农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究  

A study on transformation and expression of the Lyz-GFP genes mediated by agrobacteria in creeping bentgrass variety Penn A-1

作　　者：安惠惠[1,2,3] 马晖玲[1,2,3] 李坚[4] 白生军[1,3] 马祥[1,3] 

机构地区：[1]草业生态系统教育部重点实验室甘肃农业大学,甘肃兰州730070 [2]甘肃省草业工程实验室,甘肃兰州730070 [3]中-美草地畜牧业可持续发展研究中心,甘肃兰州730070 [4]陆地水循环及地表过程重点实验室、中国科学院地理科学与资源研究所,北京100101

出　　处：《草业学报》2012年第2期141-148,共8页Acta Prataculturae Sinica

基　　金：草业生态系统教育部重点实验室(甘肃农业大学)开放课题(CYZS-2011-002);甘肃省作物遗传改良与种质创新重点实验室项目-匍匐翦股颖抗病转基因植株的选育(033160)资助

摘　　要：以匍匐翦股颖Penn A-1成熟胚为供试材料,建立了其植株高效再生体系。利用农杆菌介导法将溶菌酶与绿色荧光蛋白Lyz-GFP双元基因转入匍匐翦股颖Penn A-1胚性愈伤组织中,经培养获得抗病转基因植株。并对Lyz-GFP双元基因转化Penn A-1的适宜条件进行了研究。研究结果表明,在2.0mg/L 2,4-D+0.1mg/L 6-BA的MS培养基上Penn A-1愈伤组织诱导率最高,可达36%,且质量最好。愈伤组织在MSO+0.5mg/L NAA上分化率最高,达42.5%;浓度为300mg/L的头孢霉素(Cef)可抑制农杆菌LBA4404的生长;Penn A-1胚性愈伤组织经携有pBI121-Lyz-GFP的农杆菌LBA4404(OD600值0.3～0.5)侵染10～15min,共培养3d后,转化愈伤组织生长状况良好,转化率达12.5%,且在后期的生长和转化苗的再生中有良好的表现,转化苗再生率为27.5%;转化植株有较强的荧光表达量,并经PCR检测,获得的2株匍匐翦股颖Penn A-1转化植株中均扩增出750bp的目标基因片断。Mature embryos of bentgrass （Agrostis stolonifera） Penn A-1 were used to establish a highly effi- cient regeneration system. The dual genes Lyz-GFP （Lysozyme gene and green fluorescence protein gene） were transformed into embryonic calli of creeping bentgrass by an agrobacterium-mediation method to obtain trans- genic plants with disease resistance capability. The best conditions for transformation of the Lyz-GFP genes in Penn A-1 were studied. The callus induction ratio of Penn A-1 was highest at 36% on MS medium supplemen- ted with 2.0 mg/L 2, 4-D and 0.1 mg/L 6-BA, and the calli had the optimum growth status. The highest dif- ferentiation ratio was 42.5% on MSO medium supplemented with 0.5 mg/L NAA. The study also showed that growth of agrobacterium LBA4404 was restrained by 300 mg/L cefotaxime （Cef）. Transformed calli performed well in later growth periods as well as in the regeneration of transgenic plants after infection with agrobacterium LBA4404 （OD600 ： 0.3-0.5） carried with pBI121-Lyz-GFP for 10-15 min, and co-cultured for 3 days. The transformation ratio of ealli was 12.5% ; the regeneration ratio of transgenic plants was 27.5% the transgenic plants strongly expressed fluorescence, and the 750 bp target fragments of the GFP gene were amplified by PCR from 2 acquired transgenic plants with expression of Penn A-1 fluorescence.

关 键 词：匍匐翦股颖 基因转化 溶菌酶Lyz基因 绿色荧光蛋白GFP基因 

分 类 号：S688.4[农业科学—观赏园艺]