Cloning And Functional Analysis Of Transcription Factors CMMYB1and CMMYB2in Chrysanthemum

The growth and metabolism of plants are regulated by controlling the expression of targeted gene on the transcript level. MYB transcription factor gene is one of the largest families in plants, which involved in regulation of secondary metabolism, responding to hormones and environmental stresses, also regulation of cell differentiation and organs morphogenesis. Chrysanthemum morifolium is one of ten most famous flowers in China and one of the top four cut flowers in the world with high ornamental and practical value. It occupies an important position in flower production. However, the discovery of new genes and the methods for molecular breeding of Chrysanthemum just started. In this study, two genes of MYB transcription factors of Chrysanthemum were cloned and characterized. These are expected to provide some basic theories for further research of MYB transcription factors on regulation of plant secondary metabolism and responding to abiotic stresses. The main results are as follows:1. Two homologous MYB genes were screened from the EST libraries of Chrysanthemum and amplified by3’and5’RACE to obtain the full-length cDNA of CmMYB1(JF795917) and CmMYB2(JF795918). CmMYB1contains an846bp ORF, encoding a281residue peptide, with a95bp5’untranslated region and a296bp3’ untranslated region with a poly(A) tail. CmMYB2contains an948bp ORF, encoding a315residue peptide, with a86bp5’untranslated region and a297bp3’untranslated region with a poly(A) tail. Comparison of deduced amino acid sequences showed that the two genes are typical R2R3-MYB transcription factors and belong to the subgroup4and22, respectively. Then the full-length DNA sequences of CmMYB1and CmMYB2were also amplified using genomic DNA as templates. The results indicated that CmMYBl contains a307bp intron, whereas the gene CmMYB2lacks intron.2. By TAIL-PCR and5’RACE, full-length promoter sequences of CmMYB1and CmMYB2were obtained and submitted into promoter database for element forecast analysis. The results showed that there were some elements for response to ABA, drought and low temperature.3. Recombinant vectors pGBKT7-CmMYB1and pGBKT7-CmMYB2were constructed and transformed into the yeast strains Y2H. The positive yeast transformants were selected using troptophan-deficient medium SD/-Trp, then transformed on SD/-His-Ade medium. The results showed that CmMYB1and CmMYB2have no significantly transcriptional activity.4. Quantitative real-time PCR (qRT-PCR) showed CmMYB1and CmMYB2were expressed in roots, stems, leaves and flowers of plants with the highest expression in stems. CmMYB1has higher expression level at the early opening and full color of the flowers, but little level in roots. The transcript product of CmMYB2was higher in leaves, but lower in roots and at any time of the flowering. The results from the responses to abiotic stress showed that the expression of CmMYB2increased rapidly in response to drought, salinity or cold stress within1h, while the expression of CmMYB1was induced only under the low temperature condition.5. The green fluorescent protein expression vectors pBI121-GFP-CmMYB1and pBI121-GFP-CmMYB2were constructed and transformed into the epidermal cells of onion by Agrobacterium-mediated transformation system. The results of instantaneous expression showed that CmMYB1and CmMYB2proteins were both localized in cell nucleus.6.CmMYB1and CmMYB2were transformed into Arabidopsis thaliana (Col-0) with Agrobacterium EHA105containing the expression vectors pCAMBIA1301-CmMTB1and pCAMBIA1301-CmMYB2, respectively. The positive Arabidopsis transformants were selected using MS medium containing hygromycin resistance, GUS activity assay and RT-PCR. There was no phenotypic difference between the CmMYB1transgenic Arabidopsis and the wild-type plants. The qRT-PCR analysis indicated that CmMYB1could down-regulate some important genes involved in phenylpropanoid pathway and lignin biosynthetic pathway, such as CAD (cinnamyl alcohol dehydrogenase)、COMT (caffeic acid o-methyl-transferase)、C4H (cinnamate4-hydroxylase) and4CL1(4-coumarate-CoA ligase), resulting the decrease of lignin content. The transgenic Arabidopsis overexpressing CmMYB2exhibited delayed flowering and the increasing of leaves number compared with the wild-type plants. Results of abiotic-stress experiments showed that heterologous expression of CmMYB2could enhance drought and salt tolerance of Arabidopsis. In line with the above findings, the expression levels of several stress-responsive genes RD22^RD29A、RAB18、COR47、ABA1and ABA2were strongly increased, while the expression of genes related to flowering, such as CO (CONSTANS)、FT (FLOWERING LOCUS T)、 SOC1(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1)、LFY(LEAFY) and AP1(APETALA1) reduced.