CN103421782A - Isolation, cloning and expression pattern identification of sheep hair follicle specific expression promoter fragment - Google Patents

Abstract

The invention belongs to the technical field of animal genetic engineering and particularly relates to the isolation, cloning and expression pattern identification of a sheep hair follicle specific expression promoter fragment. A promoter with hair follicle tissue expression specificity is isolated from, cloned and identified in a sheep genome. The nucleotide sequence of the promoter is shown as SEQ ID NO:10. A eukaryotic expression vector sKAP11.1p-GFP is constructed through the sheep hair follicle specific expression promoter fragment obtained by cloning; a green fluorescent report gene ZsGeen1 is inserted behind the promoter. The constructed eukaryotic expression vector sKAP11.1p-GFP is integrated into the genome of a transgenic mouse through a pronucleus microinjection method. At the individual level, the fact that the promoter has expression activity in mammals and shows hair follicle tissue specificity is verified. The promoter can be applied to improvement on the wool producing capacity and hair properties of animals.

Description

The separation of sheep hair follicle specific expressing promoter fragment and expression pattern are identified

Technical field

The invention belongs to animal gene engineering technology field, the separating clone and the expression pattern that are specifically related to sheep hair follicle specific expressing promoter fragment are identified.

Background technology

Promotor (Promoter) is the important component part of gene, refers to one section thymus nucleic acid that can make gene be transcribed (DNA) sequence.Promotor is usually located at the upstream of gene, can be by Yeast Nucleic Acid (RNA) polysaccharase, and the identification such as transcription regulaton factor is also with it in conjunction with forming transcription initiation mixture zone, thus initial gene is transcribed.In eukaryote, according to the mode of action of promotor and the difference of function, can be divided into 3 types: constitutive promoter, inducible promoter, and organizing specific type promotor.Under the regulation and control of constitutive promoter, the expression of gene is constant has continuity on a level, does not present Space-time speciality, also is not subject to inducing of extraneous factor; Inducible promoter need, under some specific physics or chemical signal stimulation, start or improve significantly the transcriptional level of gene; Tissue-specific promoter can express in some specific organ or tissue by regulatory gene, and generally possesses the developmental regulation characteristic.Because tissue-specific promoter has the characteristics of regulate gene expression space-time, so this type of promotor can be in biotechnology, for realizing the targeting of exogenous gene expression, reduce loss and the foreign gene of human body energy and material and arbitrarily express the impact on the organism metabolism activity.Also Just because of this, tissue-specific promoter has great significance to genetically engineered.

In eukaryotic cell, utilizing genetic engineering technique to build the expression that multiple eukaryotic expression system carries out foreign gene has been a technology commonly used.CMV and SV40 promotor are widely used promotors in the present stage genetically engineered, and both all can foreign gene-carrying, and in the cell of nearly all type high efficient expression.But, because this class promotor does not possess the ability of Targeted-control genetic expression, therefore tend to cause that the normal growth of transgenosis individuality grows is abnormal and do not reach experiment purpose.But tissue-specific promoter can well solve this difficult problem, it can control the expression of foreign gene in specific tissue or cell type, and the growth of its hetero-organization is not impacted, so tissue-specific promoter more and more has been subject to scientist's favor.But analyze and find from the tissue-specific promoter obtained plant and animal at present, the ubiquity specificity is not very high; Number is less; The problems such as activity is low.These have limited the application of tissue-specific promoter in genetically engineered greatly.

The present invention be take sheep as research material.Sheep is one of important economic animal of China, is the Important Economic source that the herdsman depends on for existence, is the important component part of the gross value of agricultural production.Wool traits, be one of Major Economic of sheep, improves and improve this economic characters, can directly improve the production performance of sheep.Utilizing genetic engineering technique to be improved by the shape economic characters sheep's wool, is to improve the sheep economic worth, increases economic income of herdsmen, improves the efficient means of people's living standard.The present invention is exactly the problem that solves current sheep's wool lens capsule tissue specificity promoter number deficiency, and separating clone has also been identified a sheep hair follicle specific expression promoter fragment, for utilizing genetic engineering technique improvement sheep's wool, by the shape economic characters, provides basis.

