Expression Of Cry1Ac Gene In Entomogenous Fungus Beaveria Bassiana

Fungal insecticides is a sort of environmental friendly environmentally safebiopesticide, with good safety to human, vertebrates and non target invertebrates, andsubstantial sustainable control effect, and easiness of mass production with artificialmedia. On the other hand, they are subjected easily to environmental factors and killinsects slowly and not steadily, which has constrained the development of the use ofthe fungi. Moleculer pathogenic mechanisms have been studied in order to thevirulent genes which can be improved by gene engineering.Bacillus thuringiensis is gram-positive bacterium, one of kinds which can formparasporal crystal protein（insecticidal crystal protein, ICP） or δ-endotoxin in thestability of the bacteria growth. It is strong poisonous force and speeding deadly forpests and that is avirulent entomopathogen for the natural enemy of pests. Cry1Acgene of B. thuringiensis is an important virulent gene, but it is unknown whether itcan enhance the virulence of a fungus. In the present study, the exogenous Cry1Acwas transferred into two wild type isolates of Beauveria bassiana, one from theMasson’rpiller, Dendrolimus punctatus, and another from the Asian corn borer,Ostrinia furcanalis，to constitute two gene engineered strains with one copiedoverexpressed Cry1Ac.A vector of pbarGPE1-Cry1Ac containing the selecting marker bar gene andexogenous gene Cry1Ac was constoituted and then transferred into RCEF0013andRCEF3383by blastospore tranform method. Transformants were derived throughsieving on SDAY medium containing100g/ml glufosinate ammonium, resulting inthe monovalent Cry1Ac transgenic engineered strains, RCEF0013-G1andRCEF3383-G1. The Cry1Ac gene was overexpressed with the engineered strains.The result of bioassay for Dendrolimus punctatus showed that as compared tothe wild type isolate RCEF0013, the LC₂₅of RCEF0013-G1to the pine caterpillarsdecreased by5.4-folds, the LD₂₅reduced by7.1times, and LT50shortened by2.2d,indicating a substantial virulence enhancement. Compared to spray treatment, thecumulative mortality of feeding treatment and feeding with spray treatment wereincreased by55.6%and63.0%, and LT50shortened by4.5d days and5.3d days. Thesedata indicated that the stomach toxicity of Bt delta-endotoxin gene enhanced thevirulence of B. bassiana greatly. And this is the first expression of Cry1Ac gene in theinsect fungi.The result of bioassay for Ostrinia furnacalis showed that as compared to the wild type isolate RCEF3383, the LC₅₀and LD₅₀of RCEF3383-G1to the O.furnacalis with spraying decreased by5-folds and7.2-folds. By2×108conidia/mlwith spraying, the LT50of RCEF3383-G1shorten1.7days than RCEF3383, indicatinga substantial virulence enhancement.Consequently, CryAc gene was expressed in the transgenic strains so as to infecthosts mainly through bioassay, and its virulence was raised substantially as a result.