[关键词]
[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kguT,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kguT,其核苷酸序列与Pseudomonas sp. CCOS191的编码2-酮基葡萄糖酸转运蛋白(KguT)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KguT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kguT,并对其进行了生物信息学分析,为变形假单胞菌的KguT的功能研究奠定了基础。
[Key word]
[Abstract]
The aim of this study was to clone the gene kguT encoding 2-ketogluconate transporter from Pseudomonas plecoglossicida JUIM01 and to find its biological information. The degenerate primers were designed on the basis of available sequences of pseudomonads and the physical maps of the 2-ketogluconate utilization operon (kgu operon), and the kguT with the entire nucleotide sequences of 1278 bp was amplified from the strain JUIM01 by TD-PCR. The kguT was predicted to share 87% sequence identity with the corresponding gene encoding 2-ketogluconate transporter (KguT) from Pseudomonas sp. CCOS191, and encode 425 amino acids. The KguT was a hydrophobic transmembrane protein with 12 membrane-spanning domains located in the cell membrane, without signal peptide, and was predicted to share 90% sequence identity with the KguT from Pseudomonas sp. M1. The predicted secondary structure of the KguT contained 75.76% of α helixes, 2.12% of extended strand and 22.12% of random coil. The kguT gene was successfully cloned for the first time from the strain used in industrial production of 2-ketogluconic acid and analyzed by the bioinformatics methods. The results of this study may provide a foundation for further study about the function of 2-ketogluconate transporter from Pseudomonas plecoglossicida JUIM01.
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[基金项目]
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)