甜瓜蔗糖磷酸合成酶(SPS)基因正反义表达载体的构建

为了改善甜瓜果实的品质及研究甜瓜蔗糖磷酸合成酶(SPS)基因的生物学功能,本试验分别构建了甜瓜果实SPS基因的正义及反义表达载体。用PCR方法将克隆到pMD18-T载体上的甜瓜SPS基因用带有KpnⅠ和XbaⅠ酶切位点的引物扩增,然后将该扩增片段克隆到pMD19-T Simple载体上,再用KpnⅠ和XbaⅠ双酶切,得到该基因编码区3.37 kb的cDNA片段,将其定向插入到植物表达载体pROK2的KpnⅠ/XbaⅠ克隆位点,构建了甜瓜果实SPS基因的正义表达载体。将已克隆到pMD18-T载体上的甜瓜SPS基因用KpnⅠ和HincⅡ双酶切,得到该基因编码区830 bp的cDNA片段,将其定向插入到植物表达载体pROK2的KpnⅠ/SmaⅠ克隆位点,构建了甜瓜果实SPS基因的反义表达载体。

Construction of Sense and Antisense Expression Vector of Sucrose Phosphate Synthase Gene from Muskmelon

In order to improve the quality of muskmelon and clarify the function of sucrose phosphate synthase gene,we constructed the sense and antisense expression vectors of sucrose phosphate synthase.In previous study we cloned a full-length cDNA of sucrose phosphate synthase from muskmelon fruit into pMD18-T vector.For constructing sense muskmelon sucrose phosphate synthase gene vector,firstly we transformed the sucrose phosphate synthase from pMD18-T vector to pMD19-T simple vector by PCR with primers added KpnⅠand XbaⅠsite sequence,and then the gene was digested with KpnⅠand XbaⅠto remove a 3.37 kb coding region fragment and ligated between KpnⅠand XbaⅠof pROK2.The results of the recombinant plasmid digested with KpnⅠand XbaⅠand PCR amplification indicated that we constructed muskmelon SPS gene sense expression vector.For constructing antisense sucrose phosphate synthase gene expression vector,the gene was digested with KpnⅠand HincⅡ to remove a 830 bp coding region fragment and ligated between KpnⅠand SmaⅠof pROK2.The results of the recombinant plasmid digested with KpnⅠand HincⅡand PCR amplification showed that we constructed the muskmelon SPS gene antisense expression vector successfully.