桑树内生枯草芽孢杆菌TasA基因的克隆及序列分析与表达产物的抑菌作用

Cloning and Sequence Analysis of TasA Gene from Mulberry Endophytic Bacteria Bacillus subtilis and Antibacterial Activity of its Expressed Product

TasA是枯草芽孢杆菌(Bacillus subtilis)在芽孢的形成过程中产生的蛋白,对多种动、植物病原菌有较强的抑制作用。从桑树内生枯草芽孢杆菌菌株ME0717基因组DNA中克隆到TasA基因的全序列,包含一个786bp的完整开放阅读框(ORF),GenBank登录号为FJ713582。该序列与来源于B.subtilis的已知同源TasA序列Z99116、AJ871386的相似性达99.0%和98.1%,与芽孢形成相关的基因YqhF(GenBank登录号:BACJH642)的序列相似性也达99.0%,而与来源于地衣芽孢杆菌(B.licheniformis)的TasA序列(GenBank登录号:AE017333)相似性仅有46.0%,与来源于其它芽孢杆菌属的金属蛋白酶基因和芽孢外壳相关蛋白基因也有较高的相似性。构建该基因的原核表达载体,并在大肠杆菌(Escherichia coli)BL21中获得表达,表达产物对桑疫病病原细菌的菌体生长以及桑漆斑病菌和桑炭疽病菌的菌丝生长均有一定程度的抑制作用。

TasA is a protein produced during sporulation of Bacillus subtilis. It has strong inhibitory effect to many animal and plant pathogens. The full-length sequence of TasA gene was cloned from the genomic DNA of mulberry endophytic bacterium Bacillus subtilis strain ME0717 （GenBank accession number FJ713582）. The full length DNA sequence includes a complete ORF of 786 base pairs. Compared with other TasA sequences previously reported, the gene had a high sequence similarity with those of B. subtilis, which was 99.0% with Z99116 and 98.1% with AJ871386, while the similarity with the sporulation-associated gene YqhF （GenBank accession number BACJH642） reached 99.0% as well. Sequence similarity with TasA gene of B. licheniformis （GenBank accession number AE017333） was only 46.0%. The gene also has a high level similarity with the genes of cell envelope-bound metalloprotease and spore coat-associated protein from Bacillus. The prokaryotic expression vector carrying TasA gene was constructed and expressed in Escherichia coil BL21. The expressed protein showed certain inhibitory activity on growth of bacterial cells of Pseudornonas syringae pv. mori and of mycelia of Colletotrichum morifolium and Myrothecium roridum.