大麦醇溶蛋白基因片段克隆和RNAi植物表达载体构建

 为了降低啤酒大麦蛋白的含量，提高啤酒大麦品质，利用甘肃推广面积最大的品种甘啤4号为材料，克隆大麦醇溶蛋白基因保守区序列，构建RNAi表达载体。根据已知大麦醇溶蛋白基因保守序列设计一对特异性引物B2PF5′-CAACCATTTCCACAGCAACCACCAT-3′，B2PR5′-GAAAGATAGAGTAGACGATTGCACG-3′，采用PCR技术从甘啤4号大麦基因组中获得了1个349bp片段（GP4）。依据载体pHANNIBAL和pART27的结构，分别将GP4按所对应酶切位点正反向插入，利用热激法转化。结果分析显示，克隆出的片段与GenBank （NCBI）中醇溶蛋白基因核苷酸序列同源性高达92%，所构建成的RNAi表达载体pART27-pHAN-GP4中含目标片段。

Cloning of Hordein Gene in Barley and Construction of RNAi Plant Expression Vector

In order to reduce the protein content and improve the quality of beer barley, this paper takes Gansu Beer Barley No. 4, as the material for study, which has a wide promotion area in Gansu Province. Hordein gene was cloned in the conserved sequence, with RNAi expression vector constructed with further improvements on the theoretical basis of barley quality. A pair of primers was designed according to the released DNA sequence of Hordein gene. B2PF5′-CAACCATTTCCACAGCAACCACCAT-3′, B2PR5′-GAAAGATAGAGTAGACGATTGCACG-3′, with PCR employed to clone a 349bp fragment(GP4) from Gansu Beer Barley No.4. Based on the structure of vector pHANNIBAL and pART27, GP4 was inserted forward and reversely according to the enzyme position. The sequence analysis shows that the cloned DNA shares 92% of identity with the data. An RNAi expression vector of the Hordein gene, named pART27-pHAN-GP4 was constructed with the RNAi expression vector pART27-pHAN-GP4 containing the object gene.