| The main component of insect exoskeleton is chitin and the function of chitin is to support the body,avoid predator’s predation,defense of pathogen invasion and so on.Chitin occupied about 40%of epidermal substances.Chitin is also present in the intestine of the foregut and hindgut,mouthparts and salivary glands of some insects.The intestinal peritrophic matrices is mainly composed of chitin and protein.Chitin synthesis and degradation need chitin synthase and chitinase,they are closely related to growth and development of insect.According to the latest research on chitinase gene structure,chitinase was divided into 8 groups,the function and the structure of each group was not the same.In order to study chitinase structure and function,in this paper,C.suppressalis was used as the research object.The full length of four chitinase genes was cloned by RACE technique,and the gene sequence and expression were analyzed.The results showed that different chitinase was expressed at different ages,and we speculated that was related to the content and the composition of chitin,so we examined the expression patterns of chitin in C.suppressalis.1)RACE clones the full length of the chitinase gene cDNA in Chilo suppressalisWe obtained four chitinase gene of the transcripts in C.suppressalis,CsCht1,CsCht2,CsCht3 and CsCht4.We acquired the full length of the four chitinase genes by RACE technique and analyzed the molecular weight and isoelectric point.The molecular weight of CsCht1,CsCht2 and CsCht3 were between 40-85 kD and the isoelectric point were between 5-7.CsCht4 molecular weight was 37.11 kD,the isoelectric point was 9.25.We also predicted the secondary structure of four chitinase proteins.The results showed that four chitinases contained alpha helix,extended strand,beta turn and random coil.2)Analyze of chitinase gene family in Chilo suppressalisThe chitinases of C.suppressalis was analyzed from three aspects:gene structure,protein domain and phylogenetic tree.The results showed that the number of exons and introns of 12 chitinase sequences was different.The length of the exons and introns was not the same.CsCht5 different from Cht5 of Bombyx mori,Tribolium castaneum,Drosophila melanogaster and Apis mellifera in exon-intron structures.The results showed that Cht5 gene was conserved in sequence but the gene structure have changed;12 chitinase sequences belonged to the GH18 in the protein domain,Chitinase CsChtl,CsCht5 and CsCht7 contained ChtBD2 and chitinase CsCht10 contained ChtBD3.From the chitinase phylogenetic tree,it could be concluded that CsCht5 and CsCht7 belonged to GroupⅠ,CsCht3,CsCht4,CsCht8,CsChtll and CsCht12 belonged to Group Ⅳ;Group Ⅵcontained the CsCht10,whereas GroupsV,Ⅶ and Ⅷ didn’t find a correlation chitinase gene in C.suppressalis.3)The expression of chitinase genes in different tissues in Chilo suppressalisThe expression of four chitinase genes in different tissues was analyzed from different tissues of C.suppressalis.The results of qRT-PCR showed that the expression levels of CsChtl and CsCht3 were higher in the epidermis,the head followed the epidermis,whereas the expression levels of the fat body,midgut and malpighian tube was low.The expression of CsCht1 and CsCht4 was higher in the midgut,while the expression of the head,fat body,midgut and malpighian tube of was low.The CsCht3 gene belonged to GroupII,and CsChtl belonged to GroupⅢ,GroupⅡ and Ⅲ played an important role in insect molt.GroupIV chitinase is expressed in insect larvae and adult stage,and the content of larvae was high in intestinal tissue,so GroupIV chitinase may regulate the content of chitin in the intestinal peritrophic matrices.4)Chitin content in different developmental stages in Chilo suppressalisChitin was an important component of insect epidermis.Chitinase from Streptomyces griseus enzyme was used to measure the chitin content of larvae-larvae,larvae-pupa,pupa-adult.The results showed that the content of chitin was the lowest in the third day and the last day,the content of chitin was the highest at the second day of thir instar larvae,the chitin content was the highest at the third day of fourth instar larvae.The content of chitin was the highest at the fourth day of the fifth instar larave,the chitin content was the highest at 3-5-days of the sixth instar larvae.and the content of chitin was the highest at 2-6 day of pupa.Prepupa and adult contained very little chitin. |
