大豆fad基因反义表达载体构建及转化烟草研究

Study on the construction of antisense expression vector of Glycine max oleic acid desaturase gene and its transfer to Nicotiana tabacum

关键词:  大豆  反义油酸脱饱和酶基因  根癌农杆菌  烟草  遗传

Key words:  Glycine max  antisense oleic acid aesaturase gene  Agrobacterium tumefaciens  tobacco(Nicotiana tabacum)  genetic transformation

摘要:

使用PCR方法从大豆基因组DNA中扩增出大豆油酸脱饱和酶基因fad2-1,连接到pMD18-T载体中,转化大肠杆菌JM109菌株。测序后,用DNAstar软件进行同源性比对。然后将正确的序列反向克隆到表达载体pBt,并转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因反向序列的农杆菌工程菌,转化烟草无菌苗叶片外植体,经过组织培养和卡那霉素抗性筛选,获得抗性烟草转化植株共75株,PCR扩增出npt-II基因,RT-PCR检测到大豆反义油酸脱饱和酶基因转录产物和GC-MS测定其油酸含量增加而亚油酸含量降低。结果表明,克隆的fad2-1基因为1196bp,基因序列与NCBI中已发表的基因fad2-1序列蛋白质相似性达到96.7%,反义大豆fad2-1基因在烟草基因组中整合表达。

Abstract:

The Glycine max oleic acid desaturase gene fad2-1 was cloned from its genomic DNA by PCR. The amplicon was linked to pMD18-T vector and transformed into E.coli JM109. After sequencing,the correct gene was reversely inserted in pBt expression vector in order to construct plant antisense expression vector,which was transformed into Agrobacterium tumefacien strains LBA4404 by freeze-thawing method. The modified strain LBA4404 was confirmed by double enzyme digestion and PCR detection. The antisense fad2-1 was introduced into tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation,and 75 kanamycin-resistant tobacco plantplets were regenerated. PCR,RT-PCR methods and GC-MS analysis were used to detect the npt-II gene,the transcript of antisense fad2-1 and oleic acid content to get the positive transgenetic plants. The results indicated that the size of the isolated gene was 1196bp,bearing 96.7% identity with the published data in NCBI database. The antisense fad2-1 expression vector was successfully constructed and transformed into Agrobacterium tumefacien strains LBA4404 and tobacco cells,and antisense fad-1 gene was successfully integrated into the genomes of tobacco cells and expressed in transgenic tobacco plantlets.