| The white-backed planthopper(WBPH),Sogatella furcifera(Horváth),is one of the major rice pests,belong to the Hemiptera,Delphacidae.It damages rice through three ways,including laying eggs,feeding,and spreading virus deseases.Recent years,the damagement from the white blackhooper has been more serious.Thus,it is urgent to develop new ways to control this kind of rice pest.It can be a good and new control strategy to disturb the regulation of chitnase which playing important role in insect development.Now,there have been some reports about the white blackhopper chitinase 5 gene and 10.However,there is limited information about other chinases.In this study,we obtained the full-length c DNA sequences of Sf Cht7 and Sf Cht8 through RT-PCR and RACE techniques,and investigated the expression patterns and functions of those two genes through RT-PCR and RNAi.The results will provide basic information for development of novel molecular targets for pest management.1.Cloning the full-length c DNA sequences of Sf Cht7 and Sf Cht8 Using RT-PCR combing with RACE,we obtained the full-length c DNA sequences of Sf Cht7 and Sf Cht8.The complete c DNA of Sf Cht7 consists of 3219 nucleotides,including an open reading frame(ORF)of 2877 nucleotides encoding 958 amino acid residues,and 142 and 200 nucleotides non-coding regions at 5′ and 3′ ends,respectively.The complete c DNA of Sf Cht8 is 2603 bp,including an ORF of 1317 nucleotides encoding 438 amino aicds,and 102 and 1184 nucleotides non-coding regions at 5′ and 3′ ends,respectively.2.Bioinformatics analysis The ORF of Sf Cht7 encoding a poplypeptide with a calculated molecular weight of 107.9 k Da and isoelectric point(p I)of 7.0 and chemical formula of C4838H7434N1294O1434S39.The ORF of Sf Cht8 encoding a poplypeptide with a calculated molecular weight of 49.1 k Da,a p I of 5.2 and chemical formula of C2215H3408N566O664S15.Besides,both of the two chitinases consist of twenty kinds of amino acids.The two genes show the highest homology with the brown planthopper chitinase genes.Both chitinases have one membrane spaning domain and signal peptide and lots of phosphorylation sites.The proportions of a spiral in Chitinase 7 and 8 are 25.8 % and 31.3 % respectively.The ratios of β-sheet are 20.5 % and 20.3 %,respectively.The proportions of random coil are 53.3 % and 47.9 %.Whatsmore,chitinase 7 has a chitin binding domine and two active sites,while chitinase 8 has only one active site.Sf Cht7 belongs to the third group chitinases member and Sf Cht8 belongs to the seventh group chitinases member.3.Expression patterns of Sf Cht7 and Sf Cht8 The quantitative real-time PCR(q PCR)assay was performed to investigate m RNA expression profiles of Sf Cht7 and Sf Cht8 in different developmental stages.The results showedthat the expression of Sf Cht8 changes seriously from 5 instar nymphy period to adult stage.The expression of the gene increase with times and reached peak in the latest of 5 instar stage,then it declines after emergence.Sf Cht7 is expressed in different development stages.4.Functional analysises of Sf Cht7 and Sf Cht8 Thefifth-instars larvae of the white-backed planthopper were used for RNAi experiment.After the injections,phenotypic change and death rate were observed and recorded.Total RNA was isolated from treatment insects and control groups after 72 h injection,and the levels of transcripts of target gene were measured by q PCR.The q PCR results showed that the expressions of Sf Cht7 and Sf Cht8 declined by 82.6 % and 37.2 %,reprectively.The results show that RNAi effectivelu reduced the expression levels of corresponding genes compared with the control.When injected with ds Cht7,the insects’ death rate and deformity rate reach 27.7 % and 58.5 %.There three deformity types,molting abnormal(25 %),being unable to molt(14.7 %)and wing type exception(60.3 %).However,injection of ds RNA for Sf Cht8 did not result in any abnormal molting or observed morphs.. |
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