E、图G、图1和图M是每种植物器官的野生型对照。 Panels A, Fig. C, Figure E, Figure G, FIGS. 1 and M are each plant organ wild type control. 比例尺表示2mm。 Scale representation 2mm.

[0021] 图6示出BASTA-抗性35S: JcWRII麻风树植物的PCR分析。 [0021] Figure 6 shows BASTA- resistance 35S: JcWRII Jatropha plant PCR analysis. 泳道M，DNA ladder ;泳道#1-#7来自BASTA抗性麻风树属枝条叶；泳道_，野生型对照；泳道+，pBA002-MYC-JcWRIl的质粒DNA。 Lane M, DNA ladder; lane # 1- # 7 BASTA resistance from Jatropha branches leaves; lane _ the wild-type control; lanes +, pBA002-MYC-JcWRIl of plasmid DNA.

[0022] 图7示出利用抗HA抗体，表达35S:RcFAH12和35S:JcDGATl的转基因麻风树属植物的叶中RcFAH12和JcDGATI水平的蛋白印迹分析。 [0022] Figure 7 illustrates the use of anti-HA antibodies, the expression of 35S: RcFAH12 and 35S: JcDGATl transgenic Jatropha plant leaves RcFAH12 and JcDGATI levels Western blot analysis. 底部:作为上样对照的RUBL(RUBISC0的大亚基)的考马斯亮蓝染色。 Bottom: As a loading control of RUBL (RUBISC0 large subunit) Coomassie staining.

[0023] 发明详述 [0023] DETAILED DESCRIPTION

[0024] 本发明涉及用于麻风树属的植物，更具体的是麻风树的再生和土壤杆菌介导的转化的方法。 [0024] The present invention relates to a Jatropha plants, more specifically a method jatropha regeneration and Agrobacterium-mediated transformation.

[0025] 在一方面，本发明提供了一种用于再生麻风树属植物的方法。 [0025] In one aspect, the present invention provides a method for the regeneration of Jatropha plants. 根据该实施方案，外植体获得自约5天至约12天龄幼苗的子叶，优选约5-7天龄幼苗。 According to this embodiment, explants obtained from about 5 days to about 12 days old seedlings of cotyledon, preferably about 5-7 days old seedlings. 培养在25°C ±2°C和光照下进行，16h光照(100ymoVm2S)/8h避光循环。 Incubated at 25 ° C ± 2 ° C and light were, 16h light (100ymoVm2S) / 8h dark cycle. 幼苗在组织培养基中生长。 Seedlings grown in tissue culture medium. 利用常规技术将麻风树的种仁表面灭菌，在避光条件下浸于28°C无菌水中过夜。 Using conventional techniques will surface sterilized seeds of Jatropha, under dark conditions at 28 ° C immersed in sterile water overnight. 使不含胚乳的胚在不含激素的发芽培养基上发芽，其中根与培养基接触。 So without endosperm embryo germination germination on hormone-free medium, in which the root contact with the media. 发芽培养基包含1/2浓度的MS无机盐、维生素B5和蔗糖。 Germination medium containing half strength MS salts, vitamins B5 and sucrose. 蔗糖浓度为约5% (w/v) 0发芽培养基还可以包含缓冲液。 Sucrose concentration of about 5% (w / v) 0 germination medium may also contain a buffer. 在一实施方案中，缓冲液为约0.5g/L的2-(4-吗啉代)乙磺酸(MES)，pH为约5.6。 In one embodiment, the buffer was about 0.5g / L of 2- (4-morpholino) ethanesulfonic acid (MES), pH of about 5.6. 发芽培养基用琼脂或植物凝胶(phytogel)固化。 Germination medium solidified with agar gel or plants (phytogel). 培养在25°C ± 1°C和光照下进行，16h光照(IOOymol/m2S) /8h避光循环。 Incubated at 25 ° C ± 1 ° C and light were, 16h light (IOOymol / m2S) / 8h dark cycle.

[0026] 将外植体在愈伤组织形成培养基中避光培养约2周至约3周，优选约3周。 [0026] The explants callus formation in the culture medium in the dark for about two weeks to about three weeks, preferably about 3 weeks. 愈伤组织形成培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-苄氨基嘌呤(6-BA)和1-萘乙酸(NAA)。 Callus formation medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, as well as a plant hormone sucrose 6-benzylaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA) . 柠檬酸的浓度为约10mg/L至约30mg/L,优选约10mg/L。 Citric acid concentration of about 10mg / L to about 30mg / L, preferably from about 10mg / L. 谷氨酰胺的浓度为约150mg/L至约200mg/L,优选约150mg/L。 Glutamine concentration of about 150mg / L and about 200mg / L, preferably from about 150mg / L. 酪蛋白水解物的浓度为约100mg/L。 Casein hydrolyzate concentration is about 100mg / L. 蔗糖的浓度为约3%。 Sucrose concentration is about 3%. 6-BA的浓度为约1.5mg/L。 6-BA concentration of about 1.5mg / L. NAA的浓度为约0.05mg/L。 NAA concentration of about 0.05mg / L. 愈伤组织形成培养基优选还包含MgCl2，其浓度为约0.5g/L至约0.95g/L，优选0.5g/L。 Callus formation medium preferably further comprises MgCl2, at a concentration of about 0.5g / L to about 0.95g / L, preferably 0.5g / L. 愈伤组织形成培养基的pH为约5.8至约6.0。 Callus formation medium pH of about 5.8 to about 6.0. 愈伤组织形成培养基用琼脂或植物凝胶(phytagel)固化,优选植物凝胶,其浓度为约2.5g/L至约3g/L,优选2.5g/L0 Callus formation medium solidified with agar or phytagel (phytagel), preferably a plant gel at a concentration of about 2.5g / L to about 3g / L, preferably 2.5g / L0

[0027] 然后将愈伤组织转移到第一枝条再生培养基，并在光照条件下培养约2周至约3周，优选约3周。 [0027] The callus is then transferred to the first shoot regeneration medium, and cultured under light condition for about 2 weeks to about 3 weeks, preferably about 3 weeks. 第一枝条再生培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、腺嘌呤、蔗糖以及作为植物激素的6-BA和3-吲哚丁酸(IBA)。 First shoot regeneration medium containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, adenine, sucrose, and as a plant hormone 6-BA and 3-indole butyric acid (IBA). 柠檬酸、谷氨酰胺、酪蛋白水解物和6-BA的浓度与愈伤组织形成培养基中的相同。 Citric acid, glutamine, casein hydrolyzate and the concentration of 6-BA and callus formation in the same medium. 腺嘌呤的浓度为约2mg/L至约4mg/L,优选约2mg/L。 Adenine concentration of about 2mg / L to about 4mg / L, preferably from about 2mg / L. IBA的浓度为约0.05mg/L。 IBA concentration is from about 0.05mg / L. 第一枝条再生培养基优选还包含MgCl2,其浓度为约0.5g/L至约0.95g/L，优选0.5g/L。 First shoot regeneration medium preferably further comprises MgCl2, at a concentration of about 0.5g / L to about 0.95g / L, preferably 0.5g / L. 第一枝条再生培养基的pH为约5.8至约6.0。 First shoot regeneration medium has a pH of from about 5.8 to about 6.0. 第一枝条再生培养基用琼脂或植物凝胶固化，优选植物凝胶，其浓度为约2.5g/L至约3g/L,优选2.5g/L。 First shoot regeneration medium solidified with agar or vegetable gel, preferably a plant gel at a concentration of about 2.5g / L to about 3g / L, preferably 2.5g / L.

[0028] 将从愈伤组织再生的任何枝条转移到第二枝条再生培养基，并在光照条件下培养约3周至约4周，优选约4周。 Any branches [0028] from callus regeneration transferred to the second shoot regeneration medium and incubated in the light conditions for about 3 weeks to about four weeks, preferably about 4 weeks. 第二枝条再生培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA、IBA和赤霉酸(GA3)。 The second shoot regeneration medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, sucrose, and as a plant hormone 6-BA, IBA and gibberellic acid (GA3). 柠檬酸、谷氨酰胺、酪蛋白水解物、6-BA和IBA的浓度与第一枝条再生培养基中的相同。 Citric acid, the concentration of glutamine, casein hydrolyzate, 6-BA and IBA is the same as the first shoot regeneration medium. GA3的浓度为约 GA3 concentration of about

0.05mg/L至约0.5mg/L,优选约0.5mg/L。 0.05mg / L to about 0.5mg / L, preferably about 0.5mg / L. 第二枝条再生培养基优选还包含浓度为约0.5g/L的MgCl2。 Second shoot regeneration medium preferably further comprises a concentration of about 0.5g / L of MgCl2. 第二枝条再生培养基的pH为约5.8至约6.0。 Second shoot regeneration medium has a pH of from about 5.8 to about 6.0. 第二枝条再生培养基用琼脂或植物凝胶固化，优选琼脂，其浓度为约6.5g/L至约7g/L，优选7g/L。 Second shoot regeneration medium solidified with agar or vegetable gel, preferably agar, at a concentration of about 6.5g / L to about 7g / L, preferably 7g / L.

[0029] 将不具有再生的枝条的愈伤组织转移到第三枝条再生培养基，并在光照条件下培养约4周至约5周，优选约4周。 [0029] The callus having no regeneration of shoots transferred to the third shoot regeneration medium, and cultured under light condition for about 4 weeks to about 5 weeks, preferably about four weeks. 第三枝条再生培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA和IBA以用于枝条的进一步再生。 The third shoot regeneration medium containing MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and further regenerated as a plant hormone 6-BA and IBA for branches. 柠檬酸、谷氨酰胺、酪蛋白水解物、6-BA和IBA的浓度与第一枝条再生培养基中的相同。 Citric acid, the concentration of glutamine, casein hydrolyzate, 6-BA and IBA is the same as the first shoot regeneration medium. 第三枝条再生培养基优选还包含MgCl2,其浓度为约0.5g/L至约0.95g/L，优选0.5g/L。 The third shoot regeneration medium preferably further comprises MgCl2, at a concentration of about 0.5g / L to about 0.95g / L, preferably 0.5g / L. 第三枝条再生培养基的PH为约5.8至约6.0。 The third shoot regeneration medium PH of about 5.8 to about 6.0. 第三枝条再生培养基用琼脂或植物凝胶固化，优选植物凝胶，其浓度为约2.5g/L至约3g/L，优选2.5g/L。 The third shoot regeneration medium solidified with agar or vegetable gel, preferably a plant gel at a concentration of about 2.5g / L to about 3g / L, preferably 2.5g / L.

