麻风树的遗传转化 Jatropha genetic transformation

[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 U.S. Provisional Patent Application Serial No. 61 December 15, 2008 submitted / 122,454, filed provisional patent application which 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（Jatropha)的再生和转化的方法。 [0004] The present invention relates to the field of plant regeneration and transformation, jatropha (Jatropha) 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 a Jatropha (Jatropha curcas) regeneration and transformation methods and media compositions of 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 [0005] As used herein publications and other materials are incorporated herein by reference, and for convenience 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 worsening problem. 亟需增加可再生能源的产生和消耗。 The urgent need to increase renewable energy production 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(X)2排放。 For many countries to find alternative energy sources, the bio-fuel has been recognized as national priorities in order to meet their energy security needs, while helping to reduce the greenhouse effect caused by the (X) 2 emissions. 对生物燃料的需要导致食物生产的压力增加。 The need for biofuels lead to pressure to 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 demand for biofuels required by the German government, all of the country's agricultural land will be used to grow bioenergy crops, and 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, we need to use marginal land to produce bio-energy.

[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, seed cost, high oil content, short gestation period, wide adaptability, drought tolerance and ability to thrive on degraded soil. 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 its plant seeds collected very convenient (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 has several disadvantages, which limits its widespread 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 plant is adversely male and female ratio limit, and the 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 of biological stress such as viruses (Narayanna et al., 2007), fungal and bacterial pathogens, and abiotic stress in particular is 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, curcin and carcinogenic phorbol ester) in plant seeds and leaves of Jatropha industry to farmers and bio-processing workers a health hazard.

[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 conventional method for improving plant quality traits by breeding superior 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) „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。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。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。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。 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) "Therefore, an urgent need, such as Alternative method of genetic transformation genetic manipulation tool to provide additional strategies for genetic improvement of crops. Agrobacterium (Agrobacterium) mediated genetic transformation has become the main choice to produce transgenic plants. However, few have been reported involving belong spurge Applications transformed plants Agrobacterium mediated transformation protocols for the only reported Jatropha's (Li et al., 2008) is in our hands can not be repeated.

[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] Thus, the urgent need for conversion of Jatropha methods in order to 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 plant Jatropha, more specifically a method Jatropha regeneration and 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 optimizes tissue culture and shoots (shoot) through regeneration conditions provides 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 for 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 rootstock 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 is not required grafting regenerated plants in tissue culture to produce roots, and significantly shorten the flowering shoots and generation of genetically modified 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, from 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 formed callus culture on a medium, the medium comprising MS salts, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as the plant hormone 6- callus formation benzylaminopurine (6-BA) and 1-naphthalene acetic 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 comprising MS salts, vitamin B5, citric acid, glutamine, casamino acids, adenine, sucrose, and 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 transfer shoots from callus regeneration to a second shoot regeneration medium comprising 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 shoots regenerated shoots are transferred to a third 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。 Already regenerated shoots are transferred to shoot elongation (elongation) medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, sucrose, and as a plant hormone 6-BA and GA3 to use to shoot elongation and 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 elongated shoots are 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。 Optional and can be elongated rhizome branches grafted to 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 of Jatropha plants for 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 and above basic schemes for jatropha reproduced the same basic 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 the conversion, the first explants with Agrobacterium cells co-cultured and 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-culture medium containing MS salts, vitamins B5, citric acid, glutamine, casein hydrolyzate, sucrose, glucose, acetosyringone well as plant hormones 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 and 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 selective agent for Agrobacterium-mediated jatropha plants 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 and the like.

附图说明 Brief Description

[0016] 图1示出本发明的土壤杆菌介导的麻风树属转化方法。 [0016] FIG. 1 shows 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 use the 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] FIG 3A-; 3K shows jatropha conversion, 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。 On the left, do not carry any carrier with 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。 On the right, with the carry the trait gene vector containing Agrobacterium inoculated cotyledons. 注意来自外植体的枝条再生。 Note that shoots from explants regeneration. 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: an enlarged top view of the brown cotyledons hygromycin resistant 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: rooting of transgenic shoots. 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 grown 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: grafted in non-transgenic transgenic shoots of the rhizome on leprosy. 白色箭头指示嫁接部位。 White arrows indicate the graft site. 图I ：转基因麻风树开花和结籽。 Figure I: transgenic Jatropha flowering and seed. 比例尺表不IOmm0 Scale table does not IOmm0

[0019] 图4示出hyg-抗性ubi ：GFP麻风树植物的PCR分析。 [0019] FIG. 4 shows hyg- resistance ubi: GFP PCR analysis of Jatropha plants. 泳道-：野生型麻风树属对照；泳道+ :P1300-GFP的质粒DNA ；泳道#1_#10来自潮霉素抗性麻风树属的枝条叶。 Lane -: wild type Jatropha control; lane +: P1300-GFP plasmid DNA; Lane # 1_ # 10 from hygromycin Jatropha branches leaves.

