中国春小麦puroindoline a基因的克隆及其真核表达载体的构建

目的:puroindoline(pin)基因在控制麦类作物的籽粒硬度中起着重要作用。构建真核表达载体pcDNA3.1( )-pina-gfp,为pina基因在哺乳细胞中的表达提供基础。方法:利用PCR方法从中国春小麦基因组中克隆到了pina基因,将其插入真核表达载体pcDNA3.1( )-gfp,用PCR和酶切鉴定重组子。结果:PCR和酶切鉴定表明,所构建的真核表达重组质粒为pcDNA3.1( )-pina-gfp;将该片段克隆到pCF-T载体中,经测序验证,表明其为目的基因。结论:构建的pina基因真核表达载体pcDNA3.1( )-pina-gfp为pina基因在哺乳动物细胞中的表达提供了基础。

Cloning and Eukaryotic Expression Vector Construction of puroindoline a Gene from Common Wheat(Triticum aestium L) Chinese Spring cv

Objective:puroindoline(pin)genes are the key genes in controlling wheat grain hardness.To construct the eukaryotic expression plasmid of pina gene as a foundation for the expression of pina gene in mammalian cell lines.Methods:The pina gene was amplified by PCR using genomic DNA template that was isolated from common wheat(Triticum aestium L)Chinese Spring(CS)and was cloned into cloning vector pcDNA3.1( )-gfp.The recombinant plasmid was digested with the restriction enzyme and verified by PCR.Results:The insert was sequenced to confirm its structural characterization.Then the pina gene was ligated to expression vector pcDNA3.1( )-gfp.pina gene was cloned and inserted to the cloning vector pCF-T,and the fragment was sequenced to confirmed structural characterization.Conclusion:The eukaryotic expression plasmid pcDNA3.1( )-pina-gfp laid a platform for pina gene expression in mammalian cell lines.