[目的]构建蛋白激发子基因peaT1的植物表达载体并转化棉花品种'CCRI24'.[方法]设计含有PstⅠ和XhoⅠ酶切位点的引物,以质粒pET28a-peaT1模板扩增得到peaT1序列,将其通过中间载体pG4AS-cup克隆到植物表达载体pCAMBIA2300上,通过农杆菌介导法转化棉花品种'CCRI24',诱导并筛选抗性愈伤和胚性愈伤,通过PCR、southern杂交和RT-PCR检测筛选的棉株.[结果]构建了含有增强子和多联终止子的植物表达载体pCAMBIA2300-peaT1,获得了大量的胚状体和4棵再生苗并嫁接成活,验证了蛋白激发子基因peaT1已经整合到再生苗2和4基因组当中.[结论]本研究为进一步开展蛋白激发子基因peaT1转化棉花的研究提供了基础材料.%[Objective] To construct the plant expression vector of the protein elicitor gene peaT1 and transfer it into 'CCRI24' . [Method] The peaT1 gene was amplified with the plasmid pET28a-peaT1 by specific primers harboring Pst Ⅰ and Xho Ⅰ sites. After cloned in the intermediate vector pG4AS-cup, peaT1 was then inserted into pCAMBIA2300 to construct the plant expression vector pCAMBIA2300-peaT1. The resulting vector was transferred into CRRI24 by using Agrobacterium-mediated method, and the resistant calli and embryonic calli were induced and screened out, and the transformation of peaT1 was confirmed by PCR, southern blot and RT-PCR.[Result] The plant expression vector pCAMBIA2300-peaT1 which contains enhancer and poly-terminator was constructed successfully, and a lot of embryoids and 4 regenerate seedlings were screened out from embryonic calli,and 4 regenerate seedlings were grafted on CRRI24. The result of molecular identification indicated that peaT1 was inserted into the genome of regenerate seedling no. 2 and no. 4. [Conclusion] This study provided some fundamental materials for the further research of cotton transformation of the protein elicitor gene peaT1.
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