创制转Mad1基因球孢白僵菌工程菌株提高对玉米螟毒力
克隆了来源于生防真菌绿僵菌的黏着蛋白基因Mad1,构建到含玉米瘤黑粉菌热激蛋白启动子hsp70和以抗萎锈灵基因carboxin为筛选标记的遗传转化载体上,利用PEG介导的原生质体转化法导入到受体球孢白僵菌菌株D1-5中,获得转化子,经过继代培养及分子检测,获得遗传稳定的基因工程菌株。利用蝗虫后翅及洋葱表皮进行吸附试验进行了工程菌株的生物学功能鉴定,结果表明,野生型和工程菌的分生孢子对于洋葱表皮的吸附量并无明显差异,而工程菌的分生孢子对蝗虫翅膀的吸附则显著高于野生型,表明Mad1基因的导入能够增加球孢白僵菌对昆虫组织的吸附,而对植物表皮组织不存在吸附能力；对亚洲玉米螟幼虫室内毒力测定结果表明转化后的工程菌株致死中时间(3.278±0.346天)比野生型(4.972±0.147天)缩短34.07%,杀虫效率显著提高。
英文摘要:
Focal adhesion protein gene Mad1 was cloned from Metarhizium anisopliae, to construct genetic transformation vector, contained the heat-shock promoter hsp70 from Ustilalgo maydis and anti-caboxin gene as screening label, and the vector was introduced into Beauveria bassiana strain D1-5 by protoplast transformation. The transgenic strain was obtained by molecular detection after subculture. Adherence assay on locust hind wings and onion epidermis was used to detect the bioactivity of genetic strain. The results showed that there was no significant difference on adherent on onion epidermis between transgenic and wild strains, while the adherent ability of genetic strain was higher than that of the later obviously. It indicated that the Mad1 gene gave an enhanced adherence on pest cuticle, but not on plant cuticle. The bioassay against the larves of Ostrinia furnacalis Guene showed that the genetic strain exhibit a higher toxicity than that of wild strain, the LT50 (3.278±0.346 days) of transgenic strain was shorten 34.07% than that of wild strain (4.972±0.147 days), the Mad 1 gene transgenic strain promoted the efficiency against pests obviously.
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