大豆种子特异性启动子的分离及结构分析

Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

采用 PCR 技术从大豆品种吉林 43 基因组 DNA 中分离到大豆β-伴球蛋白α-亚基基因启动子片段(BCSP666)。序列比较和分析表明,这一启动子片段与其它两个大豆种子特异性启动子——β-伴球蛋白α′-亚基基因启动子片段和β-伴球蛋白β-亚基基因启动子——片段在序列结构上有很大的相似性,并具有多种种子特异性启动子所特有的序列元件,据此推测该启动子片段具有种子特异性启动子活性。Δ6-脂肪酸脱氢酶是亚油酸脱氢形成γ-亚麻酸的限速酶。将启动子片段 BCSP666 与深黄被孢霉Δ6-脂肪酸脱氢酶基因(MI D6D)连接,构建种子特异性表达载体 pBMI666。通过农杆菌介导法转化大豆子叶节,在转基因愈伤组织中表达了脂肪酸脱氢酶基因(MI D6D),获得γ-亚麻酸,初步验证了启动子片段 BCSP666 的启动子活性。

The promoter region(BCSP666) of β-conglycinin α-subunit gene from the genomic DNA of soybean Jilin 43 was isolated by PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure of the soybean seed-specific promoter β-conglycinin α′-subunit gene promoter and β-conglycinin β-subunit gene promoter, and it also contains many of the motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, it was deduced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constracted the seed-specific expression vector pBMI666 with the promoter fragment BCSP666 and ?6-fatty acid desaturase gene from Mortierella isabellina. The ?6- fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in production of an essential fatty acid, γ- linolenic acid (GLA). The production of γ-linolenic acid (GLA) was observed in soybean callus cells, which were transformed with this vector. This confirmed the promoter activity of the promoter fragment BCSP666.