绿盲蝽G蛋白偶联受体激酶2基因(AlGRK2)的表达分析及在绿盲蝽生长发育中的功能  

Expression Profile of G Protein-Coupled Receptor Kinase 2 Gene(AlGRK2)and Its Function in the Development of Apolygus lucorum

作　　者：谭永安[1] 姜义平 赵静[1] 肖留斌[1] TAN YongAn;JIANG YiPing;ZHAO Jing;XIAO LiuBin(Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014)

机构地区：[1]江苏省农业科学院植物保护研究所,南京210014

出　　处：《中国农业科学》2021年第22期4813-4825,共13页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2017YFD0201900);国家现代农业产业技术体系建设专项资金(CARS-15-20);国家自然科学基金(31301668);转基因棉花环境安全性评价技术(2016ZX08011);江苏省农业科学院院基金(611613)。

摘　　要：【目的】克隆绿盲蝽(Apolygus lucorum)G蛋白偶联受体激酶2基因(AlGRK2)cDNA序列,明确其时空表达谱,阐明外源蜕皮激素(20E)对AlGRK2表达的影响,分析AlGRK2在绿盲蝽生长发育中的作用,为进一步研究其在蜕皮激素信号转导通路中的功能打下基础。【方法】RACE法克隆获得AlGRK2全长,实时荧光定量PCR(qRT-PCR)分析不同日龄绿盲蝽及雌成虫不同组织中AlGRK2的表达谱,分析外源20E诱导及RNAi处理后,AlGRK2 mRNA表达的应答反应及对绿盲蝽生长发育主要参数(发育历期、若虫体重及成虫羽化率)的影响。【结果】AlGRK2 cDNA序列全长2715 bp,开放阅读框2106 bp,编码701个氨基酸,ExPASy预测其蛋白分子量为80.2 kD,理论等电点为6.56;蛋白结构分析显示AlGRK2包含4个结构域,即G蛋白信号调节区(RGS,54—175 aa)、丝氨酸/苏氨酸激酶结构域(S-TKc,191—454 aa)、丝氨酸/苏氨酸型蛋白激酶的伸展部分(S-TK-X,455—534 aa)和PH结构域(PH,558—655 aa),其中PH结构域是GRK2蛋白的典型结构域;系统发育分析结果表明,绿盲蝽GRK2与茶翅蝽GRK2亲缘关系最近;AlGRK2在绿盲蝽1—16日龄虫体内均有表达,mRNA表达量呈现出波动式下降的模式,在绿盲蝽初始龄期的表达量较高,而在末龄期的表达量显著下降;AlGRK2在绿盲蝽雌成虫卵巢和脂肪体中高表达,在胸与足中的表达量较低;外源20E处理后,AlGRK2在绿盲蝽1日龄和3日龄表达量显著下调,AlGRK2在雌成虫各组织中均表达上调,在卵巢及脂肪体中上调幅度最大,相反的是,20E信号通路中PLC抑制剂U73122处理的AlGRK2表达量下调;绿盲蝽若虫发育历期、末龄若虫体重和成虫羽化率均显著下降,相反的是,U73122处理组若虫期的发育历期显著延长;此外,与注射dsGFP处理组相比,注射dsAlGRK2处理后绿盲蝽的AlGRK2表达水平显著下降,若虫死亡率及发育历期显著增加,而成虫羽化率和5龄若虫体重均显著下降。【结论】AlGRK2在绿盲【Objective】The objective of this study is to clone the full-length cDNA of the G protein-coupled receptor kinase 2(AlGRK2)in Apolygus lucorum,clarify its expression profiles and the effect of exogenous ecdysterone hormone 20E on the expression of AlGRK2,analyze the role of AlGRK2 in the growth and development of A.lucorum,and to provide a preliminary research basis for further study of its function in the ecdysone signaling transduction pathway.【Method】The AlGRK2 was cloned and obtained by RACE method.Using the qRT-PCR method,the relative expression levels of AlGRK2 in different days(1-16 day-old)and tissues(head,thorax,wing,leg,midgut,ovary and fat body)in female adults in A.lucorum were determined.Finally,the response of the AlGRK2 mRNA expression in A.lucorum after exogenous 20E induction and RNAi treatment and their effects on the main parameters(development progress,nymphs weight and adult emergence rate)in the growth and development of the A.lucorum were analyzed.【Result】The full length of AlGRK2 cDNA is 2715 bp,and ORF is 2106 bp,which encodes 701 amino acids.ExPASy predicts that the protein molecular weight is 80.2 kD and the theoretical isoelectric point is 6.56.Protein structure analysis shows that AlGRK2 contains 4 domains:G protein signalling domain(RGS,54-175 aa),serine/threonine protein kinases domain(S-TKc,191-454 aa),extension to Ser/Thr-type protein kinases(S-TK-X,455-534 aa)and pleckstrin homology domain(PH,558-655 aa),and the PH domain is a typical protein feature in the GRK2 subtype.Phylogenetic analysis showed that GRK2 in A.lucorum had the closest genetic relationship with Halyomorpha halys GRK2.qRT-PCR results showed that AlGRK2 was expressed in 1-day-old to 16-day-old nymphs of A.lucorum,and the mRNA expression showed a fluctuating downward pattern.The expression level of AlGRK2 was higher in the initial instar,but decreased significantly in the last instar of A.lucorum.The AlGRK2 was highly expressed in the ovary and fat body of female adults and less expressed in the thorax a

关 键 词：绿盲蝽 G蛋白偶联受体激酶2 蜕皮激素 基因克隆 表达谱 RNA干扰 

分 类 号：S433.3[农业科学—农业昆虫与害虫防治]