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为获得抗草甘膦苜蓿株系,本研究在抗草甘膦基因GR79Mt序列前加叶绿体转运肽基因CTP2序列,并在启动子和终止子两端分别加入PvuⅠ酶切位点,插入到载体pCAMBIA2300中,成功构建植物表达载体pCA-GM。采用CaCl2冻融法将其转入农杆菌中,进一步基于农杆菌介导法进行了目的基因的拟南芥和紫花苜蓿转化。经PCR和RT-PCR验证,目的基因已转入拟南芥中。本研究共得到12株苜蓿抗性苗,经PCR分析鉴定,其中两株转化成功,结果初步表明已将目的片段基因转入苜蓿中。本研究可为抗除草剂苜蓿的应用提供了种质资源和参考方法。
In order to obtain a glyphosate-resistant alfalfa line, in this study, a chloroplast transit peptide CTP2 gene was added to the glyphosate resistant GR79Mt sequence. PvuI restriction sites were added to both ends of the promoter and terminator, and inserted into the vector pCAMBIA2300 The plant expression vector pCA-GM was successfully constructed. The Agrobacterium tumefaciens strain was transformed into Agrobacterium by CaCl2 freeze-thaw method, and the transformation of Arabidopsis thaliana and alfalfa was further carried out based on Agrobacterium-mediated method. The target gene was transferred into Arabidopsis by PCR and RT-PCR. A total of 12 alfalfa resistant seedlings were obtained in this study, which were identified by PCR analysis. Two of them were transformed successfully, and the result indicated that the target gene was transferred into alfalfa. This study provides germplasm resources and reference methods for the application of herbicide-tolerant alfalfa.