茶树儿茶素合成酶基因DFR的表达特性

Expression characteristics of the key gene dihydroflavonol 4-reductase(DFR)involved in catechin synthesis in tea plants

[目的]研究茶树儿茶素积累与二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase, DFR)基因表达特性的关系。[方法]选取南昆山毛叶茶(Camellia ptilophylla Chang)和英红九号(Camellia sinensis Yinghong 9)茶树为材料,通过反转录PCR克隆茶树DFR基因,借助烟草瞬时表达技术对其表达蛋白进行亚细胞定位。经生物信息学分析,用特异性合成肽和匙孔血蓝蛋白偶联,通过免疫技术制备了该基因的特异性抗体。应用实时荧光定量PCR和Western blot技术测定DFR转录和翻译情况,并比较2种茶树中DFR基因在转录及翻译水平的差异。[结果]MY-DFR(南昆山毛叶茶DFR基因,Genbank登录号为MT875201)和YH-DFR(英红九号DFR基因,Genbank登录号为MT876617)间存在5个核苷酸碱基及相应5个编码氨基酸的差异,其表达蛋白亚细胞定位于细胞质;制备的DFR基因特异性兔源抗体效价为1∶80 000;DFR基因在南昆山毛叶茶中的转录水平极显著低于英红九号,但表达蛋白量显著高于英红九号。[结论]DFR基因在2种茶树中转录水平和翻译水平的差异体现了其表达的复杂性,推测茶树体内DFR基因的表达可能受到相关调控基因的调控。

[Purpose]To study the relationship between the accumulation of catechins and the expression characteristics of di-hydroflavonol 4-reductase(DFR)gene in tea plants.[Method]Two tea varieties,Camellia ptilophylla Chang and Camellia sinensis Yinghong 9,were selected as the materials.The DFR eneinvolved in catechin synthesis was cloned from both varieties by reverse transcription PCR(RT-PCR)and the subcellular location of DFR enzyme were determined by tobacco transient expression tech-nique.DFR-specific antibodies were prepared by immunizing rabbits with specific synthetic peptides based on bioinformatics analysis and keyhole limpet hemacyanin modification.The transcription and translation levels of DFR expression were analyzed by fluores-cence quantitative real-time PCR and Western blot,respectively.The differences in transcription and translation levels of the DFR gene in the two tea varieties were compared.[Result]The DFR genes from the two tea varieties,MY-DFR(GenBank accesison number MT875201)and YH-DFR(GenBank accesison number MT876617),differed by five nucleotides and the resulting DFR en-zyme proteins differed by five amino acids.The DFR enzyme proteins were detected in the cytoplasm.Polyclonal antibody against DFR was developed with effective titer reaching 1∶80000.The transcription level of DFR was significantly lower in C.ptilophylla Chang than that in C.sinensis Yinghong 9.However,at the translation level,C.ptilophylla Chang had significantly higher amount of DFR enzyme protein than C.sinensis Yinghong 9.[Conclusion]The differences in transcription and translation levels of the DFR gene expression between the two selected tea varieties reflect some complexity of DFR gene expression,and it is speculated that the expression of DFR gene in tea plants may be regulated by some regulatory genes.