[0020] 图5A-5P示出Ttl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0、图P)中GFP的表达。 [0020] Figure 5A-5P shows Ttl plant roots (Fig. 5B, 5D), male (FIG. 5F, FIG. 5H), and three weeks after fertilization T1 seeds (Fig J, Fig. K, Fig L, Fig. N, 0 FIG. Fig. P) in GFP expression. 图A、图C、图E、图G、图I和图M是每种植物器官的野生型对照。 Figure A, Figure C, Figure E, Figure G, Figure I and M are each plant organ of the wild type control. 比例尺表示2mm。 Scale representation 2mm.

[0021] 图6示出BASTA-抗性;35S: JcWRIl麻风树植物的PCR分析。 [0021] FIG. 6 shows BASTA- resistance; 35S: JcWRIl 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 to # 7 from BASTA resistant Jatropha branches leaves; lane _ the wild-type control; lane +, 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 antibody expressing 35S: RcFAH12 and 35S: JcDGATl transgenic Jatropha plant leaves in 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 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 plant Jatropha, 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 regenerating 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, the explants were 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 士2°C和光照下进行，1¾光照（100 μ mol/tfS)/»!避光循环。 Incubated at 25 ° C with disabilities 2 ° C and light were, 1¾ light (100 μ mol / tfS) / »! 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，在避光条件下浸于无菌水中过夜。 Using conventional techniques will surface sterilized seeds of Jatropha, immersed in sterile water under dark conditions 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 embryos germinated on hormone-free germination medium, which root in 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 inorganic 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 is about 0.5g / L 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 by agar or vegetable gel (Phytogel). 培养在25°C 士1°C和光照下进行，1¾光照（lOOymol/ m2S) /8h避光循环。 Incubated at 25 ° C 1 ° C and light disabilities were, 1¾ illumination (lOOymol / 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 in callus formation medium incubated in the dark for about 2 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, vitamin B5, citric acid, glutamine, casamino acids, sucrose, and as a plant hormone 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 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 is about 150mg / L to about 200mg / L, preferably 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 of about 3%. 6-BA的浓度为约1. 5mg/L。 The concentration of 6-BA was 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 by agar or vegetable gel (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 incubated in the light conditions for about 2 weeks to about three 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 comprising 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 6-BA concentration and callus formation in the same medium. 腺嘌呤的浓度为约ang/ L至约％ig/L，优选约ang/L。 Adenine concentration of about ang / L to about% ig / L, preferably about ang / L. IBA的浓度为约0. 05mg/L。 IBA concentration of 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 pH of 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 by agar gel or plants, 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 branch [0028] from Calluses transferred to the second shoot regeneration medium and incubated in the light conditions for about 3 weeks to about four weeks, preferably about four 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, glutamine concentration, casein hydrolyzate, 6-BA and IBA same with the first shoot regeneration medium. GA3的浓度为约0. 05mg/L至约0. 5mg/L，优选约0. 5mg/L。 GA3 concentration of about 0. 05mg / L to about 0. 5mg / L, preferably from 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。 The second shoot regeneration medium preferably also contains 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。 The second shoot regeneration medium pH of 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 plant agar or 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 not have regenerated shoots are transferred to third shoot regeneration medium and incubated in the light conditions of about 4 weeks to about five 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, vitamins B5, citric acid, glutamine, casein hydrolyzate, sucrose, and further regeneration as a plant hormone 6-BA and IBA for shoots. 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, glutamine concentration, casein hydrolyzate, 6-BA and IBA same with 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。 PH third shoot regeneration medium is 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 by agar gel or plants, 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] will be transferred in the second shoot regeneration medium regenerated shoots to shoot elongation medium and incubated in the light conditions for about 2 weeks to about three 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, sucrose, and as a plant hormone 6-BA and GA3 for elongation and bud 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 and the first shoots of regeneration of the same medium. 6-BA的浓度为约0. 3mg/L。 6-BA concentration of about 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 from 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 from 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 plant agar or 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 in the light conditions for about 3 weeks to about four weeks, preferably about four weeks. 生根培养基包含MS无机盐、维生素B5、蔗糖和IBA。 MS rooting medium containing inorganic salts, vitamins B5, sucrose and IBA. 蔗糖的浓度为约3%。 Sucrose concentration of 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。 Or plant rooting medium with agar solidified gel, preferably in a concentration of about 2. 2g / L gel plants. 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, using conventional techniques elongated branches grafted onto jatropha rhizomes, transferred to rooting medium in place.

