Construction of Plant Expression Vector of the proBA Gene from Bacillus subtilis Mutant and Analysis of the Salt Tolerance of Transgenic Arabidopsis thaliana

从枯草芽孢杆菌(Bacillussubtilis)抗脯氨酸结构类似物突变株中克隆得到脯氨酸合成途径中的关键酶基因proB(编码γ-谷氨酰胺激酶)和proA(编码谷氨酰胺-γ-半醛脱氢酶),同时设计引物从拟南芥基因组中扩增得到吡咯啉-5-羧酸合成酶B基因(p5csB)的第一个内含子序列,将其分别与proB和proA基因通过PCR拼接后,酶切连接构建得到植物双元表达载体pBI121pro。以LBA4404为介导,采用真空抽滤的方法转化得到转proBA基因拟南芥;第三代的纯合子(T3)通过半巢式PCR的方法验证外源基因已整合到拟南芥基因组中,GUS活性分析表明外源基因在叶片中表达最强,茎部其次,根部最弱;对600mmol.L-1致死浓度NaCl耐受能力的分析表明,转基因拟南芥的平均存活时间(37.8min)明显高于野生型拟南芥(26min)。The genes proB and proA encoding the two enzymes, -GK (-glutamyl kinase) and -GSADH (Glu-5-semialdehyde dehydrogenase), were amplified from the Bacillus subtilis mutant to the proline analogue.The first intron of p5csB gene was also retrieved from Arabidopsis thaliana and linked with proB and proA respectively through PCR. After the endonuclease digestion and fragment ligation, the recombinant plant binary expression vector (pBI121pro) was obtained. The transformation of Arabidopsis thaliana was mediated by Agrobacterium tumfaciens LBA4404 by using the vacuum infiltration method. Half nested PCR was performed to verify the insertion of exotic genes into the genome of T3 homozygous plants. GUS activity assay indicated that the exotic genes were expressed most highly in the leaves, secondly in the stems and least in the roots. The analysis of tolerance to the deadly salt concentration (600 mmolL~(-1) NaCl) showed that the average survival time of transgenic plants (37.8 min) was obviously higher than that of wild type controls (26 min). In this research, the key gene proBA involved in the proline biosynthesis of highly salt-tolerant microorganism were cloned, and after removal of the feedback inhibition, it was, for the first time, transferred into the model plant Arabidopsis thaliana,which is of highly theoretical and practical significance.教育部十五“211”项目和武汉大学科技创新项目资助