深黄被孢霉Δ~6-脂肪酸脱氢酶基因在转基因烟草中的表达  

Expression of Mortierella isabellina Δ~6-fatty Acid Desaturase Gene in γ-linolenic Acid Production in Transgenic Tobacco

作　　者：李明春[1] 刘莉[1] 胡国武[1] 邢来君[1] 

机构地区：[1]南开大学微生物系,天津300071

出　　处：《生物工程学报》2003年第2期178-184,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金项目 (No .39870 0 2 0 );高等学校骨干教师资助计划项目。~~

摘　　要：γ 亚麻酸 (GLA)是人体和动物饮食中具有营养作用的重要的多烯不饱和脂肪酸 ,在大多数油料作物种子中不含有GLA ,而只含有其前体物亚油酸 ,只有少数油料植物种子中含有GLA ,如夜来香 (Oenotheraspp) ,琉璃苣(Boragoofficinalis)等。Δ6 脂肪酸脱氢酶可将亚油酸转化为γ 亚麻酸 ,为了能够在传统的油料作物种子中产生GLA ,我们将从深黄被孢霉中克隆的Δ6 脂肪酸脱氢酶基因 ,与植物表达载体pGA6 43连接 ,构建了重组质粒pGAMICL6 ,将其通过农杆菌介导法 ,导入模式植物烟草中。经PCR和Southern杂交分析表明该基因已导入并整合到烟草的基因组中 ,Northern杂交结果表明该基因在转基因烟草的mRNA水平上获得表达。对转基因植株进行脂肪酸分析 ,结果显示 ,GLA和十八碳四烯酸 (OTA)分别占总脂肪酸含量的 19 7%和 3 5 %。linolenic acid (GLA, C18:3Δ 6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18∶2Δ 9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose (Oenotheera spp.), borage (Borago officinalis) and etc. Δ6-fatty acid desaturase(D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking XbaⅠ and BglⅡ restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains Δ6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28℃, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco (Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100μg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids w

关 键 词：烟草 脂肪酸脱氢酶基因 亚麻酸 转基因植株 深黄被孢霉 

分 类 号：Q786[生物学—分子生物学] S572[农业科学—烟草工业]