绿僵菌不同转化子毒力差异与胞外酶的相关性分析

Correlation between Extracellular Enzyme Activity and Virulence from Different Transformants Containing cry8E Gene

为了明确含有cry8E基因的绿僵菌Metarhizium anisopliae不同转化子存在毒力差异的原因,揭示胞外酶与毒力之间的关系,本研究测定了6株含cry8E基因绿僵菌阳性转化子的酸性蛋白酶、中性蛋白酶、碱性蛋白酶、几丁质酶和磷脂酶活性及其对暗黑鳃金龟幼虫Holotrichia parallela毒力,并分析了各个酶活指标与菌株毒力之间的相关性。生测结果表明,不同转化子间毒力差异较大,LT50的变化范围为14.48~21.26 d,校正累计死亡率变化范围为64.50%~92.86%。酶活测定结果表明,不同菌株之间活力差异较大,且不同酶的活力变化规律不一致。将不同转化子的酶活分别与校正累积死亡率和LT50值进行一元回归分析,发现各转化菌株酸性蛋白酶和几丁质酶与毒力相关显著。为了进一步确定酸性蛋白酶和几丁质酶对毒力的综合影响,将不同转化子的酶活与校正累积死亡率、LT50进行二元回归分析,发现仅有几丁质酶活性与菌株毒力相关性显著。因此,推测不同转化子对寄主毒力存在差异主要是由于菌株间几丁质酶活性不同,几丁质酶可以作为大量转化子初步筛选的参考性指标,但是不能完全替代。

In this study, we tested the acid protease, neutral protease, alkaline protease, chitinase, phospholipase enzyme activities of six Metarhizium anisopliae transformants with cry8E gene insertion and virulence against Holotrichia parallela in order to clarify the correlationship between extracellular enzyme activity and virulence of each strain. The results showed that the virulence among six transformants assayed was significantly different, and the ranges of the estimated LT50 and the corrected cumulative mortality were 14.48~21.26 d, 64.50%~92.86%, respectively. There also has significant difference in the enzyme activities among six transformants. Regression analysis also revealed that acid protease and chitinase were significantly correlated to the corrected cumulative mortality and LT50. Binary regression analysis of combination effects of acid protease and chitinase on virlence also revealed that only chitinase was significantly correlated to the corrected cumulative mortality and LT50. It suggested that the difference of virulence among transformants may be mainly derived from the activity of chitinase, and it could be used as virulence index for large scale of early candidate transformants selection, however, but it could not completely replace the virulence measurement in future.