Summary of the invention

The object of the invention is to overcome the existing organizing specific expression promotor number deficiency that can be used for the research of sheep genetically engineered, separating clone identify a promotor with hair follicle tissue's expression specificity from the tibetan sheep genome, be expected to the genetically engineered improvement for sheep by this promotor, the promoter fragment that utilizes the clone to obtain has built the sKAP11.1p-GFP carrier for expression of eukaryon, and has inserted ZsGreen1 green fluorescence reporter gene after promotor.Utilize the method for pronuclear microinjection to be incorporated in the transgenic mice genome in the carrier built, verified expression activity and the tissue specificity of this promotor on individual level, lay a good foundation for utilize this promotor in the future.

The technical scheme that realizes task of the present invention is as described below:

1. tissue specific expression gene checking: gather 16 kinds of tissues that tibetan sheep comprises hair follicle, skin, the heart, liver, spleen, lung, kidney, cud, abomasum, small intestine, large intestine, lymph (mesentery), fat, muscle, testis, ovary.Extract total RNA of these tissues, and verify the expression of candidate gene KRTAP11.1 in each tissue with reverse transcription PCR (RT-PCR).Result proof KRTAP11.1 only has expression in hair follicle and skin, by hybridization in situ experiment, further verifies discovery, and the KRTAP11.1 gene is only expressed in hair shaft, is a kind of hair follicle specific expression gene.

2. promotor is cloned and vector construction: by Auele Specific Primer, from the tibetan sheep genome, increase and obtain the promoter region fragment of 5954KB size, called after sKAP11.1p.And this promoter region is connected in the promoter region of promoterless pZsGreen1-1 green fluorescent protein report carrier, by the carrier called after sKAP11.1p-GFP built.

3. promoter expression model validation: utilize micro-procaryotic injection fabrication techniques transgenic mice, linearizing sKAP11.1p-GFP carrier sequence is incorporated in the transgenic mice genome.During 4 week age, gather mouse hair, skin, the heart, liver, spleen, lung, kidney, stomach, intestines, muscle, tongue, testis, cerebral tissue until positive mouse, extract RNA.Only in mouse hair, express through Phenotypic Observation and RT-PCR checking ZsGreen1 gene, prove that the sKAP11.1p promotor has activity in mammalian body, and be hair follicle tissue's specific expressing promoter.

The invention has the advantages that:

(1) separating clone of the present invention sheep hair follicle specific expression promoter fragment sKAP11.1p, and on individual level, identified and activity and tissue specificity, for sheep genetically engineered and molecular breeding provide new specifically expressing to start resource.

(2) the constructed sheep hair follicle specific expression promoter sKAP11.1p-GFP carrier of the present invention, can be further used for starting the functional study that foreign gene is expressed in hair follicle.

The accompanying drawing explanation

Sequence table SEQ ID NO:1 and 2 is for the primer of the tibetan sheep KRTAP11.1 Gene Partial nucleotide sequence that increases in Application Example.

Sequence table SEQ ID NO:3 is the partial nucleotide sequence of the tibetan sheep KRTAP11.1 gene that increases in Application Example.Sequence length is 127bp.

Sequence table SEQ ID NO:4 and 5 is for the primer sequence of the tibetan sheep sKAP11.1p promotor nucleotide sequence of the present invention name of increasing in Application Example.

Sequence table SEQ ID NO:6 and 7 is for the primer sequence of the partial nucleotide sequence of the tibetan sheep sKAP11.1p promotor of the present invention name of increasing in Application Example.

Sequence table SEQ ID NO:8 is the partial nucleotide sequence of the tibetan sheep sKAP11.1p promotor of the present invention name of increasing in Application Example.Sequence length is 561bp.

Sequence table SEQ IDNO:9 is the nucleotide sequence of tibetan sheep KRTAP11.1 gene hybridization in situ probe in Application Example.Sequence length is 796bp.

