桑丁香假单胞菌糖基水解酶基因的克隆及表达分析

桑疫病的病原是一种丁香假单胞菌(Pseudomonas syringae),其感染造成桑树幼枝断裂,但分解桑枝的机制尚未发现。以GenBank中同种的另一丁香假单胞菌的序列为参照,通过PCR法克隆了桑疫病病原细菌的糖基水解酶基因。该基因的读码框为1 173 bp,编码390个氨基酸,与已公布的丁香假单胞菌属细菌(pv.syringae B728 a)的纤维素酶基因的核苷酸同源性为94%,氨基酸的同源性为97%。对该肽段的序列进行分析,确定了保守位点和各个功能域的序列。将该基因克隆到原核表达载体pET28 a中,在大肠杆菌中进行了表达。Western blotting检测的结果表明,该基因在大肠杆菌中获得高效表达,酶活力为2.3×103U/L,说明该基因产物具有分解纤维素的酶活性。

Cloning of Putative Cellulase Gene of Pseudomonas syringae pv.mori and its Expression Analysis

Pseudomonas syringae pv.mori is a pathogen of the buds and tender stems of mulberry tree and causes rupture.However,the contributing mechanism is unknown.The specific primers were designed to amplify the putative cellulase gene of P.syringae pv.mori according to the sequence from Pseudomonas syringae pv.syringae B728a in GenBank(CP000075).The PCR product was cloned into the vector pET28a and sequenced.It showed that the open reading frame of the gene has 1 173 base pairs long,and encode 390 amino acids.The similarity is 94 percent between the genes and 97 percent between the proteins from P.syringae pv.mori and P.syringae pv.syringae B728a,respectively.The functional motif and conservative sites of this peptide were estimated by online analysis.The gene was expressed in the E.coli strain DE3 by the inducer IPTG.The result showed that the cellulase gene could be highly expressed by SDS-PAGE and Western Blotting.The cellulase activity of the gene was detected as 2.3×103 U/L in the bacteria lysate.It indicated that the gene had cellulase activity.