棉花GhTGA2.1基因的克隆表达及功能的初步分析  

Cloning, Expression and Functions Preliminary Analysis of GhTGA 2.1 Gene inCotton

作　　者：李潇玲[1,2] 赵曾强 张析 郭文婷[1] 张薇 Li Xiaoling;Zhao Zengqiang;Zhang Xi;Guo Wenting;Zhang Wei(College of Agronomy,Shihezi University,Shihezi,8320oo;Gansu Key Laboratory of Functional Genomics and Molecular Diagnostics,Lanzhou,730030;Xinjiang Production&Construction Group Key Laboratory of Crop Germplasm Enhancement and Gene Resources Utilization,Biotechnolo-gy Research Institute,Xinjiang Academy of Agricultural Reclamation Sciences,Shihezi,832000;College of Life Science and Technology,Xinjiang University,Urumqi,830046)

机构地区：[1]石河子大学农学院,石河子832000 [2]甘肃省功能基因组与分子诊断重点实验室,兰州730030 [3]新疆农垦科学院生物技术研究所,作物种质创新与基因资源利用兵团重点实验室,石河子832000 [4]新疆大学生命科学与技术学院,乌鲁木齐830046

出　　处：《分子植物育种》2023年第1期46-54,共9页Molecular Plant Breeding

基　　金：国家自然基金项目(31260358;31560-407)资助。

摘　　要：为了获得棉花GhTGA2.1基因并分析其生物学功能,本研究通过分析枯萎病菌诱导的棉花转录因子,获得1个TGA转录因子基因,命名为GhTGA2.1。该基因ORF全长为1389bp,编码462个氨基酸,序列比对发现GhTGA2.1具有碱性结构域(N-x7-R/K-x9)、亮氨酸拉链结构域(L-x6-L-x6-L)和1个保守的DOG1结构。GhTGA2.1基因编码蛋白分子量为51.12kD,理论等电点为7.88,为亲水的不稳定蛋白,二级结构主要为无规则卷曲和α螺旋。利用实时荧光定量RCR技术(RT-qRCR)分析基因表达水平,发现棉花枯萎病菌诱导GhTGA2.1基因的下调表达;经SA、JA和ET处理后,GhTGA2.1基因表达量相对于mock发生显著变化。为进一步研究该基因功能,构建GhTGA2.1基因过表达载体并进行烟草遗传转化,获得6株转GhTGA2.1基因阳性植株,其抗病相关基因NtPR1、NtPR5、NtNPR1和NtERF1表达量明显低于野生型株系,其中NtPR1和Nt-PR5的相对表达量下降幅度较大;而NtPR4和NtWRKY70的表达量升高,NtPR2的表达量较野生型没有明显差异。本研究通过对棉花的GhTGA2.1转录因子的表达特性以及转烟草的相关基因进行分析,为今后进一步研究该基因的功能,揭示其在棉花抗病中的作用奠定基础。In order to obtain the cotton GhTGA2.1 gene and analyze its biological function, in this study, a TGA transcription factor gene was obtained from Gossypium hirsutum by sequence alignment and PCR, and named as GhTGA2.1. Analysis of its sequence showed that the full-length ORF of the gene was 1 389 bp, encoding 462 amino acids, and sequence alignment revealed that GhTGA2.1 had a basic domain(N-x7-R/K-x9) and a leucine zipper domain(L-x6-L-x6-L) and a conserved DOG1 structure. The molecular weight of the protein was 51.12 kD, and the theoretical isoelectric point was 7.88. It was a hydrophilic unstable protein, the major components of the secondary structure of the protein were random coils and alpha helices. Real-time fluorescent quantitative RCR technology(RT-qRCR) was used to analyze gene expression characteristics, and the results showed that GhTGA2.1 gene could be induced by Fusarium wilt, and the relative expression of GhTGA2.1 was lower than that of the parallel control mock, showing down-regulated expression. After SA, JA and ET treatment, the expression of GhTGA2.1 gene significantly changed compared to mock. Construction of GhTGA2.1 gene overexpression vector, Agrobacterium-mediated transformation of tobacco, PCR and RT-PCR detection, and 6 GhTGA2.1 gene positive plants were obtained. Real-time fluorescence quantitative analysis of the expression of disease resistance-related genes in transgenic tobacco. Compared with wild-type tobacco, under normal growth conditions, the expression of NtPR1, NtPR5, NtNPR1, and NtERF1 was significantly lower in GhTGA2.1 transgenic lines, and the relative expression levels of NtPR1 and NtPR5 decreased significantly. In wild-type lines, the relative expression of NtPR1 and NtPR5decreased significantly, while the expression of NtPR4 and NtWRKY70 increased, and the expression of NtPR2did not change much compared with the wild type. This study analyzed the expression characteristics of the GhTGA2.1 transcription factor in cotton and related disease resistance genes in transgeni

关 键 词：GhTGA2.1 基因 克隆 表达功能分析 

分 类 号：S562[农业科学—作物学]