大豆热激蛋白基因HSP17.4的耐热功能鉴定
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摘要
为研究小热激蛋白基因HSP17. 4在大豆热生物胁迫耐受过程中的作用,利用农杆菌介导法,将植物过表达载体p CPB-HSP17. 4和RNA干扰表达载体p CPB-HSP17. 4-RNAi导入受体大豆JN18中。经PCR检测,获得T0代阳性植株13株,T1代阳性植株26株,T2代阳性植株39株。T1、T2代转基因植株Southern blotting结果显示,目标基因以单拷贝形式整合到基因组中。荧光定量PCR结果显示,HSP17. 4基因在转化植株的叶、茎中均有表达。在42℃高温胁迫下,与未转化植株相比,T1、T2转过表达植株叶片中相对表达量分别为未转化植株的813%和793%,转RNAi表达植株叶片中相对表达量分别为未转化植株的49. 77%和52. 81%。耐高温鉴定结果表明,转过表达HSP17. 4基因提高了植株的耐高温能力。
To study the function of a small heat shock protein HSP17. 4 in thermotolerance of soybean( JN18),HSP17. 4 gene was cloned from the JN18. An overexpression vector pCPB-HSP17. 4 and an RNA expression vector pCPB-HSP17. 4-RNAi were constructed. Detection of PCR showed that we obtained thirteen transformed plants in T0 generation,twenty-six transformed plants in T1 generation and thirty-nine transformed plants in T2 generation of JN18. Southern blotting of T1 and T2 generations indicated that target gene was integrated into the genome of soybean with single copy.The quantitative real-time PCR showed that the HSP17. 4 gene was expressed in the leaves and stems of transformed plants. Under high temperature stress at 42,compared with the non-transformed plants,the relative expression of T1 and T2 transformed plants in pCPB-HSP17. 4 was 813% and793%,respectively. The relative expression of T1 and T2 transformed plants in pCPB-HSP17. 4-RNAi was 49. 77% and 52. 81% that of non-transformed plants in leaves,respectively. The results showed that the overexpression of HSP17. 4 gene could improve the high temperature resistance of soybean.
To study the function of a small heat shock protein HSP17. 4 in thermotolerance of soybean( JN18),HSP17. 4 gene was cloned from the JN18. An overexpression vector pCPB-HSP17. 4 and an RNA expression vector pCPB-HSP17. 4-RNAi were constructed. Detection of PCR showed that we obtained thirteen transformed plants in T0 generation,twenty-six transformed plants in T1 generation and thirty-nine transformed plants in T2 generation of JN18. Southern blotting of T1 and T2 generations indicated that target gene was integrated into the genome of soybean with single copy.The quantitative real-time PCR showed that the HSP17. 4 gene was expressed in the leaves and stems of transformed plants. Under high temperature stress at 42,compared with the non-transformed plants,the relative expression of T1 and T2 transformed plants in pCPB-HSP17. 4 was 813% and793%,respectively. The relative expression of T1 and T2 transformed plants in pCPB-HSP17. 4-RNAi was 49. 77% and 52. 81% that of non-transformed plants in leaves,respectively. The results showed that the overexpression of HSP17. 4 gene could improve the high temperature resistance of soybean.
引文
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[4]何欢,高嵩,韩丹,等.抗虫基因CrylIem转化大豆的研究[J].吉林农业大学学报,2017,39(6):665-669.
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[9]李忠光,龚明.愈创木酚法测定植物过氧化物酶活性的改进[J].植物生理学通讯,2008,44(2):323-324.
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[11] Tissieres A,Mitchell H K,Tracy U M. Protein synthesis in salivary glands of Drosophila melanogaster:relation to chromosome puffs[J]. Journal of Molecular Biology,1974,84(3):389-398.
[12]栗振义,龙瑞才,张铁军,等.植物热激蛋白研究进展[J].生物技术通报,2016,32(2):7-13.
[13]郭虹霞,王创云,赵丽,等.小分子热激蛋白的研究进展[J].山西农业科学,2013,41(12):1421-1423.
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[15] Baniwal S K,Bharti K,Chan K Y. Heat stress response in plants:a complex game with chaperones and more than twenty heat stress transcription factors[J]. Journal of Biosciences,2004,29(4):471-487.
[16]郭文丽.热激蛋白在作物抗逆境胁迫中的研究进展[J].教育教学论坛,2010(6):142.
[17]王涛,唐颂豪,何梅,等.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79.
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[19] Sharma-Natu P,Sumesh K V,Ghildiyal M C.Heat shock protein in developing grains in relation to thermotolerance for grain growth in wheat.[J].Journal of Agronomy Crop Science,2010,196(1):76-80.
