携带AaHIT1杀虫蛋白基因的白僵菌改造菌株的构建及应用

Construction and application of modified Beauveria bassiana strains carrying the AaHIT1 insecticidal protein gene

摘要

农业害虫通过直接取食作物、传播病害等方式,对作物产量、品质及农业生态安全构成严重威胁。当前依赖化学杀虫剂的防治模式导致害虫抗药性加剧、生态平衡破坏、环境污染等问题,亟需开发环境友好型防控技术。以北非蝎AaHIT1蝎毒蛋白基因为模板,克隆构建携带该基因的重组质粒,通过醋酸锂转化法导入野生型球孢白僵菌,经蛋白质和转录水平的检测,得到目标基因正常表达的重组白僵菌菌株。通过对转化菌株生长速率、杀虫效果的检测分析,证实导入AaHIT1蝎毒蛋白基因可提升球孢白僵菌的杀虫效力;以玉米螟为靶标生物的测定结果表明,SP-nAaH 及nAaH 转化菌株相对于野生型,对玉米螟的致死时间分别提前14.2%和16.5%,最终病死率分别提高19.6%和16.2%,表现出较好的杀虫效果。研究发现,信号肽对蝎毒蛋白的外排及杀虫效果作用不大。所得转化菌株在农业害虫防控及工业生产方面具有一定的应用价值。

Abstract

Agricultural pests pose severe threats to crop yield, quality, and agro-ecological security through direct crop consumption, disease transmission, and degradation of agricultural product quality. The current reliance on chemical pesticides has intensified issues including pest resistance, ecological imbalance, and environmental pollution, necessitating the development of eco-friendly control technologies. Using the AaHIT1 scorpion venom protein gene from the North African scorpion as a template, recombinant plasmids carrying the AaHIT1 gene were cloned and constructed. These plasmids were introduced into wild-type Beauveria bassiana via lithium acetate transformation, yielding recombinant strains with normal target gene expression confirmed through protein and transcriptional level analyses. Evaluations of growth rates and insecticidal efficacy of the transformed strains demonstrated that the integration of the AaHIT1 gene enhanced the entomopathogenic activity of B.bassiana. Bioassays targeting Ostrinia furnacalis revealed that SP-nAaH and nAaH transformed strains reduced lethal time by 14.2% and 16.5%, respectively, compared to the wild-type strain, while achieving 19.6% and 16.2% higher final mortality rates. The signal peptide showed negligible effects on venom protein secretion or insecticidal performance. The resulting transformed strains exhibit potential applications in agricultural pest control and industrial production.

关键词

白僵菌 / 蝎毒蛋白 / 玉米螟 / 毒力测定 / 质粒构建

Key words

Beauveria bassiana / scorpion venom protein / Ostrinia furnacalis / virulence assay / plasmid construction

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基金资助

国家自然科学基金(31570106)