Sequence table SEQ ID NO:10 is the nucleotide sequence of the tibetan sheep sKAP11.1p promotor of the present invention name of increasing in Application Example.Sequence length is 5954bp.

Fig. 1: the expression of tibetan sheep KRTAP11.1 gene in each tissue of tibetan sheep.In figure: swimming lane 1 is 100bp Marker; Swimming lane 2-17 is followed successively by and organizes hair follicle, skin (containing hair follicle), the heart, liver, spleen, lung, kidney, cud, abomasum, small intestine, large intestine, lymph (mesentery), fat, muscle, testis, ovary; Swimming lane 18 is without the template negative control.

The in situ hybridization result of Fig. 2 tibetan sheep KRTAP11.1 gene in tibetan sheep skin.The zone of arrow (white) indication is the probe hybridization signal.

Fig. 3: sKAP11.1p amplified fragments.In figure: swimming lane 1 is DL15000+2000Marker; Swimming lane 2 is a sKAP11.1p amplification fragment; Swimming lane 3 is without the masterplate negative control.

Fig. 4: sKAP11.1p-GFP carrier structure schematic diagram.This carrier is carrier for expression of eukaryon; The sKAP11.1p promotor connects ZsGreen1 green fluorescence reporter gene; This carrier has kantlex and neomycin resistance.

Fig. 5: the hair Phenotypic examination result of sKAP11.1p-GFP transgenic positive mouse and negative control mouse.Fig. 5 A is light field.In figure, upper left is sKAP11.1p-GFP transgenic positive mouse, the negative control mice in bottom right; Fig. 5 B is the fluorescence field, wavelength 500nm.In figure, upper left is sKAP11.1p-GFP transgenic positive mouse, the negative control mice in bottom right.

Fig. 6: sKAP11.1p promoter fragment tissue specificity detected result.Fig. 6 A is light field; Fig. 6 B is the fluorescence field, wavelength 500nm.Tissue is followed successively by: 1 hair; 2 spleens; 3 livers; 4 hearts; 5 lungs; 6 stomaches; 7 intestines; 8 kidneys; 9 testis; 10 brains; 11 muscle; 12 tongues.

Embodiment

Embodiment 1: the checking of tibetan sheep hair follicle specific expression gene KRTAP11.1 distribution expression pattern

This experiment agents useful for same and instrument comprise: TRIzol buys the (article No.: 15596026) in Invitrogen company; RNase-FreeddH2O buys the (article No.: RT121) in sky root biochemical technology company limited; Precious biotechnology Dalian company limited " PrimeScript RT reagent Kit With gDNA Eraser " test kit is used in reverse transcription, and (article No.: DRR047S), the TaKaRarTaq(article No. of precious biotechnology Dalian company limited is used in the PCR reaction: DR001); The DEPC(diethylpyrocarbonate) buy (article No.: D5758) in Sigma company; In situ hybridization use Affymetrix company QuantiGene ViewRNA ISH Tissue Assay in situ hybridization test kit (article No.: QVT0050); Test all consumptive materials used and all use the 0.1%DEPC soaked overnight, and in 120 ℃ of autoclaving 30min.The MyCycler PCR instrument that instrument is Bio-red company.(1) RT-PCR detects the expression of tibetan sheep KRTAP11.1 gene in each tissue

Gather 16 kinds of tissue samples that tibetan sheep (Linzhi Area of Tibet Yang Chang) comprises hair follicle, skin (containing hair follicle), the heart, liver, spleen, lung, kidney, cud, abomasum, small intestine, large intestine, lymph (mesentery), fat, muscle, testis, ovary.Utilize the TRIzol method to extract each total tissue RNA and (buy in the reagent of Invitrogen company article No.: 15596026), operate according to the operated products specification sheets.It is good that the RNA extracted detects integrity through 1% agarose gel electrophoresis, through spectrophotometer, detects OD260/OD280 between 1.8-2.2, can be used for further experiment and use.Take out 1 μ g from total RNA of each tissue, utilize "

RT reagent Kit With gDNAEraser " test kit (purchased from precious biotechnology Dalian company limited) respectively reverse transcription become cDNA, concrete reaction conditions and operation are with reference to product description.Utilize round pcr to detect the expression of KRTAP11.1 gene (NCBI accession number: NM 001080740.1) in each tissue, primer sequence is shown in SEQID NO:1 and 2.The fragment sequence that amplification obtains is shown in SEQ ID NO:3.

