沙棘GPD1和DGAT1双基因载体的构建及表达验证

Construction and Expression Validation of GPD1 and DGAT1 Double-gene Expression Vector of Sea Buckthorn

为了提高沙棘种子的品质,选取大连民族大学校园内的沙棘‘实优1号’的种子为研究对象,用野生型拟南芥(Col-0)作为目的基因的转化受体,以沙棘种子油脂合成关键基因GPD1和DGAT1为载体,通过In-fusion连接技术构建HrGPD1和HrDGAT1基因双价表达载体pCGD,采用农杆菌蘸花法获取转基因拟南芥植株,利用甲酯化和氯仿甲醇法对和野生型的拟南芥植株种子进行脂肪酸不同组份的比例及含油率检测。结果表明:拟南芥转基因2代与野生型相比,拟南芥2代种子油酸、亚油酸的比例分别提高了46.70%和9.35%,但种子含油率未明显提高。因此,同时表达沙棘HrGPD1和HrDGAT1基因提高了油酸、亚油酸含量,为高油酸沙棘优良品种的培育和改良提供了基因材料。

In order to improve the quality of sea buckthorn seeds,with the seeds of sea buckthorn‘Shiyou 1’on the campus of Dalian University for Nationalities were selected as the research object,wild-type Arabidopsis thaliana(Col-0)as the transformation receptor of the target gene,the key genes GPD1 and DGAT1 for oil synthesis from sea buckthorn seeds were used as vectors,and the bivalent expression vectors pCGD of HrGPD1 and HrDGAT1 genes were constructed by in-fusion ligation technology,transgenic Arabidopsis thaliana plants were obtained by Agrobacterium floral dipping method,and the proportion of fatty acids and oil content of different components of A.thaliana plants and wild-type Arabidopsis plants were detected by methylation and chloroform methanol method.Compared with wild type,the proportions of oleic acid and linoleic acid in A.thaliana 2 nd generation seeds increased by 46.70%and 9.35%,respectively,but the oil content of seeds was not improved significantly.Therefore,the simultaneous expression of HrGPD1 and HrDGAT1 genes of sea buckthorn increased the content of oleic acid and linoleic acid,which provided genetic material for the cultivation and improvement of excellent varieties of high oleic acid sea buckthorn.