大豆GmTIP1-1基因克隆及功能研究  

Cloning and functional analysis of GmTIP1-1 in soybean

作　　者：李双飞 黄颜众 轩慧冬 黄鹭[1] 赵晋铭[1] 王海棠[1] 郭娜[1] 邢邯[1] LI Shuangfei;HUANG Yanzhong;XUAN Huidong;HUANG Lu;ZHAO Jinming;WANG Haitang;GUO Na;XING Han(National Center for Soybean Improvement/Key Laboratory of Biology and Genetics and Breeding for Soybean,Ministry of Agriculture and Rural Affairs/State Key Laboratory of Crop Genetics and Germplasm Enhancement/College of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学国家大豆改良中心/农业农村部大豆生物学与遗传育种重点实验室/作物遗传与种质创新国家重点实验室/农学院

出　　处：《南京农业大学学报》2019年第5期793-801,共9页Journal of Nanjing Agricultural University

基　　金：国家转基因生物新品种培育重大专项(2016ZX08004002-005);大豆现代产业技术体系(CARS-004-PS10);长江学者和创新团队发展计划(PCSIRT_17R55)

摘　　要：[目的]本研究通过对大豆水通道蛋白基因GmTIP1-1的克隆及其在不同胁迫处理下的差异表达分析,探究水通道蛋白在大豆耐受非生物胁迫中的作用机制。[方法]从大豆品种‘天隆1号’中克隆出GmTIP1-1的CDS序列,采用生物信息学方法对该基因及其编码蛋白进行分析。采用实时荧光定量PCR检测在不同逆境处理下GmTIP1-1在大豆根、茎、叶等组织的表达情况。利用基因枪轰击法对GmTIP1-1蛋白全长及不同跨膜结构域进行亚细胞定位分析。通过酵母双杂试验确定GmTIP1-1蛋白的互作蛋白,并利用实时荧光定量PCR检测在干旱处理下与GmTIP1-1互作蛋白基因的表达情况。[结果]GmTIP1-1的CDS序列全长为753bp,编码250个氨基酸,等电点为6.51。系统进化分析发现,GmTIP1-1与苜蓿(Medicago truncatula)和黄瓜(Cucumis sativus)的亲缘关系十分相近。亚细胞定位结果显示,GmTIP1-1定位在细胞膜上。实时荧光定量PCR结果显示:GmTIP1-1在根中的表达量最高,在茎中次之,叶中最低;在干旱、ABA处理下均能诱导GmTIP1-1基因在大豆根、茎、叶中的表达;在干旱和ABA处理下,GmTIP1-1基因在根中的表达水平均高于在茎和叶中的表达水平。酵母双杂试验表明,GmTIP1-1与参与非生物胁迫调节的GmSNARE蛋白以及响应逆境胁迫相关蛋白GmF-box均存在互作。在干旱处理下,大豆根和茎中GmSNARE和GmF-box基因都会受到干旱诱导表达。[结论]GmTIP1-1蛋白定位于细胞膜,通过与参与非生物胁迫调节的GmSNARE蛋白以及响应逆境胁迫相关蛋白GmF-box互作来增强大豆对非生物胁迫的抗性。[Objectives] In this study,the mechanism of aquaporin in abiotic stress tolerance of soybean was explored by cloning the soybean aquaporin gene GmTIP1-1 and analyzing its differential expression under different stress treatments.[Methods] The CDS sequence of GmTIP1-1 was cloned from the soybean variety‘Tianlong No.1’,and its encoded protein were analyzed by bioinformatics method.Quantitative real-time PCR was used to detect the expression of GmTIP1-1 in roots,stems and leaves of soybean under different stress treatments.Subcellular localization analysis of GmTIP1-1 protein full-length and its different transmembrane domains was performed by gene gun mediated transformation.The interaction protein of GmTIP1-1 protein was determined by yeast double-hybrid assay,and the expression of GmTIP1-1 interaction protein gene under drought treatment was detected by quantitative real-time PCR.[Results] The full length of the CDS sequence of GmTIP1-1 was 753 bp,encoding 250 amino acids,and the isoelectric point was 6.51.Phylogenetic analysis found that the relationship between GmTIP1-1 and Medicago truncatula and Cucumis sativus was very similar.Subcellular localization results showed that GmTIP1-1 was localizated on the cell membrane.The results of quantitative real-time PCR showed that GmTIP1-1 had the highest expression in roots,followed by stems and leaves.And under drought and ABA treatment,the expression level of GmTIP1-1 gene in roots was higher than that of GmTIP1-1 gene in stems and leaves.Yeast double-hybrid results indicated that GmTIP1-1 interacted with GmSNARE protein involved in the regulation of abiotic stress regulation and GmF-box protein in response to stress.Under drought treatment,GmSNARE and GmF-box genes in soybean roots and stems were all induced.[ Conclusions ] GmTIP1-1 protein localizes to the cell membrane and enhance the resistance of soybean to abiotic stress by interacting with GmSNARE protein involved in abiotic stress regulation and GmF-box protein in response to stress.

关 键 词：大豆 水通道蛋白 干旱 互作蛋白 

分 类 号：S565.1[农业科学—作物学]