[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 of Jatropha plants for 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 and above basic schemes for jatropha reproduced the same basic 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 utilize conventional techniques such as electroporation containing Agrobacterium import interest. 在使用前，利用常规技术培养转化的土壤杆菌细胞。 Before use, the use of 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苄西林（carbicillin)的LB培养基。 According to one such technique, the Agrobacterium cells were inoculated into kanamycin and added carbenicillin (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 to about 100mg / L, preferably about 50mg / L. 羧苄西林的浓度为约50mg/L至约100mg/L，优选约100mg/L。 Carbenicillin concentration was about 50mg / L to about 100mg / L, preferably about 100mg / L. 使土壤杆菌细胞在^°C，250rpm条件下生长过夜。 Agrobacterium cells under ^ ° 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。 The concentration of glucose is 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 from 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] In order to be transformed, first explants were co-cultured with 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 callus formation 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/L。 The concentration of 6-BA was about 1. 5mg / L. NAA的浓度为约0. 05mg/L至约0. lmg/L，优选约0. 05mg/L。 NAA concentration of about 0. 05mg / L to about 0. lmg / L, preferably from 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 from 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] Callus for Agrobacterium-mediated transformation of Jatropha callus culture medium and for reproducing the same form of media, except that it also 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。 Such 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 hygromycin at a concentration of from about 3mg / L and 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, the glufosinate selection agent, at 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 eradicate agent may be 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 eradicate agent at a concentration of about 100mg / L to about 150mg / L, preferably 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 of culture medium is formed on the conditions in the dark for about 2 weeks to about three 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, in accordance with 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 branches of Jatropha for Agrobacterium-mediated transformation and regeneration medium for regenerating the first shoot regeneration medium of the same, except that it also 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。 Such 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 hygromycin at a concentration of approximately: 3mg / L to approximately 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, the glufosinate selection agent, at 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 eradicate agent may be 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 eradicate agent at a concentration of about 100mg / L to about 150mg / L, preferably 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 the second shoot regeneration medium of the same, except that it also 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。 Such 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， 其浓度为约細g/L至约5mg/L，优选％ig/L。 In one embodiment, the selection agent hygromycin at a concentration of from about fine g / L and about 5mg / L, preferably% ig / 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, the glufosinate selection agent, at 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 eradicate agent may be 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 eradicate agent at a concentration of about 100mg / L to about 150mg / L, preferably 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 third shoot regeneration medium identical, except that it also 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。 Such 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 hygromycin at a concentration of approximately: 3mg / L to approximately 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, the glufosinate selection agent, at 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 eradicate agent may be 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 eradicate agent at a concentration of about 100mg / L to about 150mg / L, preferably 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 conversion method. 通常，导入植物的DNA是构建体的一部分。 Typically, DNA is introduced into the 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节基因表达的序列，例如反义序列、正义抑制序列或miRNA序列。 DNA may be the gene of interest, such as the coding sequence of the protein; or it may be capable of regulating gene expression sequences, such as antisense sequences, sense suppression sequence or miRNA sequences. 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 the DNA of interest' end of the construct typically comprises regulatory region operably linked to the DNA of interest 5. 含有所有这些元件的盒在本文中也称为表达盒。 Cassette containing all of these elements are 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'前导序列。 In the expression cassette construct, expression cassette may also contain 5 'leader sequences. 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 (ie promoters, transcriptional regulatory regions, and translational termination regions) and / or encoded signal anchor polynucleotide to a host cell 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 anchor polynucleotide of host cells may be heterologous 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2006/0M8616。 See, U.S. Patent No. 7,205,453 and US Patent Application Publication No. 2006/0218670 and 2006 / 0M8616. 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/(^48616。 See, US Patent No. 7,205, 453 and US Patent Application Publication No. 2006/0218670 and 2006 / (^ 48616.

[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 comprising 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 selectable marker gene by an appropriate antibiotic resistance gene encodes a suitable genes 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) gene, 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Q，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 a sulfonylurea type herbicides, glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetic acid Q, 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2006/0M8616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 US Patent Application Publication No. 2006 / 0M8616 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。 The selectable marker gene list 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 number of promoters can be used to practice 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 禾口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); CaMV35S core promoter (Odell et al, 1985.); Rice actin (McElroy et . al, 1990); ubiquitin (Christensen and Quail, 1989 Wo mouth Christensen et al, 1992);. pEMU (Last et al, 1991);. MAS (Velten et al, 1984);. ALS promoter (U.S. Patent No. 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 of 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, especially a pathogen inducible promoter. 这类启动子包括来自致病相关蛋白（ra蛋白）的那些启动子，其在病原体感染后被诱导；例如，ra蛋白、SAR 蛋白、β-1，3-葡聚糖酶、壳多糖酶等。 Such promoters include those from pathogenesis-related protein promoter (ra protein), which is induced after infection the pathogen; e.g., ra proteins, SAR proteins, β-1,3- glucanases, chitin enzyme . 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 sites or those close to local expression promoter. 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, can object to the use of chemical regulation of the promoter to regulate gene expression in plants by applying an exogenous chemical regulation. 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。 The promoter may be chemically inducible promoter, where the application of chemicals to induce gene expression; or chemical inhibition promoter, wherein the application of chemicals to 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 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 each described in U.S. 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] In appropriate cases, the DNA of interest can be optimized in order 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, using plant preferred codons were synthesized in order to improve the expression of the coding sequence. 本领域提供合成植物优选基因的方法。 Gene synthesis plant is preferably provided 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幵号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 Jian.

