| 摘要: |
| 【目的】 从“皇家嘎拉”苹果幼果外果皮中克隆L-半乳糖脱氢酶(L-galactose dehydrogenase,GalDH) cDNA，进行生物信息学分析并构建其RNAi植物表达载体，为该基因功能鉴定奠定基础。【方法】 应用同源序列克隆法克隆“皇家嘎拉”苹果GalDH基因，并进行生物信息学分析；利用噬菌体同源重组原理构建其RNAi植物表达载体pMDR-GalDH，并导入农杆菌EHA105。【结果】 通过RT-PCR扩增得到1条约1.1 kb的条带，序列分析表明，该片段长为1 111 bp，包含一个975 bp的开放阅读框，可编码324个氨基酸残基，包含有醛酮还原酶的保守结构域和3个跨膜螺旋结构，其理论上的等电点pI为5.44，蛋白分子质量为34.99 ku；聚类分析显示,苹果GalDH核苷酸与已知其他植物GalDH核苷酸的相似性均在95%以上，判断其为苹果GalDH基因cDNA全长，命名为MdGalDH(GenBank登录号为：GQ131419)。另外，通过RT-PCR获得GalDH基因保守区段，成功构建了其RNAi植物表达载体pMDR-GalDH。【结论】 从“皇家嘎拉”苹果中克隆了GalDH cDNA的编码区全长，并构建了该基因的RNAi植物表达载体。 |
| 关键词: 苹果 L-半乳糖脱氢酶 基因克隆 RNAi植物表达载体 |
| DOI： |
| 分类号: |
| 基金项目:陕西省自然科学基金项目（2006C101）；陕西省重大科技专项计划项目（2006kz05-G4） |
|
| Cloning of L-galactose dehydrogenase cDNA from fruits of Malus domestica Borkch.and construction of its RNAi plant expression vector |
|
GAO Jing,SHANG Zeng-zhen,WANG Xiao-hua,MA Feng-wang and LIANG Dong,GAO Jing,SHANG Zeng-zhen,WANG Xiao-hua,MA Feng-wang and LIANG Dong,GAO Jing,SHANG Zeng-zhen,WANG Xiao-hua,MA Feng-wang and LIANG Dong,GAO Jing,SHANG Zeng-zhen,WANG Xiao-hua,MA Feng-wang and LIANG Dong,GAO Jing,SHANG Zeng-zhen,WANG Xiao-hua,MA Feng-wang and LIANG Dong
|
| Abstract: |
| 【Objective】 L-galactose dehydrogenase (GalDH) cDNA was isolated from young fruit of Malus domestica Borkch cv.Royal Gala.According to its bioinformatics analysis,RNAi plant expression vector was constructed,which will establish basis for further functional identification.【Method】 GalDH gene was amplified by homology-based candidate gene technique.Its expression vector pMDR-GalDH was constructed through phage homologous recombination,and introduced into agrobacterium strain EHA105.【Result】 One fragment about 1.1 kb was amplified by RT-PCR method.Sequence analysis indicated that the cDNA contained an open reading frame of 975 bp,encoded a polypeptide of 324 amino acids residues,had an putative isoelectric point of 5.44 and a calculated molecular weight of 34.99 ku.And meanwhile the conserved domains of aldo-keto reductase (AKRs) and three transmembrane helix structure were discovered.Homology analysis demonstrated that the similarity of nucleotides with those from other plant species was over 95%.We presumed that the fragment cDNA was the full-length cDNA of GalDH of apple,and named MdGalDH(GeneBank accession number:GQ131419).In addition,RNAi plant expression vector pMDR-GalDH was constructed with GalDH conservative fragments by PCR.【Conclusion】 One full-length cDNA of GalDH was amplified and its RNAi expression vector pMDR-GalDH was successfully constructed. |
| Key words: Malus domestica Borkch.cv.Royal Gala L-Galactose dehydrogenase(GalDH) gene cloning RNAi plant expression vector |