[0030] 将在第二枝条再生培养基上再生的枝条转移到枝条伸长培养基，并在光照条件下培养约2周至约3周，优选约2周。 [0030] In the second transfer shoot regeneration medium regenerated shoots to a shoot elongation medium, and cultured under light condition for about 2 weeks to about 3 weeks, preferably about 2 weeks. 枝条伸长培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖以及作为植物激素的6-BA和GA3以用于伸长和芽增殖。 Shoot elongation medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, as well as a plant hormone sucrose 6-BA and GA3 for elongation and shoot proliferation. 柠檬酸、谷氨酰胺和酪蛋白水解物的浓度与第一枝条再生培养基中的相同。 Concentration of citric acid, glutamine and casein hydrolyzate with the same first shoot regeneration medium. 6-BA的浓度为约 The concentration of 6-BA is about

0.3mg/L。 0.3mg / L. GA3的浓度为约0.lmg/L至约0.5mg/L,优选约0.lmg/L。 GA3 concentration of about 0.lmg / L to about 0.5mg / L, preferably about 0.lmg / L. 枝条伸长培养基的pH为约5.8至约6.0。 Shoot elongation medium pH of about 5.8 to about 6.0. 枝条伸长培养基用琼脂或植物凝胶固化，优选琼脂，其浓度为约6.5g/L至约7g/L,优选7g/L。 Shoot elongation medium solidified with agar or vegetable gel, preferably agar, at a concentration of about 6.5g / L to about 7g / L, preferably 7g / L.

[0031] 将伸长的枝条转移到生根培养基，并在光照条件下培养约3周至约4周，优选约4周。 [0031] The elongated shoots are transferred to rooting medium and incubated under light conditions for about 3 weeks to about 4 weeks, preferably about four weeks. 生根培养基包含MS无机盐、维生素B5、蔗糖和IBA。 Rooting medium containing MS salts, vitamins B5, sucrose and IBA. 蔗糖的浓度为约3%。 Sucrose concentration is about 3%. IBA的浓度为约0.07mg/L。 IBA concentration of about 0.07mg / L. 生根培养基的pH为约5.6。 Rooting medium pH of about 5.6. 生根培养基用琼脂或植物凝胶固化，优选浓度为约2.2g/L的植物凝胶。 Rooting medium solidified with agar or vegetable gel, preferably a concentration of about 2.2g / L of the plant gel. 生根后，将幼苗转移到土壤中。 After rooting, the seedlings were transferred to soil. 可选地，可以利用常规技术将伸长的枝条嫁接到麻风树根状茎，以代替转移到生根培养基。 Alternatively, the use of conventional techniques elongated branches grafted to jatropha rhizomes, instead transferred to rooting medium.

[0032] 在第二方面，本发明提供了一种用于土壤杆菌介导的麻风树植物转化的方法。 [0032] In a second aspect, the present invention provides a method for Jatropha curcas plant Agrobacterium-mediated transformation. 根据该实施方案，土壤杆菌介导的麻风树转化利用与上述用于麻风树再生的基本方案相同的基本方案。 According to this embodiment, Agrobacterium-mediated transformation and utilization of Jatropha for Jatropha to the basic program of the same basic regeneration scheme. 利用诸如电穿孔的常规技术将含有所关注的DNA的载体导入土壤杆菌。 DNA vectors utilizing conventional techniques such as electroporation of interest containing the introduced Agrobacterium. 在使用前，利用常规技术培养转化的土壤杆菌细胞。 Prior to use, using conventional techniques culturing the transformed Agrobacterium cells. 根据一种这样的技术，将土壤杆菌细胞接种到添加了卡那霉素和羧节西林(carbicillin)的LB培养基。 According to one such technique, the Agrobacterium cells were inoculated into kanamycin was added and carboxymethyl section amoxicillin (carbicillin) LB medium. 卡那霉素的浓度为约25mg/L至约100mg/L,优选约50mg/L。 Kanamycin concentration of about 25mg / L and about 100mg / L, preferably from about 50mg / L. 羧节西林的浓度为约50mg/L至约100mg/L,优选约100mg/L。 Carboxylic section amoxicillin concentration of about 50mg / L and about 100mg / L, preferably from about 100mg / L. 使土壤杆菌细胞在28°C，250rpm条件下生长过夜。 Agrobacterium cells at 28 ° C, 250rpm grown overnight. 通过离心收集土壤杆菌细胞，再将其重悬浮于添加了蔗糖、葡萄糖、乙酰丁香酮(AS)、6-BA和NAA的液体MS培养基中。 Agrobacterium cells were collected by centrifugation, and then resuspended in added sucrose, glucose, acetosyringone (AS), 6-BA and NAA liquid MS medium. 蔗糖的浓度为约30g/L。 Sucrose concentration of about 30g / L. 葡萄糖的浓度为约10g/L。 Glucose concentration of about 10g / L. AS的浓度为约20mg/L。 AS concentration of about 20mg / L. 6-BA的浓度为约1.5mg/L0 NAA的浓度为约0.05mg/L至约0.lmg/L,优选约0.lmg/L。 The concentration of 6-BA is a concentration of about 1.5mg / L0 NAA is about 0.05mg / L to about 0.lmg / L, preferably about 0.lmg / L.

[0033]为了进行转化，首先将外植体与土壤杆菌细胞共培养，然后转移到愈伤组织形成培养基，随后转移到如上所述的枝条再生培养基、枝条伸长培养基和生根培养基。 [0033] For the transformation, first of all the explants co-cultured with the Agrobacterium cells, then transferred to callus formation medium, then transferred to shoot regeneration medium as described above, shoot elongation medium and rooting medium . 共培养在避光条件下进行约2-3天。 Co-cultured under dark conditions for about 2-3 days. 共培养培养基包含MS无机盐、维生素B5、柠檬酸、谷氨酰胺、酪蛋白水解物、蔗糖、AS以及作为植物激素的6-BA和NAA。 Co-culture medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, sucrose, AS well as plant hormones 6-BA and NAA. 柠檬酸、谷氨酰胺、酪蛋白水解物和蔗糖的浓度与愈伤组织形成培养基中的相同。 Citric acid, glutamine, casein hydrolyzate and sucrose concentrations and the formation of callus in the same medium. AS的浓度为约20mg/L。 AS concentration of about 20mg / L. 6-BA的浓度为约1.5mg/Lo NAA的浓度为约0.05mg/L至约0.lmg/L,优选约0.05mg/L。 6-BA concentration of about 1.5mg / Lo NAA at a concentration of from about 0.05mg / L to about 0.lmg / L, preferably about 0.05mg / L. 共培养培养基还可以包含合适的缓冲液。 Co-culture medium may also contain a suitable buffer. 在一实施方案中，缓冲液为MES。 In one embodiment, the buffer is MES. MES的浓度为约0.5g/L，pH为约5.0至约5.2。 MES concentration of about 0.5g / L, pH of about 5.0 to about 5.2.

[0034] 用于土壤杆菌介导的麻风树转化的愈伤组织形成培养基与用于再生的愈伤组织形成培养基相同，除了它还包含选择剂和土壤杆菌铲除剂。 [0034] The callus used for Agrobacterium-mediated transformation of Jatropha callus culture and regeneration used in the same formation medium, except that it also contains a selection agent and Agrobacterium eradicant. 例如下文所述，选择剂可以为针对包含在转化的土壤杆菌内的标记基因的任何选择剂。 E.g., as described below, the selection agent for the marker gene may be included in the Agrobacterium transformation of any selection agent. 在一实施方案中，选择剂为潮霉素，其浓度为约3mg/L至约5mg/L,优选3.5mg/L。 In one embodiment, the selection agent is hygromycin at a concentration of about 3mg / L to about 5mg / L, preferably 3.5mg / L. 在另一实施方案中，选择剂为草铵膦,其浓度为约lmg/L。 In another embodiment, for glufosinate selection agent, a concentration of about lmg / L. 土壤杆菌铲除剂可以为任何常规铲除剂，例如头孢噻肟(cefotaxinme)等。 Agrobacterium eradicate agents can eradicate any conventional agents, such as cefotaxime (cefotaxinme) and so on. 在一实施方案中，土壤杆菌铲除剂为头孢噻肟，其浓度为约100mg/L至约150mg/L，优选100mg/L。 In one embodiment, the Agrobacterium is cefotaxime eradication agent, a concentration of about 100mg / L and about 150mg / L, preferably from 100mg / L. 用于土壤杆菌介导的转化的愈伤组织形成培养基上的培养在避光条件下进行约2周至约3周,优选约3周。 Callus for Agrobacterium-mediated transformation is formed on the culture medium under the exclusion of light, for about 2 weeks to about 3 weeks, preferably about 3 weeks.

[0035] 然后如上文所述，按照麻风树再生的描述处理愈伤组织，转移到第一枝条再生培养基、第二枝条再生培养基、第三枝条再生培养基、枝条伸长培养基、生根培养基上在光照条件下培养或嫁接。 [0035] Then, as described above, according to the processing described jatropha regenerated calli, transferred to shoot regeneration medium first, second shoot regeneration medium, the third shoot regeneration medium, shoot elongation medium rooting the culture medium or grafting in the light conditions. 用于土壤杆菌介导的麻风树转化的第一枝条再生培养基与用于再生的第一枝条再生培养基相同，除了它还包含选择剂和土壤杆菌铲除剂。 The first branch of Jatropha for Agrobacterium-mediated transformation and regeneration medium for regenerating the first shoot regeneration medium except that it contains a selection agent and Agrobacterium eradicate agent. 例如下文所述，选择剂可以为针对包含在转化的土壤杆菌内的标记基因的任何选择剂。 E.g., as described below, the selection agent for the marker gene may be included in the Agrobacterium transformation of any selection agent. 在一实施方案中，选择剂为潮霉素，其浓度为约3mg/L至约5mg/L,优选3.5mg/L。 In one embodiment, the selection agent is hygromycin at a concentration of about 3mg / L to about 5mg / L, preferably 3.5mg / L. 在另一实施方案中，选择剂为草铵膦，其浓度为约lmg/L。 In another embodiment, for glufosinate selection agent, a concentration of about lmg / L. 土壤杆菌铲除剂可以为任何常规铲除剂，例如头孢噻肟。 Agrobacterium eradicate agents can eradicate any conventional agents, such as cefotaxime. 在一实施方案中，土壤杆菌铲除剂为头孢噻肟，其浓度为约100mg/L至约150mg/L，优选100mg/L。 In one embodiment, the Agrobacterium is cefotaxime eradication agent, a concentration of about 100mg / L and about 150mg / L, preferably from 100mg / L.

