摘要
根据GenBank登录的牡丹ACS基因DNA全长序列(FJ769773),设计一对特异性引物,在优化的PCR扩增体系的基础上,应用高保真DNA聚合酶KOD-Plus从洛阳红牡丹叶片总DNA中扩增出牡丹ACC合成酶基因片段PsACS-4。测序结果表明:克隆序列长970 bp,不包含牡丹ACS基因内含子序列,与目标序列同源性达100%;用SacI和SmaI对重组质粒和空载体pBI121双酶切、连接,将PsACS-4基因片段反身插入到植物表达载体pBI121的35S启动子下游,成功构建了牡丹ACS反义基因植物表达载体。
According to the DNA full-length sequence of ACC synthase gene fragment of Paeonia suffruticosa（FJ769773）in GenBank database,a pair of specific primers,P1 and P2,were designed and synthesized.Based on the optimized PCR amplification system and high fidelity DNA polymerase KOD-Plus,ACS gene fragment PsACS-4 was successfully amplified from the total DNA which was extracted from leaves of peony cultivar Luo Yang Hong.Sequencing result indicated that the length of PsACS-4 fragment was 970 bp,and the similarity of the sequence of PsACS-4 and the target sequence was 100%.Two sequences alignment analysis revealed that sequence of PsACS-4 didn′t contain any intron sequence.The recombinant plasmid and the plant expression vector pBI121 were digested with the Sac I and Sma I digest enzyme.After the fragment of ACC synthase gene and pBI121 were recovered and ligated,PsACS-4 gene fragment was inserted into 35S promoter downstream of plant expression vector pBI121 reverse in orientation,and an antisense plant expression vector pBI121-anti-PsACS-4 was constructed successfully.
出处
《华北农学报》
CSCD
北大核心
2010年第6期34-37,共4页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金(30740013)
教育部重点实验室开放基金(05-03)
河南省教育厅自然基金(2008B210003)
洛阳市重点实验室专项(0901063A)
河南省重大科技攻关项目(0122012400)
关键词
牡丹
ACC合成酶
反义植物表达载体
Paeonia suffruticosa
ACS
Antisense plant expression vector
