Studies On Cloning And Expression Of McMYB16and McMYB12Genes In Malus Crabapple

Anthocyanin is one type of flavonoid widely existing in plants, and is a plantpigment from red to blue, to purple. Anthocyanin plays a critical role in the formationof flower, leaf and fruit colors. There has been intensive study on its metabolicpathway. The biosynthesis of plant anthocyanin is mainly regulated by two types ofgenes: structural genes which directly encode the enzymes in its synthetic route andare shared by different plants; and regulatory genes which regulate the expressingactivity and degree of structural genes. It is the latter's regulation on the former'sspatio-temporal expression that decides the occurrence and distribution ofanthocyanin synthesis. In particular, MYB protein is the most extensive regulatorduring the synthetic route of plant anthocyanin, so the cloning of MYB transcriptionfactor is critical for studying the synthetic route of anthocyanin and the formationmechanism of plant colors.Using the total leaf RNA from the Malus crabapples cultivar 'Royalty' astemplate, we cloned the sequence of one1350bp MYB gene cDNA by means ofRT-PCR and RACE amplification. The length its coding region is768bp, with255amino acids to be coded, and this gene was named McMYB16. Multiple sequencealignment and phylogenetic tree were used for protein analysis for McMYB16.Real-time quantitative PCR and HPLC were used for measuring and analyzing theexpression level of McMYB16and McC4H, as well as anthocyanin content, for Maluscrabapples cultivars 'Royalty',' Radiant ' and 'Flame' with different leaf colors and atdifferent times. The results show that: the homologies of conserved protein domainsof McMYB1with apple MdMYB16and Arabidopsis AtMYB4were94.12%and61.13%, respectively. McMYB16was located at the same branch with MdMYB16and was related to AtMYB4. McMYB16was correlated negatively to the expressionlevel of McC4H and to anthocyanin content. Presumably, this gene is a reversetranscription factor in the synthetic route of phenylpropane, and restrains itsdownstream route by regulating the expression opening and closing of the keyenzyme gene McC4H in the public phenylpropane route.Using the total leaf RNA from the Malus crabapples cultivar 'Royalty' as template,we cloned the sequence of one1632bp MYB gene cDNA by means of RT-PCR and RACE amplification. The length of its coding region is1047bp, with348amino acidsto be coded, and this gene was named McMYB12. Multiple sequence alignment andphylogenetic tree were used for protein analysis of McMYB12. The results show that:McMYB12was related to AtMYB12, and its tertiary protein structure was identical toAtMYB12. It was stably expressed in the selected Malus crabapples cultivars at anyperiod. In the new colored leaf category ' Radiant ' and in the green leaf category'Flame', its relative expression level was negatively correlated to anthocyanin content.Presumably, this gene was related to the metabolic pathway of flavonoid, and was aspecific gene in the metabolic and synthetic route of flavonols, regulating the similarfunctions as AtMYB12.