松材线虫伴生菌中产纤维素酶细菌的筛选、鉴定及其相关基因的克隆

摘要

To screen, identify bacterial strains with high capability to degrade cellulose from bacteria associated with Bursaphelenchus xylophilus and to clone related genes. [Methods] First, we collected B. xylophilus samples from pine wood nematode disease areas in Nanyang, Henan province, China. Then, we obtained the bacterial strains with high cellulase activities by primarily screening according to Congo red plate methods. The bacterial strain was classified by phenotypic and genotypic characteristics. We designed degenerate primes according to the known endoglucanase gene sequences in GenBank to carry out PCR, and analyzed the cloned sequence. [Results] We obtained seven bacterial strains with high cellulase activities. Among them, the bacterial strain numbered C8 showed the highest cellulase activities. The bacterium was classified to be Enterobacter genus. The full length of a cellulase gene cDNA (1104 bp) (GenBank JQ845065) coding region was successfully cloned. The homogeneous analysis demonstrated that the deduced nucleotide and amino acid of the gene separately shared 97% and 92% with the cellulase from E. aerogenes KCTC 2190, and 82% with the endo-1,4-D-glucanase gene from Klebsiella pneumoniae, and 82% with the a cellulase gene from unculturable bacteria. [Conclusion] It was a novel cellulose gene cloned from B. xylophilus associated bacteria.%[目的]从松材线虫伴生菌中筛选出高效降解纤维素的细菌菌株,初步鉴定后,对其相应的纤维素酶基因尝试克隆.[方法]首先从河南南阳松材线虫病疫区采集到的木材样本中,分离获得松材线虫.采用刚果红平板初筛法,从松材线虫伴生菌中获得具有分泌较高活性纤维素酶的细菌菌株.基于该菌株的形态学、生理学及16 s rDNA序列特征等对高活性菌株进行分类鉴定.设计兼并引物,从高活性菌株中克隆该菌株的纤维素酶基因,并进行序列分析.[结果]获得7株具有分泌较高活性纤维素酶的细菌菌株,其中编号为C8的菌株酶活最高.经鉴定该菌株归为肠杆菌属,命名为Enterobacter sp.C8.进一步从C8菌株中成功克隆出该菌株的一个全长1104 bp的纤维素酶基因(GenBank JQ845065),在NCBI比对后发现该基因分别与产气肠杆菌( Enterobacter aerogenes) KCTC 2190的纤维素合成酶亚单位BcsC的核苷酸序列有97％的同源性,氨基酸序列有92％的同源性 ;与克雷白氏肺炎菌(Klebsiella pneumoniae)的endo-1,4-D-glucanase基因有82％的同源性,与不可培养的细菌内切纤维素酶基因有82％的同源性.[结论]本文首次从松材线虫伴生菌中筛选到了一株简单的产纤维素酶的细菌菌株并从中克隆出了一个新型纤维素酶基因,为下一步进行新能源的利用奠定了理论基础.