Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (4): 651-660.doi: 10.3864/j.issn.0578-1752.2019.04.007

• PLANT PROTECTION • Previous Articles     Next Articles

Expression and Function Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-2 in Locusta migratoria

JIA Pan1,2,ZHANG Jing1,2,YANG Yang1,2,LIU WeiMin1,ZHANG JianZhen1,ZHAO XiaoMing1()   

  1. 1Research Institute of Applied Biology, Shanxi University, Taiyuan 030006
    2College of Life Science, Shanxi University, Taiyuan 030006
  • Received:2018-09-29 Accepted:2018-11-15 Online:2019-02-16 Published:2019-02-27
  • Contact: XiaoMing ZHAO E-mail:zxming@sxu.edu.cn

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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

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
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

Table 2

Primer sequences used in this study"

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
GFP F:taatacgactcactatagggTGGAGAGGGTGAAGG dsGFP 合成
dsGFP synthesis		 571
R:taatacgactcactatagggGGGCAGATTGTGTGGAC
LmAbd-2 F:GTGTTTGACCCTGCTGGTG RT-qPCR 106
R:CCGTTGCCGTTTCGTATT
β-actin F:CGAAGCACAGTCAAAGAGAGGTA 156
R:GCTTCAGTCAAGAGAACAGGATG

Fig. 1

Sequence analysis of LmAbd-2"

Fig. 2

Multiple sequence alignments and phylogenetic analysis of LmAbd-2 and Abd-2 from different insect species"

Fig. 3

The relative expression of LmAbd-2 in different tissues and different developmental stages"

Fig. 4

Effect of LmAbd-2 on the formation of cuticle by H&E staining and TEM after injection of dsRNA"

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