麻疯树JcFATA基因干涉表达载体构建和功能初步分析
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摘要
植物脂酰-酰基载体蛋白硫酯酶A(fatty acyl-acyl carrier protein thioesterase,FATA)是脂肪酸积累过程中的关键酶,直接调控脂肪酸的含量与组成。目前有关麻疯树(Jatropha curcas)FATA基因(JcFATA)(Gen Bank登录号:EU267122.2)的功能研究还未见报道。本研究首先构建JcFATA基因RNA干涉(RNA interference,RNAi)表达载体,进行下调表达研究,以麻疯树叶片cDNA为模板,克隆出JcFATA基因正向片段FATA1(439 bp),然后将其连接到含内含子的干涉表达载体p YLRNAi.2-35S上,构建JcFATA基因RNAi表达载体pYLRNAi.2-35S-FATA1-FATA2;再通过电击法将其转入到根癌农杆菌(Agrobacterium tumefaciens)EHA105感受态细胞中,然后利用花序侵染法进行拟南芥(Arabidopsis thaliana)遗传转化,最后通过PCR检测和Southern blot验证。对转基因拟南芥植株进行了表型观察、表达量分析和脂肪酸的含量测定,结果显示,JcFATA基因干涉表达的转基因植物生长发育可能受到抑制。本研究结果可为进一步探讨JcFATA基因的功能及该基因在麻疯树遗传改良中的应用提供理论依据。
The fatty acyl-acyl carrier protein thioesterase(FATA) is a key enzyme in the accumulation of fatty acids, which directly regulates the content and composition of fatty acids. The function of the acyl-ACP thioesterase A gene(Gen Bank accession number: EU267122.2) in Jatropha curcas has not been reported before. In this study, the JcFATA gene RNAi expression vector had been constructed and down-regulation of JcFATA gene expression was accomplished, the positive fragment FATA1(439 bp) of JcFATA gene was cloned from Jatropha curcas leaf cDNA, which was then inserted into p YL RNAi.2-35 S contained intron to construct the intermediate vector pYLRNAi.2-35 S-FATA1; the reverse fragment FATA2(439 bp) was amplified by PCRused the intermediate vector as a template, and then inserted into the intermediate vector to construct the JcFATA gene RNAi expression vector p YLRNAi.2-35 S-FATA1-FATA2; it was transformed into Agrobacterium tumefaciens EHA105 competent cells by electroporation, and then transformed into Arabidopsis thaliana by inflorescence infection method. Finally, the transformation was confirmed by PCR and Southern blot. In addition, preliminary phenotypic observation, expression analysis and determination of fatty acid content of transgenic Arabidopsis plants was carried out in this study, the results indicated that the growth and development of transgenic plants might be inhibited by RNAi expression of JcFATA gene. The results of this study can provide a theoretical basis for further discussion of the function of JcFATA gene and its application in the genetic improvement of Jatropha curcas.
The fatty acyl-acyl carrier protein thioesterase(FATA) is a key enzyme in the accumulation of fatty acids, which directly regulates the content and composition of fatty acids. The function of the acyl-ACP thioesterase A gene(Gen Bank accession number: EU267122.2) in Jatropha curcas has not been reported before. In this study, the JcFATA gene RNAi expression vector had been constructed and down-regulation of JcFATA gene expression was accomplished, the positive fragment FATA1(439 bp) of JcFATA gene was cloned from Jatropha curcas leaf cDNA, which was then inserted into p YL RNAi.2-35 S contained intron to construct the intermediate vector pYLRNAi.2-35 S-FATA1; the reverse fragment FATA2(439 bp) was amplified by PCRused the intermediate vector as a template, and then inserted into the intermediate vector to construct the JcFATA gene RNAi expression vector p YLRNAi.2-35 S-FATA1-FATA2; it was transformed into Agrobacterium tumefaciens EHA105 competent cells by electroporation, and then transformed into Arabidopsis thaliana by inflorescence infection method. Finally, the transformation was confirmed by PCR and Southern blot. In addition, preliminary phenotypic observation, expression analysis and determination of fatty acid content of transgenic Arabidopsis plants was carried out in this study, the results indicated that the growth and development of transgenic plants might be inhibited by RNAi expression of JcFATA gene. The results of this study can provide a theoretical basis for further discussion of the function of JcFATA gene and its application in the genetic improvement of Jatropha curcas.
