苏云金芽孢杆菌芽孢萌发相关基因gerM的克隆及功能研究

Cloning and characterization of gerM gene involved in germination in Bacillus thuringiensis

摘要:

依照蜡状芽孢杆菌gerM基因的保守序列设计引物，从苏云金芽孢杆菌中扩增出640bp的DNA片段。以此为探针，从苏云金芽孢杆菌部分基因组酶切文库中成功地克隆到了一个4.5kb的DNA片段。序列分析表明，该片段包含一个完整的开放阅读框，其预测的编码产物与枯草芽孢杆菌GerM蛋白具有很高的同源性，将该基因命名为gerM。RT-PCR分析表明，gerM基因仅在芽孢形成的过程中表达。通过同源重组的策略构建了gerM基因的阻断突变株。研究表明，gerM基因的破坏影响苏云金芽孢杆菌芽孢萌发的速率和比例。

Abstract:

In Bacilli, gerM is a very conservative gene. Primers were designed according to the gerM gene sequence of Bacillus cereus, and a 640bp DNA fragment was obtained from Bacillus thuringiensis subsp. kustaki 1.175 by PCR. Using this fragment as a probe, a 4.5kb DNA fragment was cloned from the partial DNA library of Bacillus thuringiensis subsp. kustaki 1.175.Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 349-amino acid (aa) protein, which has high homology with GerM protein from Bacillus subtilis. This gene was designated gerM (GenBank Accession No. DQ537381). RT-PCR analysis showed that gerM gene was only expressed in the process of sporulation, suggesting gerM is not required for the vegetative growth. The function of the gerM gene was studied by a strategy of gene disruption, and the resulting gerM disruption mutant did show normal growth and sporulation. However, gerM disruption mutant spores germinate slower than wild-type spores when triggered by L-alanine or inosine, indicating that gerM is required for the spore normal germination initiated by L-alanine or inosine in Bacillus thuringiensis.