[20] ASSAB E,RAMPINO P,MITA G,et al.Heat shock response in olive twigs:identification and analysis of a c DNA coding a classⅠsmall heat shock protein.[J].Plant Biosystems,2011,145(2):419-425.
[21]张路,胡伟娟,张启翔,等.报春PF s HSP 17-1基因转化拟南芥及耐热鉴定[J].生物技术通报,2011,11(14):177-181.
[22]李春子,成善汉.烟草细胞质小分子热激蛋白基因NtHsp17. 8的克隆及胁迫诱导表达特性分析[J].华北农学报,2011,26(4):202-206.
[23]匡洁.水稻小热激蛋白s HSP17. 5和热激因子抗白叶枯病的功能研究[D].金华:浙江师范大学,2013.
[24]牛洪斌,陈小霞,白润娥,等.小麦16. 9kD热激蛋白c DNA克隆及其表达分析[J].麦类作物学报,2008,28(5):719-723.
[25]王丹,王丕武,姚丹.大豆hsp17. 4基因克隆及植物表达载体的构建[J].吉林农业大学学报,2015,37(6):643-648.
[26]贺添艳,刘亚娟,徐文选,等. T4 DNA连接酶性质及其平端连接功能[J].河南科学,2016,34(7):1058-1062.
[27]汪由,吴禹,王岩,等.五种常用的植物转基因技术[J].杂粮作物,2010,30(3):186-189.
[28]金宇良. PCR技术的研究进展[J].现代农业科技,2012,37(10):47-48.
[29]杨金慧,王丕武,曲静,等.BADH/pepB双价基因无选择标记表达载体转化苜蓿的初步研究[J].中国草地学报,2009,31(4):20-23.
[30]陈旭,齐凤坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010,41(8):148-155.
[31]杨怡姝,孙晓娜,王小利,等.实时荧光定量PCR技术的操作实践[J].实验室研究与探索,2011,30(7):15-19.
[32]孙爱清,葛淑娟,董伟,等.玉米小分子热激蛋白Zm HSP17. 7基因的克隆与功能分析[J].作物学报,2015,41(3):414-421.
[33] Ul Haw N,Raza S,Luthe DOS,et al.A dual role for the chloroplast small heat shock protein of Chenopodium album including protection from-both heat and metal stress[J].Plant Molecular Biology Reporter,2013,31(2):398-408.
[34] CHAUHAN H,KHURANA N,NIJHAVANA A,et al. The wheat chloroplastic small heat shock protein(s HSP26)is involved in seed maturation and germination and imparts tolerance to heat stress[J]. Plant Cell and Environment,2011,35(11):1912-1931.
[35]杨传燕,王翠,张景霞,等.过表达番茄叶绿体小分子热激蛋白提高植株的耐热性[J].山东师范大学学报(自然科学版),2008,23(4):106-108.
[36]陈晓军,叶春江,吕慧颖,等. Gm HSFA1基因克隆及其过量表达提高转基因大豆的耐热性[J].遗传,2006,28(11):1411-1420.
[37] Li P S,Yu T F,He G H,et al. Genome-wide analysis of the Hsf family in soybean and functional identification of Gm Hsf-34 involvement in drought and heat stresses.[J]. BMC Genomics,2014,15:998-1009.
[2]刘清玮,高延辉,王希.大豆皂苷脂质体的制备及稳定性研究[J].吉林农业大学学报,2017,39(5):619-623.
[3]林春晶,张春宝,赵洪锟,等.同核异质大豆叶绿体DNA的SNP分析[J].吉林农业大学学报,2017,39(2):134-138.
[4]何欢,高嵩,韩丹,等.抗虫基因CrylIem转化大豆的研究[J].吉林农业大学学报,2017,39(6):665-669.
[5]解文娟,杨晓光,杨婕,等.气候变化背景下东北三省大豆干旱时空特征[J].生态学报,2014,34(21):6232-6243.
[6]杨如萍,包振贤,陈光荣,等.大豆抗旱性研究进展[J].作物杂志,2012(5):8-12.
[7]徐新娟,李勇超. 2种植物相对电导率测定方法比较.[J].江苏农业科学,2014,42(7):311-312.
[8]张秋萍,吴霞红,郑剑恒,等.生物样本中丙二醛测定方法的研究进展[J].理化检验(化学分册),2016,52(8):979-985.
[9]李忠光,龚明.愈创木酚法测定植物过氧化物酶活性的改进[J].植物生理学通讯,2008,44(2):323-324.
[10] Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila[J]. Experientia,1962,18(12):571-573.
[11] Tissieres A,Mitchell H K,Tracy U M. Protein synthesis in salivary glands of Drosophila melanogaster:relation to chromosome puffs[J]. Journal of Molecular Biology,1974,84(3):389-398.
