CDNA Sequence Cloning,Characterization And Functional Research Of Chitin Synthase B From The Oriental Armyworm,Mythimna Separata Walker

When it occurs a large area of M.separata,it will cause a huge number of crops to be reduced or even disappeared.It is one of the most important agricultural pests in our country.At present,the main method to control M.separata is chemical pesticide.However,it not only cause serious damage to non-target organisms and environment pollution,but also enhances the resistance of M.separata.In the future,the use of molecular biology technology is one of the most effective and environmental friendly biological control methods.Chitin is the main component of the insect peritrophic membrane,and its synthesis plays a crucial role in the development and metamorphosis of insects.Because chitin is not found in plants and animals,it is considered to be the ideal design target for environmental friendly pesticides.Chitin synthase B?CHSBs?is responsible for synthesis of chitin on peritrophic membrane of midgut,ensuring the re-formation of peritrophic membrane during insect growth and development.When the expression of CHSBs gene is inhibited,the structure of peritrophic membrane will be destroyed and in turn affects the digestion and absorption of M.separata.Therefore,the functional study of CHSB,which is a chitin in midgut of the synthetic mucoid,is the focus of the development of biotechnology methods to control M.separata.In this study,afull-length cDNA sequence of CHSB gene in M.separata was obtained by high-throughput sequencing.Bioinformatics analysis,the expression patterns of MsCHSB at different developmental stages and in different tissues and functional analysis were analyzed in order to revealthe expression characteristics of CHSB gene in M.separata and the function of synthesizing chitin of peritrophic membrane in midgut at molecular level.The results were obtained as follows:?1?A full-length cDNA sequence of CHSB in M.separata was 4617 bp,containing an open reading frame encoding a polypeptide of 1538 amino acids.The molecular weight was about 175.629 ku and its theoretical isoelectric point was 5.96.There are 4 tag sequences of CHS,CATMWHET,DGD,EDR and QRRRW,and one putative catalytic domain.The cDNA sequence was designated as MsCHSB and has been deposited in Gen Bank,accession No.KY348776.?2?MsCHSB gene m RNA expression showed that the gene was expressed at different developmental stages and in different tissues.Defining the expression pattern of MsCHSB gene.MsCHSB gene was expressed on the first and last days of 1-6^th instar,the prepapuestage,the papue stage and the adult stage.And the expression level was highest on the first day of 3^rd instar and the first day of papue.The expression level was lowest on the last day of 1^st instar.MsCHSB gene was differently expressed in seven tissues of foregut,midgut,hindgut,body wall,malpighian tubes,fat bodies and salivary glands,and the highest expression in the midgut.The lowest expression level was in body wall.?3?Starvation treatment has a certain inhibitory effect on the expression of MsCHSB gene,further verifying that hunger has a certain influence on the feeding of M.separata and the digestion and absorption of midgut.The second day of 4^th instar were selected.After feeding for 24 h,the expression of MsCHSB gene was significantly lower than that of continuous feeding for 48 h and continuous hunger for 48 h.The difference was significant.After starving for 24 h and feeding for 24 h,The expression level of MsCHSB gene was significantly higher than that of continuous feeding for 48 h and continuous hunger for 48 h,and the differences were significant.The change of the expression level of MsCHSB gene after the treatment with validamycin has the same trend as the change of chitin content in midgut.?4?20 E ecdysone has a certain induction effect on the expression of MsCHSB gene,and the induction effect is most obvious after injected 10 ?g/?L of 20 E for 6 h.20 E was injected into the second day of 4^th instar,and it was found that the expression of MsCHSB gene increased by 32.0% and 30.4% after injected 10 ?g/?L of 20 E for 6 h and 12 h,respectively,which was significantly different from the control.After injected other concentrations of 20 E for other times,the expression of the gene decreased,and the difference was significant.?5?Validamycin inhibited the expression of MsCHSB gene.When the concentration of validamycin was 20 ?g/?L after 24 h and 48 h treatmenton the second day of 4^th instar,the expression of MsCHSB gene was lowest,and was lower than that of the control group,decreacing 95.2% and 96.4%,the inhibition of this gene is most obvious.The change of the expression level of MsCHSB gene in M.separata after the treatment with validamycin has the same trend as the change of chitin content in midgut.It also showed that there was a positive correlation between the expression level of MsCHSB gene and the content of chitin in the midgut during the growth and development of M.separata.?6?The expression of MsCHSB gene was decreased by RNA interference,indicating that RNA interference successfully inhibited the expression of MsCHSB gene.Gene silencing affects the synthesis of chitin in the midgut of M.separata.The permeability of peritrophic membrane in midgut maybe destroied,and effect the growth and development of armyworm and eventually die.The ds MsCHSB was injected into the second day of 4^th instar.After 24 h and 48 h treatments,the expression levels of MsCHSB gene decreased by 82.1% and 86% which the interference effects was the best.After RNA interference,some of the M.separata stopped eating,could not properly molt,and produced abnormalities and even death.After 96 h of interference,the mortality rate reached 46.7%.In this experiment,the sequence of the MsCHSB cDNA was obtained,which was differentially expressed at different developmental stages and in different tissues of M.separata,and the effects of starvation treatment,20 E ecdysone treatment,effectivemycin treatment and RNA interference on the expression of the gene were studied.To research whether the CHSB can complete the function of chitin in the synthetic midgut peritrophic membrane under these treatments.In order to further clarify the mechanism of CHSB,it provides a new idea for the biological to control M.separata.