PCR system and reaction conditions are as follows:

PCR reaction system (cumulative volume) is 10 μ L:

The PCR reaction conditions:

94 ℃ of 5min; (94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 10s) * 28 circulations; 72 ℃ of 3min

The PCR product is detected through 1% agarose gel electrophoresis, PCR product loading 4 μ L, 100bpMarker loading 4 μ L, 100V constant voltage electrophoresis 30min, take pictures under ultraviolet gel imaging instrument, and result is as Fig. 1.

As shown in Figure 1, KRTAP11.1 only has amplified band in hair follicle and skin (containing hair follicle) sample, illustrates that KRTAP11.1 is in detected tissue, only in hair follicle and skin (containing hair follicle), expresses.

(2) in situ hybridization detects the expression position of tibetan sheep KRTAP11.1 in tibetan sheep skin

In order further to determine the expression position of tibetan sheep KRTAP11.1 gene in skin, we locate it by hybridization in situ experiment.Use QuantiGene ViewRNA ISH Tissue Assay in situ hybridization test kit (purchased from Affymetrix company, article No.: QVT0050).By Affymetrix company according to the sequence synthesising probing needle shown in SEQ ID NO9.Gather fresh tibetan sheep skin samples, fixing 48h in 4% paraformaldehyde (containing 0.1%DEPC).The skin histology embedding is become to cured also with anticreep microsection manufacture Skin slice, and thickness is 4 μ m.In situ hybridization operation reference reagent box specification sheets.Result as shown in Figure 2.

As shown in Figure 2, red probe signals is present among the tibetan sheep hair shaft, illustrates that tibetan sheep KRTAP11.1 is the hair follicle specific expression gene.

Embodiment 2: tibetan sheep hair follicle specific expression gene KRTAP11.1 promoter region sKAP11.1p clone

The present embodiment agents useful for same and instrument comprise: the PrimeSTARHS DNAPolymerase(article No. that high-fidelity amplification enzyme is precious biotechnology Dalian company limited: DR010A); The DNA purification kit is used the TIANquick Midi Purification Kit test kit (article No.: DP204) of TIANGEN Biotech (Beijing) Co., Ltd..The MyCyclerPCR instrument that instrument is Bio-red company.

According to Australian CSIRO(British Commonwealth of Nations's science and industrial research tissue) issue tibetan sheep genome v2.0 version (network address: http://www.livestockgenomics.csiro.au/sheep/oar2.0.php), at tibetan sheep KRTAP11.1 gene 5 ' end upstream design primer, this gene promoter region 5954KB size that increases fragment, and called after sKAP11.1p(SEQ ID NO:10).In order to carry out next step construction of eukaryotic expression vector, at 5 ' of amplimer, hold and added respectively the SacII(forward primer) and the BamHI(reverse primer) restriction enzyme site.Primer sequence is as SEQ ID NO:4,5.Concrete reaction system and condition are as follows:

PCR reaction system (cumulative volume) is 50 μ L:

Reaction conditions:

94 ℃ of 5min; (98 ℃ of 10s, 68 ℃ of 7min) * 35 circulations; 72 ℃ of 10min

The PCR product detects through 0.8% agarose gel electrophoresis, purified product loading 4 μ L, DL15,000+2000Marker(TIANGEN Biotech (Beijing) Co., Ltd., article No.: MD116) loading 4 μ L, 100V constant voltage electrophoresis 40min, under ultraviolet gel imaging instrument, take pictures, result is as Fig. 3.

As shown in Figure 3, amplified fragments is special and clear, and size conforms to expection, can be used for next step experiment.

