马铃薯StTrxF基因的克隆与功能分析
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摘要
为探究马铃薯硫氧还蛋白基因(StTrxF)的耐盐生理机制,通过RT-PCR方法从马铃薯品种中薯5号中克隆得到全长549bp硫氧还蛋白基因(StTrxF)。该基因编码182个氨基酸,预测蛋白质分子量为44.17ku,理论等电点(pI)为5.23,具有典型的Thioredoxin结构域。由StTrxF基因推导的氨基酸序列与番茄、拟南芥、芝麻和赤霞珠等TrxF蛋白质氨基酸序列的同源性为96.02%～59.28%。构建植物过表达载体,导入拟南芥中,获得转基因拟南芥纯系植株。通过对转基因拟南芥植株的耐盐离体和盆栽鉴定,表明转基因植株的耐盐性显著提高。同时盐胁迫下,转基因植株的超氧化物歧化酶(SOD)活性和脯氨酸含量显著提高,丙二醛(MDA)含量显著降低。结果表明,表达StTtxF基因通过增加转基因植株脯氨酸含量,提高SOD活性,降低MDA含量,以维持细胞渗透平衡并激活ROS清除系统,具有提高转基因拟南芥植株耐盐性的显著效果。
To explore the salt-tolerant physiological mechanism of potato thioredoxin gene(StTrxF),StTrxF gene was cloned from potato No.5 by RT-PCR.The ORF of StTrxF was 549 bp in length and encoded 182 amino acids with a molecular weight of 44.17 ku and a theoretical isoelectric point(pI)of 5.23 with a typical thioredoxin domain.The amino acid sequence deduced from StTrxF gene was 96.02%-59.28%homology to the TrxF proteins of Arabidopsis thaliana,sesame and Cabernet sauvignon.A plant expression vector was constructed and the StTrxF gene was introduced into Arabidopsis by dipping method to obtain transgenic Arabidopsis homozygous lines.The salt tolerance of transgenic Arabidopsis plants was identified by pot experiment.The results indicated that the salt tolerance of transgenic plants was significantly improved.At the same time,under salt stress,the superoxide dismutase(SOD)activity and proline content were significantly increased,and the content of malondialdehyde(MDA)was significantly decreased.These results indicated that the expression of StTtxF gene had significant effect on enhancing the salt tolerance of transgenic Arabidopsis plants by increasing proline content and SOD activity,decreasing MDA content in the transgenic plants,maintaining cell osmotic balance and activating ROS scavenging system.This study would provide an experimental basis for further research of salt-tolerant crops,and a theoretical reference for plant salttolerant bio-genetic engineering.
To explore the salt-tolerant physiological mechanism of potato thioredoxin gene(StTrxF),StTrxF gene was cloned from potato No.5 by RT-PCR.The ORF of StTrxF was 549 bp in length and encoded 182 amino acids with a molecular weight of 44.17 ku and a theoretical isoelectric point(pI)of 5.23 with a typical thioredoxin domain.The amino acid sequence deduced from StTrxF gene was 96.02%-59.28%homology to the TrxF proteins of Arabidopsis thaliana,sesame and Cabernet sauvignon.A plant expression vector was constructed and the StTrxF gene was introduced into Arabidopsis by dipping method to obtain transgenic Arabidopsis homozygous lines.The salt tolerance of transgenic Arabidopsis plants was identified by pot experiment.The results indicated that the salt tolerance of transgenic plants was significantly improved.At the same time,under salt stress,the superoxide dismutase(SOD)activity and proline content were significantly increased,and the content of malondialdehyde(MDA)was significantly decreased.These results indicated that the expression of StTtxF gene had significant effect on enhancing the salt tolerance of transgenic Arabidopsis plants by increasing proline content and SOD activity,decreasing MDA content in the transgenic plants,maintaining cell osmotic balance and activating ROS scavenging system.This study would provide an experimental basis for further research of salt-tolerant crops,and a theoretical reference for plant salttolerant bio-genetic engineering.
