飞蝗内表皮结构糖蛋白基因LmAbd-2的表达与功能分析

doi:10.3864/j.issn.0578-1752.2019.04.007

摘要/Abstract

摘要：

【目的】分析飞蝗（Locusta migratoria）内表皮结构糖蛋白（endocuticle structural glycoprotein）基因LmAbd-2的序列特征和表达特性,利用RNA干扰（RNA interference,RNAi）技术结合Hematoxylin and eosin（H&E）染色方法以及透射电镜（transmission electron microscopy,TEM）技术探究其生物学功能,探讨其对飞蝗内表皮发育的影响。【方法】基于课题组飞蝗转录组数据库获得LmAbd-2的cDNA编码序列,并克隆验证。利用ExPASy工具翻译LmAbd-2氨基酸序列,然后利用SignaIP4.1Server和SMART软件分别对其信号肽和结构域进行分析;利用NetOGlyc4.0 Server对其糖基化位点进行预测,使用GENEDOC软件进行多序列比对,并利用MEGA 6.0软件中neighbor-joining（NJ）方法与其他昆虫同源序列进行聚类分析。采用荧光定量PCR（reverse- transcription quantitative PCR,RT-qPCR）分析LmAbd-2在飞蝗5龄第2天不同组织以及4龄（N4D1—N4D6）、5龄（N5D1—N5D8）和成虫（AD1—AD4）表皮不同发育时期表达模式。采用RNAi技术结合H&E染色方法以及TEM技术观察干扰LmAbd-2后对飞蝗生长发育和表皮结构的影响。【结果】序列分析结果显示,飞蝗LmAbd-2 cDNA编码序列长为683 bp,开放阅读框为459 bp,编码152个氨基酸;功能域分析发现LmAbd-2含有1个信号肽和1个几丁质结合域（chitin binding domain 4,ChtBD4）,属于CPR家族RR-1亚类;序列比对分析发现其在物种间具有较高的保守性,其中与沙漠蝗（Schistocerca gregaria）SgAbd-2序列相似度为92.5%,且在N末端含有一个保守基序PTPPPIP,其中第2位苏氨酸为糖基化位点;系统发育分析结果显示LmAbd-2与SgAbd-2聚为一支,亲缘关系最近;RT-qPCR分析结果显示LmAbd-2在体壁中高表达,且在蜕皮后（内表皮形成时期）高表达;沉默LmAbd-2后飞蝗没有肉眼可见的表型,但H&E染色结果显示与对照组相比飞蝗表皮层变薄,进一步超微结构分析发现内表皮片层结构减少。【结论】LmAbd-2是一种内表皮结构糖蛋白,属于CPR家族RR-1亚类,其编码基因主要在体壁中高表达,而且在飞蝗4龄、5龄和成虫内表皮形成时期呈周期性表达;RNAi试验表明,LmAbd-2在飞蝗蜕皮过程中参与内表皮的形成。

关键词: 飞蝗, 内表皮, 糖蛋白, LmAbd-2, RNA干扰

Abstract:

【Objective】The objective of this study is to analyze the sequence and expression characteristics of endocuticle structural glycoprotein gene LmAbd-2 that identified from Locusta migratoria transcriptome, explore its function by Hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) based on RNAi with dsLmAbd-2, and to discuss the effect on the formation of endocuticle during L. migratoria molting.【Method】The cDNA sequence of LmAbd-2 was obtained according to the transcriptome database of L. migratoria, and was cloned by reverse-transcription PCR (RT-PCR) and sequenced. The amino acid sequence of LmAbd-2 was translated by ExPASy tool, and its signal peptide and structural domain were analyzed by using SignaIP4.1Server and SMART software, respectively. Meanwhile, the O-linked glycosylation sites were predicted by using NetOGlyc4.0 Server. The homologous sequences of Abd-2 from locust and other insect species were aligned by GENEDOC software and evolutionary tree was constructed by using the MEGA 6.0 software with the neighbor-joining (NJ) method. Expression profiles of LmAbd-2 in different tissues at day 2 of 5th instar nymphs and developmental days of 4th instar nymphs (N4D1-N4D6), 5th instar nymphs (N5D1-N5D8), and the early stage of adults (AD1-AD4) were assayed by using reverse-transcription quantitative PCR (RT-qPCR). The biological function of LmAbd-2 was analyzed by combination of RNAi and H&E staining and TEM methods. 【Result】The results of sequence analysis showed that the length of the LmAbd-2 cDNA is 683 bp and full length of its ORF is 459 bp, encoding 152 amino acids. The functional domain analysis showed that the protein encoded by LmAbd-2 contains one signal peptide and one chitin binding domain (ChtBD4), which belongs to RR-1 subclass of the CPR family. Sequence alignment analysis showed that it is highly conserved among species, and the similarity with SgAbd-2 is 92.5%, and it contains conserved motif (PTPPPIP) in N-terminal and has a potential O-linked glycosylation site (T116) at which glycosylation modification may occur. Phylogenetic tree analysis showed that LmAbd-2 has a close genetic relationship with SgAbd-2 of Schistocerca gregaria. The results of RT-qPCR revealed that LmAbd-2 was highly expressed in the integument, but lower or no expression in other tested tissues, and showed periodic expression during molting. After injection of dsLmAbd-2 on day 3 of 4th instar nymphs and day 4 of 5th instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than those in the control.【Conclusion】An endocuticle structural glycoprotein gene LmAbd-2 was obtained according to the L. migratoria transcriptome database. The protein encoded by LmAbd-2 belongs to RR-1 subclass of CPR family. LmAbd-2 was mainly expressed in integument among all the tested tissues and showed periodic expression at the early stage of 4th instar nymphs, 5th instar nymphs and adults. The results of RNAi suggested that LmAbd-2 was involved in the formation of endocuticle during L. migratoria molting.

