| 绿盲蝽G蛋白偶联受体激酶2基因（Al GRK2）的表达分析及在绿盲蝽生长发育中的功能 |
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| 引用本文： | 谭永安,姜义平,赵静,肖留斌. 绿盲蝽G蛋白偶联受体激酶2基因（Al GRK2）的表达分析及在绿盲蝽生长发育中的功能[J]. 中国农业科学, 2021, 54(22): 4813-4825. DOI: 10.3864/j.issn.0578-1752.2021.22.009 |
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| 作者姓名： | 谭永安 姜义平 赵静 肖留斌 |
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| 作者单位： | 江苏省农业科学院植物保护研究所,南京 210014 |
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| 基金项目： | 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) |
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| 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2 715 bp,开放阅读框2 106 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...
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| 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 |
| 收稿时间： | 2021-04-16 |
Expression Profile of G Protein-Coupled Receptor Kinase 2 Gene (AlGRK2) and Its Function in the Development of Apolygus lucorum |
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| TAN YongAn,JIANG YiPing,ZHAO Jing,XIAO LiuBin. Expression Profile of G Protein-Coupled Receptor Kinase 2 Gene (AlGRK2) and Its Function in the Development of Apolygus lucorum[J]. Scientia Agricultura Sinica, 2021, 54(22): 4813-4825. DOI: 10.3864/j.issn.0578-1752.2021.22.009 |
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| Authors: | TAN YongAn JIANG YiPing ZHAO Jing XIAO LiuBin |
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| Affiliation: | Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 |
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| Abstract: | 【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 2 715 bp, and ORF is 2 106 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 and leg. After exogenous 20E treatment, the expression of AlGRK2 was significantly down-regulated in nymphs at the 1-day-old and 3-day-old. The relative expression of AlGRK2 in all tissues of females was up-regulated after treating with 20E which compared with ethanol control, especially in ovary and fat body. In contrast, the expression of AlGRK2 treated with U73122 (PLC inhibitor) was down-regulated. Compared with ethanol control, the developmental duration of nymph, the body weight of the last instar nymph and the emergence rate of adult in A. lucorum were significantly decreased after 20E treatment. On the contrary, the development duration of nymph stage in U73122 treatment group was significantly prolonged. In addition, compared with the dsGFP group, AlGRK2 expression level of A. lucorum was significantly decreased after injection of dsAlGRK2, and nymphs mortality and developmental stages were significantly increased, while adult emergence rate and weight of the 5th instar nymph were significantly decreased. 【Conclusion】The expression profile of AlGRK2 in A. lucorum showed the specificity of the developmental stage and the tissue. Exogenous 20E inhibitor and RNAi treatment can inhibit the expression of AlGRK2, and have adverse effects on the growth and development of A. lucorum, such as delaying the development progress of A. lucorum, reducing the weight of 5th instar nymph and adult emergence rate. |
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| Keywords: | Apolygus lucorum G protein-coupled receptor kinase 2 (GRK2) ecdysterone hormone gene cloning expression profile RNAi |
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