草苷膦抗性新基因的克隆、序列分析及其蛋白高级结构预测

利用错误倾向PCR（error-prone PCR）突变技术，以可变盐单胞菌（Halomonas variabilis）HTG7的5-烯醇丙酮莽草酸-3-磷酸(EPSP)合酶基因为模板进行随机PCR扩增，得到目的基因片段（约1.35 kb）.将该基因片段与pACYC184载体连接后转化EPSP合酶缺陷型菌株大肠杆菌ER2799.利用功能互补筛选法得到了2株不具有草苷膦抗性的EPSP合酶阳性克隆突变株，记为Pmu1和Pmu2.序列分析表明，突变体Pum1的EPSP合酶编码区与突变前基因相比，核苷酸有2处发生突变，导致氨基酸残基1处发生了改变；突变体Pmu2的EPSP合酶编码区与突变前基因相比，核苷酸有5处发生突变，导致氨基酸残基2处发生了改变.对突变前后EPSP合酶进行比较预测发现，其三级结构及蛋白中心骨架是大致相同的，但突变前后氨基酸位点肽平面和Cα相连的N键之间形成的扭转角度存在一定的差别.这些结果表明，酶的功能主要由蛋白的构象决定，二肽链形成后肽平面和N键之间角度的变化，造成高级结构构象细微的差别，致使草苷膦抗性功能丢失.

Cloning, Sequencing and Protein Tertiary Structure Prediction of a Novel EPSPs-encoding Gene with High Glyphosate Tolerance

Using error-prone PCR technique to find some glyphosate-tolerant sites,the novel EPSPs(5-enolpyruvyl-shikimate-3-phosphate synthase)-encoding gene from Halomonas variabilis strain HTG7 was amplified. Two EPSPs mutants that exhibit significantly reduced tolerance to glyphosate were identified using complement of E.coli ER2799. Two mutants carry one or more de novo point mutations. Tertiary structure of the three EPSPs was predicted and aligned. The backbone was same. But the torsion angle between peptide plane and N atom linking with Cα were quite different in the original and mutated amino acid position. All above suggested that the backbone decided the EPSPs function, and torsion angle maybe induced delicate change of tertiary structure led to the loss of glyphosate tolerance of EPSPs.