飞蝗核受体基因LmE75的分子特性和功能分析

doi:10.3864/j.issn.0578-1752.2020.11.008

摘要/Abstract

摘要：

【目的】核受体（nuclear receptor,NR）是一类配体依赖型的转录因子,在昆虫生长发育过程中发挥重要作用,本文以重要农业害虫飞蝗（Locusta migratoria）为研究对象,分析核受体基因LmE75的分子特性以及功能,为害虫防治提供新分子靶标。【方法】基于飞蝗转录组数据库获得3条LmE75的cDNA序列,运用Blast和ExPASy网站中相关软件分析各个基因的开放阅读框、预测氨基酸序列、理化特性及保守结构域;使用MEGA 6.0 软件中邻接法（neighbor-joining,NJ）与其他昆虫同源序列进行聚类分析。利用Primer 3.0软件设计3个LmE75亚型的特异性表达引物,运用 reverse transcription-quantitative PCR（RT-qPCR）技术分析3个LmE75亚型在5龄若虫不同组织及4—5龄不同发育日龄的表达特性。体内注射蜕皮激素（20-hydroxyecdysone,20E）诱导和干扰20E受体基因LmEcR的表达后,RT-qPCR检测LmE75的表达,明确LmE75是否受20E调控。利用RNAi（RNA interference）技术,将体外合成的LmE75亚型共同区域的dsRNA注射至5龄飞蝗若虫体内,48 h后提取体壁RNA,反转录为cDNA,运用RT-qPCR技术检测LmE75亚型的沉默效率并观察表型;之后进一步利用H&E（hematoxylin and eosin）染色方法观察LmE75对飞蝗表皮结构的影响。【结果】基于飞蝗转录组数据库获得3条LmE75的cDNA序列,根据其结构特征命名为LmE75A、LmE75B和LmE75C,GenBank登录号分别为MN584732、 MN584733和MN584734。3个LmE75亚型均具有典型的核受体结构域,其中DBD（DNA binding domain）和LBD（ligand binding domain）在各昆虫中高度保守;E75聚类分析结果显示不同目的昆虫类群E75亚型优先聚为一支;RT-qPCR结果显示3个LmE75亚型具有不同的组织和发育表达特性。LmE75A在体壁和肌肉中高表达;LmE75B在体壁中高表达,其次是前肠;LmE75C特异性在体壁高表达;LmE75A在4龄和5龄期均呈现先升高后降低的表达趋势;LmE75B在N4D3和N5D5表达量最高,蜕皮前后表达量均较低;LmE75C在4龄和5龄期均呈现逐渐升高的表达趋势,在蜕皮前表达量最高。20E诱导3 h和6 h后,处理组LmE75的表达量较对照组分别上调了4.2倍和4.5倍。利用RNAi技术干扰LmEcR 48 h后LmE75的表达量较对照组下调59%,表明LmE75受20E调控。在5龄第1天注射dsLmE75后的飞蝗具有明显的发育延迟现象,若虫无法蜕皮至成虫并最终100%死亡。体壁H＆E染色结果显示,注射dsLmE75组飞蝗没有发生皮层溶离现象。【结论】飞蝗中有3个LmE75亚型,三者具有不同的组织和发育表达特性;RNAi试验表明,LmE75在飞蝗蜕皮过程中发挥重要作用。

关键词: 飞蝗, 核受体, LmE75, 蜕皮激素, RNA干扰

Abstract:

