Changes In Expression Level Of Neutral Trehalase Suggest A Role For Thermotolerance In Metarhizium Anisopliae

In the worldwide, the locust is a vital pest of agriculture. The population happens in the most countries of world, bringing serious economic loss to agriculture. Metarhizium anisopliae, an entomopathogenic fungus, as biological pesticides, applies in biological control of locust and has good foreground in the domain of biocontrol as its unique merit. But its main disadvantage is the rigorous require when product,storage,transit and use, it costs lot to store in cold. These problems limited M. anisopliae insecticide in a great deal of usage on the production and make its research hard to make breakthrough progress.Trehalose, a non-reducing disaccharide, occurs in a large range of organisms, such as bacteria,fungi,animals and plants. In addition to its function as a storage carbohydrate and transport sugar, trehalose plays an important role in stress protection, especially during heat stress, dehydration, drought, freeze, high saturation, heavy metal and noxious reagent. It can be treated as protectant and preservation of bacterium. Trehalase, as trehalose hydrolase, degrades trehalose into two molecules of glucoses. In most fungal species, trehalose hydrolysis is carried out by the trehalase. The single known exception is Pichia fermentans, in which the trehalase has phosphorylase activity. Trehalose is strict specifically substrate. We speculated that increasing trehalose content in conidia, enhancing its environmental stability may be feasible through changing trehalose metabolism by application of genetic engineering.We have isolated a strain of M.anisopliae var acridum CQMa102 exhibiting a high degree of locust specificity from natural environment, and registered its conidial powder and oil suspending preparation in China. The neutral trehalase gene (ntl) in M. anisopliae has been cloned, NCBI accession number is AY557613. We constructed an ntl RNA interference (RNAi) vector of neutral trehalase with dual promoter system and an over-expression vector, which could randomly integrate into the fungal genomic DNA, transformed into strain CQMa102 by bombardment. After pestanal and PCR selection, four RNA interference transformants and four over-expression transformants have been obtained. The main results were as follows by analysis of these mutants:1. Compared to the wild-type, ntl mRNA reduced to 35-66% in RNAi mutants, while increased to 2-3 folds in the over-expression mutants.2. In RNAi conidiospores accumulated more trehalose, had less trehalase activity, and were much more tolerable to heat stress than the wild-type. Opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants.3. Pathogenicity was not altered with the changes in ntl mRNA and activity compared to the wild-type.These results indicated that ntl controlled trehalose concentration in M. anisopliae by degrading trehalose, which indirectly increased conidiospore thermotolerance. These results offered a new strategy to increased conidiospore thermotolerance of entomopathogenic fungus without affecting its virulence. Additionally, the dual promoter system developed for RNAi was proved as an efficient tool for functional analysis of entomopathogenic fungal genes.