引文
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[11]吴剑,宋宝安,胡德禹,杨松.植物耐盐的信号转导途径及相关基因研究进展[J].山地农业生物学报,2011,30(5):443-447Wu J,Song B A,Hu D Y,Yang S.Progress on signal transduction pathways and related genes of plant salttolerance in plant[J].Journal of Mountain Agriculture and Biology,2011,30(5):443-447(in Chinese)
[12] Wang F B,Zhu H,Kong W L,Peng R H,Liu Q C,Yao Q H.The Antirrhinum AmDEL gene enhances flavonoids accumulation and salt and drought tolerance in transgenic Arabidopsis[J].Planta,2016,244(1):59-73
[13] Wang F B,Kong W L,Niu Y,Ye Y X,Fan S,Wang Y J,Chen X H,Zhou Q.StTrxF,apotato plastidic thioredoxin F-type protein gene,is involved in starch accumulation in transgenic Arabidopsis thaliana[J].Biotechnology&Biotechnological Equipment,2017,31(3):486-492
[14] Jiang W,Yang S P,Yu D Y,Gai J Y.Cloning and characterization of a novel gene GmMF1in soybean(Glycine max L merr)[J].Agricultural Sciences in China,2011,10(12):1834-1841
[15]许红梅,张立军,刘淳.农杆菌蘸花法侵染拟南芥的研究[J].北方园艺2010(14):143-146Xu H M,Zhang L J,Liu C.Study of infection Arabidopsis thaliana by agrobacterium dipping flower method[J].Northern Horticulture,2010(14):143-146(in Chinese)
[16] Wang F B,Zhu H,Chen D H,Li Z J,Peng R H,Yao Q H.A grape bHLH transcription factor gene,VvbHLH1,increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana[J].Plant Cell,Tissue and Organ Culture,2016,125:387-398
[17] Wang F B,Kong W L,Wong G,Gary Wong,Fu L F,Peng R H,Li Z J,Yao Q H.AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana[J].Molecular Genetics and Genomics,2016,291(4):1545-1559
[18]王学奎.植物生理生化实验原理和技术(第2版)[M].北京:高等教育出版社,2007Wang X K.Plant Physiology And Biochemistry Experimental Principles And Techniques 2nd[M].Beijing:Higher Education Press,2007(in Chinese)
[19]李合生,孙群,赵氏杰.植物生理生化实验原理与技术[M].北京:高等教育出版社,2000Li H S,Sun Q,Zhao J.Plant Physiology And Biochemistry Experiment Principle and Technology[M].Beijing:Higher Education Press,2000(in Chinese)
[20]王伟旗,侯文胜.大豆硫氧还蛋白基因的克隆与分析[J].大豆科学,2011,30(3):351-355Wang W Q,Hou W S.Clone and analysis of thioredoxin gene soybean[J].Soybean Science,2011,30(3):351-355(in Chinese)
[21]曹军,胡忠,庄东红,陈尚万,邹湘辉.花生“汕油523”在盐胁迫下应激反应的初步研究[J].汕头大学学报:自然科学版,2004,19(1):29-33Cao J,Hu Z,Zhuang D H,Chen S W,Zou X H.Studies on the shock responses of arachis hypogaea L cv shanyou 523under salt stress[J].Journal of Shantou University:Nature Science,2004,19(1):29-33(in Chinese)
[22]高树涛,石生伟.盐胁迫对5种抗旱型小麦苗期生理特性的影响[J].南方农业学报,2017,48(8):1374-1380Gao S T,Shi S W.Effects of salt stress on physiological characteristics of five drought-resistant wheat cultivars at seedling stage[J].Journal of Southern Agriculture,2017,48(8):1374-1380(in Chinese)
[23]余文兰,王朵朵,郭剑英,胡莲美.硫氧还蛋白对过氧化氢诱导的BRL-3A细胞损伤的保护作用[J].中国畜牧兽医,2014,41(6):154-159Yu W L,Wang D D,Guo J Y,Hu L M.Protective effect of thioredoxin on hydrogen H2O2-induced injury in BRL-3A cells[J].China Animal Husbandry and Veterinary Medicine,2014,41(6):154-158(in Chinese)