【Objective】Nuclear receptors (NRs) act as ligand-inducible transcription factors, which play an important role during growth and development in insects. Locusta migratoria is an important agricultural pest. The objective of this study is to analyze the molecular characteristics and biological function of the nuclear receptor gene LmE75 in L. migratoria, and to provide a new molecular target for pest control. 【Method】The cDNA sequences of three LmE75 genes were obtained based on the transcriptome database of L. migratoria. The open reading frames, predicted amino acid sequences, physicochemical properties and conserved domains of each gene were analyzed using Blast and ExPASy websites. Using MEGA 6.0 software, the neighbor-joining (NJ) method was used to construct a phylogenetic tree with the homologous sequences of E75 from other insects. Specific primers of three LmE75 isoforms were designed using Primer 3.0 software. Reverse transcription-quantitative PCR (RT-qPCR) was used to analyze the expression characteristics of three LmE75 isoforms in different tissues and developmental days from 4th to 5th instar nymphs. To further test whether LmE75 was regulated by 20-hydroxyecdysone (20E), the expression of LmE75 was detected by RT-qPCR after injected with 20E in vivo and interfered with the 20E receptor gene LmEcR by RNAi. The double-stranded RNA (dsRNA) of the common region of three LmE75 isoforms was synthesized in vitro, and then was injected into the 5th instar nymphs. The total RNA of integument was extracted after 48 h, and the first strand cDNA was synthesized as the template of RT-qPCR. The silencing efficiency of LmE75 was detected by RT-qPCR technology and the phenotype was observed. The effect on the structure of cuticle after injected with dsLmE75 was analyzed by H&E staining method. 【Result】Three cDNA sequences of LmE75 were obtained based on the transcriptome database of L. migratoria. According to structural characteristics, they were named LmE75A, LmE75B and LmE75C, respectively. The GenBank accession numbers were MN584732, MN584733 and MN584734. Each of the three LmE75 isoforms had typical nuclear receptor conserved domains, and DBD (DNA binding domain) and LBD (ligand binding domain) were highly conserved among insects. Phylogenetic tree analysis showed that E75 isoforms of different orders of insects were preferentially clustered into one group. RT-qPCR results showed that three LmE75 isoforms had different tissue and developmental expression characteristics. LmE75A was highly expressed in the integument and muscle. LmE75B was highly expressed in the integument, followed by the foregut. LmE75C was specifically expressed in the integument. The expression level of LmE75A increased first and then decreased at the 4th and 5th instar nymphs. The expression levels of LmE75B were the highest in N4D3 and N5D5, and lower before and after molting. The expression level of LmE75C increased gradually at both the 4th and 5th instar nymphs, with the highest expression before molting. Compared with the control group, the expression level of LmE75 significantly increased by 4.2 and 4.5 times after 20E induction for 3 h and 6 h, respectively. The expression level of LmE75 was significantly down-regulated by 59% after interference with LmEcR for 48 h by RNAi. After injection of dsLmE75 on the 1st day of 5th instar nymphs, L. migratoria exhibited obvious developmental delay phenotype and all L. migratoria could not molt to adults. The H&E staining of the integument found that the cuticle of dsLmE75-injected L. migratoria showed no apolysis. 【Conclusion】There are three LmE75 isoforms in L. migratoria, and they have different tissue and developmental expression characteristics. RNAi results show that LmE75 play an important role in the molting of L. migratoria.

Key words: Locusta migratoria, nuclear receptor (NR), LmE75, 20-hydroxyecdysone (20E), RNA interference (RNAi)

刘晓健,郭俊,张学尧,马恩波,张建珍. 飞蝗核受体基因LmE75的分子特性和功能分析[J]. 中国农业科学, 2020, 53(11): 2219-2231.

LIU XiaoJian,GUO Jun,ZHANG XueYao,MA EnBo,ZHANG JianZhen. Molecular Characteristics and Function Analysis of Nuclear Receptor Gene LmE75 in Locusta migratoria[J]. Scientia Agricultura Sinica, 2020, 53(11): 2219-2231.