引文
安建平,王小非,刘鑫,等.2016.苹果生长素输入载体Md AUX1的基因克隆及在拟南芥和烟草中的遗传转化[J].植物研究,36(6):902-908.(An J P,Wang X F,Liu X,et al.2016.Molecular cloning and genetic transformation analysis of an apple auxin influx transporter gene Md AUX1 in Arabidopsis thaliana and tobacco[J].Bulletin of Botanical Research,36(6):902-908.)
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霍雪寒,张景霞,高阳,等.2013.陆地棉Gh BI-1基因在拟南芥中的异位表达及耐盐性研究[J].山东农业科学,45(4):1-3.(Huo X H,Zhang J X,Gao Y,et al.2013.Ectopic expression of Gh BI-1 gene from upland cotton in Arabidopsis thaliana and study on its salt tolerance[J].Shandong Agricultural Sciences,45(4):1-3.)
胡旭霞,刘耀光.2006.植物RNA干涉载体的构建及其在水稻基因表达沉默中的应用[J].分子植物育种,4(5):621-626.(Hu X X,Liu Y G.2006.The construction of RNAi vectors and the use for gene silencing in rice[J].Molecular Plant Breeding,4(5):621-626.)
李云,宋云枝,朱常香,等.2008.Hp RNA的茎环比例对RNA介导的病毒抗性产生的影响[J].植物病理学报,38(5):468-477.(Li Y,Song Y Z,Zhu C X,et al.2008.Effect of stem-loop proportion in hp RNA on the RNA-mediated virus resistanc[J].Acta Phytopathologica Sinica,38(5):468-477.)
刘燕霞,彭廷,赵亚帆,等.2014.水稻简易RNAi载体构建及沉默效果鉴定[J].农业生物技术学报,22(7):832-840.(Liu Y X,Peng T,Zhao Y F,et al.2008.Construction of simple RNAi vector and identification of silencing effect in rice(Oryza sativa ssp.japonica)[J].Journal of Agricultural Biotechnology,22(7):832-840.)
刘颖,杨亚利,殷学贵,等.2017.麻疯树Jc FATA基因组织表达特性及其启动子克隆与表达分析[J].农业生物技术学报,25(2):214-221.(Liu Y,Yang Y L,Yin X G,et al.2017.Expression of Jc FATA gene in Jatropha curcas and its promoter cloning and analysis[J].Journal of Agricultural Biotechnology,25(2):214-221)
柳忠玉,赵树进.2015.虎杖查耳酮合酶基因RNAi载体的构建及其遗传转化[J].中草药,46(3):412-417.(Liu ZY,Zhao S J.2015.Construction of RNA interference vector of Polygonum cuspidatum chalcone synthase gene and its genetic transformation[J].Chinese Traditiona and Herbal Drugs,46(3):412-417.)
倪雪峰.2014.利用DGAT1、SLC、FAD2和FAD3基因改良拟南芥和甘蓝型油菜的含油量和脂肪酸不饱和性[D].硕士学位论文,西北农林科技大学,导师:张猛,pp 20-21.(Ni X F.2014.The oil content and fatty acid unsaturation of Arabidopsis thaliana and Brassica napus L.were improved by DGAT1,SLC,FAD2 and FAD3 genes[D].Thesis for M.S.,Technology of Northwest A&F University,Supervisor:Zhang M,pp.20-21.)
仇键,刘彤,王帆,等.2016.巴西橡胶树Hb MYB52基因的克隆及其在拟南芥中的表达[J].热带亚热带植物学报24(6):671-679.(Qiu J,Liu T,Wang F,et al.2016.Cloning of Hb MYB52 from rubber tree(Hevea brasiliensis)and its heterogenous expression in Arabidopsis thaliana[J].Journal of Tropical and Subtropical Botany,24(6)671-679.)
陶芳,韩国民,项艳,等.2006.玉米FAD2基因RNA干涉表达载体的构建及转化[J].激光生物学报,15(5):488-495.(Tao F,Han G M,Xiang Y,et al.2006.Construction of maize FAD2-hp RNAi expression vector and its transformation[J].Acta Laser Biology Sinica,15(5):488-495.)