[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 following embodiments of the present invention uses conventional technology: chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are in this field technology within range. 参见，例如Maniatis et al. ，1982，Molecular Cloning (Cold Spring Harbor Laboratory Press，Cold Spring Harbor，New York) ；Sambrook et al.，1989，Molecular Cloning，2nd Ed. (Cold Spring Harbor Laboratory Press， Cold Spring Harbor， New York) ；Sambrook and Russell，2001，Molecular Cloning，3rd Ed. (Cold Spring Harbor 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 of plants :a laboratory course manual (Cold Spring Harbor Laboratory Press，Cold Spring Harbor，NY) ；Anand, Techniques for the Analysis of Complex Genomes， (Academic Press， New York，1992) ；Guthrie and Fink, Guide to Yeast Genetics and 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 (BD Hames & SJ Higgins eds.1984) ；Transcription And Translation (BD Hames & SJ Higgins eds. 1984) ；Culture Of Animal Cells (RIFreshney, 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 Molecular Biology(Mayer and Walker, eds.，Academic Press，London，1987) ；Handbook Of Experimental Immunology, Volumes I-IV(DM Weir and CC Blackwell, eds.，1986)； Riott， Essential Immunology,6th Edition， Blackwell Scientific Publications， Oxford, 1988 ；Fire et al.，RNA Interference Technology :From Basic Science to Drug Development, Cambridge University Press，Cambridge，2005 ；Schepers，RNA Interference in Practice，Wiley-VCH,2005 ；Engelke, RNA Interference (RNAi) :The Nuts & Bolts of siRNA Technology，DNA Press,2003 ；Gott,RNA Interference，Editing， and Modification :Methods and Protocols(Methods in Molecular Biology), Human Press,Totowa,NJ, 2004 ；SohailiGene Silencing by RNA Interference-Technology and Application，CRC，2004。 See, e.g., Maniatis et al, 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York);. Sambrook et al, 1989, Molecular Cloning, 2nd Ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor.. , New York);. Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York);. Ausubel et al, 1992), Current Protocols in Molecular Biology (John Wiley & Sons , including the current update); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and 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 (BD Hames & SJ Higgins eds.1984);. Transcription And Translation (BD Hames & SJ Higgins eds 1984); Culture Of Animal Cells (RIFreshney, 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 Molecular Biology (Mayer and Walker, eds, Academic Press, London, 1987.); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds, 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al, RNA Interference Technology:. From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005. ; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; SohailiGene Silencing by RNA Interference-Technology and Application, 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 invention. 使用本领域公知的标准技术或下文具体描述的技术。 It is known in the art using standard techniques or 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% (ν/ν)乙醇表面灭菌60秒，随后浸入10% (v/v)H2O2中lh，然后用无菌水淋洗两次，最后于下在避光条件下浸入无菌水中过夜。 After removal of the testa, the seeds with 75% (ν / ν) ethanol surface sterilization 60 seconds, then immersed in 10% (v / v) H2O2 in lh, then rinsed with sterile water twice, the last at immersed in sterile water overnight in the dark condition. 使不含胚乳的胚在无激素的半浓度Murashige 和Skoog 盐（1/2MQ 培养基(Murashige and Skoog, 1962)上发芽，并在组织培养室中于25°C 士2°C、l^i光照（100 μ mol/tfS)/»!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Β5 (Gamborg et al.，1968)、5g/L蔗糖、0. 5g/L2_ (4-吗啉代） 乙磺酸（MES)和2. 2g/L 植物凝胶（Sigma)，ρΗ 5. 6。 So without endosperm embryo in hormone-free half-strength Murashige and Skoog salts (1 / 2MQ medium (Murashige and Skoog, 1962) on germination, and 25 ° C Case disabilities 2 ° C in tissue culture chamber, l ^ i Light (100 μ mol / tfS) / »! dark cycle under culture conditions, wherein the medium in contact with the root, the medium containing vitamin Β5 (Gamborg et al., 1968), 5g / L sucrose, 0. 5g / L2_ (4- morpholino) ethanesulfonic acid (MES), and 2. 2g / L plant gel (Sigma), ρΗ 5. 6.

[0048] 培养基：本发明使用的培养基如下所示。 [0048] Medium: the medium of the present invention are shown below.

[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)蔗糖、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) sucrose, 0. 5g / L MgCl2 (only for gel medium containing a plant) in combination with plant growth regulators. 用IN KOH将培养基I调节为ρΗ 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。 With IN KOH was adjusted to the medium I ρΗ 5.8-6.0, with 2.5g / L gel curing plant, 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。 Before adding to autoclaved medium, all the plant growth regulator filter sterilization.

[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/L 6-苄氨基嘌呤（6-BA)和0. 05mg/L 1-萘乙酸（NAA)，ρΗ 5. 0-5. 2。 [0050] co-cultivation medium: base medium plus 20mg / L acetosyringone (AS), 0 5g / L MES, 1 5mg / L 6- benzylaminopurine (6-BA) and 0. 05mg / L.. 1- naphthalene acetic acid (NAA), ρΗ 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，A. G scientific, SanDiego, CA)或用于清除土壤杆菌(Agrobacteria)细胞的lmg/L 草铵膦(BASTA,Crescent Chemical,NY)和100mg/L 头孢噻肟（Cef)。 [0051] callus formation medium: base medium plus 1. 5mg / L 6_BA, 0 05mg / L NAA, as the plant transformation selection agent 3. 5mg / L hygromycin (hyg, A G scientific,.. SanDiego, CA) or to remove Agrobacterium (Agrobacteria) cells lmg / L glufosinate (BASTA, Crescent Chemical, NY) and 100mg / L cefotaxime (Cef).

