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

Abstract

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)

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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Figures/Tables 7

Table 1

Species and GenBank accession number for phylogenetic tree used in this study"

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

Molecular Characteristics and Function Analysis of Nuclear Receptor Gene LmE75 in Locusta migratoria

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