Cloning And Primary Characterization Of ChiMYB In Chrysanthemum Indicum

The medicinal value of the herb Chrysanthemum indicum has been recorded for hundreds of years,which possess particularly potent effects on bacteria and viruses as well as having antioxidant,anti-inflammatory,and immunomodulatory properties.Chrysanthemum indicum is a garden application plant with yellow flowers,strong resistanced and is easy to breed.Genetic engineering research is of great significance to improve the ornamental and quality traits of ornamental plants,and to cultivate new varieties of Chrysanthemum indicum.MYB transcription factor plays an important role in the regulation of plant growth and development,The regulatory potential of ChiMYB has been studied inNicotiana tabacum by developing transgenic plants constitutively expressing ChiMYB.The ChiMYB gene was cloned by using the specific primers obtained from the earlier stage transcriptome data and the plant expression vector was constructed and transferred into Nicotiana tabacum,and the phenotype and photosynthetic characteristicsanalysis of all lines was carried out.The preliminary result shows that the expression of photosynthetic enzyme gene and photosynthetic pigment gene were inhibited on the photosynthetic pathway and the synthesis of related enzymes and pigments were blocked by ChiMYB gene,which resulting in a decrease in net photosynthetic rate.That is,the ChiMYB gene has the function of a transcription repressor.Not only do we hope to play the multi-channel value and improve the quality of Chrysanthemum indicum,but also lay the foundation for Chrysanthemum indicum molecular breeding.The results are as follows:1.The total RNA of leaves of Chrysanthemum morifolium was used as template,a MYB transcription factor gene was amplified by RT-PCR,named ChiMYB.The amino acid sequence encoded by ChiMYB gene was analyzed through bioinformatics method.The results show that: the ChiMYB cDNA was about 1200 bp,and the open reading frame(ORF)was 948 bp,encoding a protein of 315 amino acid.There was a specific conserved sequence,SANT superfamily,a SANT domain,with function of the binding domain.And the ChiMYB gene may have the function of transcriptional inhibitory factor.Phylogenetic tree revealed that the close relatives of ChiMYB were MYB from Dendrathema grandiflora and Dendranthema indicum var.aromaticum,followed by the homology of the homologous domain of the Cynara cardunculus var.scolymus.2.We constructed the plant expression vector of pBI121-MYB with the method of double enzyme digestion.The pBI121-MYB recombinant plasmid and pBI121 empty vector plasmid were successfully introduced into Agrobacterium tumefaciens EHA105 by electroporation.ChiMYB gene and pBI121 into tobacco plants through Agrobacterium-mediated transformation.The results showed that the positive lines of ChiMYB gene and pBI121 empty vector had been successfully obtained by resistance detection and PCR detection of resistant plant DNA.Three transgenic lines expressing ChiMYB and one transgenic pBI121 lines were selected.3.There are a series of changes in phenotypic and photosynthetic characteristics of ChiMYB expressing tobacco lines(L1,L2,L3)compared with wild type and empty vector.The leaves of the transgenic lines showed a yellowish color,and the leaf area decreased significantly,and the stems were laterally bent,and the sprouts side buds.The maximum net photosynthetic rate,stomatal conductance,transpiration rate and apparent quantum efficiency of transgenic lines were significantly lower than those of WT and EV,and light saturation point drop is not significant.The chlorophyll a,chlorophyll b,total chlorophyll and carotenoid contents of the transgenic lines were significantly decreased(the average values were53.8%、46.5%、52.1%、49.1% less thanWT and 47.6%、46.1%、47.2%、48.5% less than EV)and the difference of chlorophyll a / b between the transgenic lines and wild type and empty vector was not significant.There was no significant difference in initial fluorescence,maximum fluorescence,maximum photochemical efficiency of PSII,and the actual photochemical efficiency of PSII between the transgenic lines and wild type and empty vector.Only the nonphotochemical quenching coefficient of chlorophyll fluorescence parameters decreased significantly(the average values were 23.8% less than WT and 23.3% less than EV),and the photochemical quenching coefficient decreased but the difference was not significant.