Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (10): 1817-1826.doi: 10.3864/j.issn.0578-1752.2017.10.007

• PLANT PROTECTION • Previous Articles     Next Articles

Expression and Functional Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-5 in Locusta migratoria

ZHAO XiaoMing1, JIA Pan1,2, GOU Xin1,2, LIU WeiMin1, MA EnBo1, ZHANG JianZhen1   

  1. 1Research Institute of Applied Biology, Shanxi University, Taiyuan 030006; 2College of Life Science, Shanxi University, Taiyuan 030006
  • Received:2016-11-28 Online:2017-05-16 Published:2017-05-16

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Abstract: 【Objective】The objective of this study is to obtain a cDNA sequence of endocuticle structural glycoprotein LmAbd-5based on Locusta migratoria transcriptome, clarify its molecular characteristics and biological function, reveal its role in the formation of cuticle in L. migratoria, and provide a new molecular target for pest control. 【Method】The full length cDNAof LmAbd-5 was searched from transcriptome database of L. migratoria using bioinformatics method. The cDNA was cloned and sequenced. The signal peptide and function domain of deduced amino acid were analyzed by SignalP and SMART, respectively. Phylogenetic tree was constructed using the sequences of amino acid from different insect species by the MEGA 7.0 software with the neighbor-joining (NJ) method. Reverse transcription quantitative PCR (RT-qPCR) was applied to reveal the expression patterns of LmAbd-5 in different tissues on day 2 of 5th instar nymph and different developmental stages of integument. The effects of LmAbd-5 on locust growth development and the structure of cuticle were investigated by using RNA interference (RNAi) and transmission electron microscopy (TEM). 【Result】The full length cDNA of LmAbd-5 was got from transcriptome database, which had 520 bp including ORF 303 bp. The gene structure analysis showed that LmAbd-5 has three exons. The deduced protein contains a signal peptide and one chitin binding domain 4 (ChtBD4) through the BLAST analysis, Abd-5 was highly conserved among insect species, and the sequence identity is as high as 81% between LmAbd-5 and SgAbd-5. Abd-5 belongs to the RR-1 class of CPR family by WebLogo analysis. The result of phylogenetic tree showed that LmAbd-5 has a close genetic relationship with SgAbd-5. RT-qPCR results showed that LmAbd-5 was predominately expressed in the tissues originated from ectoderm, such as the foregut, hindgut, trachea and integument, and lower expressed or not detected in the gastric caecum, midgut, Malpighian tube, fat body and wing pads. The expression at different stages showed that LmAbd-5 mainly expressed at early of 5th instar (0-72 h after ecdysis from 4th instar nymph), and up to the peak at 72 h after molting, then markedly decreased at 96-168 h. The expression pattern is related with the formation of endocuticle. Compared with dsGFP injected control, the nymphs with the injection of dsLmAbd-5 could normally molt, and no visible abnormal phenotypes was found although the expression of LmAbd-5 was decreased significantly after dsLmAbd-5 injection. However, compared to the control group, the lamellar structure from adult cuticle with injection of dsLmAbd-5 was loose, and lamellar became thicker, finally led to the endocuticle thickening. 【Conclusion】LmAbd-5 was obtained from locust transcriptome database, which contains a signal peptide and ChtBD4, belonging to the RR-1 class of CPR family. LmAbd-5 mainly expressed in the tissues derived from ectoderm and in integument at early of 5th instar. Although there was no visible phenotypes after silencing LmAbd-5, but it was found that the lamellar structure of endocuticle is loose and endocuticle becomes thicken from ultrastructure by TEM, suggesting it may be participated in the formation of endocuticle in L. migratoria.

Key words: Locusta migratoria, cuticular proteins, LmAbd-5, RNAi, transmission electron microscopy

