大豆E3泛素连接酶基因GmPUB1的克隆及功能研究  

Cloning and functional analysis of soybean E3 ubiquitin ligase gene GmPUB1

作　　者：宫宇 毛卓卓 史贵霞 杨中义 喻德跃[1] 黄方[1] GONG Yu;MAO Zhuozhuo;SHI Guixia;YANG Zhongyi;YU Deyue;HUANG Fang(National Center for Soybean Improvement/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/作物遗传与种质创新国家重点实验室/农学院,江苏南京210095

出　　处：《南京农业大学学报》2020年第5期820-828,共9页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31871649)。

摘　　要：[目的]本研究通过对大豆E3泛素连接酶基因GmPUB1的克隆及在拟南芥中异源表达,探究E3泛素连接酶在大豆生长发育和非生物胁迫中的作用。[方法]克隆大豆E3泛素连接酶基因GmPUB1,并对其进行生物信息学分析。利用qRT-PCR技术分析该基因在不同组织中以及不同胁迫处理下的表达模式。通过洋葱表皮细胞瞬时表达系统对GmPUB1蛋白进行亚细胞定位分析。在拟南芥中异源表达GmPUB1并对其表型进行分析。[结果]GmPUB1基因(Glyma.14g212200)的CDS序列为1320 bp,编码439个氨基酸且该蛋白相对分子质量和等电点分别为48.63×10^3和8.35。qRT-PCR分析发现GmPUB1在开花后30 d的种子中表达最高。在PEG3350、NaCl、4℃及JA等非生物胁迫诱导下,GmPUB1均上调表达。GmPUB1蛋白在整个细胞内分布。过表达GmPUB1的转基因拟南芥株系的千粒质量显著提高,氨基酸含量发生改变,尤其甲硫氨酸含量显著升高,种子萌发率降低。转基因拟南芥株系具有耐盐性,但对干旱以及ABA敏感。[结论]GmPUB1基因可参与调控种子生长发育并响应逆境胁迫。[Objectives]In this study,the mechanism of E3 ubiquitin ligase in soybean growth and abiotic stress was explored by cloning the soybean E3 ubiquitin ligase gene GmPUB1 and heterologous expression in Arabidopsis thaliana.[Methods]The E3 ubiquitin ligase gene GmPUB1 was cloned and analyzed by bioinformatics.The expression pattern of the gene under different tissues and different stress treatments was analyzed by qRT-PCR.Subcellular localization analysis of GmPUB1 protein was performed by the onion epidermal cell transient expression system.Heterologous expression of GmPUB1 in A.thaliana and analysis of its phenotype were conducted.[Results]The CDS sequence of GmPUB1(Glyma.14g212200)was 1320 bp,encoding 439 amino acids.What’s more,the molecular weight and isoelectric point of the protein were 48.63×10^3 and 8.35,respectively.qRT-PCR analysis showed that GmPUB1 was expressed with the highest level in seeds 30 days after flowering.GmPUB1 was up-regulated under the induction of PEG3350,NaCl,4℃and JA abiotic stress.GmPUB1 protein was lacalized into cells.The 1000-grain weight of the transgenic lines increased significantly,and the amino acid content changed,especially the methionone significantly increased,but the germination rate decreased.Transgenic A.thaliana lines were salt tolerant but sensitive to drought and ABA.[Conclusions]GmPUB1 gene can be involved in regulating seed growth and development and respond to abiotic stress.

关 键 词：大豆 E3泛素连接酶 GmPUB1基因 功能分析 

分 类 号：S565.1[农业科学—作物学]