几丁质酶基因在球孢白僵菌中的超表达提高其对马尾松毛虫的毒力  

Over-expression of Chitinase Gene in Beauveria bassiana Enhanced Its Virulence on Dendrolimus punctatus

作　　者：郝蕾蕾[1] 陈珊珊[1] 谢翎[1] 李增智[1] 

机构地区：[1]安徽农业大学微生物防治省重点实验室,合肥230036

出　　处：《中国生物防治》2010年第3期267-273,共7页Chinese Journal of Biological Control

基　　金：国家"863"项目(2006AA10A212);教育部高等学校博士点专项科研基金项目(200503644001);安徽省高校省级自然科学研究重点项目(TD200708)

摘　　要：几丁质酶在虫生真菌穿透寄主体壁的过程中起一定作用,但其与真菌毒力的关系尚不清楚。本研究通过几丁质酶基因在球孢白僵菌Beauveria bassiana中的超表达以及超表达菌株的毒力变化研究了该基因与毒力的关系。将几丁质酶基因Bbchit1通过芽生孢子转入野生型球孢白僵菌中,获得多拷贝的转化子;转化菌株在胶体几丁质诱导培养基中酶活最大值高于出发株4.2倍,表明该几丁质酶基因在转化菌株中得到超表达。毒力测定发现转化子对马尾松毛虫Dendrolimus punctatus的致死中浓度比出发株降低84.5%,在2.5×107孢子/ml浓度下的致死中量减少84.5%,致死中时缩短0.7d,因此毒力大大提高。第2~9d感病死虫数与转化子第1~8d酶活呈极显著正相关（P〈0.01）,而与出发株不相关（P〉0.05）。这些证据表明,几丁质酶与毒力的关系是复杂的,在分泌达到一定阈值后与毒力才有明显的相关关系。Chintinase plays a role in the penetration by entomopathogenic fungi,but its relation to virulence of the fungi remains unclear.In the present paper,the relation of chitinase of Beauveria bassiana to its virulence was tested by over-expression of the chitinase gene and detection of virulence change in a transgenic B.bassiana strain.Chitinase gene Bbchit1 was transferred into a wildtype strain of B.bassiana by blastospore transform,resulting in a multicopy transformant.The results revealed that the maximal activity of chitinase of the transformant on an inducing medium containing colloidal chitin was 4.2 times higher than that of wild type strain,suggesting an over-expression of chitinase gene in the transgenic multicopy strain.Bioassay against the Masson＇s pine caterpillar,Dendrolimus punctatus,showed that LC50 of the transformant was reduced by 84.5%,LD50 was decreased by 84.5%,and LT50 shortened by 0.7 d at the concentration of 2.5×107 conidia/ml,indicating a substantial virulence enhancement.The daily mortality of the caterpillar on the 2nd to 9th day was positively correlated to the chitinase activity of the transformant on the 1st to 8th day significantly（P0.01）,but not to that of the original wild-type strain（P0.05）.All these results suggest that the relationship between chitinase and virulence is complicated.Only to a threshold of the chitinase secretion,does the correlation become obvious.

关 键 词：几丁质酶 球孢白僵菌 超表达 马尾松毛虫 毒力 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治] Q786[农业科学—植物保护]