蛋白激发子基因peaT1植物表达载体的构建及其转化棉花的研究*

［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为模板扩增得到 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。

Construction of plant expression vector for elicitor gene peaT1 and its transformation into cotton

［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 PstⅠand XhoⅠ 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.