中间锦鸡儿fad2基因片段克隆、反义表达载体构建及遗传转化初步研究  被引量:2

Cloning of fad2 Gene Fragments from Caragana intermedia,Constructing of Anisence fad2 Expression Vector and Its Genetic Transformation in Tobacco

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作  者:汪阳东[1] 李春秀[1] 齐力旺[1] 张守攻[1] 

机构地区:[1]中国林业科学研究院林业研究所国家林业局林木培育重点实验室

出  处:《林业科学研究》2007年第1期6-9,共4页Forest Research

基  金:国家高技术研究发展项目("863")"抗逆优质柠条新品种选育"(2002)资助

摘  要:以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据GenBank中已经发表fad2基因的同源序列,利用PCR技术克隆得到基因片段。在GenBank中Blast(GenBank登录号AY957393)所得基因片段和同属豆科的Glycine max Gmfad2-2a同源性高达88%,位于fad2基因编码区中部。将所得片段经BamHI和SacI酶切后插入表达载体质粒pBI121,构建了反义表达载体pBI121fad2,并利用农杆菌介导法转入烟草叶片,获得了抗卡那霉素和氨苄青霉素的再生烟草植株。初步分析结果表明:与对照烟草相比,转基因烟草种子脂肪酸含量没有明显差异,而亚油酸则减少10.3%。△-12 fatty acid desaturase is an important enzyme in the route of production of polyunsaturated lipids. Control the gene expression of △-12 fatty acid desaturase can change plant fatty acid for good plant biodiesel. Two cDNA fragments of fad2 gene were cloned using degenerate primers from Caragana intermedia ,an important biodiesel plant. The DNA sequence analysis indicated that the fragments contained 452 and 480 bp respectively, and shared 88% of homology with Glycine max Gmfad2-2a in GenBank. Both of the gene fragments were registered in GenBank (AY957393). The fragment AY957394 (452 bp)was digested with the enzyme BamHI and SacI, and ligated to the corresponding sites of the pBI121 in the antisense orientation. The pBI121fad2 vector was introduced into Agrobacteriurn tumefaciens strain LB4404 by electroporation and transformed to leaves of tobacco via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing Ampicillin and Kanamycin. PCR amplification with primer designed according to the fad2 gene fragment and NPTII gene in pBI121 demonstrated that antisense fad2 was integrated into tobacco genomes. The analysis of fatty acids of transgenic tobacco plants with antisensefad2 showed that the content of linoleic acid was 10.3% less than that of the un-transgenic tobacco plants.

关 键 词:中间锦鸡儿 生物柴油 FAD2基因 反义表达载体 遗传转化 

分 类 号:Q75[生物学—分子生物学]

 

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