目的 克隆杭菊Chrysanthemum morifolium二氢黄酮醇还原酶(dihydroflavonol 4-reductase,DFR)基因,并研究其在花芽分化期及开花期不同时期的表达特征。方法 根据已报道的菊花Chrysanthemum morifolium DFR基因序列设计引物,通过荧光定量PCR(RT-PCR)技术从杭菊头状花序中扩增出目的基因片段,连接、转化、挑取阳性克隆测序并拼接;采用基于内参基因的相对定量PCR方法,分析该基因表达量特征。结果 得到全长1152 bp的杭菊DFR基因,最大开放阅读框1029 bp,共编码342个氨基酸。NCBI上进行Blast比对,该基因与其他植物的DFR基因有很高的同源性。RT-PCR表明该基因在花芽分化时期不同阶段以及开花期不同时期花不同部位中表达量均有差异。花芽分化时期杭菊DFR基因在50 d表达量最高;开花期该基因在时期I的管状花中表达量最高,在时期Ⅱ的花序托中表达量最低。结论 克隆得到杭菊DFR基因cDNA序列,该基因表达存在时间和空间上的差异,表达量在花蕾膨大成熟到舌状花开放30%的时间段内最高。该结果为进一步进行该基因在杭菊中的表达与催化产物的关系和调控机制研究奠定了基础。

Objective To clone the dihydroflavonol 4-reductase(DFR) gene from the capitulum of Chrysanthemum morifolium and study its expression at different stages of flower bud differentiation period, and in different periods of flowering in different tissues. Methods Two pairs of primers were designed according to the reported DFR gene sequences of C. morifolium, target fragments were amplified by RT-PCR, connected, and transformated, then positive cloning was detected by PCR and then sequenced. Results Sequencing results showed that 1152 bp sequence was acquired with the largest open reading frame of 1029 bp, which encoded 342 amino acids. Blast comparison was carried out on NCBI. The gene had higher homology compared with the DFR from other species. The sequence of DFR gene was cloned from C. morifolium. Quantitative PCR showed that the gene expressions were different at different stages of flower bud differentiation period, and in different periods of flowering in different tissues. The highest expression of DFR gene was at day 50 of stages of flower bud differentiation period; And in flowering the highest expression of DFR gene is in tubular flowers in the period I and the lowest expression is in the receptacle in period Ⅱ. Conclusion The DFR gene of C. morifolium cv.‘Hangju’is cloned. The expression of DFR gene is different both in time and in space. The highest expression is in the period from the buds mature to the capitulum open completely.

国家自然科学基金资助项目(81503180)