GhWRKY44基因在烟草中遗传转化及功能分析

Genetic Transformation and Functional Analysis of GhWRKY44 Gene in Tobacco

为了进一步验证GhWRKY44(登录号:KJ801807)基因在烟草异位表达后的功能,构建了Ca MV35S启动子调控、Nos为终止子的棉花抗枯萎病相关基因(GhWRKY44)的过表达载体;电击法将其导入农杆菌GV3101,菌液PCR筛选鉴定阳性菌株,采用农杆菌介导法转化烟草;T0转化植株经卡那霉素筛选、PCR检测后,随机从转基因T0中选取5株进行RT-PCR检测,均为阳性株,说明GhWRKY44基因不仅整合到烟草基因组中而且还能正常转录。对过表达GhWRKY44基因的转基因烟草T1株系进行实时荧光定量PCR(qRT-PCR)分析。结果表明,在正常生长情况下,PR5和NPR1的表达量在转GhWRKY44株系中明显增加,枯萎病菌侵染后,在转GhWRKY44株系中,PR1a、PR2和NPR1的表达量迅速增加,明显高于野生型株系,而PR5的表达量则低于野生型株系,说明该目的基因能在烟草中异位表达并能激活病程相关蛋白基因的表达,但其功能仍需进一步研究。

By Ca MV35 S promoter and Nos as the terminator,the overexpression vector of GhWRKY44 gene and verified the function of GhWRKY44（ Accession number： KJ801807） gene in tobacco ectopic expression were constucted. Overexpression vector was introduced into Agrobacterium GV3101 by electroporation method,positive bacteria strain was identified by screening PCR and positive colony was transferred into tobacco by Agrobacterium-mediated. After T0 of transformed plants are detected by kanamycin,PCR detection,5 strains were detected by RT-PCR technique randomly selected from T0 of transformed plants and 5 strains were positive. The results showed that not only GhWRKY44 gene was transferred into the tobacco genome but also the normal transcripted. T1 lines of transgenic tobacco were analysed by using Real-time quantitative PCR（ qRT-PCR） technique. The result showed that the wild type and transgenic GhWRKY44 tobacco were not inoculated with Fusarium oxysporum,the expression of PR5 and NPR1 expression obviously increased in transgenic GhWRKY44 plant. Wild type and transgenic plant after were infected by Fusarium oxysporum,the expression of PR1 a,PR2 and NPR1 significantly increased in transgenic lines,but the expression of PR5 was higher in wild type than transgenic lines. The gene could be expressed in the tobacco and could activate parts of PRs＇ expression. But the function of GhWRKY44 still needs further exploration.