陆地棉E3泛素连接酶基因GhPUB 19D的克隆与功能分析  

Cloning and functional analysis of E3 ubiquitin ligase gene GhPUB 19D

作　　者：金姝雯 安晓晖 葛冬冬[1] 刘康[1] JIN Shuwen;AN Xiaohui;GE Dongdong;LIU Kang(State Key Laboratory of Crop Genetics and Germplasm Enhancement/Collaborative Innovation Center for Modern Crop Production Co-sponsored by Province and Ministry,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/现代作物生产省部共建协同创新中心,江苏南京210095

出　　处：《南京农业大学学报》2021年第5期823-832,共10页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31371672)。

摘　　要：[目的]本文旨在克隆陆地棉U-box E3泛素连接酶基因GhPUB 19D并分析其在抵御生物和非生物胁迫中的功能及其作用机制。[方法]利用PCR从棉花cDNA文库中扩增盐胁迫和大丽轮枝菌处理均上调表达的GhPUB 19D全长cDNA;qRT-PCR分析GhPUB 19D的组织表达特征及其对不同非生物胁迫处理的响应;构建pBIN-GhPUB 19D-GFP载体并导入烟草叶片中,分析GhPUB19D的亚细胞定位;利用病毒介导的基因沉默(VIGS)和转基因过表达GhPUB 19D基因分析该基因对植物的耐盐性和抗病性的影响。[结果]GhPUB 19D基因ORF全长2043 bp,编码680个氨基酸,其编码的蛋白质含有1个U-Box和3个ARM结构域,定位于高尔基体。GhPUB 19D基因启动子区存在多种逆境胁迫响应顺式作用元件。GhPUB 19D在棉花根和叶片中优势表达。聚乙二醇(PEG)、NaCl、脱落酸(ABA)、乙烯(ET)、水杨酸(SA)、赤霉素(GA3)处理后,均可诱导GhPUB 19D在1 h达到表达峰值。VIGS沉默GhPUB 19D棉花在接种大丽轮枝菌Vd991之后,叶片黄化和枯萎较正常植物更为严重,植株发病率和病情指数均极显著增加。转GhPUB 19D基因可以提高拟南芥在ABA处理下的种子萌发率,但不能减轻ABA对幼苗生长的抑制作用;转GhPUB 19D基因可以提高拟南芥幼苗的耐盐性,ABA的SnRK2.3/2.6信号通路下游的AtABF 3和AtABF4对盐胁迫的响应增强,而AtRD26、AtMYB44、AtRAB18、SLAH3等对盐胁迫的响应减弱。转GhPUB19D基因可以显著减轻大丽轮枝菌病症,显著降低拟南芥植株中大丽轮枝菌含量,提高AtPR1、AtPR2、AtPR3、AtPR4、AtPR5、AtNPR1等系统获得抗性相关基因的表达及其对大丽轮枝菌的响应。[结论]过量表达GhPUB 19D可显著提高植物的耐盐性和黄萎病抗性。[Objectives]The U-box E3 ligase gene GhPUB 19D of upland cotton was molecularly cloned,and its roles and molecular mechanisms in resistance to abiotic and biotic stress were analyzed.[Methods]The full-length cDNA of GhPUB 19D up-regulated by salt stress and Verticillium dahliae was amplified from cotton cDNA library by PCR technology;qRT-PCR was used to analyze the tissue expression characteristics of GhPUB 19D and its responses to different abiotic stresses.The pBIN-GhPUB 19D-GFP vector was constructed and introduced into tobacco leaves to analyze the subcellular localization of GhPUB19D.Virus mediated gene silencing(VIGS)and transgenic overexpression of GhPUB 19D gene were used to analyze the effects of this gene on salt tolerance and disease resistance of plants.[Results]The ORF of GhPUB 19D gene was 2043 bp in length,encoding 680 amino acids.The encoded protein contained one U-box and three ARM repeats,located in Golgi apparatus.There were many tress responsive cis-acting elements in the promoter region of GhPUB 19D gene.GhPUB 19D was preferentially expressed in roots and leaves of cotton,and polyethylene glycol(PEG),NaCl,abscisic acid(ABA),ethylene(ET),salicylic acid(SA),and gibberellin 3(GA3)could induce the peak expression of GhPUB 19D in 1 h.After inoculation with Verticillium dahliae Vd991,the leaf chlorosis and wilt were more serious in the VIGS-silencing GhPUB 19D cotton than the normal plants,the incidence rate and disease index were significantly increased in the VIGS plants.Transgenic GhPUB 19D gene could increase the germination rate of Arabidopsis seeds under ABA treatment,but could not compromise the inhibition of ABA on seedling growth.Transgenic Arabidopsis seedlings with GhPUB 19D gene improved salt tolerance.AtABF 3 and AtABF 4 in the downstream of ABA snrk2.3/2.6 signaling pathway were enhanced in response to salt stress,while AtRD 26,AtMYB44,AtRAB18 and SLAH 3 were attenuated in response to salt stress.Transgenic GhPUB 19D significantly alleviated the disease of Verticillium wilt,and signif

关 键 词：陆地棉 U-box E3泛素连接酶 GhPUB19 耐盐性 抗病性 

分 类 号：S562[农业科学—作物学]