变形假单胞菌2-酮基葡萄糖酸转运蛋白基因的克隆与分析  

Cloning and Bioinformatic Analysis of a 2-Ketogluconate Transporter Gene from Pseudomonas plecoglossicida

作　　者：孙文敬[1,2] 栾方[1] 王大明[2,3] 张晓飞[1] 崔凤杰[1,2] 李运哲[1] 

机构地区：[1]江苏大学食品与生物工程学院,江苏镇江212013 [2]百勤异VC钠有限公司,江西德兴334221 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《现代食品科技》2016年第6期50-55,共6页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31571885);国家高技术研究发展计划项目(2012AA022103);江西省科技计划项目(赣知发[2015]64号);江苏大学研究生科研创新计划项目(KYXX_0046);江苏大学大学生科研立项项目(13A083);德兴市科技计划项目(德科发[2015]44号)

摘　　要：该研究旨在克隆变形假单胞菌JUIM01的2-酮基葡萄糖酸转运蛋白基因kgu T,并明确其基本的生物学信息。根据已报道的假单胞菌的基因组信息及其2-酮基葡萄糖酸操纵子的物理图谱设计简并引物,采用TD-PCR技术从变形假单胞菌中克隆到全长为1278 bp的kgu T,其核苷酸序列与Pseudomonas sp.CCOS191的编码2-酮基葡萄糖酸转运蛋白(Kgu T)的核苷酸序列的一致性为87%,编码一个由425个氨基酸残基组成的蛋白。该蛋白与Pseudomonas sp.M1的Kgu T在氨基酸序列上的一致性达90%,定位于细胞膜,是一个具有12个跨膜结构的疏水性的跨膜蛋白,无信号肽,其二级结构中α螺旋、延伸链和无规卷曲所占的比例分别为75.76%、2.12%和22.12%。本研究首次从2-酮基葡萄糖酸的工业生产用菌中克隆到基因kgu T,并对其进行了生物信息学分析,为变形假单胞菌的Kgu T的功能研究奠定了基础。The gene kgu T in Pseudomonas plecoglossicida JUIM01, encoding a 2-ketogluconate transporter, was cloned to understand its general biological properties. The degenerate primers were designed based on the available genomic information of pseudomonads and physical maps of the 2-ketogluconate utilization operon（kgu operon）. The entire 1278-bp nucleotide sequence of kgu T was amplified from strain JUIM01 by touchdown polymerase chain reaction（TD-PCR）. The kgu T gene shared 87% sequence identity with the corresponding gene encoding a 2-ketogluconate transporter（Kgu T） from Pseudomonas sp. CCOS191, and encoded a protein consisting of 425 amino acid residues. This Kgu T was a hydrophobic transmembrane protein with 12 membrane-spanning domains located in the cell membrane, without a signal peptide, and shared 90% sequence identity with the Kgu T from Pseudomonas sp. M1. The proportions of α-helices, extended strands, and random coils in the secondary structure of Kgu T were 75.76%, 2.12%, and 22.12%, respectively. Therefore, the kgu T gene was successfully cloned for the first time from the strain used in industrial production of 2-ketogluconic acid and the corresponding bioinformatics were analyzed. The results of this study may provide a foundation for studying the function of 2-ketogluconate transporter from Pseudomonas plecoglossicida JUIM01.

关 键 词：变形假单胞菌 2-酮基葡萄糖酸转运蛋白 kgu T基因 克隆 生物信息学 

分 类 号：Q933[生物学—微生物学]