Identifying And Expression Analysis Of Enzymes In Chitin Biosynthetic Pathway Genes From Cnaphalocrocis Medinalis

Rice as the main grain,the stability of yield plays an important role to the grain reserves of our country.The rice leaf folder,Cnaphalocrocis medinalis(C.medinalis),is one of the major rice pest insect which cause serious damage,has always been important object of pest control in China.Chemical control is the major method of pests control so far,which goes against environmental protection and food safety.Chitin is an important structural component of insect exoskeleton,which is widely distributed in the integument,trachea,head capsule,foregut,hind gut,crops,gland as well as peritrophic matrix.As chitin is not found in mammals and plants,insect chitin synthase has attracted much research interest because of its potential as specific target for designing eco-friendly pesticides.Chitin biosynthesis is a complex process that involves a variety of enzyme interact with each other.Chitin biosynthetic pathway begins withN-acetyl-glucosamine-6-Phosphate.Phos-poacetylglucosamine Mutase(PGM)converts N-acetyl-glucosamine-6-Phosphate to N-acetyl-glucosamine-1-phosphate,UDP-N-acetylglucosamine pyrophosphorylase(UAP)converts N-acetyl-glucosamine-1-phosphate to UDP-N-acetylglucosamine,and Chitin synthase converts UDP-N-acetylglucosamine to chitin.Insect chitin synthase includes two classes,class A(CHSA)and class B(CHSB).Cloning and spatio-temporal expression of two chitin synthases and other two key enzymes in chitin biosynthetic pathway encoding genes in C.medinalis were conducted to reveal the function of them.Based on the transcriptome data,we cloned the full length cDNA sequences of 4 key enzymes in chitin biosynthetic pathway by PCR and RACE techniques.The structure prediction,sequence alignment and phylogenetic analysis of the coding products of these 4 genes were performed using different bioinformatics software.The relative expression levels of the 4 genes in different developmental stages and various larval tissues of C.medinalis were determined by Quantitative Real-time PCR.The major findings obtained this work are as follow:Two full-length cDNA sequences encoding chitin synthase and two full-length cDNA sequences encoding other two key enzymes related in chitin biosynthetic pathway were obtained.They are Chitin Synthase A(CHSA),Chitin Synthase B(CHSB),Phosphoacetylglucosamine Mutase(PGM)and UDP-N-acetylglucosamine pyrophosphorylase(UAP),and they are named CmCHSA,CmCHSB,CmPGM and CmUAP.Sequence analysis showed that the full length of CmCHSA gene is 4868 bp,which encodes a polypeptide of 1564 amino acids,the full length of CmCHSB gene is 4651 bp,which encodes a polypeptide of 1525 amino acids,the full length of CmPGM gene is 1934 bp,which encodes a polypeptide of 548 amino acids,and the full length of CmUAP gene is 1837 bp,which encodes a polypeptide of 487 amino acids.Analysis of amino acid sequences of CmCHSA,CmCHSB,Cm UAP and CmPGM,the results show that the 4 amino acid sequences has the characteristics of CmCHSA,CmCHSB,CmUAP and CmPGM respectively.The amino acid sequence of CmCHSA and the amino acid sequence of CmCHSB have high similarity,which contains 6 highly conserved motifs that are speculated to contribute differently to the catalytic processes.The amino acid sequence similarity between different species CHSA above CHSB similarity between amino acids,the reasons for this phenomenon may be related to their functions.Tissue and developmental gene expression analysis indicated that CmUAP and CmPGM showed higher expression in hemolymph.While,CmCHSA showed higher expression in head and integument than other tissues,and CmCHSB showed higher expression in midgut than other tissues.The expression levels of CmCHSA,CmCHSB,CmPGM and CmUAP are significantly higher in adults than in larvae.It is notable that chitin synthase A and chitin synthase B tissue specific expression significantly correlated with their function.In this study,4 key enzymes in chitin biosynthetic pathway genes were cloned in C.medinalis.They have significantly differently spatio-temporal expression patterns in different developmental and tissues.This work will take a foundation for future research of the function of CmCHS,and chitin biosynthetic pathway.