发根农杆菌介导甜瓜转基因过表达体系的建立

Establishment of Agrobacterium rhizogenes-mediated gene overexpression system in melon

遗传转化是实现基因功能验证和基因快速转育的重要手段,但目前甜瓜的遗传转化依旧存在众多技术难题。笔者利用野生型发根农杆菌K599侵染甜瓜’E31’,成功诱导甜瓜产生了不定根。借助该技术,将含有GUS基因和EGF基因的过表达载体pCAMBIA3301和pCAMBIA1300-ProSuper导入发根农杆菌K599,利用转化后的农杆菌侵染甜瓜’E31’,成功诱导出了不定根。PCR检测结果表明,新生不定根的阳性率达到100%。通过GOS染色和激光共聚焦显微镜检测,在不定根中检测到GOS基因和EGFP基因的大量表达,成功实现了外源基因在甜瓜新生不定根内过表达。该方法周期短、操作方便、转化率高,可实现基因功能的快速验证,为甜瓜根系抗病抗逆方面的研究提供技术支持。

Genetic transformation is an important means to verify gene function and rapid gene transfer,but there are still many technical difficulties for melon.In this study,Agrobacterium rhizogenes K599 was used to infect melon ’E31’ and successfully induced the formation of adventitious roots.With this technique,the overexpression vectors pCAMBIA3301 and pCAMBIA1300-ProSuper containing GUS gene and EGFP gene,respectively,were introduced into A.rhizogenes K599.The modified A.tumefaciens K599 was used to infect melon ’E31’ and successfully induced the formation of adventitious roots.The results of PCR showed that the positive rate of new adventitious roots was 100%.The expression of GUS and EGFP genes in adventitious roots were successfully detected by GUS staining and confocal laser microscopy.This method has the advantages of short cycle,easy operation,high transformation rate,and can verify gene function in melon rapidly.It will provide technical support for the research on the mechanism of disease or stress resistance in melon root.