Functional Analysis Of Lm-TSP Required For The Molting Of Locusta Migratoria Manilensis And Cloning Of Its Alternative Splicing Isoforms

Molting is an important physiological behavior during development of insects. Because of the hardening of exocutitle, insect cuticle lost its extensibility, then developmental growth of larvae are hindered. So insect larvae must experience cyclic molting in which they enlarge their exoskeleton to accommodate internal growth. During molting insects stop feeding, endangerment to crop is reduced. If ecdysis couldn't be completed successfully, larvae wouldn't become adult, and generate abnormal individuals which have no vitality or no reproductive performance or food consumption reduced. Therefor molting has always been a research hotspot in the fields of insect pest control. Research molting is helpful to understand ecdysis development of insects and find some new biological control targets. Early research focus on the expression mechanism of some transcription factors which are regulated by ecdysone and purifacation of enzymes in molting fluid. However, reports of genes required for insect molting and their roles have been quite limited.Zhang Wei et al. have cloned a novel gene from Locusta migratoria manilensis, designated as Lm-TSP. Its encoding protein may be trypsin-like serine protease. RNA interference study shows that the silencing of Lm-TSP led to molt defect from the fourth instar larvae to fifth instar larvae and molt defect from the fifth instar larvae to adult and finally led to death. The knock-down of Lm-TSP also caused reduction in protease activity and cuticle degrading activity of the molting fluid. These results suggest that Lm-TSP is correlated with Locusta migratoria manilensis ecdysis. Then Lm-TSP might be considered as a target for Locusta migratoria control. So we take Lm-TSP as research project, and further analyze its function. The expression profile of Lm-TSP is analyzed, the expression of Lm-TSP in Pichia pastoris KM71 is achieved and its alternative splicing isoforms are cloned. This research is helpful to understand ecdysis development of insects and find some new insecticidal targets. The main results are as follows:1. Lm-TSP expression profile in different stages of the fifth-instar larvae development is identified. Lm-TSP is maimly expressed at 72, 24, 16 hours before fledging.2. Lm-TSP expression profile in different tissues of the fifth instar larvae is analyzed. Lm-TSP is maimly expressed in trunk, head and hind leg and there is little expression product in haemocytes, midgut and fat body. 3. Expression of Lm-TSP in eggs of different incubation time can be detected, but expression level is low. As incubation time goes on, expression level get higher.4. Lm-TSP is secretively expressed in Pichia pastoris KM71. Compared with controls, the product of Lm-TSP recombinant added a new band at about 45 kDa by SDS-PAGE.5. Five alternative splicing isoforms of Lm-TSP are cloned, designated as Lm-TSPA, Lm-TSPB, Lm-TSPC, Lm-TSPD, Lm-TSPE.