杨丽,陈东亮,彭镇华,等.2014.麻竹转录因子Dl SCL6的基因克隆及在拟南芥中异位表达[J].林业科学,50(11):52-57.(Yang L,Chen D L,Peng Z H,et al.2014Cloning of transcription factor Dl SCL6 from Dendrocalamus latiflorus and its ectopic expression in Arabidopsis thaliana[J].Scientia Silvae Sinicae,50(11):52-57.)
Buhr T,Sato S,Ebrahim F,et al.2002.Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean[J].The Plant Journal,30(2):155-163.
Carlsson A S,Yilmaz J L,Green A G,et al.2011.Replacing fossil oil with fresh oil-with what and for what?[J].European Journal of Lipid Science and Technology,113(7):812-831.
Chen M,Liu H,Kong J,et al.2011.Rop GEF7 regulatesPLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].The Plant Cell,23(8):2880-2894.
Dong S,Liu Y,Xiong B,et al.2016.Transcriptomic analysis of a potential bioenergy tree,Pistacia chinensis Bunge,and identification of candidate genes involved in the biosynthesis of oil[J].Bio Energy Research,9(3):740-749.
Helliwell C,Waterhouse P.2003.Constructs and methods for high-throughput gene silencing in plants[J].Methods,30(4):289-295.
Jones A,Davies H M,Voelker T A.1995.Palmitoyl-acyl carrier protein(ACP)thioesterase and the evolutionary origin of plant acyl-ACP thioesterases[J].The Plant Cell,7(3):359-371.
Joshi M,Mishra A,Jha B.2011.Efficient genetic transformation of Jatropha curcas L.by microprojectile bombardment using embryo axes[J].Industrial Crops and Products,33(1):67-77.
Knutzon D S,Thompson G A,Radke S E,et al.1992.Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene[J].Proceedings of the National Academy of Sciences,89(7):2624-2628.
Liu Q,Singh S P,Green A G.2002.High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing[J].Plant Physiology,129(4):1732-1743.
Moreno-Pérez A J,Venegas-Calerón M,Vaistij F E,et al.2012.Reduced expression of Fat A thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds[J].Planta,235(3):629-639.
Qu J,Mao H Z,Chen W,et al.2012.Development of markerfree transgenic Jatropha plants with increased levels of seed oleic acid[J].Biotechnology for Biofuels,5(1):1-11.
Sánchez-García A,Moreno-Pérez A J,Muro-Pastor A M,et al.2010.Acyl-ACP thioesterases from castor(Ricinus communis L.):An enzymatic system appropriate for high rates of oil synthesis and accumulation[J].Phytochemistry,71(8):860-869.
Shi D Q,Zhou Y H,Zhang L H,et al.2000.Cloning the promoter of Bc NA1 from Brassica napus and Fad2 gene from Arabidopsis thaliana and construction of the plant expression vector[J].High Technology Letters,6(1):83-90.
Sidahmed A M E,Wilkie B.2010.Endogenous antiviral mechanisms of RNA interference:A comparative biolo-gy perspective[J].RNA Interference:From Biology to Clinical Applications,623:3-19.
Siomi H,Siomi M C.2009.On the road to reading the RNA-interference code[J].Nature,457(22):396-404.
Southern E M.1975.Detection of specific sequences among DNA fragments separated by gel electrophoresis[J].Journal of Molecular Biology,98(3):503-517.
Stoutjesdijk P A,Singh S P,Liu Q,et al.2002.Hp RNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing[J].Plant Physiology,129(4):1723-1731.
Tamura K,Peterson D,Peterson N,et al.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,28(10):2731-2739.
Tan X L,Huang Q,Tan R K,et al.2015.Cloning and func-tional characterization of a fatty acyl-acyl carrier protein thioesterase gene(Bn Fat B)in Brassica napus L.[J].Journal of Agricultural Science and Technology,17(4):987-997.
Thelen J J,Ohlrogge J B.2002.Metabolic engineering of fatty acid biosynthesis in plants[J].Metabolic Engineering,4(1):12-21.
Uemura H.2012.Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast:Current state and perspectives[J].Applied Microbiology and Biotechnology,95(1):1-12.
Wesley S V,Helliwell C A,Smith N A,et al.2001.Construct design for efficient,effective and high-throughput gene silencing in plants[J].The Plant Journal,27(6):581-590.
Wesley S V,Liu Q,Wielopolska A,et al.2003.Custom knockouts with hairpin RNA-mediated gene silencing[J].Plant Functional Genomics,236:273-286.