Key words: Locusta migratoria, endocuticle, glycoprotein, LmAbd-2, RNAi

贾盼,张晶,杨洋,刘卫敏,张建珍,赵小明. 飞蝗内表皮结构糖蛋白基因LmAbd-2的表达与功能分析[J]. 中国农业科学, 2019, 52(4): 651-660.

JIA Pan,ZHANG Jing,YANG Yang,LIU WeiMin,ZHANG JianZhen,ZHAO XiaoMing. Expression and Function Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-2 in Locusta migratoria[J]. Scientia Agricultura Sinica, 2019, 52(4): 651-660.

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目Order	物种Species	基因Gene	GenBank登录号 GenBank accession number
鳞翅目Lepidoptera	玉带凤蝶Papilio polytes	PpAbd-2	XP_013141188.1
	柑橘凤蝶Papilio xuthus	PxAbd-2	NP_001299525.1
	棉铃虫Helicoverpa armigera	HaAbd-2	XP_021201029.1
	斜纹夜蛾Spodoptera litura	SlAbd-2	XP_022816544.1
半翅目Hemiptera	臭虫Cimex lectularius	ClAbd-2	XP_014247089.1
	褐飞虱Nilaparvata lugens	NlAbd-2	XP_022195400.1
直翅目Orthoptera	飞蝗Locusta migratoria	LmAbd-2	ASQ42722.1
	沙漠蝗Schistocerca gregaria	SgAbd-2	Q7M4F3.1
蜚蠊目Blattaria	堆砂白蚁Cryptotermes secundus	CsAbd-2	XP_023704658.1
	内华达古白蚁Zootermopsis nevadensis	ZnAbd-2	XP_021932412.1
	德国小蠊Blattella germanica	BgAbd-2	PSN37147.1
鞘翅目Coleoptera	山松甲虫Dendroctonus ponderosae	DpAbd-2	XP_019764279.1
	蜣螂Onthophagus taurus	OtAbd-2	XP_022906571.1
	天牛Anoplophora glabripennis	AgAbd-2	XP_018574122.1
	赤拟谷盗Tribolium castaneum	TcAbd-2	XP_974125.1

基因 Gene	引物序列Primer sequence (5′-3′)	用途 Application	产物长度 Product length (bp)
LmAbd-2	F:taatacgactcactatagggATGCAGCTGTTGGTATGCCT	dsLmAbd-2合成 dsLmAbd-2 synthesis	196
	R:taatacgactcactatagggGCTCCTGGGCGGCGATGCCG	dsLmAbd-2合成 dsLmAbd-2 synthesis	196
GFP	F:taatacgactcactatagggTGGAGAGGGTGAAGG	dsGFP 合成 dsGFP synthesis	571
	R:taatacgactcactatagggGGGCAGATTGTGTGGAC	dsGFP 合成 dsGFP synthesis	571
LmAbd-2	F:GTGTTTGACCCTGCTGGTG	RT-qPCR	106
	R:CCGTTGCCGTTTCGTATT		106
β-actin	F:CGAAGCACAGTCAAAGAGAGGTA		156
	R:GCTTCAGTCAAGAGAACAGGATG		156