[24]张春宵,杨书华,刘文国,李晓辉.脯氨酸含量与玉米耐盐碱鉴定[J].吉林农业科学,2011,36(4):12-17Zhang C X,Yang S H,Liu W G,Li X H.Indentification of proline content and salt tolerance of maize[J].Journal of Jilin Agricultural Sciences,2011,36(4):12-17(in Chinese)
[2]裴怀弟,李忠旺,张艳萍,陈玉梁.马铃薯转GhABF2转录因子苗耐盐性研究[J].干旱地区农业研究,2015,33(5):90-95Pei H D,Li Z W,Zhang Y P,Chen Y L.Characteristics of salt tolerance capability of potato seedlings overexpressing GhABF2[J].Agrarian Regional Agriculture Research,2015,33(5):90-95(in Chinese)
[3]卢兴国,张贵,田再民,侯丁一,赵君,张之为.马铃薯StBAG3基因cDNA的克隆及晚疫菌对其诱导表达[J].华北农学报,2017,32(5):142-148Lu X G,Zhang G,Tian Z M,Hou D Y,Zhao J,Zhang Z W.Cloning of cDNAStBAG3and induced express by pathogen of potato late blight[J].Acta Agriculturae Boreali-Sinica,2017,32(5):142-148(in Chinese)
[4]卢肖平.马铃薯主粮化战略意义、瓶颈与政策建议[J].华中农业大学学报:社会科学版,2015(3):1-7Lu X P.Strategy of potato as staple food:significance,bottlenecks and policy suggestions[J].Journal of Huazhong Agricultural University:Social Science,2015(3):1-7(in Chinese)
[5]李丽霞,刘玉汇,王丽,余斌,张俊莲,王蒂.转AtNHX1基因马铃薯田间盐胁迫下的生理反应及耐盐性的综合评价[J].干旱地区农业研究,2017,35(3):130-138Li L X,Liu Y H,Wang L,Yu B,Zhang J L,Wang D.A comprehensive evaluation of salt-tolerance and physiological response of transgenic potato to salt stress in field by the introduction of AtNHX1gene[J].Agricultural Research in the Arid Areas,2017,35(3):130-137,150(in Chinese)
[6]杨欣,梁爱华,吴家和.抗虫转基因马铃薯研究进展[J].生物学杂志,2010,27(3):66-68Yang X,Liang A H,Wu JH.Progress of transgenic insectresistant potato[J].Acta Biologica Sinica,2010,27(3):66-68(in Chinese)
[7]巩檑,甘晓燕,张丽,陈虞超,聂峰杰,石磊,郭志乾,宋玉霞.马铃薯StNAC72基因克隆及表达分析[J].分子植物育种,2016,14(10):2589-2595Gong L,Gan X Y,Zhang L,Chen Y C,Nie F J,Shi L,Guo Z G,Song Y X.Cloning and expression analysis of StNAC72gene in potato[J].Molecular Plant Breeding,2016,14(10):2589-2595(in Chinese)
[8]张小莹,唐玉瑾,王刚,王跃进,张朝红.葡萄硫氧还蛋白基因(VvTrx)的克隆、表达和原核表达[J].农业生物技术学报,2015,23(9):1131-1140Zhang X Y,Tang Y J,Wang G,Wang YJ,Zhang C H.Cloning,expression and fusion expression of thioredoxin gene(trx)in grape(vitis vinifera)[J].Journal of Agricultural Biotechnology,2015,23(9):1131-1140(in Chinese)
[9]郑琼,马旭俊,杨传平.硫氧还蛋白(Trx)的研究进展[J].分子植物育种,2006,4(S2):78-82Zheng Q,Ma X J,Yang C P.Functional roles of thioredoxin(trx)[J].Molecular Plant Breeding,2006,4(S2):78-82(in Chinese)
[10]王全富,苗苗,侯艳华,史永磊,韩涵,吴莹莹,严天奇,曲俊杰.硫氧还蛋白研究进展[J].生物技术进展,2015,5(3):196-200Wang Q F,Miao M,Hou Y H,Shi Y L,Han H,Wu Y Y,Yan T Q,Qu J J.Recent advances in thioredoxin[J].Current Biotechnology,2015,5(3):196-200(in Chinese)
[11]吴剑,宋宝安,胡德禹,杨松.植物耐盐的信号转导途径及相关基因研究进展[J].山地农业生物学报,2011,30(5):443-447Wu J,Song B A,Hu D Y,Yang S.Progress on signal transduction pathways and related genes of plant salttolerance in plant[J].Journal of Mountain Agriculture and Biology,2011,30(5):443-447(in Chinese)
