麻疯树P5CS基因的克隆及原核表达

Molecular cloning and procaryotic expression of of P5CS gene from Jatropha curcas

使用RACE技术，从麻疯树叶片中克隆了△L吡咯啉-5-羧酸合成酶（PSCS）基因，命名为，fP5CS．JcP5CS的cDNA全长2675bp，包含一个2148bp的开放阅读框，一个117bp的5’-非翻译区以及一个410bp的3L非翻译区．开放阅读框编码716个氨基酸多肽链，分子量为77．54kD，预测等电点为6．11．通过与其它物种的P5CS氨基酸序列比对，结果显示，与其他高等植物的P5CS氨基酸序列有很高的同源性．包含谷氨酸激酶和谷氨酸-γ-半醛脱氢酶两个结构域以及ATP及NAD（P）H结合位点及亮氨酸拉链等保守位点．原核表达JcP5CS，融合蛋白的分子量与预测分子量大小相同的．活性测定结果显示获得了具有活性的麻疯树P5CS酶．

The P5CS from the leaves of J. curcas was cloned by RACE and named JcP5CS. The full- length cDNA of JcP5CS is 2675 bp, containing a 2148 bp open reading frame, a 117 bp 5＇-untranslated region and a 410 bp 3＇-untranslated region. The ORF encoded a 716 amino acid polypeptide with the molecular weight of 77.54 kD and the pI value of 6.11. Multiple sequence alignments showed that JcPSCS shared high homology in amino acid sequence with P5CS of other higher plants. The conserved Glu-5-kinase, glutamic-γ-semialdehyde dehydrogenase （GSA-DH） domains, conserved leu zipper and the putative ATP and NAD（P）H-binding sites was also been found. The JcP5CS protein was successfully expressed in Escherichia coll. The molecular weight of the expressed protein in E. coli was identified with the deduced protein. The result of enzyme activity assay indicated that we obtained the functional JcP5CS enzyme.