Embodiment 3: carrier for expression of eukaryon sKAP11.1p-GFP builds

The present embodiment agents useful for same and instrument comprise: restriction enzyme SacII and BamHI are purchased from Fermentas company; The ZsGreen1-1 carrier framework is purchased from the Clontech(article No.: 632473); Gel reclaims the TIANgel Midi Purification Kit(article No. that test kit is used TIANGEN Biotech (Beijing) Co., Ltd.: DP209); Ligase enzyme is used the DNA Ligation Kit LONG(article No. of precious biotechnology Dalian company limited: D6024); Competent cell DH5 α is purchased from precious biotechnology Dalian company limited (article No.: D9057).The TaKaRa rTaq(article No. of precious biotechnology Dalian company limited is used in the PCR reaction: DR001).Plasmid extraction is used the TIANprep Mini Plasmid Kit test kit (article No.: the MyCyclerPCR instrument that DP103) instrument is Bio-red company of TIANGEN Biotech (Beijing) Co., Ltd..Concrete operations are as follows:

(1) sKAP11.1p promoter region fragment pcr amplification in above-described embodiment 2 obtained is carried out enzyme and is cut, and its objective is the restriction enzyme site that cuts the fragment two ends, forms sticky end, in order to be connected in carrier.

(2) reaction system and condition:

Cumulative volume 20ul system:

Reaction conditions: 37 ℃ are spent the night.

Enzyme is cut product after 0.8% agarose gel electrophoresis, and the fragment of the about 6000bp size of sKAP11.1p is cut to glue purification (use TIANgel Midi Purification Kit, concrete operations are with reference to specification sheets).

(2) digested plasmid ZsGreen1-1.Purpose is to cut and the corresponding restriction enzyme site in sKAP11.1p fragment two ends at the plasmid promoter region, forms sticky end, in order to the sKAP11.1p fragment is connected on carrier.Concrete system and reaction conditions are as follows:

Cumulative volume 20 μ l systems:

Reaction conditions: 37 ℃ are spent the night

Enzyme is cut product after 0.8% agarose gel electrophoresis, and the about 4500bp size of linearizing ZsGreen1-1 fragment is cut to glue purification (use TIANgel Midi Purification Kit, concrete operations are with reference to specification sheets).

(3) connect.Purpose is by the sKAP11.1p fragment, connects into the promoter region of ZsGreen1-1 carrier, finally is built into the sKAP11.1p-GFP carrier.Use DNALigation Kit LONG test kit.Concrete system and reaction conditions are as follows:

Cumulative volume 49 μ L systems:

(1) the ZsGreen1-1 enzyme obtained in is cut purified product 10 μ L

(2) the sKAP11.1p fragment enzyme obtained in is cut purified product 1 μ L

10X LONG Ligation Buffer 5μL

DdH ₂O to 49 μ L

Reaction conditions: after 65 ℃ of 3min, chilling in ice; Add DNALigase<LONG in reaction system > 1 μ L, the slight mixing; 16 ℃ of 15h.

(4) vector plasmid transforms and extracts.Purpose is to screen positive plasmid enlarged culturing.Concrete operations are as follows:

1. the DH5 α competent cell 100 μ L that thaw on ice, add 10 μ L to connect product, standing 30min on ice wherein;

2. put into 42 ℃ of water-bath heat shock 60s, place 3min on ice;

3. the LB substratum (not containing microbiotic) that adds 37 ℃ of preheatings of 900 μ L, 37 ℃ of shaking culture 1h(225rpm)

4. get 200 μ L bacterium liquid coating LB solid plate substratum (containing kantlex, concentration 30 μ g/mL), 37 ℃ of incubated overnight.

5. the picking white colony is put in the 1.5mL centrifuge tube that contains 1mLLB substratum (containing kantlex, concentration 30 μ g/mL)), 37 ℃ of shaking culture 6h(225rpm).

6. the bacterium liquid of take carries out the PCR detection as template, and the positive (the sKAP11.1p fragment is template) and negative (without template) contrast are set.Primer sequence is SEQ ID NO:6,7; The product sequence is SEQ ID NO:8, and size is 561bp.Concrete system and condition are as follows:

PCR reaction system (cumulative volume) is 10 μ L:

The PCR reaction conditions:

94 ℃ of 5min; (94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 40s) * 30 circulations; 72 ℃ of 3min

Use 1% agarose gel electrophoresis to detect the PCR product, have the sample of 561bp amplified band to be positive bacterium colony.Illustrate in this bacterium colony and comprise the sKAP11.1p-GFP carrier built.

