摘要
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。
Chitosanase gene （csn） of Bacillus subtilis XF-1, which was amplified with primers designed according to its reported homologous sequence, was cloned and sequenced. The gene had an 83 lbp coding sequence, encoding a protein with 30 kD molecular weight composed of 277 amino acids. Similarity of XF-1 csn sequence with that of B. subtilis 168 was 99% and homolog of amino acid sequence was 98%. The differences of amino acid sequence from that of B168 appeared in the 19th, 47th, 60th, 256th and 257th amino acids, and the five amino acids of methionine, glutamic acid, isoleucine, glutamic acid, asparagine were replaced with isoleucine, valine, threonine, aspartic acid and lysine, respectively. The XF- 1 strain was inhibitory to Magnaporthe oryzae and Plasmodiophora brassicae, but B168 was not. The expressed proteins of csn from XF-1 and B168 in Escherichia coli both had chitosanase activity, but the former had inhibitory effect on M. oryzae and P. brassicae compared with the latter. Theresults indicated that chitosanase is one of mechanisms of XF-1 against P. brassicae and the replacements of 5 amino acids above may contribute to its antimicrobial activity.
出处
《中国生物防治学报》
CSCD
北大核心
2011年第4期504-509,共6页
Chinese Journal of Biological Control
基金
科技部国际科技合作项目(2009DFA32360)
农业部公益性行业(农业)科研专项(201003029)
云南省自然科学基金重点项目(2008CC024)
云南省科技强省专项(2009EB060)
云南省教育厅科学研究基金项目(09J0073)