曹毅,陈英,蒋彦,等.2001.利用电击法转化玉米胚性愈伤组织获得GUS基因瞬时表达的研究[J].四川大学学报,38(6):913-918.(Cao Y,Chen Y,Jiang Y,et al.2001.Study of tissue culture of embrogenic calli of maize and transient expression of GUS gene by electroporation[J].Journal of Sichuan Univevsity,38(6):913-918.)
霍雪寒,张景霞,高阳,等.2013.陆地棉Gh BI-1基因在拟南芥中的异位表达及耐盐性研究[J].山东农业科学,45(4):1-3.(Huo X H,Zhang J X,Gao Y,et al.2013.Ectopic expression of Gh BI-1 gene from upland cotton in Arabidopsis thaliana and study on its salt tolerance[J].Shandong Agricultural Sciences,45(4):1-3.)
胡旭霞,刘耀光.2006.植物RNA干涉载体的构建及其在水稻基因表达沉默中的应用[J].分子植物育种,4(5):621-626.(Hu X X,Liu Y G.2006.The construction of RNAi vectors and the use for gene silencing in rice[J].Molecular Plant Breeding,4(5):621-626.)
李云,宋云枝,朱常香,等.2008.Hp RNA的茎环比例对RNA介导的病毒抗性产生的影响[J].植物病理学报,38(5):468-477.(Li Y,Song Y Z,Zhu C X,et al.2008.Effect of stem-loop proportion in hp RNA on the RNA-mediated virus resistanc[J].Acta Phytopathologica Sinica,38(5):468-477.)
刘燕霞,彭廷,赵亚帆,等.2014.水稻简易RNAi载体构建及沉默效果鉴定[J].农业生物技术学报,22(7):832-840.(Liu Y X,Peng T,Zhao Y F,et al.2008.Construction of simple RNAi vector and identification of silencing effect in rice(Oryza sativa ssp.japonica)[J].Journal of Agricultural Biotechnology,22(7):832-840.)
刘颖,杨亚利,殷学贵,等.2017.麻疯树Jc FATA基因组织表达特性及其启动子克隆与表达分析[J].农业生物技术学报,25(2):214-221.(Liu Y,Yang Y L,Yin X G,et al.2017.Expression of Jc FATA gene in Jatropha curcas and its promoter cloning and analysis[J].Journal of Agricultural Biotechnology,25(2):214-221)
柳忠玉,赵树进.2015.虎杖查耳酮合酶基因RNAi载体的构建及其遗传转化[J].中草药,46(3):412-417.(Liu ZY,Zhao S J.2015.Construction of RNA interference vector of Polygonum cuspidatum chalcone synthase gene and its genetic transformation[J].Chinese Traditiona and Herbal Drugs,46(3):412-417.)
倪雪峰.2014.利用DGAT1、SLC、FAD2和FAD3基因改良拟南芥和甘蓝型油菜的含油量和脂肪酸不饱和性[D].硕士学位论文,西北农林科技大学,导师:张猛,pp 20-21.(Ni X F.2014.The oil content and fatty acid unsaturation of Arabidopsis thaliana and Brassica napus L.were improved by DGAT1,SLC,FAD2 and FAD3 genes[D].Thesis for M.S.,Technology of Northwest A&F University,Supervisor:Zhang M,pp.20-21.)
仇键,刘彤,王帆,等.2016.巴西橡胶树Hb MYB52基因的克隆及其在拟南芥中的表达[J].热带亚热带植物学报24(6):671-679.(Qiu J,Liu T,Wang F,et al.2016.Cloning of Hb MYB52 from rubber tree(Hevea brasiliensis)and its heterogenous expression in Arabidopsis thaliana[J].Journal of Tropical and Subtropical Botany,24(6)671-679.)
陶芳,韩国民,项艳,等.2006.玉米FAD2基因RNA干涉表达载体的构建及转化[J].激光生物学报,15(5):488-495.(Tao F,Han G M,Xiang Y,et al.2006.Construction of maize FAD2-hp RNAi expression vector and its transformation[J].Acta Laser Biology Sinica,15(5):488-495.)
