棉花CBF基因的克隆及其转基因烟草的抗寒性分析

Cloning of Cotton CBF Gene and Its Cold Tolerance Expression in Transgenic Tobacco

从中棉12(Gh12)、中棉36(Gh36)和海岛棉7124(Gb7124)品种中克隆并鉴定了棉花的CBF基因,该基因编码一个由184个氨基酸组成的蛋白CBF,该蛋白具有CBF转录因子典型的序列标签"PKRRAGRKKFQETRHP"和"FADSAW"。Southern杂交表明,CBF基因在3个棉花品种中均以基因家族的形式存在。围绕海岛棉7124的CBF基因(GbCBF1)开展的逆境表达谱分析表明,GbCBF1基因受低温、干旱、盐和ABA等多种逆境条件的诱导表达。将GbCBF1基因构建到由强启动子35S和弱启动子NOS这2种启动子控制的植物表达载体pCambia2301上并转化烟草NC89,经过筛选及PCR鉴定,共获得26株转基因烟草。对部分T1代植株进行的PCR和RT-PCR检测表明,GbCBF1基因可以在烟草中正常转录并遗传。在低温下,转基因烟草的电解质渗漏率普遍低于野生型烟草,而游离脯氨酸含量和可溶性糖含量均高于野生型烟草,说明转GbCBF1基因提高了烟草的耐寒性。

Low temperature is an adverse environment condition affecting the growth and productivity of crops.It is also one of the limiting factors for cotton yeild and quality in China.CBF is a kind of transcription factor that can regulate expression of a number of genes related to abiotic stresses.Therefore,it is important to study the characteristics of cotton CBFs and their response to abiotic stresses.In this study,three CBF gene sequences were isolated from the genomic DNA of cotton cultivars Gh12,Gh36,and Gb7124.Cotton CBF gene encodes 184 amino acids,containing CBF-family signature ＂PKRRAGRKKFQETRHP＂ and ＂FADSAW＂.Southern blotting result showed that CBF genes were presented as the form of gene family in the genome of cotton.Northern blotting result indicated that GbCBF1 gene was induced by low temperature,drought,salt,and ABA.GbCBF1 was constructed into plant expression vector pCambia2301,in which the gene was driven by 35S and NOS promoters separately.Plant expression vectors were then transferred into tobacco NC89 using Agrobacterium-mediated transformation method.Twenty-six trangentic tobacco lines were obtained after kanamycin screening and PCR detection.PCR and reverse tanscription PCR methods were used to analyze part of T1 transgenic tobacco,the results showed that GbCBF1 gene could be transcripted and inherit in off-spring normally.The analytical result demonstrateted that the electrolytic leakage rate of transgenic tobacco was lower than that of wild type tobacco generally,however,free proline content and soluble sugar content of transgenic tobacco were higher than those of wild type tobacco under low temperature stress.In conclusion,GbCBF1 enhances cold tolerance in transgenic tobacco.