枯草芽孢杆菌HAS中TasA基因的克隆与分析

Cloning and Analysis of TasA in Bacillus subtilis HAS

为弄清枯草芽孢杆菌HAS对甘蔗黑穗病菌的抑制作用机理,选取可对多种植物病原真菌有抑制作用的抗菌蛋白TasA基因进行克隆与分析。结果表明:在HAS菌株中含有TasA基因,克隆编码抗菌蛋白TasA的基因全长,序列分析其有786个碱基组成,该序列与GenBank上登录的TasA(AJ871386.1)序列同源性达99%,推测蛋白分子量大小大约28KD,其中第104、164、169、250、399、623、627位等7个碱基发生碱基转换或颠换,而且这些变异分别发生在密码子的第2、2、3、1、3、2、3位碱基上,引起多肽链氨基酸的错义突变。经氨基酸序列比对,同Bacillus subtilis subsp.subtilis str.168编码的TasA蛋白(NP_390342.1)在第150位和第209位上的氨基酸发生变化,由苏氨酸、天冬酰胺分别代替丙氨酸和谷氨酸。

To explore the inhibition mechanism of B. subtilis HAS on sugarcane smut fungus, antifungal protein TasA was cloned and analyzed, which could inhibit various plant pathogenic fungi. Results： The TasA open reading frame （ORF） was PCR-amplified from HAS. Sequence analysis of the clone indicated that the TasA ORF consisted of 786 nucleotides, and shared 99% homology at nucleotide sequence, and 99% identity at amino acid sequence between it and the published gene TasA（corresponded with B. subtilis subsp, subtilis str. 168, Accession No：NP390342.1）. The protein molecular weight was about 28 KD, transitions or transposes occurred on seven bases, including 104, 164, 169, 250, 399, 623 and 627. The nucleotide sequence variations were respectively on their 2, 2, 3, 1, 3, 2, 3 base of TasA codons in the ORF. These changes of seven bases maybe cause the missense mutation of polypeptide chain AA. Through comparing the results of AA translation, we knew that there were changes of AA on the 150 and 209 site of TasA which were coded by B. subtilis subsp, subtilis str. 168, the Threonine replaced alanine and the Asparagine replaced Glutamicacid.