海岛棉病程相关蛋白GbPR10基因的克隆及其在干旱胁迫下的功能分析  

Cloning and Drought Stress Function Analysis of Pathogenesis-related Proteins GbPR10 Gene in Sea-island Cotton(Gossypium barbadense)

作　　者：刘建光[1] 豆海宽 赵贵元[1] 耿昭 韩硕 安泽彤 张寒霜[1] 王永强[1] LIU Jian-Guang;DOU Hai-Kuan;ZHAO Gui-Yuan;GENG Zhao;HAN Shuo;AN Ze-Tong;ZHANG Han-Shuang;WANG Yong-Qiang(National Cotton Improvement Center Hebei Branch,Institute of Cotton,Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Cotton in Huanghuaiha Semiarid Area,Ministry of Agriculture,Shijiazhuang 050051,China;Insititue of Open Education,Xingtai Open University,Xingtai 054000,China)

机构地区：[1]河北省农林科学院棉花研究所国家棉花改良中心河北分中心/农业部黄淮海半干旱区棉花生物学与遗传育种重点实验室,石家庄050051 [2]邢台开放大学开放教育学院,邢台054000

出　　处：《农业生物技术学报》2022年第2期272-283,共12页Journal of Agricultural Biotechnology

基　　金：棉花生物学国家重点实验室开放课题(CB2020A11);河北省现代农业产业技术体系棉花创新团队建设项目(HBCT2018040203);河北省“三三三人才工程”资助项目(A202105012)。

摘　　要：病程相关蛋白10(pathogensis related protein 10,PR10)基因在植物抵御生物胁迫和非生物胁迫过程中起到重要作用。基于本课题组前期干旱胁迫转录组数据,本研究从海岛棉(Gossypium barbadense)‘比马90-53’(‘Pima90-53’)中克隆得到1个干旱胁迫相关PR10基因,命名为GbPR10(GenBank No.MT612460);利用qPCR揭示GbPR10基因的组织表达特异性和外源激素诱导、不同逆境诱导条件下的表达特征;同时克隆并分析GbPR10基因启动子序列特征;构建GbPR10基因的过表达载体PCAMBIA1301-GbPR10转化拟南芥(Arabidopsis thaliana),分析过表达GbPR10基因拟南芥在干旱胁迫条件下的耐受能力。结果显示GbPR10基因开放阅读框为486 bp,编码161个氨基酸,包含1个Bet_v1-like结构域,具有Ploop保守结构域,属于病程相关蛋白Bet_v1家族;组织特异性表达结果表明,GbPR10基因在海岛棉苗期和花铃期的根中优势表达,并受外源脱落酸(abscisic acid,ABA)、乙烯(ethylene,ET)、茉莉酸甲酯(methyl jasmonate,MeJA)和水杨酸(salicylic acid,SA)激素的响应上调表达,在20%聚乙二醇6000(polyethylene glycol 6000,PEG6000)和200 mmol/L NaCl非生物胁迫下上调表达;通过启动子序列分析克隆并获得了2176 bp的启动子片段,该序列存在ET和ABA等激素响应元件及多个干旱相关调控元件;过表达GbPR10基因拟南芥在干旱胁迫下根长显著高于野生型(P<0.05),并且抽薹后植株在自然干旱处理下转GbPR10基因植株的耐旱性显著高于野生型(P<0.05)。由此可见,海岛棉GbPR10基因在棉花应答干旱过程中具有重要作用。本研究为深入研究棉花抗干旱基因的功能机制提供了理论参考。The pathogenesis-related protein 10(PR10)gene plays an important role in plants when resistance to biological and abiotic stresses.In this study,a PR10 gene was cloned from island cotton(Gossypium barbadense)‘Pima90-53’and named GbPR10(GenBank No.MT612460)based on the transcriptome data of drought stress;To reveal tissue expression specificity,exogenous hormones induction,expression characteristics under different stress for GbPR10 gene by qPCR;Meanwhile the promoter of GbPR10 gene was cloned and analyzed;GbPR10 gene overexpression vector PCAMBIA 1301-GbPR10 was transformed into Arabidopsis thaliana to analyze the tolerance of over expression of GbPR10 gene in drought stress conditions.The results showed that the open reading frame of GbPR10 gene was 486 bp,encoding 161 amino acids,and contained a Bet_v1-like domain with P-loop conserved domain,which belonged to the Bet_v1 family of pathrelated proteins.The sequence blast analysis showed that the cloned GbPR10 was located on chromosome D02 and exist a homologous gene on chomesome A02 with the similarity of 96.07%.Phylogenetic tree analysis with the reported PR10 sequence showed that GbPR10 was closer with GbPR10-5 and NtPR10.Protein phosphorylation site prediction analysis showed that GbPR10 contains 8 serine phosphorylation sites,4 threonine phosphorylation sites and 3 tyrosine phosphorylation sites.Tissue specific expression results showed that GbPR10 gene was predominance expressed in seedling and bolling roots,especially in 4 leaf age,which the expression level in 4 leaf age root was more than 1000 folds than that in leaf.Exogenous hormones induction expression of GbPR10 showed that GbPR10 was significant up-regulated by exogenous ABA(abscisic acid),ET(ethylene),MeJA(salicylic acid)and SA(salicylic acid),and meanwhile GbPR10 was also up-regulated under 20%PEG6000(polyethylene glycol 6000)as well as 200 mmol/L NaCl stress treament.All these induction expression of GbPR10 indicated that GbPR10 may be involved in abiotic stress responses.The GbPR10 promoter

关 键 词：棉花 病程相关蛋白 海岛棉病程相关蛋白10(GbPR10)基因 干旱胁迫 

分 类 号：S562[农业科学—作物学]