矮牵牛F3′,5′H全长cDNA的克隆及花特异启动子介导表达载体的构建

Cloning of Flavonoid-3′,5′-hydroxylase gene(F3′,5′H) of Petunia hybrida and Construction of a Novel Vector Expression F3′,5′H Driven by Flower-specific Promoter

通过RT-PCR从蓝色矮牵牛(Petunia hybrida)花瓣中克隆类黄酮-3',5'-羟基化酶基因(F3',5'H)全长cDNA(GenBank登记号EF371021)。生物信息学分析表明克隆的F3',5'H基因与GenBank登记号D14588、Z22545和DQ352142等的F3',5'H基因序列高度一致;其编码的氨基酸序列与GenBank登记号BAA03438.1(矮牵牛)、BAC10997.1(银杯草)和AAV85470.1(马铃薯)的高度同源,同源率分别为99%、88%和87%。利用pBIN19、pBluescript SK(+)和pMD18-PchsA质粒成功构建了花特异性启动子PchsA驱动的F3',5'H基因的表达载体pBIN19-PchsA-F3'5'H,并导入到野生型发根农杆菌K599中。利用K599诱导菊花生根,不定根的诱导率可达30.7%。这为利用转基因技术创造蓝色花卉提供了重要的基础。

The full-length eDNA of flavonoid-3′,5′-hydroxylase gene （F3 ′,5 ′H） was cloned by RT-PCR from blue color pedal of Petunia hybrida （GenBank Accession EF371021 ）. Bioinformatics analysis showed that the gene sequence was highly consistent with previously reported F3 ′,5 ′H genes, such as GenBank Accession D14588, Z22545, and DQ352142. The encoded amino acid sequence was highly homology with that of GenBank Accession BAA03438.1 （Petunia hybrida）, BAC10997.1 （Nierembergia sp. NB17） and AV85470.1 （Solarium tuberosum） at 99%, 88% and 87%, respectively. A novel vector pBIN19-PchsA-F3 ′ 5′H was constructed from plasmids of pBIN19, pBluescript SK（＋） and pMD18-PchsA, which was driven by flower-specific promoter of ehalcone synthase A gene （PchsA）. Furthermore, the vector was successfully transformed into the wild-type Agrobacterium rhizogenes K599. The hairy roots of Dendranthema morifolium were induced by K599 harboring pBIN19-PchsA-F3′5′H with the frequency of 30.7%. The results provided an important foundation for blue flower breeding by gene engineering technique.