摘要
参考GenBank登录的植物胞壁转化酶基因序列,设计1对PCR引物,以番茄(‘中蔬四号’)叶片总RNA的反转录产物为模板,扩增出长为416bp的cDNA片段,Blast结果表明,其为番茄胞壁转化酶基因LIN6片段;将其反向插入双元载体pBinAR的CaMV 35S启动子和OCS终止子间,构建反义植物表达载体pBinAR-aLIN6。通过农杆菌EHA105介导转化番茄MicroTom,PCR和Southern斑点杂交检测表明,共获得5株转基因番茄;生化检测表明,5个转化植株叶片胞壁转化酶活性分别比未转化植株下降67.9%、13.4%、73.5%、85.6%和58.0%。
Total RNA was isolated from tomato （cv. ＇Zhongshu No. 4＇） leaves using the specific primers de- signed according to the sequence of cell wall invertase gene in GenBank. A fragment about 416bp was am- plified by reverse transcription and polymerase chain reaction. Sequence analysis showed the amplified frag- ment was the LIN6 cDNA fragment of the cell wall invertase gene. The fragment was inserted into plant expression vector pBinAR between CaMV 35S promoter and OCS terminator to form an antisense plant ex- pression vector pBinAR-aLIN6. Five transgenic tomato plants （cv. MicroTom）, identified by PCR and Southern hybrids detection, were obtained through transferring of EHA105/pBinAR-aLIN6. The activity of cell-wall invertase in the leaves of those five antisense plants was found decreasing by 67. 9%, 13. 4%, 73.5G ,85.6% and 58.0G ,respectively.
出处
《西北植物学报》
CAS
CSCD
北大核心
2012年第2期241-245,共5页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金项目(30370977)
国家"十一五"科技支撑计划(2007BAD79B01)
河南科技大学博士科研基金(09001216)
关键词
番茄
胞壁转化酶
RT-PCR
反义沉默
tomato
cell-wall invertase
reverse transcription PCR
antisense silencing
作者简介
吴正景（1974-），男，博士，副教授，主要从事园艺植物种质资源与生物技术研究。E-mail：wzjhaust@gmail．com
通讯作者：程智慧，博士，教授，博士生导师，主要从事园艺植物栽培生理生态和生物技术方面的研究。E-mail：chengzh@nwsuaf．edu．cn