[12] Wang F B,Zhu H,Kong W L,Peng R H,Liu Q C,Yao Q H.The Antirrhinum AmDEL gene enhances flavonoids accumulation and salt and drought tolerance in transgenic Arabidopsis[J].Planta,2016,244(1):59-73
[13] Wang F B,Kong W L,Niu Y,Ye Y X,Fan S,Wang Y J,Chen X H,Zhou Q.StTrxF,apotato plastidic thioredoxin F-type protein gene,is involved in starch accumulation in transgenic Arabidopsis thaliana[J].Biotechnology&Biotechnological Equipment,2017,31(3):486-492
[14] Jiang W,Yang S P,Yu D Y,Gai J Y.Cloning and characterization of a novel gene GmMF1in soybean(Glycine max L merr)[J].Agricultural Sciences in China,2011,10(12):1834-1841
[15]许红梅,张立军,刘淳.农杆菌蘸花法侵染拟南芥的研究[J].北方园艺2010(14):143-146Xu H M,Zhang L J,Liu C.Study of infection Arabidopsis thaliana by agrobacterium dipping flower method[J].Northern Horticulture,2010(14):143-146(in Chinese)
[16] Wang F B,Zhu H,Chen D H,Li Z J,Peng R H,Yao Q H.A grape bHLH transcription factor gene,VvbHLH1,increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana[J].Plant Cell,Tissue and Organ Culture,2016,125:387-398
[17] Wang F B,Kong W L,Wong G,Gary Wong,Fu L F,Peng R H,Li Z J,Yao Q H.AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana[J].Molecular Genetics and Genomics,2016,291(4):1545-1559
[18]王学奎.植物生理生化实验原理和技术(第2版)[M].北京:高等教育出版社,2007Wang X K.Plant Physiology And Biochemistry Experimental Principles And Techniques 2nd[M].Beijing:Higher Education Press,2007(in Chinese)
[19]李合生,孙群,赵氏杰.植物生理生化实验原理与技术[M].北京:高等教育出版社,2000Li H S,Sun Q,Zhao J.Plant Physiology And Biochemistry Experiment Principle and Technology[M].Beijing:Higher Education Press,2000(in Chinese)
[20]王伟旗,侯文胜.大豆硫氧还蛋白基因的克隆与分析[J].大豆科学,2011,30(3):351-355Wang W Q,Hou W S.Clone and analysis of thioredoxin gene soybean[J].Soybean Science,2011,30(3):351-355(in Chinese)
[21]曹军,胡忠,庄东红,陈尚万,邹湘辉.花生“汕油523”在盐胁迫下应激反应的初步研究[J].汕头大学学报:自然科学版,2004,19(1):29-33Cao J,Hu Z,Zhuang D H,Chen S W,Zou X H.Studies on the shock responses of arachis hypogaea L cv shanyou 523under salt stress[J].Journal of Shantou University:Nature Science,2004,19(1):29-33(in Chinese)
[22]高树涛,石生伟.盐胁迫对5种抗旱型小麦苗期生理特性的影响[J].南方农业学报,2017,48(8):1374-1380Gao S T,Shi S W.Effects of salt stress on physiological characteristics of five drought-resistant wheat cultivars at seedling stage[J].Journal of Southern Agriculture,2017,48(8):1374-1380(in Chinese)
[23]余文兰,王朵朵,郭剑英,胡莲美.硫氧还蛋白对过氧化氢诱导的BRL-3A细胞损伤的保护作用[J].中国畜牧兽医,2014,41(6):154-159Yu W L,Wang D D,Guo J Y,Hu L M.Protective effect of thioredoxin on hydrogen H2O2-induced injury in BRL-3A cells[J].China Animal Husbandry and Veterinary Medicine,2014,41(6):154-158(in Chinese)
[24]张春宵,杨书华,刘文国,李晓辉.脯氨酸含量与玉米耐盐碱鉴定[J].吉林农业科学,2011,36(4):12-17Zhang C X,Yang S H,Liu W G,Li X H.Indentification of proline content and salt tolerance of maize[J].Journal of Jilin Agricultural Sciences,2011,36(4):12-17(in Chinese)