Functional Study On Group â… and Group â…¡ Chitinases During Development Of Lymantria Dispar

Chitin is a polysaccharide long-chain polymer comprised of β-(1,4)-N-acetylglucosamine residues and is an important biopolymer natural biosphere, and widely exited in fungi, algae, nematodes, crustaceans and insects. In insects, chitin is one of the key components of the cuticle and peritrophic membrane (PM). Insect chitinases are one class of hydrolases involving in hydrolyzing β-1,4-glycosidic linkages of chitin and are encoded by single gene and belong to family 18 of the glycohydrolase superfamily. Chitinases have important function in digestion, arthropod molting, metamorphosis, infection and other physiological processes of insects. Individual insect contains multiple genes encoding chitinases, which differ in domain architectures, biochemical properties and physiological function. At present, in view of its ability to degrade chitin, insect chitinases are mainly used in the insect pest control and disease control, and so forth.In this study, the complete cycle system of development and passage, from egg hatching, larvae development, pupae to adult and passaged, has been established. Subsequently, the numbers of developmental days for each stage have been recorded during gypsy moth larvae feeding with the artificial diet and specific day of every instar larval has been obtained to provide the basic data about expression pattern studies of Group I chitinase gene (LdCht5) and Group ⅡI (LdCht10) chitinase gene.The key sequence of Lymantria dispar Group II chitinase gene was cloned and was 2 057 bp in length, encoding a polypeptide of 685 amino acid residues with a complete catalytic domain and chitin binding domain. In addition, the successful cloning of a 18S rRNA and β-actin sequences has great role in subsequent gene primer designing of control gene during studies of relative expression of the target gene. The transcriptional level of LdCht5 and LdCht10 in the fourth and fifth instar larvae as well as different tissue of sixth instar larvae were proceeded with Real-time PCR and the results showed that the expression of LdCht5 and LdCht10 have been detected almost every day, and their transcription is relatively active in two periods of time before and after molting, which might indicate that they probably participated in the chitin degradation in these two stages. The expression studies of different tissues of sixth instar larvae revealed that both LdCht5 and LdCht10 had higher expression level in the epidermis, followed by foregut, which presumably implied that two types of enzymes play a key role in the degradation process of the epidermis and foregut in molting process. Then the biological function of LdCht5 and LdCht10 have been performed by RNAi and the results showed that sixth-instar larvae or pupae could complete metamorphosis from larval to pupal after pre-injection LdCht5 dsRNA, but could not be completely separated from pupal cuticle during metamorphosis from the pupa to adult and ultimately dead. After LdCht10 dsRNA has been injected into larvae, sixth-instar larvae could not separated its old skin to finish metamorphosis from larval to pupal.In this study, the chitinase genes of gypsy moth have been studied on molecular biology level. The time and tissue expression pattern of Group I and Group II chitinase genes was conducted by Real-time PCR, the biological function of chitinases during development of gypsy moth was performed by RNAi technique, these enrich the current research findings on insect Group I and Group II chitinase and have important role in the foundational studies of chitinases gene function.