[0052] 枝条再生培养基I ：基础培养基加1. 5mg/L 6_ΒΑ、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 I: Basic medium plus 1. 5mg / L 6_ΒΑ, 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II ：基础培养基加1. 5mg/L 6_BA、0. 05mg/L IBA、0. 5mg/L赤霉酸(GA3) ,4mg/L Hyg或lmg/L草铵膦和lOOmg/L Cef 100，将植物凝胶变为7g/L琼脂。 [0053] shoot regeneration medium II: base medium plus 1. 5mg / L 6_BA, 0 05mg / L IBA, 0 5mg / L gibberellic acid (GA3), 4mg / L Hyg or lmg / L glufosinate. and lOOmg / L Cef 100, the plant gel becomes 7g / L agar.

[0054]枝条再生培养基 III ：基础培养基加1. 5mg/L 6_BA、0. 05mg/L IBA、3. 5mg/L Hyg 或lmg/L 草铵膦和lOOmg/L Cef。 [0054] shoot regeneration medium III: base medium plus 1. 5mg / L 6_BA, 0 05mg / L IBA, 3 5mg / L Hyg or lmg / L glufosinate and lOOmg / 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: base 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/L ΙΒΑ、2· 2g/L 植物凝胶，ρΗ5· 6。 [0056] The rooting medium:.. MS major salts, MS minor salts and vitamins B5,3% sucrose, 0 5g / LMES, 0 07mg / L ΙΒΑ, 2 · 2g / L phytagar, ρΗ5 · 6.

[0057]培养基 II ：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 II: 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Gnvitrogen)提取。 [0058] RNA extraction and analysis: fresh leaf or seed tissue (IOOmg) ground in liquid nitrogen and extracted with plant RNA purification kit Gnvitrogen). 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) described below.

[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 1 (JcffRI 1)和DGATl序列。 [0059] Agrobacterium strains and vectors: Jatropha seeds by sequencing of cDNA libraries to identify Jatropha WRINKLE 1 (JcffRI 1) and DGATl sequences. JcWRI 1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 1 full-length cDNA from the first strand cDNA jatropha seeds amplified product, the use of two primers: 5, -AATCGGATCCTAATGAAGAGGTCTTCTGCT-3 '(SEQ ID NO: 1) and 5, -TCATGTTAATT AATCAAACAGAATAGTTACAAGAAA-3' (SEQ ID NO : 2) (underlined nucleotide recognition site for the enzyme). 再将PCR产物插入用BamHI和I3ac I处理的pBAOO2-MYC载体以形成pBA002-MYC-JcffRI 1。 Then PCR products were inserted with BamHI and pBAOO2-MYC carrier I3ac I treated to form pBA002-MYC-JcffRI 1. JcDGAT 1全长cDNA从麻风树种子第一链cDNA产物扩增，所用的两条弓I 物：5'-CAATATCTAGACCATGACGATTTTGGAGACCACT-3' (SEQ ID NO ：3)和5?-TATTAGATCTGGTCT TAATTCAGCATTGCC-3，(SEQ ID NO :4)(下划线的核苷酸为酶识别位点）。 JcDGAT 1 full-length cDNA from the first strand cDNA jatropha seeds amplified product, the use of two bow I was: 5'-CAATATCTAGACCATGACGATTTTGGAGACCACT-3 '(SEQ ID NO: 3) and 5 -TATTAGATCTGGTCT TAATTCAGCATTGCC-3, (SEQ? ID NO: 4) (underlined nucleotide recognition site for the enzyme). 再将PCR产物插入用XbaI 和BamHI 处理的pBA002_HA 载体以形成pBA002-JcDGATl_HA。 Then PCR products were inserted with XbaI and BamHI 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 castor bean seed of the first strand cDNA amplification products, the use of two primers: 5 '-CAATATCTAGACCATGGGAGGTGGTGGTC -3, (SEQ ID NO: 5) and 5' -TGTAGGATCCGGATACTTGTTCCGGTACCAG-3 '(SEQ ID NO: 6 ) (underlined nucleotide recognition site for the enzyme). 再将PCR产物插入用)(bal和BamHI处理的pBA002_HA载体以形成pBA002-RcFAH12-HA。通过电穿孔（BIO-RAD，CA，USA)将载体导入土壤杆菌菌株AGLl。使用转化的土壤杆菌细胞接种液体LB培养基，并在，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 1，pBA002-JcDGATl_HA，pBA002-RcFAH12_HA)和100mg/L羧苄西林。通过在20°C下以4200rpm离心IOmin来收集土壤杆菌细胞。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)。 PCR product was then inserted by) (pBA002_HA bal and BamHI treated vector to form pBA002-RcFAH12-HA. By electroporation (BIO-RAD, CA, USA) the vector into Agrobacterium strain AGLl. Agrobacterium transformed cells were inoculated using liquid LB medium, and, under 250rpm overnight grown to a final OD595 = 0. 7-1, the culture medium was added 50mg / L Kanamycin (for pCAMBIA 1300-GFP) or 50mg / L spectinomycin Su (spectimycin) (for pBA002-MYC_WRI 1, pBA002-JcDGATl_HA, pBA002-RcFAH12_HA) and 100mg / L carbenicillin. By 4200rpm at 20 ° C in order to be collected by centrifugation IOmin Agrobacterium cells prior to co-cultivation, 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1小时。 [0060] Isolation and genotyping of DNA leaves leprosy: leprosy 50mg of fresh leaves crushed in liquid nitrogen and add 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 for 1 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 precooling chloroform extracted twice and the DNA 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' -ACTTCTACACAGCCATCGGT CC-3，(SEQ ID NO :8)。 For genotyping hygromycin gene, primers used were hyg5: 5 '-CGATGTAGGAGGGCGTGG-3' (SEQ ID NO: 7), hyg3: 5 '-ACTTCTACACAGCCATCGGT CC-3, (SEQ ID NO: 8). 对于bar基因基因型分析，所用的引物为bar5 :5，-GTCTGCAC CATCGTCAACC-3，(SEQ ID NO ：9), bar3 ：5' -GAAGTCCAGCTGCCAGAAAC-3，(SEQ ID NO: 10)。 For the bar gene genotyping, primers used for bar5: 5, -GTCTGCAC CATCGTCAACC-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。 [0061] antibody and protein gel blot analysis: curcin antibody manufactured by Dr. Yin Zhongcao laboratory. 按照先前的描述（Qu et al. ,2007)进行蛋白印迹分析。 As previously described (Qu et al., 2007) for western blot analysis. 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。 By 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis separation of total plant protein. 使用ECL过氧化物酶偶联的驴抗兔免疫球蛋白G作为二抗。 The ECL peroxidase-conjugated donkey anti-rabbit immunoglobulin G as a secondary antibody. 使用ECL蛋白印迹检测试剂（GE healthcare)将免疫反应性条带显影。 Using ECL Western blotting detection reagents (GE healthcare) The immunoreactive bands developed.

