转天山雪莲sikPIP_3基因烟草的获得及抗逆性鉴定

Transformation of Saussurea involucrata sikPIP_3 Gene into Tobacco and Evaluation of Transgenic Plant Stress-resistance

利用PCR技术从实验室建立的天山雪莲DNA文库中克隆了天山雪莲质膜水孔蛋白基因sikPIP3,构建了植物表达载体pBI121-sikPIP3,通过农杆菌介导法转化烟草品种NC89,经PCR和RT-PCR检测证明目的基因成功导入并得到了表达,采用水分胁迫进行抗旱分析和采用冷冻胁迫进行抗寒性分析。结果显示:(1)克隆出具有水孔蛋白基因特性的sikPIP3基因。(2)经断水干旱处理,转基因烟草的生长表型优于野生型烟草,特别是在断水9d的情况下,野生型烟草已经完全萎蔫,转基因烟草萎蔫症状较轻;生理指标测量结果显示,转基因烟草的相对电导率和MDA的含量低于野生型烟草,相对含水量和CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗旱性高于野生型烟草。(3)经不同温度胁迫处理,转基因烟草的生长表型优于野生型烟草,特别是在-4℃冷处理6h情况下,野生型烟草已经完全萎蔫,转基因烟草只出现少量伤斑;生理指标分析结果表明,转基因烟草的相对电导率和MDA的含量低于野生型烟草,CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗寒性高于野生型烟草。综合结果表明:sikPIP3基因在抗逆基因工程方面具有较高的应用前景。

Saussurea involucrata plasma membrane aquaporin protein gene sikPIP3 was cloned from S.involucrata DNA library which was prepared in laboratory by PCR technique.Then a plant expression vector pBI121-sikPIP3 was constructed using sikPIP3 gene.The transgenic tobacco was further obtained through Agrobacterium tumefaciens transformation of tobacco NC89.PCR and RT-PCR analysis indicated that the plasma membrane aquaporin protein gene sikPIP3 had been integrated into the genome.Water stress method was introduced to analyze drought resistance and freezing stress method was used to analyze cold resistance.The results showed that：（1）Plasma membrane aquaporin protein gene sikPIP3 was cloned;（2）Growth phenotype of transgenic tobacco is better than that of wild-type after drought treatment,especially under the circumstances without water for 9 days.The wild-type tobacco is wilting completely,and transgenic ones presented mild wilting symptoms;Physiological index analysis results showed that relative conductivity and MDA content of transgenic tobacco is lower than that of wild-type,but relative water content is higher.The drought resistance of sikPIP3 transgenic tobacco is better than that of wild-type.（3）Growth phenotype of transgenic tobacco is better than that of wild-type after stress treatment with different temperatures,specially under the condition of-4℃ cold treatment for 6 h.The wild-type was wilting completely,however the transgenic-type only emerged little injure spot.Physiological index analysis results showed that relative conductivity and MDA content of genetic tobacco is lower than that of wild-type.The cold resistance of sikPIP3 transgenic tobacco is better than that of wild-type.Comprehensive results indicated that sikPIP3 gene has nice applications in resistance genetic engineering.