番木瓜β-Gal基因的克隆分析与植物表达载体构建  

Isolation of a Papaya β-Gal Gene and Construction of Plant Expression Vector

作　　者：何玮毅[1,2,3] 陈晓静[1,2] 申艳红[1,2] 卢秉国[1] 

机构地区：[1]福建农林大学园艺学院,福州350002 [2]福建农林大学园艺植物遗传育种研究所,福州350002 [3]福建农林大学应用生态研究所,福州350002

出　　处：《热带作物学报》2010年第2期217-223,共7页Chinese Journal of Tropical Crops

基　　金：福建省科技厅重点项目(No.2008N0008);福建省自然科学基金项目(No.B0610010)资助

摘　　要：番木瓜果实采后贮藏期间的迅速软化与β-Gal基因的表达密切相关。利用常规PCR结合反向PCR技术,分离了一条4501bp的番木瓜β-Gal基因组序列。其共含有17个内含子,外显子部分与相应的cDNA序列只有1个碱基的差异。生物信息学分析结果表明,该番木瓜β-GAL属于糖苷水解酶超级家族42中家族35的成员,在进化过程中与拟南芥的亲缘关系较近,与鳄梨和北美云杉则较远。同时,它还具有一段定位于胞外的信号肽,再次表明其可能参与了果肉细胞壁的降解和果实软化。进一步分离了β-Gal基因启动子,初步验证了其果实表达特异性。将该启动子替换载体p2301/TTRG上的CaMV 35S启动子,构建出RNAi-β-Gal双T-DNA植物表达载体。酶切分析和PCR检测结果表明,载体p2301/BPTTRG已被成功导入农杆菌,可用于后续的遗传转化研究。Rapid softening of papaya fruit（Carica papaya L.）during postharvest storage is closely related to the expression of β-Gal gene.By using conventional and inverse PCR,a 4501 bp genomic sequence coding for papaya β-Gal gene was isolated,containing 17 introns and exons with one base different from the cDNA sequence.Bioinformatic analysis of this gene revealed that this protein belonged to the member of family 35 of glycoside hydrolyase superfamily 42,and had a closer genetic relationship with Arabidopsis thaliana,but farther with Persea americana and Picea sitchensis.Additionally,the predicted protein included a signal peptide located extracellularly,indicating again the possible involvement of this enzyme in the degradation of cell wall matrix and fruit softening.The corresponding promoter of β-Gal was also isolated,and proved to be of expression specificity in the fruit.CaMV 35S promoter on the vector p2301/TTRG was replaced by β-Gal promoter,leading to the construction of β-Gal-RNAi Two-T-DNA plant expression vector p2301/BPTTRG.The transformation of p2301/BPTTRG into Agrobactrium tumefaciens was confirmed by restriction enzyme analysis and PCR assay.

关 键 词：番木瓜 Β-半乳糖苷酶 果实特异性启动子 双T-DNA植物表达载体 

分 类 号：S667.9[农业科学—果树学] Q789[农业科学—园艺学]