[0036]用于土壤杆菌介导的麻风树转化的第二枝条再生培养基与用于再生的第二枝条再生培养基相同，除了它还包含选择剂和土壤杆菌铲除剂。 [0036] The second branch for Agrobacterium-mediated transformation of Jatropha regeneration medium and for reproducing a second shoot regeneration medium of the same, except that it also contains a selection agent and Agrobacterium eradicant. 例如下文所述，选择剂可以为针对包含在转化的土壤杆菌内的标记基因的任何选择剂。 E.g., as described below, the selection agent for the marker gene may be included in the Agrobacterium transformation of any selection agent. 在一实施方案中，选择剂为潮霉素，其浓度为约4mg/L至约5mg/L，优选4mg/L。 In one embodiment, the selection agent is hygromycin at a concentration of from about 4mg / L to about 5mg / L, preferably 4mg / L. 在另一实施方案中，选择剂为草铵膦，其浓度为约lmg/L。 In another embodiment, for glufosinate selection agent, a concentration of about lmg / L. 土壤杆菌铲除剂可以为任何常规铲除剂，例如头孢噻肟。 Agrobacterium eradicate agents can eradicate any conventional agents, such as cefotaxime. 在一实施方案中，土壤杆菌铲除剂为头孢噻肟，其浓度为约100mg/L至约150mg/L，优选100mg/L。 In one embodiment, the Agrobacterium is cefotaxime eradication agent, a concentration of about 100mg / L and about 150mg / L, preferably from 100mg / L.

[0037]用于土壤杆菌介导的麻风树转化的第三枝条再生培养基与用于再生的第三枝条再生培养基相同，除了它还包含选择剂和土壤杆菌铲除剂。 [0037] for Agrobacterium-mediated transformation of Jatropha third shoot regeneration medium and for reproducing the same third shoot regeneration medium, except that it also contains a selection agent and Agrobacterium eradicant. 例如下文所述，选择剂可以为针对包含在转化的土壤杆菌内的标记基因的任何选择剂。 E.g., as described below, the selection agent for the marker gene may be included in the Agrobacterium transformation of any selection agent. 在一实施方案中，选择剂为潮霉素，其浓度为约3mg/L至约5mg/L，优选3.5mg/L。 In one embodiment, the selection agent is hygromycin at a concentration of about 3mg / L to about 5mg / L, preferably 3.5mg / L. 在另一实施方案中，选择剂为草铵膦，其浓度为约lmg/L。 In another embodiment, for glufosinate selection agent, a concentration of about lmg / L. 土壤杆菌铲除剂可以为任何常规铲除剂，例如头孢噻肟。 Agrobacterium eradicate agents can eradicate any conventional agents, such as cefotaxime. 在一实施方案中，土壤杆菌铲除剂为头孢噻肟，其浓度为约100mg/L至约150mg/L，优选100mg/L。 In one embodiment, the Agrobacterium is cefotaxime eradication agent, a concentration of about 100mg / L and about 150mg / L, preferably from 100mg / L.

[0038] 用于土壤杆菌介导的麻风树转化的枝条伸长培养基和生根培养基与用于再生的枝条伸长培养基和生根培养基相同。 [0038] Jatropha for Agrobacterium-mediated transformation of shoot elongation medium and rooting medium for regeneration and shoot elongation medium and rooting the same medium.

[0039] 插入麻风树属植物的DNA(所关注的DNA)对于转化方法不重要。 [0039] DNA insert Jatropha plant (concerning DNA) is not important for the transformation methods. 通常，导入植物的DNA是构建体的一部分。 Typically, DNA is introduced into a plant part constructs. DNA可以是所关注的基因，例如蛋白的编码序列；或者它可以是能够调苄基因表达的序列，例如反义序列、正义抑制序列或miRNA序列。 DNA may be the gene of interest, e.g., protein coding sequence; or it may be able to adjust benzyl gene expression sequences, e.g., an antisense sequence, sense suppression sequence or a miRNA sequence. 构建体通常包含可操作地连接至所关注的DNA的5'端和/或所关注的DNA的3'端的调节区。 '3 end and / or DNA of interest' end of the construct typically comprises regulatory region operably linked to a DNA of interest 5. 含有所有这些兀件的盒在本文中也称为表达盒。 Containing all of these Wu member cartridge also referred to herein as an expression cassette. 在表达盒构建体中，表达盒还可以含有5'如导序列。 In the expression cassette construct, the expression cassette may also contain 5 'leader sequence, such as. 调节区(即启动子、转录调节区和翻译终止区)和/或编码信号锚定的多核苷酸对宿主细胞可以是天然的/类似的，或彼此是天然的/类似的。 Regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and / or anchoring of the encoded signal to the host cell a polynucleotide may be native / analogous, or another natural / similar. 可选地，调节区和/或编码信号锚定的多核苷酸对宿主细胞可以是异源的，或彼此是异源的。 Alternatively, the regulatory regions and / or encoded signals on the anchor polynucleotide may be heterologous to the host cell or heterologous to each other. 参见，美国专利号7，205，453和美国专利申请公开号2006/0218670和2006/0248616。 See, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616. 表达盒还可以含有选择标记基因。 The expression cassette may also contain a selectable marker gene. 参见，美国专利号7，205, 453和美国专利申请公开号2006/0218670和2006/0248616。 See, U.S. Patent No. 7,205, 453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.

[0040] 通常，表达盒包含用于选择转化的细胞的选择标记基因。 [0040] Typically, the expression cassette comprises a selectable marker gene for selection of transformed cells. 选择标记基因用于选择转化的细胞或组织。 Selectable marker gene for selection of transformed cells or tissues. 通常，植物选择标记基因通过合适的基因编码抗生素抗性，所述合适的基因包括至少一组编码抗生素壮观霉素抗性的基因、编码链霉素抗性的链霉素磷酸转移酶(spt)基因、编码卡那霉素或遗传霉素抗性的新霉素磷酸转移酶(nptll)基因、编码潮霉素抗性的潮霉素磷酸转移酶(hpt或aphiv)基因、乙酰乳酸合酶(als)基因。 Typically, plant selection marker gene by appropriate antibiotic resistance genes encoding the appropriate gene include genes encoding at least one group of antibiotics spectinomycin resistance, streptomycin resistance encoding streptomycin phosphotransferase (spt) gene encoding kanamycin or geneticin resistance to neomycin phosphotransferase (nptll) gene, encoding hygromycin resistance, hygromycin phosphotransferase (hpt or aphiv) genes, acetolactate synthase ( als) gene. 或者，植物选择标记基因编码除草剂抗性，如磺酰脲型除草剂、草铵膦、草甘膦、铵、溴苯腈、咪唑啉酮和2，4-二氯苯氧基乙酸(2，4_D)抗性，所述选择标记基因包括编码诸如膦丝菌素或basta的抑制谷氨酰胺合成酶活动的除草剂抗性的基因(例如bar基因)。 Alternatively, the plant selectable marker gene encoding herbicide resistance, such as sulfonylurea herbicides, glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxy acetic acid (2 , 4_D) resistance, the selectable marker genes include genes encoding such as phosphinothricin or basta inhibiting glutamine synthetase activity of herbicide resistance (such as the bar gene). 一般见WO 02/36782、美国专利号7，205, 453及美国专利申请公开号2006/0248616和2007/0143880，以及其中引用的那些参考文献。 See generally WO 02/36782, U.S. Patent No. 7,205, 453 and U.S. Patent Application Publication No. 2006/0248616 and 2007/0143880, and those references cited therein. 这个选择标记基因的列表不意味着限制。 This list of selectable marker genes is not meant to be limiting. 可以使用任何选择标记基因。 You can use any selection marker gene. [0041] 大量启动子可用于实施本发明。 [0041] a large number of promoters can be used in the practice of the invention. 可以基于期望的结果选择启动子。 You can select the desired result based promoter. 也就是说，核酸可以与组成型、组织优选型或其他启动子组合以用于在所关注的宿主细胞中表达。 That is, the nucleic acid may be constitutive, tissue-type or preferably in combination with other promoters for expression in a host cell of interest. 这类组成型启动子包括例如，Rsyn7的核心启动子(W0 99/48338和美国专利号6，072，050)；核心CaMV35S 启动子(Odell et al., 1985);稻米肌动蛋白(McElroy et al., 1990);泛素(Christensen and Quail,1989 和Christensen et al.,1992) ；pEMU(Last et al.,1991) ；MAS(Velten et al.，1984) ;ALS 启动子(美国专利号5，659，026)等。 Such constitutive promoters include, for example, Rsyn7 the core promoter (W0 99/48338 and U.S. Patent No. 6,072,050); the core of the CaMV35S promoter (Odell et al, 1985.); Rice actin (McElroy et . al, 1990); ubiquitin (Christensen and Quail, 1989 and Christensen et al, 1992);. pEMU (Last et al, 1991);. MAS (Velten et al, 1984);. ALS promoter (U.S. Patent No. 5,659,026) and the like. 其他组成型启动子包括例如，在美国专利号5，608，149,5, 608，144,5, 604，121,5, 569，597,5, 466，785、5，399，680,5, 268，463和5，608，142中公开的那些启动子。 Other constitutive promoters include, for example, in U.S. Patent No. 5,608,149,5, 608,144,5, 604,121,5, 569,597,5, 466,785,5,399,680,5, 268,463 and 5,608,142 in those promoters disclosed.