Get 100 μ L from the bacterium liquid of positive bacterium colony, join in the 15mL centrifuge tube that 6mLLB liquid nutrient medium (containing kantlex, concentration 30 μ g/mL) is housed.37 ℃ of enlarged culturing of spending the night.

Bacterium liquid after using TIANprep Mini Plasmid Kit test kit to enlarged culturing carries out plasmid extraction, the final sKAP11.1p-GFP hair follicle specific eucaryon expression vector built that obtains.SKAP11.1p-GFP carrier structure schematic diagram as shown in Figure 4.

Embodiment 4:sKAP11.1p-GFP vector expression activity and specificity checking

The present embodiment laboratory animal used is that laboratory animal is kunming mice (white is provided by BGI-Shenzhen).

(1) make transgenic mice

The present embodiment adopts the method (Xu Yimei of micro-procaryotic injection, Deng. the expression [J] of green fluorescence protein gene in the transgenic mice body. biotechnology communication .2005) linearizing (is utilized to the digestion with restriction enzyme circular vectors, make carrier be become the linear structure of open loop by closed ring texture) the sKAP11.1p-GFP vector injection enter Mouse Pronuclear phase embryo, by by homologous recombination, the sKAP11.1p-GFP fragment being incorporated in mouse genome, be made into transgenic mice, activity and the specificity of checking sKAP11.1p promoter sequence on individual level.

The making of transgenic mice is entrusted in BGI-Shenzhen and is completed.The making flow process comprises: 1. utilize restriction enzyme A fl II single endonuclease digestion sKAP11.1p-GFP carrier, make the linearizing of carrier; Utilize DNA purification kit purifying enzyme to cut product;

2. utilize microinjection technique that the linearizing sKAP11.1p-GFP fragment after purifying is injected in the early stage protokaryon embryo of mouse;

3. the protokaryon embryo transfer after injection is entered in the uterus of the female mouse of replace-conceive acceptor; 4. after the female mouse of replace-conceive produces son, identify the sub-mouse of transgenic positive.

(2) sKAP11.1p-GFP transgenic positive mouse Phenotypic examination

In the sKAP11.1p-GFP carrier, be connected with ZsGreen1 green fluorescent protein reporter gene after the sKAP11.1p promotor, so we observe the hair phenotypic difference of sKAP11.1p-GFP transgenic positive mouse and negative control mouse by the living imaging system under the exciting light of 500nm wavelength.Result is as Fig. 4.

As shown in Figure 4, upper left is sKAP11.1p-GFP transgenic positive mouse, the negative control mice in bottom right.SKAP11.1p-GFP transgenic positive mouse table emitting fluorescence signal under the exciting light of 500nm wavelength, and negative mouse is invisible after the match at fluorescence.Illustrate that the sKAP11.1p promoter fragment has good startup activity in mouse hair.

(3) sKAP11.1p-GFP promotor tissue specificity checking

Gather sKAP11.1p-GFP transgenic positive mouse and negative hair, the heart, liver, spleen, lung, kidney, stomach, intestines, muscle, tongue, testis, cerebral tissue, by a organized way together in the living imaging system excitation light irradiation with the 500nm wavelength detect the luminous situation of each histofluorescence.Result is as Fig. 5.

1. as shown in Figure 5, in the fluorescence field, only have the hair emitting fluorescence of sKAP11.1p-GFP transgenic positive mouse, its hetero-organization is all invisible.Illustrate that the sKAP11.1p promoter fragment has hair follicle tissue's expression specificity.

Claims (2)

1. a sheep hair follicle specific expressing promoter fragment sKAP11.1, its nucleotide sequence is as shown in SEQ ID NO:10.

2. the promoter fragment sKAP11.1p claimed in claim 1 application that controlling gene is expressed in animal.

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