梅花‘南京红须’F3'H全长基于gDNA的TAIL-PCR法克隆

根据从基因组DNA扩增到的梅花‘南京红须’类黄酮3’-羟化酶基因片段（469bp）设计3条嵌套的特异性引物．与6条短的随机简并引物组成的引物库分别用热不对称交错PCR法从‘南京红须’基因组DNA扩增该片段的5’和3’旁侧序列。获得的5’和3’旁侧序列分别长1443bp和1200bp。将两个旁侧序列在469bp片段的基础上拼接得到‘南京红须’全长为2lrl4bp的类黄酮3’-羟化酶基因，被命名为pmhxF3’H。序列分析表明：该基因与11条正式发表的、已递交到GenBank的类黄酮3’-羟化酶基因的eDNA序列在总体上有52．21％的一致性．具有3个内含子。其启动子含有1个“AGGA盒”、1个“GC盒”和3个“TATA盒”。这是首次用热不对称交错PCR法从木本植物的基因组DNA克隆到类黄酮3’-羟化酶基因。本研究将为梅花花色的分子生物学机理探索、花色的基因工程改良提供参考。

Cloning of Full-length F3'H of Nanjinghongxu (Prunus mume) by gDNA-based TAIL-PCR

Three interbedded specific primers were designed according to the band (469 bp) of flavonoid 3'-hydroxylase gene amplified from genomic DNA of Nanjinghongxu (Prunus mume) ,and they were combined with a primer bank consisting of six short random degenerate primers to respectively amplify 5' and 3' flanking sequences of the band from genomic DNA of Nanjinghongxu by thermal asymmetric interlaced PCR (TAIL-PCR). The obtained 5' and 3' flanking sequences were 1 443 bp and 1 200 bp,respectively. These two sequences were assembled with the 469 bp band to result in flavonoid 3'-hydroxylase gene of Nanjinghongxu with its full length at 2 144 bp,which was named pmhxF3'H. The sequencing of the gene showed that as a whole,the gene was identical with eleven cDNA sequences of flavonoid 3'-hydroxylase gene that had reported and presented to Genbank by 52. 21%,and contain three introns whose promoters had 1 AGGA box,1 GC box and three TATA box. This was the first time to clone flavonoid 3'-hydroxy-lase gene from genomic DNA of woody plants by thermal asymmetric interlaced PCR. This study will provide a reference to the research of molecular biological mechanisms and bioengineering improvements of Prunus mume flower color.