Cloning,Expression And Function Analysis Of Eight Chitinase Genes In Striped Rice Borer,Chilo Suppressalis

Chitin forms epidermis,peritrophic membrane(PM)and some other insect structures.The degradation of chitin by chitinase was a key step in the life cycle of insects because insects should undergo periodic ecdysis and metamorphosis.Chitinase can be used to degrade chitin froms insect epidermis and peritrophic membrane(PM).This destroys the protective barrier of insects,thus facilitating the intrusion of other insect pathogens into their body,improving insect susceptibility and mortality.And it is closely related to the molting,pupate and emergence process.Here,we chose rice striped stem borer,Chilo suppressalis,as the research target.1.Cloning of eight C.suppressalis chitinase genesWe obtained partial sequences of eight chitinase genes in C.suppressalis by bioinformatics methods.Then we got the total length of the transcripts by RACE(CsCht5,CsCht6,CsCht7,CsCht8,CsCht9,CsCht10,CsCht11and CsCht12).The eight chitinase sequences were used to BLASTX against the GenBank database of NCBI,showing that they were highly similar with other species.The eight chitinase gene sequences have been submitted to the NCBI database.And then analyzed the molecular weight and isoelectric point of eight chitinase genes.The molecular weight of CsCht5,CsCht6,CsCht7,CsCht9,CsCht10 and CsCht11 were between 45-70 kD and the isoelectric point were between 5-7.CsCht8 molecular weight was 32.09 kD,the isoelectric point was 4.58.CsChtl2 molecular weight was 44.42 kD,the isoelectric point was 4.28.We also predicted the secondary structure of four chitinase proteins.The results showed that eight chitinases contained alpha helix,extended strand,beta turn and random coil.2.Expression of eight C.suppressalis chitinase genesWe collected samples at different developmental stages to study the expression profile of eight chitinase genes.qPCR results revealed that,CsCht5,CsCht6 and CsCht10 were highly expressed at pupa stage.CsCht8 and CsCht12 were highly expressed at larval stage.CsCht7 cyclical changes with the instar changes.CsCht9 and CsCht11 were highly expressed at 6L.These results showed that CsCht5,CsCht6 and CsCht10 might related with eclosion.CsCht7,CsCht8 and CsCht12 might related with molting and defense.CsCht9 might related with pupation and CsChtll might related with defence.Tissue-specific expression analysis of eight chitinase genes.C.suppressalis CsCht5,CsCht6 and CsCht7 were highly expressed in the epidermis.CsCht8 was highly expressed in the head.CsCht9 was highly expressed in the malpighian tube.CsCht10,CsChtll and CsCht12 were specifically abundant in the midgut.3.Interference of eight C.suppressalis chitinase genesRNAi-mediated knockdown of eight chitinase genes was performed by injecting small interfering RNA(siRNA).The result showed that CsCht5,CsCht6 and CsCht8 play important roles at eclosion rate and eclosion time.After injection of siCsCht5,siCsCht6 and siCsCht8,the eclosion rate of treated group was lower than the NC group,and the pupae of the treated group become black and then death.We conclude that CsCht5,CsCht6 and CsCht8 play important role at defense and eclosion.CsCht7,CsCht9,CsCht10,CsChtll and CsCht12 have no influences at pupation rate and pupation time.4.Beauveria bassiana(Bb84)infection the interferenced C.suppressalisTo explore the applification of eight chitinase genes in pest control,exogenous microbial was used in this experiment.We chose B.bassiana as an exogenous microorganism to infect C.suppressalis.The results showed that the death time of the test group in the CsCht5 and CsCht6 groups was earlier than that in the control group,and the death rate also increased.There were no significant differences in death rate and death time between the treatment group and the control group in CsCht7,CsCht8,CsCht9,CsChtll and CsCht12 groups.When the CsCht10 was interferenced,it did not induced death of treated individuals.However,when the RNAi-treated insects were treated with B.bassiana,the death time of the treated group was significantly higher than that of the control group.The results showed that CsCht10 was associated with the defense in C,suppressalis.