swnR基因在金龟子绿僵菌合成苦马豆素中的作用

doi:10.11843/j.issn.0366-6964.2021.05.030

摘要/Abstract

摘要： 苦马豆素（swainsonine， SW）是由内生真菌产生的疯草类植物的主要有毒成分，目前有关其生物合成通路及关键催化酶基因尚不十分清楚。有研究表明，SW的合成主要依赖于真菌中SWN基因簇的基因，为探明该基因簇中编码NADB Rossmann-fold还原酶的swnR基因的作用，本文以金龟子绿僵菌（Metarhizium anisopliae）为研究对象，使用PEG介导的同源重组（HR）敲除金龟子绿僵菌swnR基因，通过高灵敏质谱仪检测野生型（WT）、突变型（MT）和回补型（CT）菌株发酵产物中SW含量。结果显示，野生型、突变型和回补型菌株发酵液中的苦马豆素含量分别为（82.91±15.92）、（5.71±2.23）和（56.42±10.82） μg·mg^-1，突变菌株发酵液中SW含量显著降低，回补菌株发酵液中的SW含量恢复明显。swnR基因是金龟子绿僵菌合成苦马豆素的关键催化酶基因，本研究为后续开展疯草内生真菌合成苦马豆素的催化酶基因筛选及其生物合成通路研究奠定理论基础。

关键词: 苦马豆素, 金龟子绿僵菌, swnR基因, 生物合成, 基因敲除

Abstract: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi. Yet the biosynthetic pathway and key catalytic enzyme genes are not quite clear. Studies have demonstrated that the biosythesis of SW requires for the presence of a variety of SWN cluster genes. In order to determine the role of the swnR gene encoding NADB Rossmann-fold reductase in this gene cluster, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain. The concentration of SW was measured in the fermentation broth of M. anisopliae wild-type strain (WT), mutant-type strain (MT) and a complemented-type (CT) strain using a Q Exactive Mass Spectrometer. The content of swainsonine in the fermentation broth of the obtained WT, MT and CT were (82.91±15.92), (5.71±2.23), (56.42±10.82) μg·mg^-1, respectively. The results showed that the content of SW decreased in the fermentation broth of the MT strain, and returned to the original level in the CT strain. These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, this will lay a theoretical foundation for the follow-up study of the catalytic enzyme gene screening and biosynthetic pathway research of swainsonine synthesis of endophytic fungi in locoweed.

Key words: swainsonine, M. anisopliae, swnR gene, biosynthesis, gene knockout

中图分类号:

S476.12

孙璐, 宋润杰, 路浩, 王敬龙, 莫重辉, 赵宝玉. swnR基因在金龟子绿僵菌合成苦马豆素中的作用[J]. 畜牧兽医学报, 2021, 52(5): 1439-1446.

SUN Lu, SONG Runjie, LU Hao, WANG Jinglong, MO Chonghui, ZHAO Baoyu. The Role of swnR Gene on the Biosynthetic Pathway of the Swainsonine in Metarhizium anisopliae[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1439-1446.

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