Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.
Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.
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DOI：
10.1111/j.1751-7915.2012.00351.x
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发表时间：
2013-01
影响因子：
5.7
通讯作者：
Feng MG
中科院分区：
文献类型：
--
作者：
Wang ZL;Ying SH;Feng MG
为了确定用于工程真菌昆虫病原体以改善异源基因表达和真菌生物防治潜力的合适启动子，通过上游截短和定点突变对白僵菌 I 类疏水蛋白基因 (hyd1) 上游的 1798 bp 启动子 (Phyd1) 进行了优化。截短的 1290 bp 片段 (Phyd1-t1) 在白僵菌中比全长 Phyd1 更有效地驱动 eGFP 表达。进一步将Phyd1-t1截短至1179、991和791 bp或突变Phyd1-t1中三个转录因子的结合域之一显着降低了eGFP（增强型绿色荧光蛋白）的表达。在Phyd1-t1控制下，eGFP在分生孢子发生细胞和分生孢子中的表达量比在菌丝体中更丰富。因此，Phyd1-t1被用来将细菌来源的昆虫中肠特异性毒素（vip3Aa1）基因整合到白僵菌中，产生转基因菌株（BbHV8），其在分生孢子中表达的毒素分子是在PgpdA（广泛用于真菌中基因表达的启动子）控制下表达毒素的对应菌株（BbV28）的9.8倍。因此，在使用用于角质层穿透和摄入的正常分生孢子或仅用于摄入的热灭活分生孢子的标准化生物测定中，BbHV8 对斜纹夜蛾幼虫表现出比 BbV28 高得多的经口毒力。总之，Phyd1-t1 是增强真菌分生孢子中有益蛋白（例如 vip3Aa1）表达的有用工具，真菌分生孢子是杀真菌剂的活性成分。
To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (Phyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (Phyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length Phyd1. Further truncating Phyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in Phyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under Phyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, Phyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of PgpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, Phyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.
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影响因子：
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