摘要
从球等鞭金藻中克隆到一个Δ5去饱和酶基因,命名为IgD5。系统发育树分析表明,该基因编码产物与来源于巴夫藻Pavlova salina的Δ5去饱和酶亲缘关系最近。此前通过酿酒酵母表达系统,证明IgD5对ω6脂肪酸具有Δ5去饱和酶活性,但并未鉴定IgD5对ω3脂肪酸的催化活性。我们将IgD5转化能够内源合成二高-γ-亚麻酸(DGLA,20:3Δ8,11,14)和二十碳四烯酸(ETA,20:4Δ8,11,14,17)的已携带2个基因的拟南芥(简称TMA2),通过PCR及RT-PCR筛选阳性植株,提取转基因阳性植株叶片总脂肪酸,用气相色谱分析法检测到花生四烯酸(AA,20:4Δ5,8,11,14)和二十碳五烯酸(EPA,20:5Δ5,8,11,14,17),含量分别达7.44%和2.07%,转化效率分别为68%和60%,证明IgD5在转基因植物中具有Δ5去饱和酶的活性,对ω3/ω6脂肪酸没有明显偏好性,并且催化活性远高于其在酵母中的活性。在转IgD5的TMA2中除了AA和EPA,没有检测到其他新脂肪酸,表明IgD5底物专一性很强,是一个可用于植物转基因替代生产鱼油的良好基因。
We have isolated a △5 desaturase gene from the alga Isochrysis galbana, namely IgD5. Phylogenetic analysis showed that it shared the highest homology with a functionally characterized △5 desaturase from Pavlova salina. Previously, we demon-strated that IgD5 has activity towards the omega-6 fatty acid substrate by heterologous expression oflgD5 in Saccharomyces cer-evisiae. However, whether IgD5 also has activity towards the potential omega-3 fatty acid substrate has not been verified. In this study, we transformed IgD5 into a double transgenic Arabidopsis thaliana （TMA2） that can already synthesize di-homoq,-linolenic acid （DGLA, 20：3^△8,11,14） and eicosatetraenoic acid （ETA, 20：4△^8,11,14,17）. Triple transgenic plants were iden-tified by PCR and RT-PCR. The total fatty acids composition of the transgenic lines was determined by gas chromatography. Novel fatty acids, arachidonic acid （AA, 20：4^△5, 8,11,14） and eicosapentaenoic acid （EPA, 20：5^△5,8,11,14,17） were detected and ac-counted for 7.44% and 2.07% of total fatty acids, with a conversion rate of 68% and 60%, respectively, which are much higher than those in yeast. In addition, IgD5 did not show a preference for either ωo3 or ωo6 substrate. Furthermore, IgD5 has strong sub-strate specificity as no fatty acid products were detected except for AA and EPA. These new findings further confirmed that IgD5 is an excellent gene for the production of fish oil in plants due to its high enzyme activity coupled with substrate specificity.
出处
《作物学报》
CAS
CSCD
北大核心
2013年第5期928-934,共7页
Acta Agronomica Sinica
基金
国家自然科学基金项目(30970222)
国家转基因生物新品种培育重大专项(2009ZX08005-024B)资助
