利用双T-DNA载体系统获得无选择标记转基因菊花  

Generating Marker-free Transgenic Chrysanthemum by Agrobacterium Mediated Transformation with Twin T-DNA Binary Vectors

作　　者：孙磊[1] 张启翔[1] 周琳[1] 陆苗[1] 蔡明[1] 

机构地区：[1]北京林业大学园林学院,国家花卉工程技术研究中心,北京100083

出　　处：《园艺学报》2008年第5期727-734,共8页Acta Horticulturae Sinica

基　　金：教育部博士点基金项目(20040022022);科技部国家转基因专项项目(JY03-B-28)

摘　　要：为获得具有无选择标记的转基因菊花,构建了一个双T-DNA超级双元载体pCAMBIA1301-gus,其中一个T-DNA结构域中含有选择标记基因hpt,另一个T-DNA结构域中含有目的基因gus,而且两个T-DNA结构顺序相连,中间没有其他插入序列。利用农杆菌介导转化菊花幼嫩茎尖薄层细胞,共获得506个抗性植株,通过PCR和Southern杂交检测表明共转化率为38.4%,对其中17个同时整合了hpt和gus基因的植株自交获得的T1代株系进行检测,发现约有15.8%的T1代植株中不含选择标记基因hpt,结果表明双T-DNA载体系统能有效地用于培育无选择标记转基因植物。In this study, we constructed a super binary vector to evaluate the potential of the twin T-DNA system for generating selectable marker-free progeny plants in chrysanthemum plants. The first T-DNA of the vector, contains the hygromycin phosphotransferase （hpt） selectable gene, while the second T-DNA, bears the β-glucuronidase gene （gus）, featuring the gene of interest. The two T-DNA regions were located adjacent to each other with no intervening region. 506 resistant chrysanthemum plants were then obtained by Agrobacterium-mediated transformation with this vector, analysis of transgene inheritance was facilitated by PCR amplification and Southern blot from leaf tissue, the primary co-transformation frequency was 38.4%. A total of seventeen hpt-resistant/gus-active To plants were evaluated for segregation in the next generation, and among these, approximately 15.8% had transgene inserts which segregated in the T1 progeny to yield chrysanthemum plants without selectable marker gene. Overall, the twin T-DNA systems appeared to be a useful approach to generate marker free transgenic plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance genes in the environment.

关 键 词：菊花 转基因 双T-DNA 共转化 无选择标记 

分 类 号：S682.11[农业科学—观赏园艺]