杨丽,陈东亮,彭镇华,等.2014.麻竹转录因子Dl SCL6的基因克隆及在拟南芥中异位表达[J].林业科学,50(11):52-57.(Yang L,Chen D L,Peng Z H,et al.2014Cloning of transcription factor Dl SCL6 from Dendrocalamus latiflorus and its ectopic expression in Arabidopsis thaliana[J].Scientia Silvae Sinicae,50(11):52-57.)
Buhr T,Sato S,Ebrahim F,et al.2002.Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean[J].The Plant Journal,30(2):155-163.
Carlsson A S,Yilmaz J L,Green A G,et al.2011.Replacing fossil oil with fresh oil-with what and for what?[J].European Journal of Lipid Science and Technology,113(7):812-831.
Chen M,Liu H,Kong J,et al.2011.Rop GEF7 regulatesPLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].The Plant Cell,23(8):2880-2894.
Dong S,Liu Y,Xiong B,et al.2016.Transcriptomic analysis of a potential bioenergy tree,Pistacia chinensis Bunge,and identification of candidate genes involved in the biosynthesis of oil[J].Bio Energy Research,9(3):740-749.
Helliwell C,Waterhouse P.2003.Constructs and methods for high-throughput gene silencing in plants[J].Methods,30(4):289-295.
Jones A,Davies H M,Voelker T A.1995.Palmitoyl-acyl carrier protein(ACP)thioesterase and the evolutionary origin of plant acyl-ACP thioesterases[J].The Plant Cell,7(3):359-371.
Joshi M,Mishra A,Jha B.2011.Efficient genetic transformation of Jatropha curcas L.by microprojectile bombardment using embryo axes[J].Industrial Crops and Products,33(1):67-77.
Knutzon D S,Thompson G A,Radke S E,et al.1992.Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene[J].Proceedings of the National Academy of Sciences,89(7):2624-2628.
Liu Q,Singh S P,Green A G.2002.High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing[J].Plant Physiology,129(4):1732-1743.
Moreno-Pérez A J,Venegas-Calerón M,Vaistij F E,et al.2012.Reduced expression of Fat A thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds[J].Planta,235(3):629-639.
Qu J,Mao H Z,Chen W,et al.2012.Development of markerfree transgenic Jatropha plants with increased levels of seed oleic acid[J].Biotechnology for Biofuels,5(1):1-11.
Sánchez-García A,Moreno-Pérez A J,Muro-Pastor A M,et al.2010.Acyl-ACP thioesterases from castor(Ricinus communis L.):An enzymatic system appropriate for high rates of oil synthesis and accumulation[J].Phytochemistry,71(8):860-869.
Shi D Q,Zhou Y H,Zhang L H,et al.2000.Cloning the promoter of Bc NA1 from Brassica napus and Fad2 gene from Arabidopsis thaliana and construction of the plant expression vector[J].High Technology Letters,6(1):83-90.
Sidahmed A M E,Wilkie B.2010.Endogenous antiviral mechanisms of RNA interference:A comparative biolo-gy perspective[J].RNA Interference:From Biology to Clinical Applications,623:3-19.
Siomi H,Siomi M C.2009.On the road to reading the RNA-interference code[J].Nature,457(22):396-404.
Southern E M.1975.Detection of specific sequences among DNA fragments separated by gel electrophoresis[J].Journal of Molecular Biology,98(3):503-517.
Stoutjesdijk P A,Singh S P,Liu Q,et al.2002.Hp RNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing[J].Plant Physiology,129(4):1723-1731.
Tamura K,Peterson D,Peterson N,et al.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,28(10):2731-2739.
Tan X L,Huang Q,Tan R K,et al.2015.Cloning and func-tional characterization of a fatty acyl-acyl carrier protein thioesterase gene(Bn Fat B)in Brassica napus L.[J].Journal of Agricultural Science and Technology,17(4):987-997.
Thelen J J,Ohlrogge J B.2002.Metabolic engineering of fatty acid biosynthesis in plants[J].Metabolic Engineering,4(1):12-21.
Uemura H.2012.Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast:Current state and perspectives[J].Applied Microbiology and Biotechnology,95(1):1-12.
Wesley S V,Helliwell C A,Smith N A,et al.2001.Construct design for efficient,effective and high-throughput gene silencing in plants[J].The Plant Journal,27(6):581-590.
Wesley S V,Liu Q,Wielopolska A,et al.2003.Custom knockouts with hairpin RNA-mediated gene silencing[J].Plant Functional Genomics,236:273-286.