Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus Guanajuato Noria Alta s/n, Colonia Noria Alta Guanajuato, Gto, CP 36050, Mexico(1).
Present address: Facultad de Ciencias Naturales, Universidad Autónoma de Queretaro Centro Universitario, Cerro de las Campanas s/n, Santiago de Queretaro, Qro, CP 76010, Mexico(2).
Microbiol Res. 2015 Mar;172:57-67. doi: 10.1016/j.micres.2014.11.006. Epub 2014 Dec 5.
Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.
在各种条件下培养的昆虫病原真菌金龟子绿僵菌（Metarhizium anisopliae）的醇脱氢酶（ADH）活性的同工酶显示，微需氧生长与 ADH 活性增加有关。主要的 ADH 蛋白 AdhIp 通过亲和层析纯化至均一性，估计分子量为 41kDa，等电点（pI）为 6.4。肽质量指纹图谱分析允许鉴定和克隆编码该蛋白的基因，Adh1，如在金龟子绿僵菌基因组数据库中注释。AdhIp 与中链脱氢酶/还原酶（MDR）/锌依赖性醇脱氢酶样家族有关，并且包含保守的 ADH 序列基序，例如含锌的 ADH 特征、FAD/NAD 结合域和在大多数微生物 ADHs 中保守的氨基酸残基。半定量 RT-PCR 分析显示，Adh1 基因在小菜蛾早期感染时低水平表达，并且 Adh1 基因主要在幼虫死亡时表达，并且当菌丝从昆虫表皮出现然后产生分生孢子时表达。反义 RNA 实验表明，NAD（+）依赖性 ADH 活性在转化体中降低了 20-75%，并且 ADH 活性较低的转化体对丙烯醇具有抗性，这表明 ADH 活性的降低也发生在体内。使用 ADH 活性较低的反义 adh1 转化体进行的生物测定表明，LC50 值比野生型高 2 至 5 倍，表明 AdhIp 是真菌穿透和/或定殖昆虫的全部能力所必需的。
