鸡β-防御素Gal-3在转基因紫花苜蓿中的表达

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pCAM-BIA1390R（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240bp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。%【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-3 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.