Cloning, Sequential Analysis Of VvmybA1 Genes And Its Expression Pattern During Grape Berry Development

MybA1 is a key factor that regulates UFGR which is the main enzyme of anthocyanin's synthesis.I select Cabernet Sauvignon as test material.Firstly,using PCR amplification producted whole gene sequence of MybA1,and than got recombinant plasmid pET-mybA1 by building cloning vector and expression vector.by IPTG inducing and Ni-NTA affinity purificating,regarding this protein as antigen immune New Zealand White Rabbit,I received the polyclonal antibody of MYBA1 which has high titer and high specificity. Using western blot skill,I studied the regulation of protein MYBA1 on each period after anthesis and each tissues of Cabernet Sauvignon.The instantaneous expression rule about each period after anthesis and each tissue of Cabernet Sauvignon was studied by fluorescent quantitation.At last,I made the correlation analysis of MybA1 and other genes LAR1?LAR2?DFR? ANS?ANR?UFGT; each gene and anthocyanin content.The experimental results as follows:(1) Achieving whole gene sequence of MybA which has intire ORF,and the length is 751bp.It codes polypetide comprised by 250 amino acids,molecular weight is about 28kDa.Successfully built recommended expression vector pET-mybA1.(2) Biological information of VvmybA1 is:MYBA1 is hydrophily,pitch outside of membrane,belongs to SNAT supergene familly;Its secondary structure is composed of Alpha helix,Random coil, Extended strand and Beta turn;ts tertiary structure shows that MYBA1 is typical R2R3-MYB protein;By contrasting the homology with other species,it reserves a high homology between Cabernet Sauvignon and Piasezkii,those species which are not Vitaceae has low homology with grape.(3)I achieved high putity protein on the condition of 0.6mmol/LIPTG,37??5h.And got high purity protein MYBA1 by Ni-NTA affinity purification. Regarding this protein as antigen I received polyclonal antibody of MYBA 1 which has well titer (1:512000) and specificity.(4) Cabernet Sauvignon fruit's anthocyanin content variation is ciimb up and then decline during days20-110 after anthesis.Having no accumulation before veraison,begin accumulate with the time of version and to the top when 80 days after anthesis,and then reached a steady.(5) Analysis result of MybA1 expression is:Its expression outclass other tissues.Its expression pattern in fruit is:hardly express before veraison,expression start with veraison and to the top when 80 days after anthesis;The western-blot result of MYBA1 is:MYBA1 expression in leaves is the highest,in fruit is lowest and almost in root,bud,stem and tendril.Its expression pattern in fruit is:hardly express before veraison,expression start with veraison and to the top when 70 days after anthesis.(6) Correlation analysis shows that MYBA1 expression and MybA1mRNA,ANSmRNA,DFRmRNA chave positive correlation;MYBA1 expression and UFGTmRNA have significant positive correlation.MybA1mRNA content and DFRmRNA?UFGTmRNA have positive correlation;MybA1mRNA content and ANSmRNA content have significant positive correlation.Anthocyanin content and MYBA1 expression,ANSmRNA content have positive correlation;Anthocyanin content and MybA1mRNA? UFGTmRNA?DFRmRNA have significant positive correlation.