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图/表 7

表1

本研究中用于构建系统进化树的物种和E75的 GenBank 登录号"

目 Order	物种 Species	基因 Gene	GenBank 登录号 GenBank accession number
半翅目 Hemiptera	绿盲蝽Apolygus lucorum	AlE75A、AlE75B、AlE75C	ATN39778.1、ATN39779.1、ATN39780.1
半翅目 Hemiptera	温带臭虫Cimex lectularius	ClE75X1、ClE75X2、ClE75X3	XP_014240379.1、XP_014240380.1、XP_014240381.1
	褐飞虱Nilaparvata lugens	NlE75X1、NlE75X2	XP_022197917.1、XP_022197918.1
膜翅目 Hymenoptera	哥伦比亚芭切叶蚁Atta colombica	AcE75	KYM84307.1
	黑毛蚁Lasius niger	LnE75	KMQ93219.1
	红胡须蚁Pogonomyrmex barbatus	PbE75X1	XP_011640767.1
	小黄家蚁Monomorium pharaonis	MpE75X1、MpE75X2、MpE75X4	XP_012534660.1、XP_028049966.1、XP_028049973.1
	西方蜜蜂Apis mellifera	AmE75X1	XP_006564380.1
	丽蝇蛹集金小蜂Nasonia vitripennis	NvE75	XP_008204671.1
双翅目 Diptera	黑腹果蝇Drosophila melanogaster	DmE75A、DmE75B、DmE75C、DmE75D	AAN11688.1、AAF49282.3、AAN11687.1、AAN11689.1
	埃及伊蚊Aedes aegypti	AaE75X1、AaE75X2、AaE75X3	XP_021704146.1、XP_011493380.2、XP_001652743.3
	橘小实蝇Bactrocera dorsalis	BdE75	AGF44569.1
鞘翅目 Coleoptera	马铃薯甲虫Leptinotarsa decemlineata	LdE75X1、LdE75X2、LdE75X3	AKN56577.1、AKN56578.1、ALU57795.1
鞘翅目 Coleoptera	赤拟谷盗Tribolium castaneum	TcE75X1、TcE75X2、TcE75X3、TcE75X4、TcE75X5	XP_008197843.1、XP_008197844.1、XP_971362.2、XP_008197845、XP_015838722.1
蜚蠊目 Blattodea	德国小蠊Blattella germanica	BgE75A、BgE75B、BgE75C、BgE75D、BgE75E	CAJ87513.1、CAJ87514.1、CAM97373.1、CAM97374.1、CAM97375.1
蜚蠊目 Blattodea	湿木白蚁Zootermopsis nevadensis	ZnE75X1、ZnE75X2、ZnE75X3	XP_021938067.1、XP_021938068.1、XP_021938069.1
鳞翅目 Lepidoptera	家蚕Bombyx mori	BmE75A、BmE75B、BmE75C、BmE75D	NP_001106079.1、NP_001106080.1、NP_001037042.1、BAR88292.1
直翅目Orthoptera	飞蝗Locusta migratoria	LmE75A、LmE75B、LmE75C	MN584732、MN584733、MN584734

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基因Gene	引物序列 Primer sequence (5′-3′)	用途 Application	产物长度 Length of product (bp)
β-actin-F/R	CGAAGCACAGTCAAAGAGAGGTA/GCTTCAGTCAAGAGAACAGGATG	RT-qPCR	156
LmE75A-F/R	TTCCGTGTCCTCCAAGCTAG/ACCTTGCACGGGATGATCTC		111
LmE75B-F/R	ATGATCCTGTCCGACGGC/CCCGTCGAACTCTATGTGGA		150
LmE75C-F/R	ATGACTGTGAAGGACCCGG/ACTCTTCTAGAACCCTGCCT		133
LmE75-F/R	CTGCTCGGTGGTGGTGAT/GTCATCTTGAAGGCGAGCAG		85
LmEcR-F/R	GACAAACTGCTACGGGAAGA/CTATCGTTTCTCCCATACCAG		172
dsLmE75-F/R	taatacgactcactatagggAGCTGGAGGACCAGTCTGC/taatacgactcactatagggGCGAAGTCGAACATGGAGTC	RNAi	460
dsLmEcR-F/R	taatacgactcactatagggCAGCAACGCCGCACCCT/taatacgactcactatagggCACTGGTACACGGCATTT		408
dsGFP-F/R	taatacgactcactatagggTGGAGAGGGTGAAGG/taatacgactcactatagggGGCAGATTGTGTGGAC		712