Eukaryotic expression and cold-resistance analysis of CsCBF1 from tea plant (Camellia sinensis) | 茶树CsCBF1基因表达载体的构建及其转基因烟草的抗寒性分析

Chino. 通过转基因烟草验证茶树CsCBF1基因抗寒性功能，为发掘、筛选茶树重要功能基因提供理论依据。利用根癌农杆菌介导法将构建的表达载体pBI121-CsCBF1导入烟草，通过PCR、 RT-PCR和GUS活性染色鉴定外源CsCBF1基因是否导入并整合到烟草基因组上转录表达。然后将转pBI121-CsCBF1型、转pBI121型和野生型烟草分别置于25,4和0 ℃下处理48 h后，检测其膜质通透性、丙二醛（MDA）含量和叶绿素荧光参数Fv/Fm，并将0 ℃处理3 d的转pBI121-CsCBF1型和野生型烟草置于25 ℃下培养1周后观测恢复表型和统计死亡率。成功构建表达载体pBI121-CsCBF1，PCR、RT-PCR和GUS活性染色证明，CsCBF1已整合到烟草基因组中并转录表达。 25 ℃处理下，转pBI121-CsCBF1型、转pBI121型和野生型烟草的膜质通透性、丙二醛含量和Fv/Fm均无显著差异，而在4和0 ℃处理下，转pBI121-CsCBF1型烟草的膜质通透性和丙二醛含量均显著低于转pBI121型和野生型烟草，但Fv/Fm显著高于转pBI121型和野生型烟草。生长恢复试验中，转pBI121-CsCBF1型的存活率为66.7％，而野生型仅为23.3％。茶树CsCBF1基因能够显著增强非低温驯化植物烟草的抗寒性，说明茶树CsCBF1基因存在于细胞抗寒的分子调控途径中，从而提高植物细胞的低温防御能力。

Inglés. The cold-resistance function of CsCBF1 gene from Camellia sinensis was identified via transgenic tobacco in order to provide theoretical basis for screening the key functional genes from tea plant (C. sinensis). The expression vector pBI121-CsCBF1 was constructed, transferred into tobacco plants through Agrobacterium mediated transform methods and determined if the exogenous CsCBF1 gene was transferred, integrated into the tobacco genome and expressed through PCR, RT-PCR and GUS staining. The plasma membrane permeability, MDA content and chlorophyll fluorescence parameter Fv/Fm of pBI121-CsCBF1 transgenic, pBI121 transgenic and wild genotype tobaccos under 25, 4 and 0 ℃ treatments were measured. Meanwhile, the recovering phenotype and survival rate of pBI121-CsCBF1 transgenic and wild genotype tobaccos treated under 0 ℃ for 3 days were observed and statisticed after recovery under 25 ℃ for a week. Expression vector pBI121-CsCBF1 was successfully constructed, and detections of PCR, RT-PCR and GUS staining indicated the CsCBF1 gene was integrated into the genome and expressed in tobaccos. There was no significant difference existing in plasma membrane permeability, MDA content and chlorophyll fluorescence parameter Fv/Fm among pBI121-CsCBF1 transgenic, pBI121 transgenic and wild genotype tobaccos under 25 ℃ treatment, but plasma membrane permeability and MDA content of former genotype were extremely lower than that of both latter genotype and Fv/Fm of the former was dramatically higher than that of both latters’. After one week′s recovery, the survival rate of pBI121-CsCBF1 transgenic lines and wild type was 66.7％ and 23.3％, respectively. All the experiments results prove that the CsCBF1 gene from tea plant could remarkably enhance the cold tolerance of tobacco, which demonstrates CsCBF1 gene could help plant cells endure low temperature stress through taking part in molecular regulation pathway of cold resistance.