论文部分内容阅读
基于实验室前期野生大豆G07256碱胁迫转录组数据,结合生物信息学方法筛选得到响应碱胁迫的cation/H+逆向转运蛋白基因Gs CHX19,克隆了Gs CHX19基因全长CDS编码序列并将其构建到p CAMBIA330035Su植物超量表达载体中。以肇东紫花苜蓿为受体材料,通过农杆菌介导法将重组的植物表达载体转化其子叶节,用含0.5mg/L草铵膦的筛选培养基进行筛选,获得20株抗性植株。采用PCR方法检测抗性植株中bar基因的表达,获得16株PCR阳性植株。对获得的PCR阳性植株进行RT-PCR及real-time PCR检测,鉴定共获得11株RT-PCR阳性植株,且证明Gs CHX19基因在各转基因植株中均有不同程度的表达。结果表明,Gs CHX19基因成功转化苜蓿,并且得到表达,该转基因植株将为耐盐碱转基因苜蓿新品种的培育提供重要的试验材料。
Based on the data of G07256 alkali-stress transcriptome of pre-laboratory wild soybean, bioinformatics method was used to screen the cation / H + antiporter gene Gs CHX19 in response to alkali stress. The full-length CDS coding sequence of Gs CHX19 gene was cloned into p CAMBIA330035Su plant overexpression vector. Zhaodong alfalfa as the receptor material, the recombinant plant expression vector was transformed into cotyledonary node by Agrobacterium-mediated method and screened with screening medium containing 0.5mg / L glufosinate to obtain 20 resistant plants. The expression of bar gene in resistant plants was detected by PCR and 16 PCR positive plants were obtained. A total of 11 RT-PCR-positive plants were obtained by RT-PCR and real-time PCR. The results showed that the Gs CHX19 gene was expressed in various degrees in all the transgenic plants. The results showed that Gs CHX19 gene was successfully transformed into alfalfa and expressed. The transgenic plants will provide important experimental materials for the cultivation of new transgenic alfalfa varieties with salt tolerance.