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pCAMBIA1300上hptⅡ基因,构建中间表达载体p1300-/MFRG,分离单T-DNA区段,与载体pCAMBIA2301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EHA105。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。
The conserved region of β-Gal gene of papaya (Caricapapaya 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, hpt II 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 pl300/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.
出处
《热带亚热带植物学报》
CAS
CSCD
北大核心
2009年第6期556-561,共6页
Journal of Tropical and Subtropical Botany
基金
福建省科技厅重点项目(2008N008)
福建省自然科学基金(B0610010)资助