烟草NtSAP5基因克隆及干旱胁迫下的功能鉴定
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摘要
为研究烟草对干旱胁迫的响应机制,克隆了烟草中锌指蛋白基因NtSAP5并对其在干旱胁迫下的表达量进行了检测。结果表明,该基因全长CDS序列为474 bp,编码的蛋白中含有保守的锌指蛋白结构域,该基因NtSAP5的表达受干旱胁迫调控,在干旱胁迫下表达量先升高,后缓慢降低。将NtSAP5的CDS序列克隆到植物过表达载体pG2BW7中,并通过叶盘法转入烟草K326中进行干旱胁迫下的功能验证。选取两个独立的转基因株系及非转基因植株进行干旱胁迫处理,过表达NtSAP5的转基因植株比非转基因植株具有较强的抵御干旱胁迫能力。结果表明,烟草NtSPA5响应干旱胁迫,并在干旱胁迫应答中起到作用。
To study the response mechanism of tobacco to drought stress, Nt SAP5 gene of zinc finger protein in tobacco was cloned and its expression under drought stress was detected in this study. The full-length coding sequence(CDS) was 474 bp and the encoding protein contains conserved zinc finger domain. The expression of Nt SAP5 was regulated by drought stress, and the expression level first increased and then decreased slowly under drought stres. The CDS sequence of Nt SAP5 gene was cloned into plant overexpression vector pG2 BW7 and transferred to tobacco variety K326 by leaf disc method for functional verification under drought stress. Two independent transgenic lines and one non-transgenic control line were treated by drought stress. The overexpressed lines exhibited stronger drought resistance than the control.Thus, Nt SPA5 played an important role in drought stress response.
To study the response mechanism of tobacco to drought stress, Nt SAP5 gene of zinc finger protein in tobacco was cloned and its expression under drought stress was detected in this study. The full-length coding sequence(CDS) was 474 bp and the encoding protein contains conserved zinc finger domain. The expression of Nt SAP5 was regulated by drought stress, and the expression level first increased and then decreased slowly under drought stres. The CDS sequence of Nt SAP5 gene was cloned into plant overexpression vector pG2 BW7 and transferred to tobacco variety K326 by leaf disc method for functional verification under drought stress. Two independent transgenic lines and one non-transgenic control line were treated by drought stress. The overexpressed lines exhibited stronger drought resistance than the control.Thus, Nt SPA5 played an important role in drought stress response.
引文
[1]Chaves M M, Maroco, Jo?o P, Pereira, Jo?o S. Understanding plant responses to drought-from genes to the whole plant[J]. Functional Plant Biology, 2003, 30(3):239-264.
[2]Shinozaki K, Yamaguchishinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses.[J].Current Opinion in Plant Biology, 2003, 6(5):410-417.
[3]Zhu J K. SALT AND DROUGHT STRESS SIGNAL TRANSDUCTION IN PLANTS-Annual Review of Plant Biology, 53(1):247[J].
[4]Nakashima K, Yamaguchi-Shinozaki K. Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants[J]. Physiologia Plantarum, 2006, 126(1):62-71.
[5]Takatsuji H. Zinc-finger proteins:the classical zinc finger emerges in contemporary plant science[J]. Plant Molecular Biology, 1999,39(6):1073-1078.
[6]Arnab M, Shubha V, Tyagi A K. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(16):6309-6314.
[7]Dansana P K, Kothari K S, Vij S, et al. OsiSAP1 overexpression improves water-deficit stress tolerance in transgenic rice by affecting expression of endogenous stress-related genes[J]. Plant Cell Reports, 2014, 33(9):1425-1440.
[8]Kanneganti V, Gupta A K. Overexpression ofOsiSAP8, a member of stress associated protein(SAP)gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice[J]. Plant Molecular Biology, 2008, 66(5):445-462.
[9]Huang J, Wang M M, Jiang Y, et al. Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177that contributes to temperature stress tolerance[J]. Gene, 2008,420(2):135-144.
[10]Kang M, Fokar M, Abdelmageed H, et al. Arabidopsis SAP5functions as a positive regulator of stress responses and exhibits E3ubiquitin ligase activity[J]. Plant Molecular Biology, 2011, 75(4/5):451-466.
[11]Giri J, Vij S, Dansana P K, et al. Rice A20/AN1 zinc-finger containing stress-associated proteins(SAP1/11)and a receptor-like cytoplasmic kinase(OsRLCK253)interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants[J].New Phytologist, 2011, 191(3):721-732.
[12]Arnab M, Shubha V, Tyagi A K. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004,101(16):6309-6314.
[2]Shinozaki K, Yamaguchishinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses.[J].Current Opinion in Plant Biology, 2003, 6(5):410-417.
[3]Zhu J K. SALT AND DROUGHT STRESS SIGNAL TRANSDUCTION IN PLANTS-Annual Review of Plant Biology, 53(1):247[J].
[4]Nakashima K, Yamaguchi-Shinozaki K. Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants[J]. Physiologia Plantarum, 2006, 126(1):62-71.
[5]Takatsuji H. Zinc-finger proteins:the classical zinc finger emerges in contemporary plant science[J]. Plant Molecular Biology, 1999,39(6):1073-1078.
[6]Arnab M, Shubha V, Tyagi A K. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(16):6309-6314.
[7]Dansana P K, Kothari K S, Vij S, et al. OsiSAP1 overexpression improves water-deficit stress tolerance in transgenic rice by affecting expression of endogenous stress-related genes[J]. Plant Cell Reports, 2014, 33(9):1425-1440.
[8]Kanneganti V, Gupta A K. Overexpression ofOsiSAP8, a member of stress associated protein(SAP)gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice[J]. Plant Molecular Biology, 2008, 66(5):445-462.
[9]Huang J, Wang M M, Jiang Y, et al. Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177that contributes to temperature stress tolerance[J]. Gene, 2008,420(2):135-144.
[10]Kang M, Fokar M, Abdelmageed H, et al. Arabidopsis SAP5functions as a positive regulator of stress responses and exhibits E3ubiquitin ligase activity[J]. Plant Molecular Biology, 2011, 75(4/5):451-466.
[11]Giri J, Vij S, Dansana P K, et al. Rice A20/AN1 zinc-finger containing stress-associated proteins(SAP1/11)and a receptor-like cytoplasmic kinase(OsRLCK253)interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants[J].New Phytologist, 2011, 191(3):721-732.
[12]Arnab M, Shubha V, Tyagi A K. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004,101(16):6309-6314.