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花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△12脂肪酸脱氢酶(FAD2)是亚油酸合成的关键酶,催化油酸(18:1)在△12位上脱氢生成亚油酸(18:2),但由于△12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△12脂肪酸脱氢酶基因(GenBank接受号为AY1006)构建高效表达载体,把花生?12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET/HO-A融合表达载体,并转化到大肠杆菌表达菌BL21(DE3)pLysS中,在IPTG诱导下,pRSET/HO-A融合表达载体在BL21(DE3)pLysS菌株中高效表达了?12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,所编码的酶具有?12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11.8%。花生△12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。
Peanuts are one of the most important oilseed crops widely grown around the world, and their seeds are rich in oleic acid and linoleic acid. △ 12 fatty acid dehydrogenase (FAD2) is a key enzyme in the synthesis of linoleic acid. It catalyzes the dehydrogenation of oleic acid (18: 1) to linoleic acid (△ 18) in △ 12 position, At present, there is no effective way to purify it and further study at the protein level. However, it is still necessary to conduct a more comprehensive and comprehensive study on the structure and function of the enzyme and the regulation of the expression. In this study, we constructed an efficient expression vector using the △ 12 fatty acid dehydrogenase gene cloned from peanut (GenBank accession number: AY1006), inserted the full-length sequence of peanut 12 fatty acid desaturase gene into Escherichia coli expression vector pRSETB, pRSET / HO-A fusion expression vector was constructed and transformed into E. coli BL21 (DE3) pLysS. The pRSET / HO-A fusion expression vector was highly expressed in BL21 (DE3) pLysS strain induced by IPTG. Dehydrogenase. The Clon-Tech protein purification Kit was used to further isolate the target protein. Meanwhile, fatty acid methyl esterification was carried out by adding exogenous substrate oleic acid at 20 ℃ for 6 h, and analyzed by gas chromatography (GC) and gas chromatography / mass spectrometry -MS) analysis showed that the encoded enzyme had the activity of? 12 fatty acid dehydrogenase and could convert exogenous oleic acid to linoleic acid with a conversion rate of 11.8%. Prokaryotic expression of △ 12 fatty acid desaturase gene in peanut has not been reported at home and abroad at present. This experiment lays the foundation for its further purification and structural and functional analysis.