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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(469 bp)没计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1443 bp和1200 bp。将两个旁侧序列在469 bp片段的基础上拼接得到‘南京红须’全长为2 144 bp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cDNA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。
According to the 3’-hydroxylase gene fragment (469 bp) amplified from genomic DNA of Plum ’Nanjing red mustard’, excluding 3 nested specific primers and 6 short random degenerate primers Primer library The 5 ’and 3’ flanking sequences of this fragment were amplified from ’Nanjing Hongsha’ genomic DNA by thermal asymmetric staggered PCR respectively. The 5 ’and 3’ flanking sequences obtained were 1443 bp and 1200 bp, respectively. The two flanking sequences were stitched together on the basis of a 469 bp fragment to obtain a flavonoid 3’-hydroxylase gene of 2 144 bp in length, which was named as pmhxF3’H. Sequence analysis showed that there was a 52.21% identity between this gene and the 11 officially published cDNA sequences of the flavonoid 3’-hydroxylase gene that has been submitted to GenBank. It has 3 introns and its promoter contains 1 “AGGA box”, 1 “GC box” and 3 “TATA boxes”. This is the first time that the flavonoid 3’-hydroxylase gene has been cloned from genomic DNA of woody plants by thermal asymmetric staggered PCR. This study will provide a reference for the exploration of the molecular biology mechanism of plum blossom flower and the improvement of the genetic engineering of flower color.