[0062] 实施例2麻风树子叶外植体转化 [0062] Example 2 Jatropha cotyledon transformation

[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] FIG. 1 shows Agrobacterium-mediated transformation of Jatropha method embodiment 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 illustrates 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] the co-culture: The cotyledons of 5-7 day old seedlings (Example 1; FIG. 3A) cut into small pieces (5 X 5mm), with a target expression cassette containing Agrobacterium cells (Example 1) in 20ml medium II 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 co-culture medium, @ 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头孢噻肟（cefotaxine)洗涤一次。 After co-cultivation, explants are rinsed several times with sterile water, followed by 300mg / L cefotaxime (cefotaxine) washed once. 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 dried absorbent cotyledon tissue, which sterilized by placing them in a 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 士1°C下保持3周。 [0065] hygromycin resistance or glufosinate resistant callus selection: After co-cultivation, explants formation medium plates inoculated in callus and transferred to the dark conditions, 25 ° C with disabilities 1 ° C lower 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 callus formation (Fig. 3B), and some callus formation (FIG: 3B, right column; Fig. 3C) in culture. 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, untransformed explants usually 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、l^i光照（100 μ HioVm2S1Va1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: explants having hygromycin resistance or glufosinate resistant callus emerging transferred to shoot regeneration medium I, at 25 ° C, l ^ i illumination (100 μ HioVm2S1Va1 Under dark cycle conditions were maintained for three weeks. The method described herein based induce direct shoot from callus transformed by the addition of adenine. The article uses the term "regeneration" to describe the whole plants from such transformed callus tissue re-creation (re-creation). Although 6_BA (6_ BA) on shoot regeneration has a similar effect, but in the methods described herein which are not used in this particular step. In addition, higher or lower concentration, too early or too late will shoot regeneration adenine add 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 gland purine derivatives, such as 2-isopentenyladenine in this time period, any regenerated shoots from callus (approximately 40-50%) was transferred to shoot regeneration medium II (Fig. 3D). would not have regenerated shoots callus was transferred to shoot regeneration medium III, for further training and shoots regeneration.

[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3¾。 [0067] shoot elongation: 4 weeks, the regenerated shoots are transferred to shoot elongation medium for shoot elongation and proliferation (Fig 3¾.

