金龟子绿僵菌发酵液中苦马豆素含量及催化酶基因mRNA表达分析

旨在探明金龟子绿僵菌发酵液中苦马豆素含量及与苦马豆素生物合成有关的催化酶基因mRNA表达的关系，将金龟子绿僵菌接种于察氏培养基进行发酵培养，运用Q Exactive高分辨质谱仪检测培养第1、3、5、7天发酵液中苦马豆素的含量，采用qRT-PCR法检测培养第1、3、5、7天发酵液中苦马豆素生物合成通路上的催化酶基因SwnA、SwnH₁、SwnH₂、SwnK、SwnN、SwnR和SwnT的mRNA转录水平。结果显示，根据苦马豆素质量浓度和峰面积的变化测算出回归方程为y=31 302.5x-45 910.5（R²=0.996 7），在培养第3天发酵液中苦马豆素含量最高为174.014 μg·mg^-1；SwnA、SwnH₁、SwnH₂、SwnK、SwnN、SwnR和SwnT基因在不同发酵时期mRNA表达差异较大，其中SwnR基因mRNA表达与发酵液中苦马豆素含量变化相一致，而SwnH₂基因mRNA表达与发酵液中苦马豆素含量变化相反。结果表明，金龟子绿僵菌SwnR基因mRNA表达与苦马豆素合成关系密切，可为后续开展苦马豆素微生物发酵工艺及其生物合成通路研究提供重要参考。

mRNA Expression Analysis of Catalytic Enzyme Gene and Content of Swainsonine in the Fermentation Broth of Metarhizium anisopliae

The aim of this study was to investigate the relationship between the content of swainsonine and the mRNA expression of catalytic enzyme gene related to the biosynthesis of swainsonine in the fermentation broth of Metarhizium anisopliae. Metarhizium anisopliae was inoculated and fermented in Czapek-Dox Medium, and then we determined the swainsonine content of fermentation broth by using Q Exactive high-resolution mass spectrometer and detected the expression of mRNA of catalytic enzyme genes, namely SwnA, SwnH₁, SwnH₂, SwnK, SwnN, SwnR, and SwnT by qRT-PCR method on the 1st, 3rd, 5th and 7th day of fermentation. The results showed that the regression equation calculated from the changes in the mass concentration and peak area of swainsonine was y=31 302.5x-45 910.5 (R²=0.996 7). The highest content of swainsonine in the fermentation broth on the 3rd day was 174.014 μg·mg^-1, the mRNA expression of SwnA, SwnH₁, SwnH₂, SwnK, SwnN, SwnR, and SwnT genes have great difference in different fermentation periods. Among them, the SwnR mRNA expression was consistent with the change of swainsonine content in the fermentation broth, and the SwnH2mRNA expression was opposite to the change of swainsonine content in the fermentation broth. The results indicated that the SwnR mRNA expression was closely related to synthesis of swainsonine in Metarhizium anisopliae, which would provide an important reference for exploring fermentation process and biosynthetic pathway of swainsonine in future.