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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 order to clarify the reasons for the difference in virulence between different transformants of Metarhizium anisopliae containing cry8E gene and to reveal the relationship between extracellular enzymes and virulence, we tested the acidity of six transformants of Metarhizium anisopliae containing cry8E gene Protease, neutral protease, alkaline protease, chitinase and phospholipase activity and their toxicity to Holotrichia parallela, and analyzed the correlation between each enzyme activity index and the strain virulence. The results of bioassay showed that the virulence of different transformants varied greatly, the range of LT50 varied from 14.48 to 21.26 days, and the corrected cumulative mortality ranged from 64.50% to 92.86%. The results of enzyme activity assay showed that there was a great difference in the vitality between different strains, and the variation of the activity of different enzymes was inconsistent. Univariate regression analysis of the enzyme activity of different transformants with the adjusted cumulative mortality and LT50 values showed that the transformation strains of acid protease and chitinase had significant correlation with virulence. In order to further determine the combined effect of acid protease and chitinase on virulence, binary regression analysis of enzyme activity and corrected cumulative mortality of different transformants, LT50, found that only chitinase activity was correlated with strain virulence Sexual significant. Therefore, it is speculated that the difference of host virulence between different transformants is mainly due to the different chitinase activity among strains. Chitinase can serve as a reference index for preliminary screening of a large number of transformants, but can not be completely replaced.