| The locust is a cosmopolitan pest and has brought serious economic loss to agriculture. Metarhizium anisopliae, an entomopathogenic fungus widely applied in domestic and abroad, has an important role in biological control of locust. At present, many strains of M. anisopliae have been registered in the world and have realized commerciality. However, these products usually show instability. The storage and environmental instability of conidiospores is a significant drawback of commercial scale use of this very promising alternative to chemical pesticides. 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, and used it on a commercial scale. Whereas, there still has the problem of prolonging storage time and enhancing environmental stability. 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. There is a strong correlation between intracellular trehalose accumulation and prolonged storage time of conidia from filamentous fungi. The activity of neutral trehalase lost and trehalose concentration has been increased three times after knocking out neutral trehalase gene in S. cerevisiae. Based on these research results, we speculated that increasing trehalose content in conidia, prolonging conidial storage time and enhancing its environmental stability may be feasible through changing trehalose metabolism pathway by application of genetic engineering. In order to improve M. anisopliae strain by changing trehalose metabolism pathway, we must understand metabolism and regulative mechanism of trehalose in M. anisopliae. Nevertheless, Little attention has been given to the research of trehalose in M. anisopliae. A neutral trehalase gene in M. anisopliae was first cloned and characterized by Xia et al, but its biological role in M. anisopliae has still remained unclear. It seemed therefore worthwhile to get more information on the role of neutral trehalase during storage of conidiospores in entomopathogenic fungus M. anisopliae. For that purpose, in our research, we will increase the copies of neutral trehalase gene in M. anisopliae to decrease the trehalose synthesis during conidiation, and will knock out or knock down the activity of neutral trehalase to increase trehalose content in conidia by knocking out technology or double-stranded RNA interference. Furthermore, in order to elucidate the role of neutral trehalase in conidiospores stability during storage and heatshock, trehalose concentration, storage duration , thermotolerence of the mutants and wild-type strain were analyzed. In our research, the neutral trehalase gene in M. anisopliae was successfully cloned by PCR, RT-PCR, RACE and panhandle PCR, and submitted to GenBank, accession number are AY557613(genomic sequence) and AY557612(cDNA sequence). According to the sequences above, an over-expression vector and two homologous vectors and a double-stranded RNA interference vector have been constructed, and transformed to strain CQMa102 by PEG-CaCl2 mediated method, electroporation and particle bombardment. After PCR selection and Southern blot verification, three over-expression transformants and three RNA interference transformants have been obtained. Analysis of these mutants revealed that the expression level of the neutral trehalase gene in RNAi mutant (Ma688 strain) was reduced to 42% of which found in wild-type strain and the over-expression mutant (Ma113 strain) showed approximatlely 2-fold higher levels of neutral trehalase mRNA than that found in wild-type control. Then the transformants Ma688 and Ma113 were chosen for closer examination, including trehalose content, storage longevity , thermotolerance and growth characteristics. The main results were as follows: ⑴During the maturation of conidiospores, the trehalose content increased rapidly in M. anisopliae. The wild-type strain accumulated trehalose more slowly than the RNAi mutant, and more quickly than the over-expression mutant. ⑵During the accelerating aged storage, the storage duration of mature conidiospores is higher than immature ones. The storage duration of wild-type conidia is lower than that of RNAi mutant conidia, and higher than over-expression mutant ones. ⑶The ability to heatshock of mature conidia is higher than that of immature ones at 45℃. The wild-type conidia show lower thermotolerence ability than RNAi mutant conidia, and higher thermotolerence ability than over-expression ones. ⑷No significant differences in growth condition and sporulation frequencies between the wild-type strain and trehalase mutants (RNAi mutant and over-expression mutant) were observed. ⑸Based on above results, we can conclude that the biological function of neutraltrehalase in M. anisopliae assists in control of trehalose concentration via degradation of trehalose, and indirectly influences the stability of conidia during storage and during heat stress. This research achievement could provide a new basis and technology reserve for improving entomopathogenic fungus strains to have longer shelflife and strong resistance to adverse conditions by ferment technique and breeding technique including genetic engineering.
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