Construction Of A Chintinase-Overexpressive Transgenic Strain And A Bivalent Transgenic Strain Of Beauveria Bassiana And Their Virulence Evaluation

Beauveria bassiana is one of the most common entomogenous fungi, with wide host range and great easiness for massproduction, and has been developed into fungal insecticide used most widely. Like other entomogenous fungi, however, it has an important drawback, slow lethal time. Consequently, mollecular pathogenic mechanism of the fungi has been focused in recent years in order to look for virulence-related genes to improve by means of gene engineering. Chintinase plays a kind of role during penetration of insect cuticle of entomopathogenic fungi, but their relation to virulence of the fungi remains unclear. In the present study, exogenous chitinase gene Bbchit1 from B. bassiana was transformed into two initial strain, RCEF0013, a wild-type strain which contains Bbchit1, and RCEF0013-A, a monovalent transgenic strain of RCEF0013 with AaIT, an insect-specific neurotoxinic polypeptide gene from a north Arican scorpion, Androctonus australis, resulting in construction of a monocopic chitinase-overexpressive transgenic engineered strain and a bivalent transgenic engineered strain of scorpion toxin-chitinase, by two step transformations using different selective markers.PbarGPE1-Bbchit1, a plasmid containing bar gene and exogenous Bbchit1 gene was constructed and transformed through blastospores into RCEF0013. A bicopic transformant RCEF0013-C was obtained through screening on SDAY medium containing 100μg /ml glufosinate ammonium. The result of chitinase activity test showed the activity of RCEF0013-C on an inducing medium containing colloidal chitin was 4.2 times higher than that of RCEF0013, suggesting an overexpression of chitinase gene in the transgenic bicopy strain. Then pbenGPS3-Bbchit1, a new plasmid containing ben gene and exogenous Bbchit1 gene was constructed and transformed through blastospores into RCEF0013-A, the monovalent transgenic isolates of B. bassiana containing AaIT. Finall, RCEF0013-A-C, a bivalent transformant was obtained through screening on SDAY medium containing 2μg /ml benomyl.A bioassay was conducted on the Masson's pine caterpillar, Dendrolimus punctatus, the original host of RCEF0013. The result showed that virulence of both the over-expressive bicopic transformant and the bivalent transformant on the caterpillar was significantly increased. For RCEF0013-C, LC50 was reduced by 5.46 times, LD50 decreased by 6.47 times, and LT50 shortened by 0.7 day at the dose of 2.5×107. For RCEF0013-A-C, LC50 was reduced by 17.31 times, LD50 decreased by 16.41 times, and LT50 shortened by 1.8 day at the dose of 2.5×107.The chitinase activity of the transformant on the 1st to 8th day was extremely correlated with the daily mortality from the 2nd to the 9th day of the caterpillar (P<0.01) , while that of the original wild-type strain not correlated (P>0.05), suggesting that the relationship between chitinase and virulence is complicated. Only to a threshold of the chitinase secretion, is the relationship obvious.