[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 the rooting medium (Fig. 3F). 通常需要超过1个月来获得如图3F所示的根。 Usually it takes more than one month to get the root 3F shown. 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 solution provides rooting about 45% of a high rooting efficiency (Fig. 3G), and a length greater than a taproot IOmm can successfully be transferred to the 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] Graft: also can be used as an elongated transgenic scion to graft branches to non-transgenic rhizomes. 挑选健康和生长旺盛的麻风树植株作为根状茎。 Choose healthy and vigorous growth of Jatropha plant 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 the cambium area so from both the phloem tissue in the back splice connector. 用封口膜（parafilm)缠绕嫁接接头（joint)，并用胶带固定。 With sealing film (parafilm) wound grafting joint (joint), and secured with tape. 将嫁接的麻风树植株在弱光强度、16h光照（50 μ HioVm2S1) /8h避光循环）和85%湿度条件下保持7天。 Jatropha plants will be grafted for 7 days at low light intensity, 16h light (50 μ HioVm2S1) / 8h dark cycle) and 85% humidity. 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。 Grafting shoots of transgenic leprosy on non-transgenic rhizomes are shown in Figures 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I)。 Transgenic Jatropha plants in the greenhouse showed normal flowering and seed set (Figure I).

[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 use of the method of the present invention is converted Jatropha and transformed cells from the regeneration BASTA or hygromycin (hygmycin) Example plants described in more detail below. 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, coding such as BASTA given DNA sequence or hygromycin resistance to the selective advantage of protein and 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 a coding imparting hygromycin or BASTA resistance to DNA encoding a protein plant promoter region, 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 construct of interest. 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 constructs may comprise such a gene, whose expression results in the transformed plant 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, it 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 this gene and the GFP easily screenable marker can be used as the other 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。 It is reasonable to expect by using standard molecular biology techniques to produce the DNA constructs of the present invention may be used to obtain almost any expression of other genes Jatropha species. 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 DNA construct comprising 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 resistance presumption (PUtative) GFP transgenic Jatropha plant transformation and shoot regeneration implement 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 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) genotyping. 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 in nine is PCR positive, rather than transforming the control lanes in CK 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 that the newly introduced expression of GFP expression cassette in Ttl Jatropha plants. 在ubi:GFP转基因麻风树属开花后，我们检查了花序中的GFP表达。 In ubi: GFP transgene after Jatropha flowering, we examined the expression of GFP 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 GFP expression three weeks after fertilization of 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 (Figure 5N, FIG. 50) or internal (Figure 5P) strong GFP expression can be observed. 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 seed 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 a plant converting solar energy as the main energy storage in the form of chemical energy after. 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。 But when the plant using a variant of glycolysis as an intermediate product, which was synthesized by standard biochemical pathway is considered to be quite wasteful. WRINKLED 1 (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 1 (WRIl) is AP2 / EREB family of transcription factors, which is more specific aspects of seed conservation process, especially transcript variants sugar control into TAG has influence, and therefore control the seed oil content showed a very important aspect 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 under control of the Arabidopsis (Arabidopsis) WRIl cDNA expression resulted in 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)。 In addition, WRINKLED1 cDNA ectopic expression leads to the accumulation of triglycerides in the development of seedlings (Cernac and Benning, 2004). 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。 We believe WRIl Jatropha Jatropha gene ectopic 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, transgenic seedlings can develop into embryos or embryo-like oil-producing organ, like lipid reactor can be for the supply of sugary liquid substrate for vegetative organs of the constitutive CaMV 35S promoter - driven The WRIl strong expression.

[0075] 我们克隆了麻风树属WRIl的全长cDNA(JcWRI 1)，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 Jatropha WRIl full length cDNA (JcWRI 1), which belongs to seed RT-PCR product was PCR amplified from jatropha, which was used for cloning JcWRIl from Jatropha seed cDNA library sequencing PCR primer sequences (SEQ ID NO: 1 and SEQ ID NO: 2). 全长JcWRIl cDNA 序列示于SEQ ID N0:11。 JcWRIl cDNA full length sequence is shown in SEQ ID N0: 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。 We have built a JcWRIl cDNA by CaMV 35S promoter-controlled over-expression vector (pBA002_MYC-JcWRIl), and transformed into Agrobacterium strain AGLl. 可以利用MYC标签抗体来检测预计的6 XMYC标签融合WRIl。 MYC-tag antibody can be used to detect the expected 6 XMYC tag fusion WRIl. 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 resistance JcWRIl presumption overexpressing transgenic Jatropha plant transformation and shoot regeneration implement 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 hygromycin resistance branches genomic DNA. 利用BASTA基因引物对（SEQ ID NO :9和SEQ ID NO=IO)进行基因型分析。 Use BASTA gene primers (SEQ ID NO: 9 and SEQ ID NO = IO) 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)。 We tested all events are PCR-positive, rather than transformation control in CK showed no band (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 the nascent fatty acid packed into TAG, which followed from the endoplasmic reticulum budding (bud off) of the oil accumulated in the body. 已经证实植物1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 et al.，2001)。 It has confirmed that the plant type 1 DGAT (DGATl) gene for seed oil content has an important contribution to this by overexpression and mutation studies confirmed down (Zou et al., 2999 Jako et al., 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 can lead to higher levels.

