番木瓜β-Gal基因RNAi双T-DNA植物表达载体的构建及其遗传转化的初步研究

Construction of RNAi Two-T-DNA Plant Expression Vector of β-Gal Gene and A Preliminary Study on the Genetic Transformation of Carica Papaya L.

摘要:

克隆了番木瓜(Carica papaya L.)果肉的细胞壁水解关键酶β-半乳糖苷酶(β-GAL)基因保守区，将其反向重复插入载体pKANNIBAL, 构建RNAi中间表达载体pKAN/RG，将其上的发夹结构取代经改造的载体pCAMBIA 1300上hptⅡ基因, 构建中间表达载体p1300-/MFRG，分离单T-DNA区段，与载体pCAMBIA 2301构建RNAi双T-DNA植物表达载体p2301/TTRG。酶切分析和PCR检测表明，p2301/TTRG已被成功导入农杆菌EHA 105。通过遗传转化，初步获得了GUS染色呈阳性且具Kan抗性的番木瓜胚性愈伤组织。

关键词:番木瓜;β-半乳糖苷酶;RNA干扰;双T-DNA植物表达载体;遗传转化

Abstract:

The conserved region of β-Gal gene of papaya (Carica papaya L.) pulp was cloned, which encoded a key enzyme of β-galactosidase involved in the cell wall degradation. The RNAi intermediate expression vector of pKAN/RG was constructed containing β-Gal gene in an inverted repeat orientation with the help of pKANNIBAL vector. hptⅡ gene of the modified pCAMBIA 1300 vector was replaced by the hairpin structure of pKAN/RG, which resulted in the formation of intermediate expression vector of p1300-/MFRG. Single T-DNA region of p1300-/MFRG was isolated and incorporated into the pCAMBIA 2301 vector to produce the RNAi Two-T-DNA plant expression vector of p2301/TTRG. The transformation of p2301/TTRG into Agrobacterium tumefaciens EHA 105 was confirmed by restriction enzyme analysis and PCR assay. The embryogenic calli of papaya which had kanamycin resistance and GUS expression were obtained by genetic transformation.

Key words:Carica papaya L.; β-galactosidase; RNA interference; Two T-DNA plant expression vector; Genetic transformation