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根据脱乙酰几丁质酶同源基因序列设计引物,扩增、克隆和测序了枯草芽孢杆菌Bacillus subtilis菌株XF-1的脱乙酰几丁质酶基因(csn)。该基因编码区为834bp,编码由277个氨基酸组成的约30kD的蛋白质,其基因序列与枯草芽孢杆菌菌株B168的脱乙酰几丁质酶基因的相似性达到99%,氨基酸序列相似性为98%,在第19、47、60、256和257位的氨基酸发生了变化,分别由异亮氨酸代替了菌株B168脱乙酰几丁质酶中的甲硫氨酸、缬氨酸代替了谷氨酸、苏氨酸代替了异亮氨酸、天冬氨酸代替了谷氨酸、赖氨酸代替了天冬酰胺。菌株XF-1对稻瘟病菌Magnaporthe oryzae和芸薹根肿菌Plasmodiophora brassicae有抑制作用,而菌株B168没有。菌株XF-1和B168的csn基因在大肠杆菌Escherichia coli中的表达产物均具有脱乙酰几丁质酶活性,但前者的表达产物对稻瘟病菌及芸薹根肿菌具有抑制作用,而后者没有。表明脱乙酰几丁质酶是XF-1抑制根肿病作用机制之一,且上述5个氨基酸替代可能与抑菌机制有关。
The chitinase gene (csn) of Bacillus subtilis strain XF-1 was amplified, cloned and sequenced based on the chitosan homologous gene sequences. The coding region of this gene was 834bp and encoded a protein of about 30kD consisting of 277 amino acids. The deduced amino acid sequence was 99% identical to the chitinase gene of B. subtilis strain B168, and the amino acid sequence similarity was 98% , The amino acids at positions 19, 47, 60, 256 and 257 changed, and methionine in strain B168 chitosan replaced by isoleucine, and valine replaced glutamic acid , Threonine instead of isoleucine, aspartic acid instead of glutamic acid, and lysine instead of asparagine. Strain XF-1 inhibited Magnaporthe oryzae and Plasmodiophora brassicae, while strain B168 did not. The expression products of csn gene of Escherichia coli in Escherichia coli of XF-1 and B168 all had chitinase activity, but the former had inhibitory effect on M. grisea and P. brassicae, while the latter did not . Chitinase is one of the mechanisms of XF-1 inhibiting clubroot disease, and the five amino acid substitutions may be related to the mechanism of antibacterial activity.