Studies On The Role Of Entomopathogenic Fungus Metarhizium Anisopliae Acid Trehalase

Metarhizium anisopliae, a species of entomopathogenic fungi widely applied at home and abroad, plays an important role in biological control of insects. Compared with chemical insecticides, mycoinsecticides are not infective or toxic to human and can not pollute environment, but the slow insecticidal speed has been recognized as a potential drawback to prevent the utilization of these fungi against insects. Researches on mechanisms of fungal pathogenesis and host defense may suggest strategies for the development of more efficient anti-insect mycoinsecticides. Trehalose (α-D-glucopy- ranosyl (1-1) -α-D- glucopyranoside), a non-reducing disaccharide, exists in almost all kinds of insects as the predominant sugar (80-90% of total sugar) in the haemolymph of insects. Trehalose plays such an important role in insects'blood physiology that its exhaustion may be detrimental to the insects, which as a consequence must be viewed as a potential nutrient source for insect pathogenic fungi like Metarhizium anisopliae once the fungus infected into the haemolymph. Trehalose is impermeable substrate for some fungi, and corresponding glycosidase (acid trehalase and /orα-glucosidase) must be external to the cell membrane to hydrolyze it into glucose before use. Metarhizium anisopliae can produce acid trehalase once they get into the haemolymph of insects and the enzyme can effectively hydrolyze trehalose in the haemolymph of insects, which may suggest that the Metarhizium anisopliae acid trehalase play an important role in the pathogenesis of insects.Much less is known about acid trehalase in fungi and at present only limited acid trehalase genes of some fungi have been cloned and identified. There has no relative report about acid trehalase and its gene in entomopathogenic fungus except that purification of Metarhizium anisopliae acid trehalase (ATM1p) was recently reported by our laboratory. Unfortunately, further characterization of ATM1p has been hampered by the low levels of ATM1p produced by M.anisopliae. Moreover, purification of M.anisopliae ATM1p is costly, time-consuming, and technically inconvenient. In this research Metarhizium anisopliae var. acridum strain CQMa102 was used, and the complete ATM1 gene encoding acid trehalase was isolated and identified (GenBank accession no. EF190950) and expressed successfully in Pichia pastoris. According to the sequences above, a double-stranded RNA interference vector has been constructed, and three RNA interference transformants have been obtained. The main results were as follows:1. According to a partial ATM1 sequence obtained by our lab, using 3'race and 5'race, the complete cDNA sequence encoding the acid trehalase was isolated (GenBank accession No. EF190950). Acid trehalase gene has three introns and its ORF encodes a 1073 amino acid protein, which has a 20 aa signal peptide sequence and 30 putative N-glycosylation sites. Comparison of the amino acid sequence with the sequences found in protein data bases using the NCBI BLAST algorithm revealed the protein shared 62%, 59% and 57% similarities with alpha, alpha-trehalose glucohydrolase in Aspergillus funigatus, acid trehalase precursor in Aspergillus nidalans and acid trehalase in Talaromyces emrsonii, respectively. This protein also shared about 25% similarities with the acid trehalases from Saccharomyces cerevisiae (Ath1p) and Candida albicans (Atc1p). So we nominated its gene as ATM1 (for acid trehalase of Metarhizium anisopliae), and the corresponding protein was ATM1p.2. The mature protein of M. anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. Optimum culture medium and culture conditions base had been optimized. The pPATM1-21,with the highest acid trehalase activity was selected for scale-up expression. The recombinant ATM1p (reATM1p) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1p in the culture supernatant reached the peak, 5.35U/mg. Enzyme with a histidine tag appended to the C terminus was still active and was purified using metal-chelate affinity chromatography.3. The purified reATM1p exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. Trehazolin, a specific inhibitor of trehalases, inhibited the reATM1p with IC50 of about 1.9×10-8,and reATM1p exhibited a high specificity for trehalose. The optimum temperature and pH of reATM1 were 30℃and 6.0, respectively, and the Km and Vmax values for reATM1 were 2.6mM and 0.305 mmol/min/mg, respectively.4. According to the sequences above, a double-stranded RNA interference vector has been constructed, and transformed to strain CQMa102 by particle bombardment. After PCR selection and Southern blot verification, three RNA interference transformants have been obtained. The expression quantities of ATM1 of three RNA interference transformants (RA14\,RA42,RA74) decrease by 98.84%,98.57%and 96.56%, respectively, compared with the wide type.