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|本期目录/Table of Contents|

大豆fad基因反义表达载体构建及转化烟草研究

《广西植物》[ISSN:1000-3142/CN:45-1134/Q]

期数:
2011年01期
页码:
111-116
栏目:
植物生理学与分子生物学
出版日期:
2011-01-15

文章信息/Info

Title:
Study on the construction of antisense expression vector of Glycine max oleic acid desaturase gene and its transfer to Nicotiana tabacum
作者:
田苗苗 刘艳菊 李敏 周延清* 姚换灵 邢延豪
河南师范大学 生命科学学院, 河南 新乡 453007
Author(s):
TIAN Miao-Miao LIU Yan-Ju LI MinZHOU Yan-Qing* YAO Huan-Ling XING Yan-Hao
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
关键词:
大豆 反义油酸脱饱和酶基因 根癌农杆菌 烟草 遗传
Keywords:
Glycine max antisense oleic acid aesaturase geneAgrobacterium tumefaciens tobacco(Nicotiana tabacum) genetic transformation
分类号:
-
DOI:
-
文献标识码:
A
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fad2-1,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fad2-1基因为1196bp,基因序列与NCBI中已发表的基因fad2-1序列蛋白质相似性达到96.7%,反义大豆fad2-1基因在烟草基因组中整合表达。
Abstract:
The Glycine max oleic acid desaturase gene fad2-1 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 fad2-1 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 fad2-1 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 fad2-1 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.

参考文献/References

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备注/Memo

备注/Memo:
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更新日期/Last Update: 2011-04-07