| 摘要: |
| 【目的】在紫花苜蓿中表达鸡β-防御素3(Gal-3)，为利用紫花苜蓿规模化生产 Gal-3 奠定基础。【方法】PCR扩增鸡Gal-3基因，以潮霉素作为转基因植物的选择标记，将鸡β-防御素Gal-3基因克隆至表达载体pCAMBIA1390R（简写为p1390R）上，构建重组表达质粒 p1390R-Gal-3；采用冻融法将质粒p1390R-Gal-3转入根瘤农杆菌EHA105中，通过农杆菌介导法转化至紫花苜蓿；采用PCR检测、Southern blot 筛选阳性转基因苜蓿植株，并对Gal-3蛋白进行抑菌活性研究。【结果】PCR扩增获得了240 bp的鸡Gal-3基因，成功构建植物表达载体 p1390R-Gal-3，在苜蓿中初步建立了Gal-3的遗传转化体系，共获得22株转基因苜蓿植株，其中4株为阳性植株，转化率为18.2%。PCR扩增和Southern blot检测表明，Gal-3基因已整合到转基因苜蓿基因组中。提取转基因苜蓿的Gal-3蛋白进行抑菌活性试验，结果显示转基因苜蓿对金黄色葡萄球菌、大肠杆菌、沙门氏菌均有抑制作用。【结论】获得了具有抑菌活性的转鸡β-防御素Gal-3基因的苜蓿植株。 |
| 关键词: 农杆菌 鸡β-防御素Gal-3 紫花苜蓿 遗传转化 |
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| 基金项目:吉林省科技发展计划项目（20090244，20100147）；吉林省教育厅“十一五”科学技术研究项目（2009297） |
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| Expression of chicken beta defensin Gallinacin-3 in transgenic alfalfa |
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JIN Libo1, LIU Xiuming1, LIU Lei,et al2
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1.Engineering Research Center of Bioreactor and Pharmaceutical Development of Ministry of Education ,Jilin Agricultural University;2.Laboratory Medical College,Jilin Medical College
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| Abstract: |
| 【Objective】This study aimed to investigate the expression of chicken beta-defensin Gallinacin-3(Gal-3) in alfalfa,and lay a foundation for large-scale production of Gal-3 protein through alfalfa chloroplast.【Method】Using hygromycin gene as a selection marker for transgenic plants,the chicken Gal-3 gene was amplified by PCR before it was cloned into the pCAMBIA1390R vector to obtain the plant expression vector p1390R-Gal-3.p1390R-Gal-3 was transferred into Agrobacterium EHA105 using freeze thawing method before being transferred Gal-3 into alfalfa via Agrobacterium mediated method.The positive transgenic alfalfas and antibacterial activities of Gal-3 protein were determined by PCR and Southern blot.【Result】Gal-33 gene with 240 bp was obtained and the plant binary expression vector p1390R-Gal-3 that carried Gal-3 selection marker was successfully constructed by PCR amplification.4 of 22 transgenic alfalfas which correctly accumulated Gal-3 protein were positive alfalfas,indicating a transformation efficiency of 18.2%.Subsequent PCR and Southern blot analyses confirmed that Gal-3 gene was integrated into the genome of transgenic alfalfa.The antibacterial activity experiment of extracted Gal-3 protein from transgenic alfalfas showed that transgenic alfalfas had antibacterial activities against Staphyloccocus,E.coli and Salmonella.【Conclusion】Gal-3 had been successfully expressed in transgenic alfalfas,and the Gal-3 protein of transgenic alfalfas had good antibacterial activities. |
| Key words: Agrobacterium tumefaciens chicken beta-defenfin Gallinacin-3 alfalfa genetic transformation |
