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为研究丹参4-羟基苯丙酮酸还原酶基因(SmHPPR)在丹酚酸类化合物生物合成中的作用,构建SmHPPR的RNAi植物表达载体。根据SmHPPR的序列,设计4对含有酶切位点的特异性引物,以丹参无菌苗叶片cDNA为模板,分别扩增两段目的基因的正反向片段(约402 bp和346 bp),经T-载体克隆并测序后,将正反义片段分别插入表达载体pART27的相应位置(正向目的片段插人中间载体pKANNIBAL内含子左侧,反向目的片段插入该载体内含子右侧),构建了含有发夹结构的RNAi植物表达载体pART27-HPPR1sa、pART27-HPPR2sa和阴性对照载体pART27-Intron。利用三亲杂交法,将这3个载体导入到发根农杆菌LBA9402中。酶切鉴定和PCR扩增结果表明RNAi表达载体构建成功并成功导入到发根农杆菌LBA9402中,为下一步研究SmHPPR基因的功能奠定基础。
In order to study the role of Salvia miltiorrhiza 4-hydroxyphenylpyruvate reductase gene (SmHPPR) in the biosynthesis of salvianolic acids, the SmHPPR RNAi plant expression vector was constructed. According to the sequence of SmHPPR, 4 pairs of specific primers containing restriction enzyme sites were designed and the positive and negative fragments (about 402 bp and 346 bp) of the two genes of interest were amplified using cDNA of Salvia miltiorrhiza as leaves. After the T-vector was cloned and sequenced, the sense and antisense fragments were respectively inserted into the corresponding positions of the expression vector pART27 (the forward fragment was inserted into the left side of the intermediate vector pKANNIBAL intron, and the reverse purpose fragment was inserted into the right side of the vector intron) RNAi plant expression vectors pART27-HPPR1sa, pART27-HPPR2sa and negative control vector pART27-Intron containing hairpin structure were constructed. The three vectors were introduced into Agrobacterium rhizogenes LBA9402 by the method of the three parents. The results of restriction enzyme digestion and PCR showed that the RNAi expression vector was successfully constructed and successfully introduced into Agrobacterium rhizogenes LBA9402, which laid the foundation for the further study on the function of SmHPPR gene.