转天蚕素AD(CAD)基因于紫花苜蓿的研究
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吉林省科技厅科技发展计划项目(20100147,20090244);高等学校科技创新工程重大项目培育资金项目(70S018);吉林农业大学校内启动基金(201032);温州市科技局科技计划项目(S20100043)


Expressions of Cecropin AD(CAD) in Transgenic Alfalfa
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    为了探索利用紫花苜蓿 Medicago sativa 作为生物反应器生产杂合抗菌肽天蚕素AD(Cecropin AD, CAD)的可行性,通过PCR搭桥方法合成了CAD全长基因,并插入对外源基因表达有一定促进作用的调控元件:Ω序列、Kozak序列及KDEL四肽序列,然后将改造后的CAD基因克隆进载体pCAMBIA1390R,构建了pCAMBIA1390R CAD植物表达载体.将重组质粒转入根癌农杆菌 Agrobacterium tumefacienLBA4404,并通过农杆菌介导方法将CAD基因转化入紫花苜蓿.在含潮霉素(20 mg/L)的筛选培养基上筛选转基因植株,通过PCR、Southern blotting检测,CAD基因已经转入紫花苜蓿中;提取蛋白质进行抑菌活性研究,结果表明转基因苜蓿蛋白提取物对沙门氏菌 Salmonella 及金黄色葡萄球菌 Staphylococcus aureus 具有一定抑菌活性.

    Abstract:

    To explore the possibility of expressing the hybrid peptide CAD in the alfalfa, Medicago sativa, the cDNA sequence encoding CAD was obtained by recursive PCR(rPCR), and fused with elements such as Ω sequence, Kozak sequence and KDEL sequence to improve the expression level in the transgenic plants, and cloned into the plant transformation vector pCAMBIA1390R. This recombinant plasmid was transformed into Agrobacterium tumefacien LBA4404 and CAD gene was transformed into alfalfa by A. tumefacien-mediated method. The regenerated alfalfa plants were selected on medium 20 mg/L hygromycin. The results of PCR and Southern blotting showed that the CAD gene was successfully inserted into alfalfa genome. Proteins extracted from transgenic alfalfa showed a certain bacteriostatic activity to Salmonella and Staphylococcus aureus.

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刘秀明,朱海林,杜淑环,万 秋,李海燕,李校堃.转天蚕素AD(CAD)基因于紫花苜蓿的研究[J].华南农业大学学报,2012,33(4):498-502

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  • 收稿日期:2011-12-27
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  • 在线发布日期: 2012-11-16