[0042] 其他启动子包括诱导型启动子,尤其是病原体诱导型启动子。 [0042] Other promoters include inducible promoters, particularly pathogen-inducible promoter. 这类启动子包括来自致病相关蛋白(PR蛋白)的那些启动子，其在病原体感染后被诱导；例如，PR蛋白、SAR蛋白、β_1，3-葡聚糖酶、壳多糖酶等。 Such promoters include those promoters from pathogenesis-related proteins (PR proteins), which is induced after pathogen infection; for example, PR proteins, SAR proteins, β_1,3- glucanase, chitinase enzymes. 其他启动子包括在病原体感染部位或其附近局部表达的那些启动子。 Other promoters include pathogen infection site or in the vicinity of the local expression of those promoters. 在其他实施方案中，启动子可以是创伤诱导型启动子。 In other embodiments, the promoter may be a wound-inducible promoter. 在其他实施方案中，可以通过应用外源化学调节物以使用化学调控的启动子来调节植物中基因的表达。 In other embodiments, the composition can be used to chemically regulatable promoter to regulate gene expression in a plant through the application of an exogenous chemical regulator. 启动子可以是化学诱导型启动子，其中化学药品的应用诱导基因表达；或化学抑制型启动子，其中化学药品的应用抑制基因表达。 Promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression; or chemical suppression promoter, wherein the application of chemicals inhibit gene expression. 此外，可以使用组织优选型启动子来靶向特定植物组织中所关注的多核苷酸的增强表达。 In addition, tissue-preferred promoter is used to target enhanced expression of a polynucleotide in a particular plant tissue of interest. 这些启动子中的每一种描述于美国专利号6，506，962,6, 575，814,6, 972，349和7，301，069 以及美国专利申请公开号2007/0061917和2007/0143880。 These promoters in each described in US Patent No. 6,506,962,6, 575,814,6, 972,349 and 7,301,069 and US Patent Application Publication No. 2007/0061917 and 2007/0143880. [0043] 在合适情况下，可以优化所关注的DNA以增加在转化的植物中的表达。 [0043] where appropriate, the DNA of interest can be optimized to increase the expression in the transformed plant. 也就是说，可以使用植物优选的密码子来合成编码序列以提高表达。 That is, the plant can be used to synthesize preferred codons to enhance expression of the coding sequence. 本领域提供合成植物优选基因的方法。 Provides a method for synthesizing plant preferred genes in the art. 参见，例如美国专利号5，380，831.5, 436，391和7，205，453以及美国专利申请公开号2006/0218670 和2006/0248616。 See, for example, US Patent No. 5,380,831.5, 436,391 and 7,205,453 and US Patent Application Publication No. 2006/0218670 and 2006/0248616. [0044] 除非另外指出，本发明的实施使用以下常规技术:化学、分子生物学、微生物学、重组DNA、遗传学、免疫学、细胞生物学、细胞培养和转基因生物学，这都在本领域技术范围内。 [0044] Unless otherwise indicated, the practice of this invention uses the following conventional techniques: chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are in the art technology within range. 参见，例如Maniatis et al., 1982, Molecular Cloning(Cold Spring HarborLaboratory Press，Cold Spring Harbor，New York) ；Sambrook et al., 1989, MolecularCloning，2nd Ed.(Cold Spring Harbor Laboratory Press， Cold Spring Harbor，New York) ；Sambrook and Russell，2001，Molecular Cloning，3rd Ed.(Cold SpringHarbor Laboratory Press， Cold Spring Harbor, New York) ；Ausubel et al.，1992)，Current Protocols in Molecular Biology (John Wiley & Sons，包括目前的更新)；Glover，1985， DNA Cloning(IRL Press, Oxford) ;Russell，1984， Molecular biology ofplants:a laboratory course manual(Cold Spring Harbor Laboratory Press， ColdSpring Harbor，NY) ;Anand，Techniques for the Analysis of Complex Genomes，(Academic Press, New York，1992) ；Guthrie and Fink， Guide to Yeast Geneticsand Molecular Biology (Academic Press, New York，1991) ；Harlow and Lane，1988，Antibodies，(Cold Spring Harbor Laboratory Press，Cold Spring Harbor, New York)；Nucleic Acid Hybridization(BDHames & SJHiggins eds.1984) ；TranscriptionAnd Translation (BDHames & SJHiggins eds.1984) ；Culture Of Animal Cells (R.1.Freshney, Alan R.Liss，Inc.,1987) ；Immobilized Cells And Enzymes(IRL Press，1986) ；B.Perbal, A Practical Guide To Molecular Cloning(1984) ；the treatise，Methods In Enzymology(Academic Press，Inc., NY) ；Methods In Enzymology,Vols.154 and 155 (Wu et al.eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds.，Academic Press，London，1987) ；Handbook OfExperimental Immunology, Volumes 1-1V(DMWeir and CCBlackwell, eds.，1986)；Riott， Essential Immunology,6th Edition， Blackwell Scientific Publications，Oxford, 1988 ；Fire et al.，RNA Interference Technology:From Basic Science toDrug Development, Cambridge University Press， Cambridge，2005 ;Schepers， RNAInterference in Practice，Wiley-VCH，2005 ;Engelke，RNA Interference (RNAi):TheNuts & Bolts of siRNA Technology，DNA Press,2003 ;Gott，RNA Interference,Editing,and Modification !Methods and Protocols(Methods in Molecular Biology)， HumanPress,Totowa,NJ,2004 ；SohaiI,Gene Silencing by RNA Interference-Technology andApplication，CRC，2004。 See, e.g., Maniatis et al, 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York);... Sambrook et al, 1989, MolecularCloning, 2nd Ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York);. Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York); Ausubel et al, 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including the current. updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology ofplants: a laboratory course manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Geneticsand Molecular Biology (Academic Press, New York, 1991); Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York); Nucleic Acid Hybridization (BDHames & SJHiggins eds.1984); TranscriptionAnd Translation (BDHames & SJHiggins eds.1984); Culture Of Animal Cells (R.1.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc, NY.); Methods In Enzymology, Vols.154 and 155 (Wu et al.eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds, Academic Press, London, 1987.); Handbook OfExperimental Immunology, Volumes 1-1V (DMWeir and CCBlackwell, eds, 1986.); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al, RNA Interference Technology: From Basic Science toDrug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNAInterference in Practice, Wiley-VCH, 2005. ; Engelke, RNA Interference (RNAi):! TheNuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification Methods and Protocols (Methods in Molecular Biology), HumanPress, Totowa, NJ, 2004; SohaiI , Gene Silencing by RNA Interference-Technology andApplication, CRC, 2004.

实施例 EXAMPLE

[0045] 本发明参考下列实施例进行描述，提供所述实施例是为了说明而不是意图以任何方式限制本发明。 [0045] The present invention will be described with reference to the following examples, these embodiments are provided for purposes of illustration and are not intended in any way to limit the present invention. 使用本领域公知的标准技术或下文具体描述的技术。 Known in the art using standard techniques or the techniques specifically described below.

[0046] 实施例1材料和方法[0047] 植物材料和培养法:麻风树(L)MD种子获得自印度尼西亚。 [0046] Example 1 Materials and Methods [0047] Plant material and culture: Jatropha (L) MD seeds obtained from Indonesia. 除去外种皮后，将种仁用75% (v/v)乙醇表面灭菌60秒，随后浸入10% (v/v)H202中lh，然后用无菌水淋洗两次，最后于28°C下在避光条件下浸入无菌水中过夜。 After removal of the testa, the seeds with 75% (v / v) ethanol surface sterilized for 60 seconds and then immersed in 10% (v / v) H202 in lh, then rinsed twice with sterile water, and finally at 28 ° C, immersed in sterile water overnight under the exclusion of light. 使不含胚乳的胚在无激素的半浓度Murashige 和Skoog 盐(1/2MS)培养基(Murashige and Skoog, 1962)上发芽，并在组织培养室中于25°C ±2°C、16h光照(100ymol/m2S)/8h避光循环条件下培养，其中根与培养基接触，所述培养基含有维生素B5 (Gamborg et al., 1968)、5g/L鹿糖、0.5g/L2_ (4-吗啉代)乙磺酸(MES)和2.2g/L 植物凝胶(Sigma)，pH 5.6。 So without endosperm embryos in hormone-free half-strength Murashige and Skoog salts (1 / 2MS) medium (Murashige and Skoog, 1962) on the germination and tissue culture chamber at 25 ° C ± 2 ° C, 16h light lower (100ymol / m2S) / 8h dark cycle cultured, wherein the culture medium in contact with the roots, the medium containing vitamin B5 (Gamborg et al., 1968), 5g / L deer sugar, 0.5g / L2_ (4- morpholino) ethanesulfonic acid (MES) and 2.2g / L phytagel (Sigma), pH 5.6.

[0048] 培养基:本发明使用的培养基如下所示。 [0048] Culture medium: culture medium of the present invention is used as follows.

[0049] 培养基I (基础培养基):使用MS主要盐、MS次要盐和维生素B5、10mg/L柠檬酸、150mg/L谷氨酰胺、100mg/L酪蛋白酶促水解物、3% (w/v)鹿糖、0.5g/L MgCl2 (只用于含有植物凝胶的培养基)与植物生长调节剂的组合。 [0049] Medium I (basal medium): Use MS major salts, MS minor salts and vitamins B5,10mg / L citric acid, 150mg / L glutamine, 100mg / L casein hydrolyzate promote, 3% ( w / v) deer sugar, 0.5g / L MgCl2 (only for the gel medium containing a plant) in combination with plant growth regulators. 用IN KOH将培养基I调节为pH 5.8-6.0，用2.5g/L植物凝胶固化，并在121°C下高压灭菌20分钟。 The medium I with IN KOH was adjusted to pH 5.8-6.0, with 2.5g / L phytagel solidified, and autoclaving at 121 ° C for 20 minutes. 在添加到高压灭菌的培养基之前，将所有植物生长调节剂过滤灭菌。 Prior to addition to the autoclaved medium, the plant growth regulator of all sterilized by filtration.

[0050] 共培养培养基:基础培养基加20mg/L乙酰丁香酮(AS)、0.5g/L MES、1.5mg/L6-苄氨基嘌呤(6-BA)和0.05mg/L 1-萘乙酸(NAA)，pH 5.0-5.2。 [0050] Co-culture medium: base medium plus 20mg / L acetosyringone (AS), 0.5g / L MES, 1.5mg / L6- benzylaminopurine (6-BA) and 0.05mg / L 1- naphthaleneacetic acid (NAA), pH 5.0-5.2.

[0051] 愈伤组织形成培养基:基础培养基加1.5mg/L 6-BA,0.05mg/L NAA、作为植物转化选择剂的3.5mg/L潮霉素(hyg, AG scientific, SanDiego, CA)或用于清除土壤杆菌(Agrobacteria)细胞的lmg/L 草铵勝(BASTA,Crescent Chemical,NY)和100mg/L 头抱噻肟(Cef)。 [0051] callus formation medium: basic medium plus 1.5mg / L 6-BA, 0.05mg / L NAA, as the plant transformation selection agent 3.5mg / L hygromycin (hyg, AG scientific, SanDiego, CA ) or to remove Agrobacterium (Agrobacteria) cells lmg / L ammonium grass wins (BASTA, Crescent Chemical, NY) and 100mg / L cephalosporin cefotaxime (Cef). [0052] 枝条再生培养基1:基础培养基加1.5mg/L 6-BA,0.05mg/L 3-吲哚丁酸(IBA)、2mg/L腺嘌呤(腺嘌呤半硫酸盐，SIGMA)、3.5mg/L Hyg或lmg/L草铵膦和Cef 100mg/L。 [0052] shoot regeneration medium 1: basic medium plus 1.5mg / L 6-BA, 0.05mg / L 3- indole butyric acid (IBA), 2mg / L adenine (adenine hemisulfate, SIGMA), 3.5mg / L Hyg or lmg / L glufosinate and Cef 100mg / L.

