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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1 152 bp的杭菊DFR基因,最大开放阅读框1 029 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c DNA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 gene of dihydroflavonol 4-reductase (DFR) from Chrysanthemum morifolium and to study its expression pattern at different stages of flower bud differentiation and flowering. Methods According to the published DFR gene sequences of Chrysanthemum morifolium, primers were designed and amplified from the flower buds of Chrysanthemum morifolium by fluorescence quantitative PCR (RT-PCR). The positive clones were sequenced and spliced. The relative quantitative PCR method based on the reference gene was used to analyze the gene expression characteristics. The full-length 1 152 bp Chrysanthemum morifolium DFR gene was obtained, with the largest open reading frame of 1029 bp encoding a total of 342 amino acids. Blast alignment was performed on NCBI, which has high homology with other plant DFR genes. The results of RT-PCR showed that there were differences in the expression of this gene in different stages of flower bud differentiation and in different parts of flower at different stages of flowering. In the flower bud differentiation stage, Chrysanthemum indicum DFR gene had the highest expression level at 50 days. In flowering stage, the expression level of DFR gene was the highest in tubular flower of period I and lowest in inflorescence of stage II. Conclusion The c DNA sequence of DFR gene from Chrysanthemum morifolium was cloned. There was a time and space difference in the expression of DFR gene between Chrysanthemum morifolium and Chrysanthemum morifolium. The results laid the foundation for the further study on the relationship between the expression of this gene and the catalytic products in Hang Ju and its regulatory mechanism.