Cloning And Transformation Of A R2R3 MYB Gene And Analysis Of Its Chlorophyll Fluorescence Parameters And Flavonoid Content In Erigeron Breviscapusus

Erigeron breviscapusus(Vant.)Hand.-Mazz,as known as herb Eerigerontis,is a kind of plant belongs to Erigeron of compositae.And it's the main effective medicinal chemical compositions are flavonoids.Due to the specific physiological activity and the diversity of the biological functions,the secondary metabolic product research of the flavonoids plants has been researched as a popular topic.Flavonoids are produced mainly through the pathway of metabolism of flavonoids,which are mainly in the pathway of phenylpropanoid metabolism and its deriveed metabolic sub-pathways.Based on the earlier transcriptome measuring of Erigeron breviscapus,we firstly filtered an annotation about the R2R3 MYB gene and cloned the full-lenth cDNA using the RACE technology.Then we built an expression vector which is fused with the GFP gene.And transform expression vector into the Erigeron breviscapus via agrobacterium-mediated which lead the over-expression about MYB gene in transgenic plant.At last,we measured the chlorophyll fluoresce and the metabolite about the transgenic plant and got the specific conclusion:We filtered a gene which may associates with the flavonoids metabolism and named it EbMYB06.Then we analyzed the physical and chemical characters of the protein in EbMYB06.And the result shows that the protein molecular mass is63.80 KDa and the electric point is 5.18.And the atom of coded polypeptide is C2377H3976N780O996S129 and the instability index 35.45.The results show that the protein is stable.The aliphatic inde is 33.21 and the GRAVY is 0.721.And the results show that it's a hydrophobic protein.Using the ProScale to analyze and predict the protein hydrophobicity,we can get that the 369 th is Thr,the 428 th is Pro and the hydrophobicity is 2.189.We can know that the hydrophobicity is the strongest.We can also know that the 715 th is Gly,the 717 th is Leu and the hydrophily is-0.600.It's the strongest.We use the TMpred to analyze the trans-membrane helix of the protein but didn't find the transmembrane structures and areas.And we find phosphorylation sites on a serine,a threonine and 2 tyrosines.And we find a brokensite of the signal peptide between 30 th amino acid and the 31 st amino acid.The secondary structure shows that this protein is composed of 46.15% random ccoil and43.85% helix.And the genealogical tree shows that the EbMYB06 and the AtMYB4?7?32?6?8 have the closest evolutionary relationship.And the EbMYB06 and the AtMYB111?11?12make up a neighboring group.So we can infer that the EbMYB06 and the AtMYB4?7?6?8?32 have similar functions and have relation to the generation of flavonoids.We do the hormones and antibiotics concentration related experiments,find the most suitable for Erigeron breviscapus culture methods is added 0.3 mg/L 6-BA and1.0 mg/L NAA,eventually we choice kanamycin and cephalosporin of 5 mg/L and200mg/L.And cloning of the gene EbMYB06 into the plants by agrobacteriummediated method,to make the MYB gene over-expression,experiment received 6plants of Erigeron breviscapus,and we measure the chlorophyll fluorescence and content of flavonoids and total phenol.Found that the total phenol and flavonoids content have obviously improved than the wild Erigeron breviscapus.And we analysis th chlorophyll fluorescence parameters,find that qP of genetically modified plants is higher than the wild plants,show that enetically modified plants absorb a higher proportion of energy used for photochemical reaction.And Fv/Fm value of genetically modified Erigeron breviscapus is lower than the wild,Fv/Fm value is a light inhibition index,maximum photochemical efficiency of PS? under dark adaptation,plant under the condition of adversity,often accompanied by strong light,prone to photoinhibition,Fv/Fm value decreased obviously,speculated that make the increases of the flavonoids content.According to the data we fonnd enetically modified plants contain the chlorophyll b content and ratio of chlorophyll a and chlorophyll b is higher than the wild,and the ratio of chlorophyll a and chlorophyll b and photosynthetic rate showed a negative correlation.Experiments show that the overexpression of EBMYB06 leads to the increase of chlorophyll b content of transgenic Erigeron breviscapus and the increase of flavonoid production.But the reason for the increase in flavonoids is not clear,and it is necessary to further study the formation of flavonoids.