[0053] 枝条再生培养基I1:基础培养基加1.5mg/L 6-BA,0.05mg/L ΙΒΑ、0.5mg/L赤霉酸(GA3)、4mg/L Hyg或lmg/L草铵膦和100mg/L Cef 100，将植物凝胶变为7g/L琼脂。 [0053] shoot regeneration medium I1: base medium plus 1.5mg / L 6-BA, 0.05mg / L ΙΒΑ, 0.5mg / L gibberellic acid (GA3), 4mg / L Hyg or lmg / L glufosinate and 100mg / L Cef 100, the plants were gel becomes 7g / L agar.

[0054]枝条再生培养基 II1:基础培养基加1.5mg/L 6_BA、0.05mg/L ΙΒΑ、3.5mg/L Hyg或lmg/L 草铵膦和100mg/L Cef。 [0054] shoot regeneration medium II1: basic medium plus 1.5mg / L 6_BA, 0.05mg / L ΙΒΑ, 3.5mg / L Hyg or lmg / L glufosinate and 100mg / L Cef.

[0055] 枝条伸长培养基:基础培养基加0.3mg/L 6-BA,0.lmg/L GA3,将植物凝胶变为7g/L琼脂。 [0055] shoot elongation medium: basic medium plus 0.3mg / L 6-BA, 0.lmg / L GA3, the plant gel becomes 7g / L agar.

[0056] 生根培养基:MS主要盐、MS次要盐和维生素B5、3%蔗糖、0.5g/LMES、0.07mg/LΙΒΑ、2.2g/L 植物凝胶，ρΗ5.6。 [0056] Rooting medium: MS major salts, MS minor salts and vitamins B5,3% sucrose, 0.5g / LMES, 0.07mg / LΙΒΑ, 2.2g / L phytagel, ρΗ5.6.

[0057]培养基 I1:液体MS 培养基，添加了10g/L 葡萄糖、0.5g/L MES、20mg/L AS、1.5mg/L 6-BA、0.lmg/L NAA, pH 5.0-5.2。 [0057] Medium I1: liquid MS medium, add 10g / L glucose, 0.5g / L MES, 20mg / L AS, 1.5mg / L 6-BA, 0.lmg / L NAA, pH 5.0-5.2.

[0058] RNA提取和分析:将新鲜叶或种子组织(IOOmg)在液氮中研磨，并用植物RNA纯化试剂(Invitrogen)提取。 [0058] RNA extraction and analysis: fresh leaf or seed tissue (IOOmg) ground in liquid nitrogen and extracted with plant RNA purification kit (Invitrogen). 利用Nanodrop (Thermo, USA)测量RNA浓度。 Use Nanodrop (Thermo, USA) measuring RNA concentration. DNase处理和逆转录(RT)反应按照(Qu et al.，2007)所述进行。 DNase treated and reverse transcription (RT) reaction in accordance with (Qu et al., 2007) were described.

[0059] 土壤杆菌菌株和载体:通过对麻风树属种子cDNA文库测序来鉴定麻风树WRINKLE I (JcffRI I)和DGATl序列。 [0059] Agrobacterium strains and vectors: Jatropha seeds by sequencing of cDNA libraries to identify Jatropha WRINKLE I (JcffRI I) and DGATl sequences. JcWRI I全长cDNA从麻风树种子第一链cDNA产物扩增，所用的两条引物:5' -AATCGGATCCTAATGAAGAGGTCTTCTGCT-3> (SEQ ID NO:1)和5' -TCATGTTAATT AATCAAACAGAATAGTTACAAGAAA-3，(SEQ ID NO:2)(下划线的核苷酸为酶识别位点)。 JcWRI I jatropha seed from full-length cDNA first strand cDNA product was amplified, two primers were used: 5 '-AATCGGATCCTAATGAAGAGGTCTTCTGCT-3> (SEQ ID NO: 1) and 5' -TCATGTTAATT AATCAAACAGAATAGTTACAAGAAA-3, (SEQ ID NO : 2) (underlined nucleotide for enzyme recognition site). 再将PCR产物插入用BamHI和Pac I处理的pBAOO2-MYC载体以形成pBA002-MYC-JcffRI I。 Then the PCR product was inserted into vector pBAOO2-MYC treated with BamHI and Pac I to form pBA002-MYC-JcffRI I. JcDGATI全长cDNA从麻风树种子第一链cDNA产物扩增，所用的两条引物:5，-CAATATCTAGACCATGACGATTTTGGAGACCACT-3，(SEQ ID NO:3)和5?-TATTAGATCTGGTCTTAATTCAGCATTGCC-3' (SEQ ID NO:4)(下划线的核苷酸为酶识别位点)。 JcDGATI jatropha seed from full-length cDNA first strand cDNA product was amplified, used two primers: 5, -CAATATCTAGACCATGACGATTTTGGAGACCACT-3, (SEQ ID NO: 3) and 5 -TATTAGATCTGGTCTTAATTCAGCATTGCC-3 '(SEQ ID NO:? 4 ) (underlined nucleotide for enzyme recognition site). 再将PCR产物插入用XbaI 和BamHI 处理的pBA002_HA 载体以形成pBA002-JcDGATl_HA。 PCR product was then inserted into XbaI and BamHI using pBA002_HA vector processing to form pBA002-JcDGATl_HA. RcFAHl2 全长cDNA 从蓖麻子种子第一链cDNA产物扩增，所用的两条引物:5' -CAATATCTAGACCATGGGAGGTGGTGGTC_3' (SEQ ID NO:5)和5，-TGTAGGATCCGGATACTTGTTCCGGTACCAG-3，(SEQ ID NO:6)(下划线的核苷酸为酶识别位点)。 RcFAHl2 full-length cDNA from the first strand cDNA product castor seed amplification, two primers were used: 5 '-CAATATCTAGACCATGGGAGGTGGTGGTC_3' (SEQ ID NO: 5) and 5, -TGTAGGATCCGGATACTTGTTCCGGTACCAG-3, (SEQ ID NO: 6) ( underlined nucleotide for enzyme recognition site). 再将PCR产物插入用XbaI和BamHI处理的pBA002_HA载体以形成pBA002-RcFAH12-HA。 PCR product was then inserted into XbaI and BamHI using pBA002_HA vector processing to form pBA002-RcFAH12-HA. 通过电穿孔(BIO-RAD，CA，USA)将载体导入土壤杆菌菌株AGLl。 By electroporation (BIO-RAD, CA, USA) vector into Agrobacterium strain AGLl. 使用转化的土壤杆菌细胞接种液体LB培养基，并在28°C，250rpm条件下生长过夜至最终OD595=0.7-1，所述培养基添加了50mg/L卡那霉素(用于pCAMBIA 1300-GFP)或50mg/L壮观霉素(spectimycin)(用于pBA002_MYC_WRI I, pBA002-JcDGATl-HA, pBA002-RcFAH12-HA)和100mg/L羧节西林。 Agrobacterium cells were used to inoculate liquid LB medium transformed, and 28 ° C, conditions under 250rpm grown overnight to a final OD595 = 0.7-1, the culture medium was added 50mg / L kanamycin (for pCAMBIA 1300- GFP) or 50mg / L spectinomycin (spectimycin) (for pBA002_MYC_WRI I, pBA002-JcDGATl-HA, pBA002-RcFAH12-HA) and 100mg / L carboxymethyl section amoxicillin. 通过在20°C下以4200rpm离心10min来收集土壤杆菌细胞。 By 20 ° C by centrifugation at 4200rpm 10min with Agrobacterium cells were collected. 在共培养前，将细胞沉淀用培养基II重悬浮，并调节至OD595为0.25-0.35 (仅土壤杆菌AGL1)。 Before co-culture, the cell pellet was resuspended with medium II, and adjusted to OD595 0.25-0.35 (only Agrobacterium AGL1).

[0060] 麻风树叶子DNA的分离和基因型分析:将50mg新鲜麻风树叶子在液氮中破碎，并在添加400yL CTAB 提取缓冲液(IOOmM Tris, pH 8.0 ；1.4M NaCl ；20mM EDTA ；2%十六烷基三甲基溴化铵(CTAB))后在65°C下孵育I小时。 [0060] Isolation and genotyping DNA from leaves leprosy: leprosy 50mg of fresh leaves in liquid nitrogen and broken, and in addition 400yL CTAB extraction buffer (IOOmM Tris, pH 8.0; 1.4M NaCl; 20mM EDTA; 2% cetyl trimethyl ammonium bromide (CTAB)) after incubation at 65 ° C under I hour. 用预冷的氯仿提取两次后，将DNA用异丙醇沉淀并通过离心收集。 After pre-cooling the chloroform extracted twice, and the DNA was precipitated with isopropanol and collected by centrifugation. 对于潮霉素基因基因型分析，所用的引物为hyg5:5' -CGATGTAGGAGGGCGTGG-3' (SEQ ID NO:7), hyg3:5' -ACTTCTACACAGCCATCGGTCC-3' (SEQ ID NO:8) „对于bar基因基因型分析，所用的引物为bar5:5'-GTCTGCACCATCGTCAACC-3' (SEQ ID NO:9)，bar3:5' -GAAGTCCAGCTGCCAGAAAC-3 ' (SEQ ID NO: 10)。 [0061] 抗体和蛋白凝胶印迹分析:由Yin Zhongcao博士的实验室制备麻风树毒蛋白抗体。按照先前的描述(Qu et al., 2007)进行蛋白印迹分析。通过12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳来分离总植物蛋白。使用ECL过氧化物酶偶联的驴抗兔免疫球蛋白G作为二抗。使用ECL蛋白印迹检测试剂(GE healthcare)将免疫反应性条带显影。 For the hygromycin gene genotyping, the primers used were hyg5: 5 '-CGATGTAGGAGGGCGTGG-3' (SEQ ID NO: 7), hyg3: 5 '-ACTTCTACACAGCCATCGGTCC-3' (SEQ ID NO: 8) "for the bar gene genotyping, the primers used were bar5: 5'-GTCTGCACCATCGTCAACC-3 '(SEQ ID NO: 9), bar3: 5' -GAAGTCCAGCTGCCAGAAAC-3 '(SEQ ID NO: 10) [0061] Antibody and protein gels. Western blot analysis: curcin antibody preparation by Dr. Yin Zhongcao laboratory as previously described in (Qu et al, 2007.) were analyzed by Western blotting 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis. total plant protein isolated using ECL peroxidase conjugated donkey anti-rabbit immunoglobulin G as the secondary antibody using ECL Western blotting detection reagents (GE healthcare) The immunoreactive bands were developed.

