含硫氨基酸基因植物表达载体的构建及对百脉根的转化  

Construction of Plant Expression Vectors for Sulphur-amino Acid Gene and Transformation to Lotus corniculatus L.

作　　者：关宁[1] 王涌鑫[1] 李聪[1] 苗丽宏[1] 张博[2] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所牧草遗传育种研究室,北京100193 [2]新疆农业大学草地资源与生态自治区重点实验室,乌鲁木齐830052

出　　处：《分子植物育种》2009年第2期257-263,共7页Molecular Plant Breeding

基　　金：国家十一五科技支撑计划(2006BAD01A19);新疆农业大学草地资源与生态自治区重点实验室开放课题(XJDX0201-2005-03)资助

摘　　要：在本研究中,用限制性内切酶NcoⅠ和BglⅡ从中间载体pMD18zeolin和pMD18zein上切下zeolin基因(约1600bp)和γ-zein基因(约720bp),将其定向连接在经相同酶切的质粒载体pCAMBIA1302上,构建成植物表达载体pCBzeolin和pCBzein。采用冻融法将pCBzeolin和pCBzein导入根癌农杆菌(Agrobac-terium tumefaciens)菌株LBA4404,利用该菌株转化百脉根(Lotus corniculatusL.)。经过共培养、筛选分化、再生,得到抗性植株。对抗性植株进行了PCR、RT-PCR检测表明,γ-zein和zeolin基因已经整合到百脉根基因组中,在核酸水平得到了表达。含硫氨基酸数据分析表明,转zeolin基因植株含硫氨基酸含量极显著高于转γ-zein基因植株和对照植株的含量(P<0.01),但转γ-zein基因植株与对照植株间差异不显著。Zeolin gene （about 1 600 bp） and γ-zein gene （about 720 bp） were got by cutting from the middle vectors pMD 18zeolin and pMD 18zein, respectively, and were linked to the expression vector pCAMBIA1302 in the same endonucleases digestion sites. The plant expression vector pCBzeolin and pCBzein were obtained. Plasmids pCBzeolin and pCBzein were transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and then were transformed into Lotus corniculatus mediated by stain LBA4404. After co-culture, selective differentiation and regeneration, resistant plants were obtained. Resistant plants were detected by PCR, RT-PCR. The results have proved that the γ-zein and zeolin genes have been transformed into the genome of Lotus corniculatus L., and expressed on nucleic acid level in the transgenic plants. Sulphur-containing amino acid of transgenic plants analysis showed that the content of sulphur-amino acid of transgenic zeolin plants was significantly higher than that of the content of the transgenic γ-zein plants and control plants （P〈0.01）, but there was no significant difference between the transgenic γ-zein plants and control plants.

关 键 词：含硫氨基酸基因 表达载体构建 百脉根 转化 

分 类 号：S541.6[农业科学—作物学]