摘要
以成熟红巴梨果皮的总 RNA为模板,根据仁果类果树的 f3h基因的保守序列设计引物,利用 RT PCR方法从果皮中克隆出花青素生成相关基因 f3h基因的全长 cDNA,并将该片段连接到 pGEM T easy载体中。核苷酸序列测定表明,该基因全长1 095 bp,与苹果的f3h基因同源性高达92%,与矮牵牛相似系数达83%,与拟南芥相似系数达到82%。利用EcoRⅤ和BglⅡ对重组质粒进行酶切,回收 1 095 bp条带,并将其连接到 pBI121载体上,通过抗性筛选和PCR检测,证实了f3h基因已经构建到植物表达载体 pBI121上。
The fragment of f3h gene of anthocyanin biosynthesis was amplified by RT-PCR method using total RNA from ripping “Red Ballet” fruit peel as template and oligonucleotides designed from the published sequence of kernel fruits as primers.Sequence analysis showed that this fragment is full-length cDNA of f3h gene and it contained 1095 base pairs.Sequence comparison revealed that the f3h gene of “Red Ballet” shared homologies of 92%,83% and 82% with those of “Fuji” apple,Petunia and Arabidopsis isolates.,respectively.Then the f3h gene was digested by EcoRⅤ and BglⅡ endoenzymes and 1095bp fragment was recovered.Vector pBI121 and 1095bp fragment,were connected using ligase.Reconstituted plasmid pBI121 included the anticipated fragment of f3h gene was obtained and was proved by antibiotic selection and PCR analysis.The reconstituted plasmid is using for the further study.
出处
《南京林业大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第2期65-68,共4页
Journal of Nanjing Forestry University：Natural Sciences Edition