[0062] 实施例2麻风树子叶外植体转化 [0062] Example 2 jatropha cotyledon explants transformed

[0063] 图1示出土壤杆菌介导的麻风树属转化方法，其在本实施例中进一步详细描述。 [0063] Figure 1 shows the Agrobacterium-mediated transformation method Jatropha, its examples are described in further detail in this embodiment. 图2示出用于本实施例的土壤杆菌转化载体。 Figure 2 shows the present embodiment Agrobacterium transformation vectors.

[0064] 共培养:将5-7天龄幼苗的子叶(实施例1 ;图3A)切成小块(5 X 5mm)，并用载有靶表达盒的土壤杆菌细胞(实施例1)在20ml培养基II中于25°C下孵育10_20min。 [0064] Co-culture: The cotyledons 5-7 day old seedlings (Example 1; FIG. 3A) cut into small pieces (5 X 5mm), and with a target expression cassette containing the Agrobacterium cells (Example 1) in 20ml II medium at 25 ° C incubated 10_20min. 然后将外植体转移到共培养培养基，在22°C下避光保持2-3天。 The explants were then transferred to a co-cultivation medium, at 22 ° C under the dark for 2-3 days. 共培养后，将外植体用无菌水淋洗若干次，之后用300mg/L头孢噻厢(cefotaxine)洗漆一次。 After co-cultivation, explants are rinsed several times with sterile water, then washed once with paint 300mg / L cefotaxime compartment (cefotaxine). 将子叶组织吸水干燥，这通过将它们置于灭菌的纸垫上以去除过量的表面水完成。 The cotyledon tissue absorbent sulfate, which are sterilized by placing them in the paper pad to remove excess surface water is complete.

[0065] 潮霉素抗性或草铵膦抗性愈伤组织的选择:共培养后，将外植体接种于愈伤组织形成培养基板上，并转移到避光条件，在25°C ±1°C下保持3周。 [0065] hygromycin or glufosinate resistant callus selection: After co-culture, the board will form the medium was inoculated explants callus and transferred to dark conditions, at 25 ° C ± at 1 ° C for 3 weeks. 未转化和转化的外植体形成愈伤组织(图3B)，而一些在培养时形成愈伤组织(图3B，右栏；图3C)。 Untransformed and transformed explants formed callus (Fig. 3B), and some callus formation in culture (Fig. 3B, right column; Fig. 3C). 当在避光条件下培养时，未转化的外植体通常会变为褐色。 When cultured under dark conditions, non-transformed explants often browned.

[0066] 枝条再生:将具有新出现的潮霉素抗性或草铵膦抗性愈伤组织的外植体转移到枝条再生培养基I，在25°C、16h光照(100 μ HioVm2S1VSh避光循环条件下保持3周。本文描述的方法基于通过添加腺嘌呤从转化的愈伤组织的直接枝条诱导。而本文使用术语“再生”来描述从这类转化的愈伤组织的完整植物的再创造(re-creation)。尽管6_BA(6_苄基腺嘌呤)对枝条再生具有类似的作用，但在本文所述方法中其并不用于这一特定步骤。此外，更高或更低的浓度，过早或过迟的腺嘌呤添加会使枝条再生更困难或异常的发芽(shooting)。在替代性实施方案中，获得枝条再生的方法包括添加2mg/L加普通6-BA或其他腺嘌呤衍生物，例如2-异戊烯腺嘌呤。在这个时间段中，将从愈伤组织再生的任何枝条(约40-50% )转移到枝条再生培养基II (图3D)。将不具有再生的枝条的愈伤组织转移到枝条再生培养基III，以用于枝条的进一步培养和再生。 [0066] shoot regeneration: having explants hygromycin or glufosinate resistant callus emerging transferred to shoot regeneration medium I, at 25 ° C, 16h light (100 μ HioVm2S1VSh dark under cyclic conditions remain three weeks. The methods described herein based on direct shoot induction from callus transformed by the addition of adenine. The article uses the term "regeneration" to describe this kind of whole plants from the transformed callus re-creation (re-creation). Although 6_BA (6_ benzyladenine) on shoot regeneration have a similar effect, but the methods described herein which are not used in this particular step. In addition, higher or lower concentrations, adenine added early or too late will shoot regeneration more difficult or abnormal germination (shooting). In an alternative embodiment, to obtain shoot regeneration method comprises adding 2mg / L 6-BA or add another ordinary adenine derivative thereof, such as 2-isopentenyl adenine. In this time period, any shoots regenerated from the callus (approximately 40-50%) was transferred to shoot regeneration medium II (Fig. 3D). will not have regenerated branches of callus was transferred to shoot regeneration medium III, for further training and regeneration branches.

[0067] 枝条伸长:4周后，将再生的枝条转移到枝条伸长培养基上以用于伸长和芽增殖(图3E)。 [0067] shoot elongation: After four weeks, the regenerated shoots are transferred to shoot elongation medium for shoot elongation and proliferation (Fig. 3E).

[0068] 生根:将长度为约2.5cm的伸长的枝条根植于生根培养基(图3F)。 [0068] root: the length of the elongated shoots about 2.5cm rooted in rooting medium (Fig. 3F). 通常需要超过I个月来获得如图3F所示的根。 Usually takes more than I month to obtain the root as shown in Figure 3F. 我们的生根方案可以提供约45%的高生根效率(图3G)，并且长度大于IOmm的一个主根可以成功转移到土壤中并获得大于90%的存活率(图3H)。 Our rooting program could provide about 45% of the high rooting efficiency (Fig. 3G), and a length greater than a main root IOmm can successfully transferred to soil and get greater than 90% survival rate (Fig. 3H).

[0069] 嫁接:还可以将伸长的转基因枝条用作接穗来嫁接到非转基因根状茎。 [0069] Grafting: can also be used as an elongation of transgenic shoots come scion grafted onto non-transgenic rhizomes. 挑选健康和生长旺盛的麻风树植株作为根状茎。 Selection of healthy and vigorous growth of Jatropha plants as rhizomes. 将接穗和根状茎切至形成层区，从而来自这两者的韧皮组织会在接合后连接。 The scion and rhizomes cut to form a layer region, thus from both phloem tissue will, after joining the connection. 用封口膜(parafilm)缠绕嫁接接头(joint),并用胶带固定。 With parafilm (parafilm) wound grafting joint (joint), and secured with tape. 将嫁接的麻风树植株在弱光强度(28°C、16h光照(50 μ mol/niS1) /8h避光循环)和85%湿度条件下保持7天。 Jatropha plants will be grafted in low light intensity (28 ° C, 16h light (50 μ mol / niS1) / 8h dark cycle) and at 85% humidity for 7 days. 嫁接到非转基因根状茎上的转基因麻风树枝条示于图31和3J。 Leprosy branches grafted onto transgenic article on non-GM rhizomes are shown in Figure 31 and 3J. 转基因麻风树植株在温室中表现出正常的开花和结籽(图3K)。 Transgenic Jatropha plants in the greenhouse showed normal flowering and seed set (Figure 3K).

[0070] 实施例3转基因麻风树的转化和分析 3 transgenic Jatropha transformation and analysis [0070] EXAMPLE

[0071] 利用本发明方法转化麻风树属以及从转化的细胞再生BASTA或潮霉素(hygmycin)植株的实施例将在下文详细描述。 [0071] The present invention is transformed by the method of Jatropha from transformed cells and regeneration of BASTA or hygromycin (hygmycin) Example plants will be described below in detail. 简单来说，该方法要求提供异源DNA构建体，其包含植物启动子、编码赋予诸如BASTA或潮霉素耐受性的选择优势的蛋白的DNA序列以及3'非翻译转录终止子区。 In simple terms, this method requires providing a heterologous DNA construct comprising a plant promoter, such as a given encoding hygromycin or BASTA resistance to DNA sequence of the protein and the selective advantage of the 3 'untranslated transcriptional terminator region. DNA构建体包含可操作地连接到编码赋予BASTA或潮霉素耐受性的蛋白的DNA编码区的植物启动子，和3'终止信号。 DNA construct comprising a polynucleotide operably linked to the encoding hygromycin or BASTA imparting tolerance protein coding region of DNA plant promoter, and 3 'termination signal. 优选地，DNA构建体编码额外的所关注的基因。 Preferably, DNA encodes an additional gene of interest construct. 例如，DNA构建体可以包含这样的基因，其表达导致转化的植物中增加的产量或改变的脂肪酸含量。 For example, DNA construct may comprise such a gene, whose expression leads to the plant transformed in increased yield or altered fatty acid content.

[0072] 在下面的实例中，从用包含GFP基因的DNA构建体转化的组织获得了表达绿色荧光蛋白(GFP)的潮霉素耐受麻风树属植物。 [0072] In the following examples, was expressed green fluorescent protein (GFP) hygromycin tolerant Jatropha plants from tissue transformed with a DNA construct containing the GFP gene. 在如下所述的一些实例中，使用这种GFP基因和可以用作容易筛选的标记的其他基因，例如GUS、萤光素酶基因，仅仅因为可以容易地在转化的植物中检测到它们的表型。 In some examples described below, the use of such may be used as the GFP gene and other easily screenable marker genes, such as GUS, luciferase gene, simply because it can be readily detected in the transformed plants to their table type. 可以合理的预期通过使用标准分子生物学技术产生的DNA构建体，可以使用本发明来获得表达几乎任何其他基因的麻风树属植物。 Can be reasonably expected by the use of standard molecular biology techniques to produce a DNA construct, the present invention may be used to obtain expression of virtually any other gene jatropha genus. 在替代性实施方案中，用于获得转化的麻风树属植物的方法涉及两种DNA构建体的共转化，其中一种DNA构建体包含选择标记，例如BASTA或潮霉素耐受标记；而另外一种DNA构建体则包含所关注的基因。 In an alternative embodiment, for obtaining a transformed plant Jatropha method involves two co-transformed with DNA constructs, one of the DNA construct comprises a selectable marker, e.g., hygromycin or BASTA tolerance marker; while the other A DNA construct contains the gene of interest.

