Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju' / 中国中药杂志

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using β-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

Prokaryotic expression and in vitro enzyme activity analysis of flavonol synthase in Chrysanthemum morifolium cv. 'Hangju' / 中国中药杂志

We cloned flavonol synthase gene (named as CmFLS) by RACE from Chrysanthemum morifolium cv. 'Hangju' based on transcriptome database. Sequencing results showed that 1 235 bp sequence was acquired with the largest open reading frame (ORF) of 1 008 bp, which encoded 335 amino acids. The predicted CmFLS encoded protein had an isoelectric point (pI) of 5.41. The phylogenetic tree analysis indicated that CmFLS was highly homologous to other FLSs, which identified from the species of Compositae. The recombinant fusion protein, with a molecular mass of 43 kDa, was successfully expressed by prokaryotic expression system. Meanwhile, Ni-NTA resin was used to purify the recombinant fusion protein, and the Ni-Native Buffer containing 250 mmol·L⁻¹ imidazole was most favorable for elution. The purified recombinant fusion protein was subjected to in vitro catalytic reaction, and then the products were extracted and analyzed by HPLC. The results showed that the recombinant fusion protein CmFLS was able to catalyze the production of quercetin by dihydroquercetin under specific buffer and reaction conditions, which indicated that the functional protein encoded by CmFLS had dioxygenase activity in the biosynthetic pathway of flavonoids biosynthesis in Ch. morifolium cv. 'Hangju'. The above results laid the foundation for further studying on CmFLS, and provided new ideas for the regulation of flavonoids metabolism from the molecular level and the catalytic synthesis of flavonols in vitro.

Effects of flood stress on anthocyanins synthesis with relative enzymes and genes of Chrysanthemum morifolium cv. 'Hangju' / 中国中药杂志

The study is aimed at determine the content of anthocyanins and expressions of relative genes and activity of relative enzymes. The effects of flood stress on anthocyanins synthesis with relative genes and enzymes of Chrysanthemum morifolium cv. 'Hangju' were analyzed. The expression of CHS and the content of CHS presented the trend of first rising and after downward with the increase of flowering degree. The content of anthocyanins, the expression of DFR and the activity of DFR presented the trend of first downward and after rising with the increase of flowering degree. There was a positive correlation among anthocyanins,DFR gene and DFR. However there was no significant correlation among anthocyanins,CHS gene and CHS. Flood stress has significant effects on anthocyanins synthesis with relative genes and enzymes of Ch. morifolium cv. 'Hangju',but don't change the patterns of genes expression and anthocyanins and enzymes accumulation. DFR and DFR are the key gene and key enzyme of anthocyanins synthesis.

Analysis on cloning of dihydroflavonol 4-reductase gene in capitulum of Chrysanthemum morifolium cv. 'Hangju' and its expression characteristics / 中草药

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 1 152 bp sequence was acquired with the largest open reading frame of 1 029 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.