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【目的】构建抗潮霉素标记基因的雪莲抗寒SiPEBP基因植物表达载体,转化新疆粳稻,为提高水稻的抗寒性奠定基础。【方法】用PCR方法扩增目的基因,连接到pMD19-T Vector载体上,测序验证,经BamHⅠ和SacⅠ双酶切,将目的片段连接到pMDC32表达载体上,通过冻融法将该载体导入根癌农杆菌EHA105中,通过根癌农杆菌介导法转化新疆粳稻品种秋田小町。【结果】得到抗寒基因SiPEBP,并构建成SiPEBP植物表达载体。获得了具有潮霉素抗性的水稻愈伤组织,经PCR技术鉴定,目的基因已整合到水稻基因组中。【结论】获得了转雪莲抗寒SiPEBP基因的水稻植株,这对深入研究该基因在水稻中的功能并探讨其在水稻耐冷分子育种中的应用价值奠定了基础。
【Objective】 The objective of this study was to construct a cryopreservation plant cryosin-resistant SiPEBP gene expression vector and transform Xinjiang japonica rice to lay a foundation for improving the cold resistance of rice. 【Method】 The target gene was amplified by PCR and ligated into vector pMD19-T. The sequence was verified by sequencing. The fragment was ligated into pMDC32 vector by restriction endonuclease BamHⅠand SacⅠ, and then introduced into pMDC32 vector by freeze-thaw method Agrobacterium tumefaciens EHA105 was transformed into Xinjiang Akita Komachi by Agrobacterium tumefaciens-mediated transformation. 【Result】 The cold resistant gene SiPEBP was obtained and constructed into SiPEBP plant expression vector. The hygromycin-resistant rice callus was obtained and identified by PCR. The target gene was integrated into the rice genome. 【Conclusion】 Obtaining transgenic rice plants resistant to cold stress by SiPEBP was the basis of further study on the function of this gene in rice and its application in cold-resistant molecular breeding in rice.