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棉花学报 ›› 2019, Vol. 31 ›› Issue (2): 89-100.doi: 10.11963/1002-7807.wjjyww.20190311

• 研究与进展 •    下一篇

陆地棉GhLEA3基因的克隆及其响应低温胁迫表达分析

王俊娟(),陆许可,阴祖军,王德龙,王帅,穆敏,陈修贵,郭丽雪,樊伟丽,陈超,叶武威*()   

  1. 中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南 安阳 455000
  • 收稿日期:2018-01-23 出版日期:2019-03-15 发布日期:2019-03-15
  • 通讯作者: 叶武威 E-mail:wjj2004liyuan@sina.com;yew158@163.com
  • 作者简介:王俊娟(1973-),女,博士,副研究员, wjj2004liyuan@sina.com
  • 基金资助:
    国家重点研发计划“七大农作物育种”(2016YFD0100203)

Isolation and Characterization of GhLEA3 Gene from Upland Cotton and Its Expression in Response to Low Temperature Stress

Wang Junjuan(),Lu Xuke,Yin Zujun,Wang Delong,Wang Shuai,Mu Min,Chen Xiugui,Guo Lixue,Fan Weili,Chen Chao,Ye Wuwei*()   

  1. Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China
  • Received:2018-01-23 Online:2019-03-15 Published:2019-03-15
  • Contact: Ye Wuwei E-mail:wjj2004liyuan@sina.com;yew158@163.com

摘要:

【目的】本研究旨在探索棉花GhLEA3基因的结构特征及其在低温胁迫下的表达模式,研究其抗逆功能。【方法】利用同源序列克隆法在陆地棉中克隆GhLEA3;采用生物信息学方法分析其蛋白质性质,构建系统进化树。采用In-Fusion连接技术构建融合蛋白表达载体35S∷GhLEA3-GFP并进行亚细胞定位;在棉花三叶期进行叶片低温表达分析;采用农杆菌介导的花序浸染法将GhLEA3基因转入拟南芥;对T3转基因拟南芥种子进行4 ℃低温萌发试验;低温处理后测转基因拟南芥叶片的电导率。【结果】GhLEA3基因编码序列全长1 218 bp,编码405个氨基酸;GhLEA3蛋白含有4个功能结构域PF02987。陆地棉GhLEA3与拟南芥AtLEA3氨基酸序列之间相似性为52.6%。GhLEA3蛋白可能定位在液泡和小泡中。GhLEA3基因在低温处理的棉花叶片中上调表达。T3转基因拟南芥低温萌发率显著高于野生型,低温处理后转基因拟南芥叶片电导率显著低于野生型。【结论】陆地棉GhLEA3属于典型的LEA3家族成员,与拟南芥AtLEA3亲缘关系最近;该基因响应低温胁迫,可能在抗冷中起重要作用。

关键词: 棉花; GhLEA3; 亚细胞定位; 低温胁迫; 功能研究

Abstract:

[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments (4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35S∷GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T3 seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T3 generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.

Key words: Gossypium hirsutum; GhLEA3; sub-cellular localization; low temperature stress; function research