【目的】克隆吡咯伯克霍尔德氏菌JK-SH007(Burkholderia pyrrocinia JK-SH007)多聚半乳糖醛酸酶基因(BpPG),并阐明BpPG基因在B. pyrrocinia JK-SH007定殖中的生物学功能。【方法】通过设计特异性引物(PG-F、PG-F)克隆BpPG基因,并对其全长基因进行生物信息学分析; 采用MEGA 5.0软件进行系统发育树分析; 将B. pyrrocinia JK-SH007接种杨树,利用实时荧光定量PCR方法,分析BpPG基因在B. pyrrocinia JK-SH007定殖过程中的差异表达。【结果】BpPG基因全长2 001 bp,编码666个氨基酸,预测蛋白质分子量69.23 ku,理论等电点为8.37; BpPG蛋白有6个蛋白质结合位点,属于Glycosyl hydrolases family 28家族,为亲水性蛋白,不具有信号肽。二级结构主要包括α-螺旋、β折叠、延伸链和无规则卷曲,三级结构预测结果与二级结构相符。系统进化分析表明,BpPG与PG(GenBank 序列号WP 034181566.1)具有同源性。实时荧光定量PCR结果显示B. pyrrocinia JK-SH007定殖过程中,不同时期的BpPG基因表达量存在差异。【结论】克隆得到BpPG基因,BpPG基因在进化上是保守的,其极有可能在B. pyrrocinia JK-SH007定殖过程中发挥作用。
Abstract
Abstract: 【Objective】In this study, we aimed to isolate the polygalacturonase gene(BpPG)from Burkholderia pyrrocinia JK-SH007 and clarify the biological characteristics of the BpPG in colonizing B. pyrrocinia JK-SH007.【Method】The BpPG was isolated from B. pyrrocinia JK-SH007 by PCR using primers PG-F and PG-F. Bioinformatics and homology analysis were conducted using various online software products and the phylogenetic tree was constructed with MEGA 5.0. The RT-qPCR method was used to analyze differences in the specific expression of the BpPG gene during B. pyrrocinia JK-SH007 colonization of poplar. 【Result】The sequence of the BpPG was 2 001 bp in length, and encoded a protein of 666 amino acids with a molecular weight of 69.23 ku and a theoretical isoelectric point of 8.37. The BpPG was homologous to polygalacturonase(WP 034181566.1), with 99% similarity, and contained six protein binding sites. BpPG is a hydrophilic protein belonging to the glycosyl hydrolase family 28, and lacks a signal peptide. The secondary structures of the protein are comprised mainly of alpha helices, beta turns, extended strands, and random coils, which contain six PbH1 motifs. The predicted tertiary structure was consistent with the secondary structure. RT-qPCR analysis revealed that the relative expression of the BpPG differed during various periods of B. pyrrocinia JK-SH007 colonization of poplar. 【Conclusion】The BpPG isolated from B. pyrrocinia JK-SH007 had a conserved sequence. In addition, the results suggest that the BpPG plays an important role during B. pyrrocinia JK-SH007 colonization of poplar.
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基金
基金项目:国家林业公益性行业专项项目(201304404); 国家自然科学基金项目(31470645); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:陈飞飞(ffchen1951@163.com)。*通信作者:叶建仁(jrye@njfu.com.cn),教授。
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