玫烟色棒束孢几丁质酶的转基因球孢白僵菌菌株的获得及其对马尾松毛虫的毒力增效作用

本文根据GenBank中已登录的蜡蚧蚧霉Lecanicillium lecanii，粉棒束孢Isaria farinosa，球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae的几丁质酶基因序列的同源性比较， 以它们高度保守的核苷酸序列设计一对引物，采用RT-PCR和3′/5′-RACE相结合的方法，首次从玫烟色棒束孢Isaria fumosorosea中克隆出几丁质酶Ifuchi1全长cDNA基因。其全序列长为1 542 bp，编码阅读框由1 275个核苷酸组成， 5′非翻译区（5′-UTR）与3′非翻译区（3′-UTR）分别为33个核苷酸和234个核苷酸。基因编码424个氨基酸， 信号肽长度为24个氨基酸， 成熟蛋白理论分子量为47.6 kDa，理论等电点为4.89。该蛋白可归于几丁质酶18族V类。运用制备芽生孢子转化法将Ifuchi1基因导入球孢白僵菌。最适产酶时间36 h条件下， 挑选的转基因菌株的几丁质酶活性相对于野生型菌株提高了86.2%；在对马尾松毛虫的生物测定中，在孢子浓度为1×10⁷个孢子/mL时，与野生型菌株Bb13相比，转基因菌株的致死中时间LT₅₀平均缩短了29%，同时死亡率提高了52.9%；野生型菌株和转基因菌株致死中浓度LC₅₀分别为4.71×10⁶ 个孢子/ mL和1.74×10⁶ 个孢子/ mL，降低了约1.7倍的剂量，故通过基因工程方法获得的转基因工程菌株显著地提高了球孢白僵菌的杀虫效率。

Generation of transgenic Beauveria bassiana strains with chitinase gene from Isaria fumosorosea and its increased virulence against Dendrolimus punctatus (Lepidoptera: Lasiocampidae)

Using SMART RACE RT-PCR, the complete full-length cDNA encoding a chitinase was cloned from the biocontrol agent Isaria fumosorosea. The gene, designated as Ifuchi1, contains an open reading frame (ORF) with 1 275 bp encoding 424 amino acids, which contains a N-terminal 24 amino acid residues displaying the characteristics of signal peptide. The mature chitinase has a molecular mass of 47.6 kDa and a calculated pI of 4.89. The protein sequence contains two highly conserved regions including a putative enzymatic active site and a potential chitin-binding domain. The chinitase belongs to the class V of family 18 of glycosyl hydrolases. The Ifuchi1 gene from I. fumosorosea was engineered into Beauveria bassiana by blastospore transformation. Compared to the wild-type strain Bb13, the chitinase activity of a selected transformant was increased by 86.2% after induction for 36 h. Bioassayed against the larvae of Masson’s pine caterpillar Dendrolimus punctatus, the median lethal time (LT₅₀) of transgenic isolates was reduced by 29%, and the mortality rate was increased by 52.9%, as compared to the wild-type at a concentration of 1×10⁷ conidia / mL. The median lethal concentration (LC₅₀) values of the wild-type and transgenic isolates were 4.71×10⁶ conidia / mL, and 1.74×10⁶ conidia / mL (approximately 1.7-fold decrease), respectively. The transgenic strain thus showed significantly improved virulence against D. punctatus.