[0073] 潮霉素抗性的推定(putative)GFP转基因麻风树属植物的转化和枝条再生根据实施例2描述的方法实现。 [0073] hygromycin presumption (putative) GFP transgenic Jatropha plant transformation and shoot regeneration realized according to the method described in Example 2. 利用实施例1描述的方法提取潮霉素抗性枝条的基因组DNA。 By the method described in Example 1 Extraction of the hygromycin resistance branches genomic DNA. 利用潮霉素基因引物对(SEQ ID NO:7和SEQ ID NO:8)进行基因型分型。 Use hygromycin gene primers (SEQ ID NO: 7 and SEQ ID NO: 8) were genotyped. 10个事件中的9个是PCR阳性的，而非转化对照在CK泳道没有表现出条带(图4)。 10 events 9 is PCR positive, rather than the transformation control in CK lane showed no band (Figure 4). 当用紫外光激发转基因麻风树属根时，快速筛选GFP表达(图3B)。 When excited by ultraviolet transgenic Jatropha roots, rapid screening of GFP expression (Figure 3B). 荧光表示，该新导入的GFP表达盒在Ttl麻风树属植物中表达。 Fluorescent said the newly imported GFP expression cassette is expressed in Ttl Jatropha plants. 在ub1:GFP转基因麻风树属开花后，我们检查了花序中的GFP表达。 In ub1: GFP transgenic Jatropha genus after flowering, we examined the expression of GFP in the inflorescence. 雄花特别是花粉具有一些弱绿色荧光(图5H)。 Especially male pollen has some weak green fluorescence (Fig. 5H). 我们还检查了受精后3周的种子中的GFP表达。 We also examined the expression of GFP three weeks after fertilization of the seeds. 在整个转基因T1种子中，从外部(图5N，图50)或内部(图5P)可以观察到强GFP表达。 Throughout the transgenic T1 seeds, from the outside (Fig. 5N, FIG. 50) or internal (Figure 5P) may be the observation of strong GFP expression. 这表明在转基因麻风树属的后代种子中GFP也较好地表达。 This shows that are better expressed in the progeny of transgenic Jatropha seeds of GFP.

[0074] 甘油三酯(TAG)是植物将太阳能转化为化学能后主要能量储存形式。 [0074] triglycerides (TAG) is the main plant will convert solar energy into chemical energy after the energy stored in the form. 但当植物使用糖酵解的变体作为中间产物时，其合成的标准生化途径被认为是相当浪费的。 However, when using a plant a variant of glycolysis as an intermediate product, which was synthesized by standard biochemical pathway is considered to be quite wasteful. WRINKLED I (WRIl)是AP2/EREB家族的转录因子，其对种子保存过程的更具体方面，特别是糖变体的转录控制转化为TAG具有影响，因此在控制种子油含量方面表现出非常重要的作用。 WRINKLED I (WRIl) is AP2 / EREB family of transcription factors, which during storage of the seed more specific aspects, in particular sugar transcript variant control into TAG having influence, and therefore in the control of seed oil content showed a very important aspect of effect. 花椰菜花叶病毒35S启动子控制下的拟南芥(Arabidopsis)WRIl cDNA表达导致种子油含量的10-20%增加。 Cauliflower mosaic virus 35S promoter in Arabidopsis under the control of (Arabidopsis) WRIl cDNA expression leads to 10-20% increase in seed oil content. 此外，WRINKLED1 cDNA的异位表达导致发育幼苗中甘油三酯的积累(Cernac and Benning, 2004) 0我们认为，麻风树属WRIl基因在麻风树属中的异位表达会导致更高的含油量。 In addition, WRINKLED1 cDNA ectopic expression leads to the accumulation of triglycerides in the development of seedlings (Cernac and Benning, 2004) 0 we believe WRIl Jatropha Jatropha ectopic gene expression leads to higher oil content. 此外，当供应糖时，转基因幼苗可以发育为胚或胚样产油器官，就像脂质反应器，可以为其供应含糖液体底物以用于营养器官中组成型CaMV 35S启动子-驱动的WRIl的强表达。 In addition, when the supply of sugar, the transgenic embryo or seedling can develop into embryoid-like oil-producing organ, like lipid reactor, the liquid may be for the supply of sugar substrates for vegetative organs constitutive CaMV 35S promoter - driven The strong expression of WRIl. [0075] 我们克隆了麻风树属WRIl的全长cDNA(JcWRIl)，其从麻风树属种子RT-PCR产物PCR扩增，这使用了用于源自麻风树属种子cDNA文库测序的JcWRIl克隆序列的PCR引物(SEQ ID NO:1 和SEQ ID NO:2)。 [0075] We cloned the full-length Jatropha WRIl cDNA (JcWRIl), which belongs to the seed RT-PCR products amplified by PCR from jatropha, which used the cloned sequence for JcWRIl from Jatropha seed cDNA library sequencing PCR primers (SEQ ID NO: 1 and SEQ ID NO: 2). 全长JcWRI I cDNA 序列示于SEQ ID NO: 11。 Full-length JcWRI I cDNA sequence is shown in SEQ ID NO: 11. 构建了具有受CaMV 35S启动子控制的JcWRIl cDNA的过表达载体(pBA002_MYC-JcWRIl)，并将其转化入土壤杆菌AGLl菌株。 Having JcWRIl cDNA was constructed by the control of the CaMV 35S promoter overexpression vector (pBA002_MYC-JcWRIl), and transformed into the Agrobacterium strain AGLl. 可以利用MYC标签抗体来检测预计的6XMYC标签融合WRII。 MYC tag antibody can be used to detect the expected 6XMYC tag fusion WRII. BASTA抗性的推定JcWRIl过表达转基因麻风树属植物的转化和枝条再生根据实施例2描述的方法实现。 BASTA resistant JcWRIl putative overexpression transgenic Jatropha plant transformation and regeneration of shoots embodiments described implement according to the method of Example 2. 利用实施例1描述的方法提取潮霉素抗性枝条的基因组DNA。 By the method described in Example 1 Extraction of the hygromycin resistance branches genomic DNA. 利用BASTA基因引物对(SEQ ID NO:9和SEQ ID NO:10)进行基因型分析。 Use BASTA gene primers (SEQ ID NO: 9 and SEQ ID NO: 10) were genotyped. 我们测试的所有事件都是PCR阳性的，而非转化对照在CK中没有表现出条带(图6)。 All events are we tested PCR positive, rather than the transformation control in CK showed no bands (Figure 6).

[0076] 植物和动物二酰甘油酰基转移酶(DGAT)负责将新生脂肪酸包装入TAG，其随后在从内质网出芽(bud off)的油体中积累。 [0076] Plant and animal diacylglycerol acyltransferase (DGAT) is responsible for packaging the nascent fatty acids into TAG, which subsequently from the endoplasmic reticulum budding (bud off) accumulated in oil bodies. 已经证实植物I型DGAT(DGATl)基因对于种子的含油量有重要贡献，这通过过表达和突变下调研究均得到证实(Zou et al.,2999 ；Jako etal.，2001)。 Type I has been demonstrated plant DGAT (DGATl) gene for the oil content of the seeds has an important contribution, which reduced by overexpression and mutation studies have confirmed (Zou et al, 2999;. Jako etal, 2001.). 我们认为，麻风树属DGATl基因在麻风树属中的异位表达会导致更高水平的含油量。 We believe that Jatropha DGATl ectopic expression of genes in Jatropha oil content will result in a higher level.

[0077] 我们根据DGATl克隆序列，利用PCR引物(SEQ ID NO:3和SEQ ID NO:4),从麻风树属种子RT-PCR产物克隆了全长麻风树属DGATl cDNA。 [0077] According DGATl we cloned sequences, using PCR primers (SEQ ID NO: 3 and SEQ ID NO: 4), the genus Jatropha seeds from RT-PCR products were cloned the full length of Jatropha DGATl cDNA. 全长JcDGATI cDNA序列示于SEQ ID NO:13o构建具有受CaMV 35S启动子控制的JcDGATl cDNA的过表达载体(pBA002-JcDGATl-HA)，并将其转化入土壤杆菌AGLl菌株。 Full-length JcDGATI cDNA sequence is shown in SEQ ID NO: 13o JcDGATl cDNA construct with the CaMV 35S promoter by the control of the over-expression vector (pBA002-JcDGATl-HA), and transformed into the Agrobacterium strain AGLl. 可以利用HA标签抗体来检测预计的3XHA标签融合DGAT1。 HA tag antibody can be used to detect the expected 3XHA tag fusion DGAT1. BASTA抗性的推定JcDGATl转基因麻风树属植物的转化和枝条再生根据实施例2描述的方法实现。 BASTA resistant putative JcDGATl transgenic Jatropha plant transformation and shoot regeneration implement a methodology described in Example 2. 利用实施例1描述的方法，通过基于HA抗体的蛋白印迹证实了35S-JcDGATl表达(图7)。 By the method described in Example 1 by Western blot-based HA antibody confirmed 35S-JcDGATl expression (Fig. 7). 在3个转基因麻风树属泳道的2条泳道中可以观察到HA特异性条带。 In the three transgenic Jatropha lane two lanes can be observed in HA-specific band.

[0078] 在植物来源的工业原料的许多情况和应用中可以使用植物油(和它们的衍生物)。 [0078] can be used vegetable oils (and their derivatives) in many cases the application of industrial raw materials and a plant-derived. 与不可再生的石油相比，可再生的性质使得它们对于环境关注是个问题的全损耗应用的许多工业应用特别具有吸引力。 Compared with the non-renewable oil, renewable nature makes them environmental concern for many industrial applications, the full application of the problem of loss particularly attractive. 蓖麻(Ricinus communis)油在运输、化妆品和医药以及制造工业中有很多应用。 Castor bean (Ricinus communis) oil has many applications in transportation, cosmetics and pharmaceuticals, and manufacturing industries. 蓖麻油含有超过90%的蓖麻油酸，其是单不饱和的18碳脂肪酸。 Castor oil containing more than 90% of ricinoleic acid, which is a mono-unsaturated 18 carbon fatty acids. 它是不寻常的，因为它在第十二碳上具有羟基功能团。 It is unusual because it has a hydroxyl functional groups in the twelfth carbon. 这个功能团造成蓖麻油酸(和蓖麻油)不寻常的极性(http colon en dot wikipedia dot org slash wiki slash Castor_oil)。 The functional groups cause ricinoleic (and castor oil) unusual polarity (http colon en dot wikipedia dot org slash wiki slash Castor_oil). 一种特定的酶:脂肪酸羟化酶12(FAH12)负责加入羟基以代替正常的FAD2功能来在第十二碳上引入不饱和带(van de Loo et al., 1995)。 A specific enzyme: fatty acid hydroxylase 12 (FAH12) responsible is added to replace the normal FAD2 hydroxy function is introduced in the unsaturated zone in the twelfth carbon (van de Loo et al, 1995.). 与缺乏羟基的其他种子油相比，蓖麻油的价格更高。 Compared with the lack of a hydroxyl group other seed oils, castor higher prices. 尽管对蓖麻油的需求广泛，但是该作物的种植受到限制，这是由于毒素(蓖麻毒蛋白)和变应原性蛋白的存在，因此蓖麻油的成本是比较高的。 Despite the widespread demand for castor oil, but the cultivation of the crop is limited, which is due to toxins (ricin) and variable should exist immunogenic proteins, and therefore the cost of castor oil is relatively high. 转基因外源FAH12可以在拟南芥种子中产生羟基蓖麻油(Lu et al.，2006)。 Transgenic exogenous FAH12 can generate hydroxyl castor oil in Arabidopsis seeds (Lu et al., 2006). 我们认为，蓖麻子FAH12基因在麻风树属中的异位表达会引起蓖麻油的产生。 We believe that the ectopic expression of genes in castor FAH12 Jatropha in the cause of castor oil production.

