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

doi:10.3864/j.issn.0578-1752.2021.22.009

摘要/Abstract

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在绿盲蝽体内的表达谱显示出发育阶段特异性和组织特异性;外源20E抑制剂及RNAi处理后,均可抑制AlGRK2的表达,同时还可对绿盲蝽生长发育产生不利影响,表现为延缓绿盲蝽的发育进度、降低5龄若虫体重及成虫羽化率。

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

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.

Key words: Apolygus lucorum, G protein-coupled receptor kinase 2 (GRK2), ecdysterone hormone, gene cloning, expression profile, RNAi

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（AlGRK2）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.

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.

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目的 Purpose	引物名称 Primer name	引物序列Primer sequence (5′ to 3′)
克隆Cloning	AlGRK-F	AGYGTNMGVAGYGTNATGCA
	AlGRK-R	TCVGCKGCRTARAAYTTCAT
	5′-AlGRK2-F	TGAAGAGAAGGAATCCCAGA
	5′-AlGRK2-R	ACTTCGTTCTTCTTTTCGAG
	3′-AlGRK2-F	GCTGGCACTCAACGAAAGGATCAT
	3′-AlGRK2-R	GTATGACTTACGCCTTCCACACGC
dsRNA	AlGRK2-F	TTCCCGACTCCTTCTCATC
	AlGRK2-R	TTTCCGTTTCTGCTCCG
	AlGRK2-T7F	TAATACGACTCACTATAGGGTTCCCGACTCCTTCTCATC
	AlGRK2-T7R	TAATACGACTCACTATAGGGTTTCCGTTTCTGCTCCG
	GFP-F	CACAAGTTCAGCGTGTCCG
	GFP-R	CACCTTGATGCCGTTC
	GFP-T7F	TAATACGACTCACTATAGGGCACAAGTTCAGCGTGTCCG
	GFP-T7R	TAATACGACTCACTATAGGGCACCTTGATGCCGTTC
qRT-PCR	AlGRK2-QF	AGGAGCGTGATGCACAAATA
	AlGRK2-QR	CGCAGTAGTCCTTGAAGAGAAG
	β-Actin-QF	ACCTGTACGCCAACACCGT
	β-Actin-QR	TGGAGAGAGAGGCGAGGAT

处理 Treatment	若虫历期Developmental period of nymphs (d)
处理 Treatment	1龄1st instar	2龄2nd instar	3龄3rd instar	4龄4th instar	5龄5th instar	若虫期Nymph
乙醇Ethanol (CK)	2.62±0.07bc	3.51±0.07b	3.60±0.07b	3.55±0.07a	3.77±0.09a	17.04±0.18b
20E	2.42±0.07c	3.17±0.05c	3.02±0.09c	2.96±0.04b	3.38±0.08b	14.94±0.19c
U73122	2.93±0.09a	3.85±0.05a	3.80±0.06a	3.67±0.07a	3.83±0.09a	18.09±0.18a
20E+U73122	2.79±0.07ab	3.66±0.07b	3.79±0.06ab	3.62±0.07a	3.89±0.11a	17.77±0.21a