[12]栗振义,龙瑞才,张铁军,等.植物热激蛋白研究进展[J].生物技术通报,2016,32(2):7-13.
[13]郭虹霞,王创云,赵丽,等.小分子热激蛋白的研究进展[J].山西农业科学,2013,41(12):1421-1423.
[14] Pratt W B,Morishima Y,Peng H M.Proposal for a role of the HSP90/HSP70-based chaperone machinery in making triage decisions when proteins undergo oxidative and toxic damage[J].Experimental Biology and Medicine,2010,235(3):278-289.
[15] Baniwal S K,Bharti K,Chan K Y. Heat stress response in plants:a complex game with chaperones and more than twenty heat stress transcription factors[J]. Journal of Biosciences,2004,29(4):471-487.
[16]郭文丽.热激蛋白在作物抗逆境胁迫中的研究进展[J].教育教学论坛,2010(6):142.
[17]王涛,唐颂豪,何梅,等.热激蛋白与植物耐热性关系的研究进展[J].西北林学院学报,2014,29(6):72-79.
[18] Yeh T S,Jan Y Y,Jeng L B,et al.Massive extraenteric gastrointestinal hemorrhage secondary to splanchnic artery aneatysms[J].Hepatogastroenterology,1997,44:1152-1156.
[19] Sharma-Natu P,Sumesh K V,Ghildiyal M C.Heat shock protein in developing grains in relation to thermotolerance for grain growth in wheat.[J].Journal of Agronomy Crop Science,2010,196(1):76-80.
[20] ASSAB E,RAMPINO P,MITA G,et al.Heat shock response in olive twigs:identification and analysis of a c DNA coding a classⅠsmall heat shock protein.[J].Plant Biosystems,2011,145(2):419-425.
[21]张路,胡伟娟,张启翔,等.报春PF s HSP 17-1基因转化拟南芥及耐热鉴定[J].生物技术通报,2011,11(14):177-181.
[22]李春子,成善汉.烟草细胞质小分子热激蛋白基因NtHsp17. 8的克隆及胁迫诱导表达特性分析[J].华北农学报,2011,26(4):202-206.
[23]匡洁.水稻小热激蛋白s HSP17. 5和热激因子抗白叶枯病的功能研究[D].金华:浙江师范大学,2013.
[24]牛洪斌,陈小霞,白润娥,等.小麦16. 9kD热激蛋白c DNA克隆及其表达分析[J].麦类作物学报,2008,28(5):719-723.
[25]王丹,王丕武,姚丹.大豆hsp17. 4基因克隆及植物表达载体的构建[J].吉林农业大学学报,2015,37(6):643-648.
[26]贺添艳,刘亚娟,徐文选,等. T4 DNA连接酶性质及其平端连接功能[J].河南科学,2016,34(7):1058-1062.
[27]汪由,吴禹,王岩,等.五种常用的植物转基因技术[J].杂粮作物,2010,30(3):186-189.
[28]金宇良. PCR技术的研究进展[J].现代农业科技,2012,37(10):47-48.
[29]杨金慧,王丕武,曲静,等.BADH/pepB双价基因无选择标记表达载体转化苜蓿的初步研究[J].中国草地学报,2009,31(4):20-23.
[30]陈旭,齐凤坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010,41(8):148-155.
[31]杨怡姝,孙晓娜,王小利,等.实时荧光定量PCR技术的操作实践[J].实验室研究与探索,2011,30(7):15-19.
[32]孙爱清,葛淑娟,董伟,等.玉米小分子热激蛋白Zm HSP17. 7基因的克隆与功能分析[J].作物学报,2015,41(3):414-421.
[33] Ul Haw N,Raza S,Luthe DOS,et al.A dual role for the chloroplast small heat shock protein of Chenopodium album including protection from-both heat and metal stress[J].Plant Molecular Biology Reporter,2013,31(2):398-408.
[34] CHAUHAN H,KHURANA N,NIJHAVANA A,et al. The wheat chloroplastic small heat shock protein(s HSP26)is involved in seed maturation and germination and imparts tolerance to heat stress[J]. Plant Cell and Environment,2011,35(11):1912-1931.
[35]杨传燕,王翠,张景霞,等.过表达番茄叶绿体小分子热激蛋白提高植株的耐热性[J].山东师范大学学报(自然科学版),2008,23(4):106-108.
[36]陈晓军,叶春江,吕慧颖,等. Gm HSFA1基因克隆及其过量表达提高转基因大豆的耐热性[J].遗传,2006,28(11):1411-1420.
[37] Li P S,Yu T F,He G H,et al. Genome-wide analysis of the Hsf family in soybean and functional identification of Gm Hsf-34 involvement in drought and heat stresses.[J]. BMC Genomics,2014,15:998-1009.