参照几种生物的甘露醇 1 磷酸脱氢酶基因 (mtlD)的序列设计引物 ,以极端耐盐的假单胞菌 (Pseudomonassp cn 4 90 2 )的总DNA为模板 ,采用PCR扩增、构建重组高效表达载体以及生物信息学研究等方法 ,进行基因克隆、表达及功能定性等研究。结果表明 ,克隆获得一长为 114 9bp的基因。经蛋白质保守区域研究 ,初步判别该基因为mtlD结构基因。将该基因与pBV2 2 0质粒构建成高效表达原核重组载体pBH。SDS PAGE电泳表明 ,含pBH的转化子产生特异的、分子量约为 4 1kD的蛋白带 ,表达量约占菌体可溶性蛋白 6 7%。转化子的耐盐水平比对照提高了约 1/5。在含 0 9mol/LNaCl的液体培养基中 ,转化子培养 2 4h后其生物量约是对照的 10 2倍 ,甘露醇含量约是对照的 4 1倍。可见 ,假单胞菌的甘露醇 1 磷酸脱氢酶基因是一个重要的耐盐相关基因 ,该基因已在GenBank登记 ,代号为AY112 6 96。According to the essential characteristic of the genome of prokaryotes that no intron can be found in the genes,an attempt was made to find a new gene of mannitol-1-phosphate dehydrogenase ( mtl D) in Pseudomonas sp. cn 4902 by means of PCR amplification. Based on the short conserved sequence of the halo-tolerant-related gene in some microorganisms,a pair of primers were designed for PCR amplification with the template of genomic DNA of Pseudomonas sp. cn 4902. An approximate 1. 1kb DNA fragment of PCR amplification product could be found in the agarose gel electropherogram. The fragment was,then,retrieved,a poly A “tail” added and finally lined together with plasmid pMD18-T overnight to form the reconstructed vectors. The white colonies were picked out,some of them were cultivated and the plasmids were extracted in the transformants and digested by two endonucleases Bam HⅠ and Sal Ⅰ for the detection of the reconstructed vectors after agarose gel electrophoresis. The inserted DNA fragment was sequenced and transferred to the multicloned site of high efficiency expression plasmid pBV220 in E.coli JM101 to constitute a new plasmid named pBH. The tolerance levels against to serious concentrations of NaCl and its protein expression products were researched. It was clear that the cloned gene was 1149bp in length and coded for 383 amino acid residuals. Using the biological informatics in GenBank we found that the nucleotide sequence and deduced amino acid residuals of this gene share more than 90% homology with the mtl D gene of other organisms,including E. coli K12. The result of protein SDS-PAGE electrophoresis of transformants indicated that a 41kD protein coincided with the molecular weight of the transferred mtl D gene,which was 6.7% of the total protein,was produced in this transformants under the condition of 42℃ hot shock. As a result,the highest tolerant level against NaCl of the transformants cultivated in the agar LB medium was raised from 0.9 to 1.1mol/L,which was 22% higher than that of the control. Meanwhile,the biomass of transformants cultivated in the liquid medium containing 0.9mol/L NaCl was about ten-fold that of the control. It was believed that the cloned gene should be a mannitol-1-phosphate dehydrogenase ( mtl D) gene,one of the key genes responsible for osmoregulation in the cells of organisms. The successful cloning and expression of the mtl D gene supplied a good foundation for cultivation of halo-tolerant-related crop plants in the future.科技部转基因植物专项基金资助 ,J0 0 B 0 14号 ;; 厦门市科委科技发展基金资助项目 ,35 0 2 2 2 0 0 0 10 4
