Molecular Characterization And Functional Analysis Of Obstructor Family Genes In Locusta Migratoria

Locusta migratoria is one of the most damaging agricultural pests of the world. In the history of China, plague of locusts, flooding and drought were regarded as three national disasters. For half a century, the application of chemical pesticides not only causes resistance of L. migratoria, but also brings out environmental pollution issues due to pesticide residue. The integument of insect plays important roles in protecting insect from the mechanical damages and environmental pathogens and performing some basic physiological functions. The integument makes a relatively closed internal environment, which protects internal structures of insect, inhibits water evaporation and serves as a protective barriers against the external environmental influence. Cuticle protein is an important component of the insect cuticle. The loss/reduction of cuticle proteins could result in abnormal physiological function or loss function of cuticle, which inevitably affect the growth and development of insect. In this project, we obtained the cDNA fragments of cuticle protein Obst family genes based on the locust transcriptome database, focused on their molecular properties, gene expression patterns, and elucidated biological functions based on RNA interference. The results will provide the new molecular targets for locust control.1. Amplify and analysis of cDNA sequence of cuticle proteins Obst family genes in L. migratoriaWe got eight cDNA fragments assumed to be Obst family genes from locust transcriptome database, and the sequences were further analyzed by blast, the candidated cDNA fragments belonging to the Obst family gene were confirmed by sequence alignmnet with other known insect Obst family genes. Then full-length cDNA sequences were further amplified by using RACE-PCR technique, the full-length cDNA sequences of obst family genes were assembled by overlap region. All the full-length cDNA sequences were translated into amino acid sequences and signal peptide were analyzed by SignalP tool, functional domains were predicted by SMART website. The functional domain analysis of eight LmObsts showed they all had a signal peptide and three chitin binding domain ChtBD2, which are in accordance with the characteristics of the representative insect Obst cuticular protein. The phylogenetic tree was constructed by using sofeware Mega 5.10 with homologous sequences from Drosophila melanogaster and Tribolium castaneum. According to the result of phylogenetic analysis, eight LmObst genes were named as LmObst-Al, LmObst-A2, LmObst-B, LmObst-C, LmObst-D1, LmObst-D2, LmObst-El and LmObst-E2, respectively.2. The temporal and spatial expression patterns of cuticle proteins Obst family genes in L. migratoriaWe designed the specific primers of eight full-length cDNA sequences, then the real-time quantitative PCR (qPCR) was applied to analyze the gene expression patterns of LmObst genes in different tissues and developmental stages of the 5th-instar nymphs. The qPCR results showed the tissue distribution of eight LmObst genes:LmObst-El and LmObst-E2 were specifically expressed in foregut and hindgut, LmObst-D1 were mainly expessed in integument and foregut, the others were highly expessed in integument, foregut and hindgut, and lowly expressed in gastric caeca, midgut, malpighian tube and fat body. Developmental expression patterns showed that they had similar trend, eight LmObst genes were highly expressed at the early stage of the 5th-instar nymphs, gradually reduced to a minimum at the middle stage, then got raised before molting.3. Functional analysis of cuticle proteins Obst family genes in L. migratoriaRNA interference (RNAi) technology was used to explore biological function of these LmObst genes.3 μg and 10 μg dsLmObsts were injected into the second day of 2nd-instar nymphs, respectively. The silencing efficiency was detected at 24 h after dsRNA injection, qPCR results showed 3 μg dsRNA injection only repressed the mRNA transcripts of LmObst-A1 and LmObst-D2,10 μg dsRNA injection significantly silenced the mRNA expression of five genes, but the expression of the remain three genes LmObst-A2、LmObst-B and LmObst-C could not be silenced well. Then we injected 10 μg dsRNA into the fifth day of 5th-instar nymphs based on the developmental expression patterns of the integument in the 5th-instar nymphs. The silencing efficiency was detected at 48 h after dsRNA injection, the mRNA expression of each gene was significantly reduced. Further phenotypic observation showed the 80% nymphs injected with dsLmObst-E1 appeared no molting and died, the rest 20% nymphs could molt to the next stage, but the molting time delayed about one day, after molting to adults, the development was retarded and died within 16 h. However, the control nymphs with dsGFP injection successfully molted to the adults and developed well. The nymphs injected the other dsObsts displayed slow development, the molting time delayed about 1-3 days, but no other visible abnormal phenotypes were found.In this study, the eight full-length cDNA sequences of Obst family genes were obtained from L. migratoria. LmObsts had typical domain structure of Obst protein with a signal peptide and three chitin binding domain (ChtBD2). The tissue distribution showed LmObsts were mainly expressed in integument, foregut and hindgut, which developed from ectoderm. The silence of LmObst-E1 affected the growth of the fifth instar nymphys and resulted in high mortality, it may help us develop better strategies for resistance management and effective control of this important insect pest.