[1]    Charles J P. The regulation of expression of insect cuticle protein genes. Insect Biochemistry and Molecular Biology, 2010, 40(3): 205-213.
[2]    Chapman R F, Simpson S J, Douglas A E. The Insects Structure and Function. 5th ed. Cambridge University Press, 2013.
[3]    王荫长. 昆虫生理学. 北京: 中国农业出版社, 2004.
Wang Y C. Insect Physiology. Beijing: China Agriculture Press, 2004. (in Chinese)
[4]    Tetreau G, Dittmer N T, Cao X, Agrawal S, Chen Y R, Muthukrishnan S, Haobo J, Blissard G W, Kanost M R, Wang P. Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin- binding domains in insects. Insect Biochemistry and Molecular Biology, 2015, 62: 127-141.
[5]    Rebers J E, Willis J H. A conserved domain in arthropod cuticular proteins binds chitin. Insect Biochemistry and Molecular Biology, 2001, 31: 1083-1093.
[6]   Willis J H. Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era. Insect Biochemistry and Molecular Biology, 2010, 40(3): 189-204.
[7]    Karouzou M V, Spyropoulos Y, Iconomidou V A, Cornman R S, Hamodrakas S J, Willis J H. Drosophila cuticular proteins with the R＆R consensus: Annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences. Insect Biochemistry and Molecular Biology, 2007, 37(8): 754-760.
[8]    Gibbs R A, Brown S J, Beeman R W, Weinstock1 G M. The genome of the model beetle and pest Tribolium castaneum. Nature, 2008, 452(7190): 949-955.
[9]    Futahashi R, Okamoto S, Kawasaki H, Zhong Y S, Iwanaga M, Mita K, Fujiwara H. Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology, 2008, 38(12): 1138-1146.
[10]   Dittmer N T, Tetreau G, Cao X, Jiang H, Wang P, Kanost M R. Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 2015, 62: 100-113.
[11]   Deng H m, Li Y, Zhang J l, Liu L, Feng Q L. Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx mori. Insect Science, 2016, 23(6): 782-790.
[12]   Arakane Y, Zhu Q, Muthukrishnan S, Matsumiy M, Kramer K J. Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect Biochemistry and Molecular Biology, 2003, 33: 631-648.
[13]   Wang P, Li G, Granados R R. Identification of two new peritrophic membrane proteins from larval Trichoplusia ni: structural characteristics and their functions in the protease rich insect gut. Insect Biochemistry and Molecular Biology, 2004, 34(3): 215-227.
[14]   Takeda M, Mita K, Quan GX, Shimada T, Okano K, Kanke E, Kawasaki H. Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations. Insect Biochemistry and Molecular Biology, 2001, 31(10): 1019-1028.
[15]   Noh M Y, Kramer K J, Muthukrishnan S, Kanost M R, Beeman R W, Arakane Y. Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum. Insect Biochemistry and Molecular Biology, 2014, 53: 22-29.
[16]   Mun S, Young Noh M, Dittmer N T, Muthukrishnan S, Kramer K J, Kanost M R, Arakane Y. Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt. Scientific Reports, 2015, 5: 10484.
[17]   Noh M Y, Muthukrishnan S, Kramer K J, Arakane Y. Tribolium castaneum RR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle. PLoS Genetics, 2015, 11(2): e1004963.
[18]   Arakane Y, Lomakin J, Gehrke S H, Hiromasa Y, Tomich J M, Muthukrishnan S, Beeman R W, Kramer K J, Kanost M R. Formation of rigid, non-flight forewings (Elytra) of a beetle requires two major cuticular proteins. PLoS Genetics, 2012, 8(4): e1002682.
[19]   Jespersen S, Hojrup P, Andersent S O, Roepstorff P. The primary structure of an endocuticular protein from two locust species, Locusta migratoria and Schistocerca gregaria, determined by a combination of mass spectrometry and automatic Edman degradation. Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology, 1994, 109(1): 125-138.
[20]   Liu S, Wei W, Chu Y, Zhang L, Shen J, An C. De novo transcriptome analysis of wing development related signaling pathways in Locusta migratoria Manilensis and Ostrinia furnacalis (Guenée). PLoS ONE, 2014, 9(9): e106770.
[21]   Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ Ct method. Methods, 2001, 25(4): 402-408.
[22]   Wang X, Fang X, Yang P, Jiang X, Jiang F, Zhao D, Li B, Cui F, Wei J, Ma C, Wang Y, He J, Luo Y, Wang Z, Guo X, Guo W, Wang X, Zhang Y, Yang M, Hao S, Chen B, Ma Z, Yu D, Xiong Z, Zhu Y, Fan D, Han L, Wang B, Chen Y, Wang J, Yang L, Zhao W, Feng Y, Chen G, Lian J, Li Q, Huang Z, Yao X, Lv N, Zhang G, Li Y, Wang J, Wang J, Zhu B, Kang L. The locust genome provides insight into swarm formation and long-distance flight. Nature Communications, 2014, 5: 2957.
[23]   Moussian B, Seifarth C, Muller U, Berger J, Schwarz H. Cuticle differentiation during Drosophila embryogenesis. Arthropod Structure & Development, 2006, 35(3): 137-152.
[24]   Rebers J E, Riddiford L M. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. Journal of Molecular Biology, 1988, 203(2): 411-423.
[25]   Nohr C, Andersen S O. Cuticular proteins from fifth instar nymphs of the migratory locust, Locusta migratoria. Insect Biochemistry and Molecular Biology, 1993, 23(4): 521-531.
[26]   Andersen S O. Amino acid sequence studies on endocuticular proteins from the desert locust, Schistocerca gregaria. Insect Biochemistry and Molecular Biology, 1998, 28: 421-434.
[27]   Noh M Y, Muthukrishnan S, Kramer K J, Arakane Y. Cuticle formation and pigmentation in beetles. Current Opinion Insect Science, 2016, 17: 1-9.
[28]   刘晓健, 崔淼, 李大琪, 张欢欢, 杨美玲, 张建珍. 飞蝗几丁质合成酶2基因的表达特性、功能及调控. 中国农业科学, 2014, 47(7): 1330-1340.
LIU X J, CUI M, LI D Q, ZHANG H H, YANG M L, ZHANG J Z. Expression, function and regulation of chitin synthase 2 gene in Locusta migratoria. Scientia Agricultura Sinica, 2014, 47(7): 1330-1340. (in Chinese)
[29]   Li D, Zhang J, Wang Y, Liu X, Ma E, Sun Y, Li S, Zhu K Y, Zhang J. Two chitinase 5 genes from Locusta migratoria: Molecular characteristics and functional differentiation. Insect Biochemistry and Molecular Biology, 2015, 58: 46-54.
[30]   王燕, 李大琪, 刘晓健, 李涛, 马恩波, 范仁俊, 张建珍. 飞蝗表皮蛋白Obstructor家族基因的分子特性及基于RNAi的功能分析. 中国农业科学, 2015, 48(1): 73-82.
WANG Y, LI D Q, LIU X J, LI T, MA E B, FAN R J, ZHANG J Z. Molecular characterization and RNAi-based functional analysis of obstructor family genes in Locusta migratoria. Scientia Agricultura Sinica, 2015, 48(1): 73-82. (in Chinese)
[31]   Magkrioti C K, Spyropoulos I C, Iconomidou V A, Willis J H, Hamodrakas S J. cuticleDB: a relational database of Arthropod cuticular proteins. BMC Bioinformatics, 2004, 5: 138.
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