[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 sequence 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JcDGAT 1 cDNA序列示于SEQ ID NO :13ο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 JcDGAT 1 cDNA sequence shown in SEQ ID NO: 13ο Build a JcDGATl cDNA by CaMV 35S promoter-controlled over-expression vector (pBA002-JcDGATl-HA), and transformed into 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 resistance presumption JcDGATl transgenic Jatropha plant transformation and shoot regeneration implement according to the method 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 demonstrated expression of the 35S-JcDGATl (Figure 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] You can use vegetable oil (and derivatives thereof) in many cases, and industrial applications of plant origin of raw materials. 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 non-renewable petroleum, renewable nature is such that they are concerned about the environment in many industrial applications for total loss applications issue is 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 contains more than 90% of ricinoleic acid, which is monounsaturated 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 group 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)。 This caused ricinoleic acid functional groups (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 for hydroxylamine in place of normal FAD2 function to introduce 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 other seed oils lack the hydroxyl group, the higher the price of castor oil. 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 become immunogenic proteins should exist, 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 produce 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弓I起蓖麻油的产生。 We believe that Jatropha ectopic expression will bow I produce castor oil of castor bean FAH12 genes.

[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] According to our FAH12 CDS sequence, using PCR primers (SEQ ID NO: 5 and SEQ ID N0: 6), from castor bean seeds RT-PCR products were cloned the full length castor bean FAH12 cDNA (RcFAHl2). 全长RcFAHl2 cDNA序列示于SEQ ID NO :15ο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: 15ο constructed by the CaMV 35S promoter has control over RcFAH12 cDNA expression vector (pBA002-RcFAH12-HA), and transformed into 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 resistance presumption RcFAH12 transgenic Jatropha plant transformation and shoot regeneration implement according to the method 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 demonstrated expression of the 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 (Figure 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 use of [0080] In describing the background of the invention (in particular in the appended claims the background), the term It should be understood to include the singular and the plural, unless otherwise indicated herein or clearly contradicted according to the 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", "including" and "comprising" should be construed as 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, numerical ranges described herein are merely intended to serve as a shorthand method belonging to the range of values ​​of each individual are involved, each individual value is included into the specification as if it were a separate described herein. 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 you open the range 10-15, 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 in any suitable order unless otherwise indicated herein, or in addition to the background clearly contradicted. 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 (eg, "such as"), is intended merely to better illustrate the invention and are not intended to limit the scope of the present invention to make. 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 element is not required to implement the present invention required.

[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 present invention may be incorporated into various forms of embodiments disclosed herein only some of them. 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 paper 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 technical personnel, as appropriate, the use of such changes, and the present inventors believe the present invention may be practiced other than as specifically described in addition to this article. 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 topics applicable law permitted recited in the appended claims all modifications and equivalents. 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/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J 40 :575-585. .. [0083] Cernac, A. and Benning, C. (2004) WRINKLED1 encodes an AP2 / EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis 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. [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 Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126:861—874. . [0085] Jako, C. et al (2001) .Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight Plant Physiol 126:. 861-874.

[0086] Jones，NMJ· (1991). Jatropha curcas-a multipurpose species for problematic sites. Land Resources Series 1 ： 1-12. [0086] Jones, NMJ · (1991) Jatropha curcas-a multipurpose species for problematic sites Land Resources Series 1:.. 1-12.

[0087] Li，MLH et al. (2008). Establishment of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tiss Org Cult 92 :173-181. [0087] Li, MLH et al (2008) Establishment of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas Plant Cell Tiss Org Cult 92:... 173-181.

[0088] Lu, C. et al. (2006). A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J 45 :847-856. [0088] Lu, C. et al (2006) A high-throughput screen for genes from castor that boost 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 and bioassays with tobacco tissue cultures. Physiol Plant 15 :473-497. [0089] Murashige, T.and Skoog, F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol Plant 15:... 473-497.

[0090] Narayana, DS A et al. (2007). Distinct Begomoviruses Closely Related to Cassava Mosaic Viruses cause Indian Jatropha Mosaic Disease. Int' 1 J Virol 3 ： 1-11. [0090] Narayana, DS A et al (2007) Distinct Begomoviruses Closely Related to Cassava Mosaic Viruses cause Indian Jatropha Mosaic Disease Int '1 J Virol 3:... 1-11.

[0091] Qu, J. et al. (2007). Artificial microRNA-mediated virus resistance in plants. J Virol 81 :6690-6699. [0091] Qu, J. et al (2007) Artificial microRNA-mediated virus resistance in plants J Virol 81:... 6690-6699.

[0092] Sujatha，M. et al. (2008). Role of biotechnological interventions in the improvement of castor (Ricinus communis L.)and Jatropha curcas L. Biotechnol Adv 26 :424-435. . [0092] Sujatha, M et al (2008) Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L. Biotechnol Adv 26:.. 424-435.

[0093] van de Loo, FJ et al. (1995). An oleate 12-hydroxylase from Ricinus communis Lis a fatty acyl desaturase homolog. Proc Natl Acad Sci USA 92: 6743-6747. [0093] van de Loo, FJ et al (1995) An oleate 12-hydroxylase from Ricinus communis Lis a fatty acyl desaturase homolog Proc Natl Acad Sci USA 92:... 6743-6747.

[0094] Zou, J. et al. (1999). The Arabidopsis thaliana TAGl mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19 [0094] Zou, J. et al. (1999). The Arabidopsis thaliana TAGl mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19