Isolation, Cloning And Function Analysis Of Trehalose-6-phosphate Synthase From Locusta Migratoria Manilensis

Trehalose is present in a wide variety of organisms, including bacteria, yeasts, fungi, insects, invertebrates, and lower and higher plants. It not only serves as a main source of energy and carbon, but also can protect organisms from various stresses such as heat, cold and drought. Trehalose-6-phosphate synthase (TPS) plays an important role in trehalose synthesis. There have been many reports about trehalose-6-phosphate synthase in fungi and bacteria, the cDNA sequences encoding TPS have also been cloned. But only a few TPS cDNA clones were obtained from insects and the researches on its function are also minority. A complete TPS cDNA clone was obtained from Locusta migratoria manilensis fat body and its tissue distribution and function are primarily studied. The main achievements are as follows:①Cloning and sequence analysis of tps gene from Locusta migratoria manilensi fat bodyA complete TPS cDNA clone (GenBank accession no: EU131894) of Locusta migratoria manilensi trehalose-6-phosphate synthase (LmTps) was obtained by means of 3'-RACE and 5'-RACE. The results showed that the cDNA was 2 806bp in length and contained an open reading frame (ORF) of 2 442bp, which encodes a protein of 813 amino acids with a calculated molecular weight of 91 976D and pI of 6.14.②The tissue expression analysis of LmTpsThrough semi-quantitative RT-PCR, we examined the tissue expression of LmTps with total RNA from fat body, gut, haemolymph and leg muscle. The result indicated that LmTps is not only transcripted in fat body, but also in gut, haemolymph and leg muscle.③Locust RNAiWe designed a 902bp fragment according the cDNA of LmTps, transcrpted into dsRNA in vitro and injected into locust migratoria. The result indicated the the concentration of trehalose decreased sharply from the 4th day to 14th day. This may contribute to the decrease of the LmTps transcript level.④Complementation experiment of Sacchromyces cerevisiaeTo further validate the function of the cDNA sequence cloned, we transformed the LmTps cDNA into Sacchromyces cerevisiae tps1 mutant by PFL61 vector. The result indicated that its character that is can't grow on glucose medium is complemented and showed that the LmTps can entirely or partly function as trehalose-6-phosphate synthase in Sacchromyces cerevisiae.⑤Expression of LmTps in Pichia pastorisThe ORF of the LmTps was inserted into pPIC9K and the recombinant of pPIC9K-LmTps plasmid was successfully transformed into Pichia pastoris KM71 by electrotransformation. In shake-flask culture induced by methanol, a 91kD lane was detected in the supernatant and may indicate the LmTps was expressed in the Pichia pastoris.