一株阴环炭团菌(香灰菌)转化子形态变异的成因

遗传转化方法是基因功能研究的重要方法之一,但在转化过程常出现转化子变异的现象,影响目标基因功能的判断。本文以阴环炭团菌菌落形态特征异常的拟基因TJAS01-V10012900敲除菌株12900-5为研究对象,通过PCR手段验证基因敲除的真实性,基因组重测序分析各转化子在基因敲除区域以及基因组其他区域的差异,转录组测序比较12900-5与其他转化子在基因表达层面的差异。比较基因组发现,12900-5与12900-6菌株在潮霉素抗性基因插入位置与片段一致,且不存在多拷贝现象。12900-5菌株有两个特异SNP位于不同的基因,一个与细胞周期S期有关,另一个与囊泡转运有关,两者均与菌丝生长有关。由此推测,转化子12900-5形态特征异常很有可能与这两个非靶标位点突变有关。转录组分析结果发现12900-5菌株在氨基酸代谢通路、翻译过程等多个代谢与信号途径基因的表达量明显下降。本研究例证了转化过程存在的非靶标变异现象,但导致这种变异的原因仍需进一步研究。

The cause of morphological mutation in a gene-deletion transformant of Annulohypoxylon stygium

Genetic transformation is one of the important methods for gene function analysis. However, some unexpected mutations occur during transformation, making results unreliable. Six knock-out like Annulohypoxylon stygium strains with TJAS01-V10012900 gene were used as experimental strains, of which 12900-5 showed obvious differences in morphology. PCR tests, genome sequencing, and RNA-seq were employed to analyze the cause of morphological variation of 12900-5 strain. The obtained results showed similarity between 12900-5 and 12900-6 at knock-out region and copy number of hygromycin resistance gene. However, 12900-5 strain had two additional SNPs within different genes. One SNP was within the gene related to the control of cell cycle at S phase, and another was within the gene related to vesicle transport, both putatively affect mycelial growth. The results suggested that morphological variations of 12900-5 might be due to the SNP mutations in the genome. Gene expression related to metabolic pathways, translation processes and other metabolic and signaling pathways were down-regulated in 12900-5 strain as compared to those in 12900-6 strain. This result is an example that non-target point mutations occurred during gene-deletion transformation. The exact mechanism responsible for this kind of point mutations remains to be determined.