[0079] 我们根据FAH12 CDS序列，利用PCR引物(SEQ ID NO:5和SEQ ID N0:6)，从蓖麻子种子RT-PCR产物克隆了全长蓖麻子FAH12 cDNA (RcFAHl2)。 [0079] We according FAH12 CDS sequence, using PCR primers (SEQ ID NO: 5 and SEQ ID N0: 6), from castor bean seeds RT-PCR product was cloned full length castor bean FAH12 cDNA (RcFAHl2). 全长RcFAHl2 cDNA序列示于SEQ ID NO:15o构建了具有受CaMV 35S启动子控制的RcFAH12 cDNA的过表达载体(pBA002-RcFAH12-HA)，并将其转化入土壤杆菌AGLl菌株。 Full-length RcFAHl2 cDNA sequence is shown in SEQ ID NO: 15o constructed by having the control of the CaMV 35S promoter RcFAH12 cDNA overexpression vector (pBA002-RcFAH12-HA), and transformed into the Agrobacterium strain AGLl. 可以利用HA标签抗体来检测预计的3XHA标签融合RcFAH12。 HA tag antibody can be used to detect the expected 3XHA tag fusion RcFAH12. BASTA抗性的推定RcFAH12转基因麻风树属植物的转化和枝条再生根据实施例2描述的方法实现。 BASTA resistant putative RcFAH12 transgenic Jatropha plant transformation and shoot regeneration implement a methodology described in Example 2. 利用实施例1描述的方法，通过基于HA抗体的蛋白印迹证实了35S-RcFAH12表达。 By the method described in Example 1 by Western blot-based HA antibody confirmed the expression of 35S-RcFAH12. 在7个转基因麻风树属泳道的5条泳道中可以观察到HA特异性条带(图7)。 In seven transgenic Jatropha lane five lanes can be observed in HA-specific band (Fig. 7). 两个泳道#2和#5，具有非常高的FAH12-HA融合表达蛋白水平。 Two lanes # 2 and # 5, with a very high FAH12-HA fusion protein levels. [0080] 在描述本发明的背景(尤其是所附权利要求的背景)下，术语“一个(a)”和“一个(an)”及“这个(the)”以及类似的所指对象的使用应理解为包括单数和复数，除非在本文中另外指出或根据背景明显抵触。 "A (a)" and "a (an)" and "this (the)" and similar referents used in the [0080] In the description of the background of the present invention (especially in the appended claims the context), the term should be understood to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted according to background. 除非另作说明，术语“包括”、“具有”、“包含”和“含有”应理解为开放式的术语(即，表示“包括但不限于”)。 Unless otherwise indicated, the term "including", "having", "contains" and "contain" should be understood as an open-ended terms (ie, meaning "including, but not limited to"). 除非本文另外指出，本文数值范围的描述仅旨在作为分别涉及属于所述范围中的每个单独值的简写方法，每个单独值被包括进说明书就如同它是在本文中单独描述的。 Unless otherwise indicated herein, describe ranges of values ​​herein are merely intended as a shorthand method belongs to the range of values ​​are involved in each individual, each individual value is included into the specification as if it is herein described separately. 例如，如果公开了范围10-15，那么11、12、13和14也被公开。 For example, if the range 10-15 is disclosed, then 11, 12 and 14 are also disclosed. 本文描述的所有方法可以按照任何合适的顺序进行，除非本文另外指出，或另外根据背景明显抵触。 All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted addition, according to the background. 除非另有要求，本文提供的任何和所有实例或示例性语言(如，“例如”)的使用仅仅是为了更好地说明本发明，而不是为了给本发明的范围做出限制。 Unless otherwise required, any and all examples, or exemplary language provided herein (e.g., "such as") is intended merely to better illuminate the invention and are not intended to limit the scope of the present invention is made. 说明书中没有语言应理解为指示任何未要求的要素是实施本发明必需的。 No language in the specification should be construed as indicating any non-claimed element is essential embodiment of the present invention.

[0081] 应理解本发明的方法和组合物可以并入各种形式的实施方案，本文只公开了其中一些。 [0081] should be understood that the methods and compositions of the invention may be incorporated in various forms of embodiments, some of which are disclosed herein only. 本文描述了本发明的实施方案，包括本发明人已知用于实施本发明的最佳方式。 This article describes the embodiments of the present invention, including the best mode known to the inventors for carrying out the invention. 在阅读上述说明书后，这些实施方案的变化对于本领域普通技术人员来说是显而易见的。 After reading the foregoing specification, variations of these embodiments to those of ordinary skill is obvious. 本发明人预期有经验的技术人员酌情使用这样的变化，并且本发明人认为可以在除本文的具体描述之外实施本发明。 The inventors expect skilled experienced such variations as appropriate, and the inventors believe that the present invention may be practiced other than as specifically described herein except. 因此，本发明包括适用法律许可的所附权利要求中叙述的主题的所有修饰和等同物。 Accordingly, the present invention includes applicable laws relating to permitted recited in the appended claims all modifications and equivalents thereof. 此外，本发明包括上述元素以其所有可能变化的任何组合，除非本文另外指出，或根据背景明显抵触。 Further, the present invention includes any combination of the above-described elements in all its possible variations, unless otherwise indicated herein or clearly contradicted according to the background.

[0082] 文献目录 [0082] Bibliography

[0083] Cernac, A.and Benning, C.(2004).WRINKLED1 encodes an AP2/EREBdomain protein involved in the control of storage compound biosynthesis inArabidopsis.Plant J 40:575-585.[0084] Gamborg, 0.L.et al.(1968).Nutrient requirements of suspension culturesof soybean root cells.Exp Cell Res 50:151—158.[0085] Jako, C.et al.(2001).Seed-specific over-expression of an ArabidopsiscDNA encoding a diacylglycerol acyItransferase enhances seed oil content andseed weight.Plant Physiol 126:861-874.[0086] Jones，NMJ(1991).Jatropha curcas-a multipurpose species forproblematic sites.Land Resources Series I: 1-12.[0087] Li, MLHet al.(2008).Establishment of an Agrobacterium-mediatedcotyledon disc transformation method for Jatropha curcas.Plant Cell Tiss OrgCult 92:173-181.[0088] Lu，C.et al.(2006).A high-throughput screen for genes from castor thatboost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis.Plant J 45:847-856.[0089] Murashige，T.and Skoog，F.(1962).A revised medium for rapid growth andbioassays with tobacco tissue cultures.Physiol Plant 15:473—497.[0090] Narayana, DSA et al.(2007).Distinct Begomoviruses Closely Related toCassava Mosaic Viruses cause Indian Jatropha Mosaic Disease.1nt' IJ Virol 3:1-11.[0091] Qu, J.et al.(2007).Artificial microRNA-mediated virus resistance inplants.J Virol 81:6690-6699.[0092] Sujatha, M.et al.(2008).Role of biotechnological interventions in theimprovement of castor (Ricinus communis L.) and Jatropha curcas L.Biotechnol Adv26:424-435.[0093] van de Loo, FJet al.(1995).An oleate 12-hydroxylase from Ricinuscommunis Lis a fatty acy I desaturase homolog.Proc Natl Acad Sci USA 92:6743-6747.[0094] Zou,J.et al.(1999).The Arabidopsis thaliana TAGl mutant has a mutationin a diacylglycerol acyItransferase gene.Plant J 19:645-653 [0083] Cernac, A.and Benning, C. (2004) .WRINKLED1 encodes an AP2 / EREBdomain protein involved in the control of storage compound biosynthesis inArabidopsis.Plant J 40:. 575-585 [0084] Gamborg, 0.L. et al (1968) .Nutrient requirements of suspension culturesof soybean root cells.Exp Cell Res 50:.. 151-158 [0085] Jako, C.et al (2001) .Seed-specific over-expression of an ArabidopsiscDNA encoding a. diacylglycerol acyItransferase enhances seed oil content andseed weight.Plant Physiol 126: 861-874 [0086] Jones, NMJ (1991) .Jatropha curcas-a multipurpose species forproblematic sites.Land Resources Series I:. 1-12 [0087] Li,. MLHet al (2008) .Establishment of an Agrobacterium-mediatedcotyledon disc transformation method for Jatropha curcas.Plant Cell Tiss OrgCult 92:... 173-181 [0088] Lu, C.et al (2006) .A high-throughput screen for genes from castor thatboost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis.Plant J 45: 847-856 [0089] Murashige, T.and Skoog, F (1962) .A revised medium for rapid growth andbioassays with tobacco tissue cultures.. .Physiol Plant 15:. 473-497 [0090] Narayana, DSA et al (2007) .Distinct Begomoviruses Closely Related toCassava Mosaic Viruses cause Indian Jatropha Mosaic Disease.1nt 'IJ Virol 3:.. 1-11 [0091] Qu, J.et al (2007) .Artificial microRNA-mediated virus resistance inplants.J Virol 81:. 6690-6699 [0092] Sujatha, M.et al (2008) .Role of biotechnological interventions in theimprovement of castor (Ricinus communis.. L.) and Jatropha curcas L.Biotechnol Adv26:. 424-435 [0093] van de Loo, FJet al (1995) .An oleate 12-hydroxylase from Ricinuscommunis Lis a fatty acy I desaturase homolog.Proc Natl Acad Sci USA 92. :.. 6743-6747 [0094] Zou, J.et al (1999) .The Arabidopsis thaliana TAGl mutant has a mutationin a diacylglycerol acyItransferase gene.Plant J 19: 645-653