Construction Of Genetic Engineering Metarhizium Anisopliae For Plutella Xylostella Control

Plutella xylostella is the worldwide vegetable pest. It has the characteristic of breeding fast,generation overlap seriously, resistant to chemical pesticides. So developing microbial insecticides withhigh pathogenicity to P. xylostella is of great value. Metarhizium is a kind of importantentomopathogenic fungi. But in the field application, it exposed the disadvangtage of long lethal time,low toxicity, narrow insecticidal spectrum, et al. Thus construction engineering M. anisopliae strainwith high virulence is one of important research directions now. In this paper, we conducted researchfrom following aspects, and made following results:Using leaf dipping method, we tested the virulence of12isolates of Metarhizium strains preservedin the laboratory against P. xylostella, and found a strain named609with high toxicity. Under thecondition of spore concentration1×10~8conidia/mL,8days after inoculation, the cumulative mortalitywas86.7%. When the condition of spore concentration was1×10~7conidia/mL, lethal time of50%（LT50）was5.7d. Based on the sequence of ITS region, strain609was identified as Metarhiziumanisopliae var. acridum.We cloned insecticidal gene cry1Ba which exhibited high virulence to lepidoptera pests, especiallyto P. xylostella, and inserted it into pET-28a to construct the vector pET-1Ba. Then it was transformedinto BL21competent cell and induced using IPTG. The Cry1Ba protein was extracted through ultrasonicbroken method and its effect on germination of strain609was investigated. The results showed thatCry1Ba protein did not affect the germination of spores from M. anisopliae strain609. Thus weconstructed the vector Camben-1Ba with resistant marker gene benA3, and target gene cry1Ba and thentransformed into strain609by Agrobacterium-mediated transformation method. The positivetransformants were verified by PCR detection and Southern hybridization, and their pathogenicity wastested on the2ndinstars larvae of P. xylostella. The bioassay results showed that compared withwild-type strain609, the virulence of transformants against P. xylostella changed by different degrees,especially the virulence of tranformant609-1increased sharply by22%, and LT50shortened by1d.We constructed vector Camben-gfp containing report gene gfp, resistant marker benA3gene andvector Camben-gfp-1Ba harboring gfp gene, benA3gene, and target gene cry1Ba, and integrated themrespectively into M. anisopliae609by the method of Agrobacterium-mediated transformation. Thepositive transformants were monitored through fluorescence microscopy. The results showed thattransformants containing gfp gene could stimulate green fluorescence. Finally we investigated thesurvival state of transformant harboring Camben-gfp vector in soil. The results showed that the numberof transformation strain609-gfp was declining,5days after the soil inoculated with spores atconcentration of1×10~7cfu/g, the number of spores decreased nearly one order of magnitude.Transformation strain can still be detected with1×104cfu/g29d after inoculation.