F3′5′H基因的克隆、表达载体构建与矮牵牛遗传转化

Cloning and Construction of Plant Expression Vector and Transformation of Flavonoid-3',5-'hydroxylase Gene in Petunia hybrida

类黄酮3′5′羟基化酶(Flavonoid-3’,5-’hydroxylase;F3′5′H)是花色素苷代谢途径中的一个关键性酶,能使花色素的合成趋向于形成蓝色的飞燕草色素。从蓝紫色矮牵牛的花瓣中提取总RNA,利用RT-PCR技术克隆得到了约1.7kb的F3′5′H片段。F3′5′H的cDNA序列分析结果表明,克隆得到的序列与原序列同源性达到99.34%,开放读码框为1 521 bp,编码507个氨基酸。将推算的氨基酸序列与NCBI登录的氨基酸序列进行分析比较,发现与文献报道的存在2个氨基酸差异,氨基酸同源率为99.6%。将此基因构建到含有35S启动子的植物表达载体PBI-F3′5′H上,并通过农杆菌介导法对粉红色矮牵牛进行了遗传转化,初步鉴定获得了转基因植株。

Flavonoid-3＇,5-＇hydroxylase（F3′5′H）is the key enzyme in the biosynthesis of anthocyanins,which can divert the production of delphinidin pigments.The total RNA was isolated from he purple flower of Petunia hybrida.A specific F3′5′H fragment of the expected size was amplified by reverse transcription polymerase chain reaction and sequenced.The DNAsequence analysis indicated that the cloned 1.7kb cDNA of F3′5′H fragment including an open reading frame of 1521bp by which encodes a predicted poly peptide of 507 amino acids.Compared with the F3′5′H gene in Gegebank,the homology of nucleotide sequence could reach 99.34%,and 99.6% at the amino acid sequence,only two amino acid differences.A construct of F3′5′H in a sense-orientation driven by CaMV 35S promoter was constructed and transformed into pink flower of Petunia hybrida by using Agrobacterium tumefacien mediated method.The transgenic plants were confimed that F3′5′H gene was interated into genome of pink Petunia hybrida by preliminary identification.