10kD玉米醇溶蛋白基因的克隆与植物表达载体构建

由于紫花苜蓿中的含硫氨基酸水平很低,采用基因工程手段将富硫蛋白基因-10kD玉米醇溶蛋白(zein)基因转入苜蓿中以提高其水平。根据该基因的保守序列设计引物,并在下游引物终止密码子前引入内质网驻留蛋白信号序列(KDEL),通过PCR技术从玉米(Zea mays L.)黄化幼苗基因组中扩增目的基因的开放阅读框(ORF)。序列测序得到全长为465bp的目的片断,该片段编码154个氨基酸,其中甲硫氨酸(M)31个占20.13%,半胱氨酸(C)6个占3.90%,含硫氨基酸共占24.03%。该基因与报道的10kD玉米醇溶蛋基因具有很高的同源性。将该基因构建到植物表达载体pCAMBIA13011上,以转化紫花苜蓿提高其含硫氨基酸的水平。

Cloning 10kD Zein Gene and Construction of Plant Expression Vector

Alfalfa was in defect of S-amino acids,so the sulf-protein gene,10kD zein gene,was introduced into alfalfa by the genetic engineering to enhance the quantity of S-amino acids.According to the,conservative sequence,primers were designed and the ER retention signal KDEL was inserted followed by the stop code.ORF was cloned from the genome DNA of maize seedling by polymerase chain reaction(PCR).The gene was 465bp in length,encoding 154 amino acid residues, of which S-amino acids were 24.03% in total.The gene had high identity to the reported ones.The plant expression vector,pCAMBIA13011,was constructed to enhance the quantity of S-amino acids in alfalfa.