Cloning And Expression Of MYB Transcription Factors Involved In Flavonoids Synthesis In Tea [Camellia Sinensis (L.) O. Kuntze]

Tea polyphenols are the main secondary metabolites of flavonoid that accumulates in tea plant ([Camellia sinensis (L.) O. Kuntze]), and play important physiological and biochemical roles. Structural genes coding interrelated enzymes and some transcription factors are involved in flavonoid biosynthetic pathway. Transcription factors can regulate the expression of functional structure genes. MYB transcription factor is one of the largest families and can be involved in phenylpropanoid secondary metabolic pathway.In order to clarify the function of MYBs in flavonoid biosynthetic pathway in tea plant, three MYB genes were full-RACE cloned and their bioinformatics analyses were performed. Different expression preferences of the three genes were analyzed by real-time fluorescent quantitative PCR. Recombinant plasmids of pET32a-CsMYB4-5, pET32a-CsMYB4-6and pET32a-CsMYB4-7were correctly constructed and successfully expressed in prokaryotic cells. Agrobacterium tumefaciens-mediated plant experssion vectors were finally constructed and had been transformated to Arabidopsis for futher gene functions.The main findings are as follows:1. Candidate genes were screened from NCBI and tea transcriptome in involved in flavonoid biosynthetic pathway. Full length sequences of CsMYB4-5, CsMYB4-6and CsMYB4-7have been cloned by RACE technology.2. Bioinformatic analysis showed that:CsMYB4-5, CsMYB4-6and CsMYB4-7proteins were all unstable proteins and hydrophilic proteins; analysis of the amino acid sequences showed that there were no signal peptides exist within them and they may localized in the nucleus; analysis of the secondary structure showed that each of the three predicted protein has the mainly a-helices and links with each other by long-chain corner, which conforms the helix-turn-helix structural characteristic of MYB domain. Further analysis showed that the three predicted proteins are all have very simple tertiary structures, with six a-helices wounding in random coil lines; CsMYB4-5showed high homologous to AmMYB330(48.45%), the highest level of homologies of CsMYB4-6were observed with the AmMYB308and AtMYB4(69.80%and62.41%, respectively), CsMYB4-7showed64.12%consistency with DkMYB4. CsMYB4-5and CsMYB4-6could be clustered with AtMYB3, AtMYB4and AtMYB7belongs to subgroup4.3. Real-time fluorescent quantitative PCR was performed to identify the different experssion levels in different organs and different matural leaves. The results showed that CsMYB4-5and CsMYB4-6both had high expression level in roots and low expression level in stems. But the CsMYB4-7had low expression level in roots and a high expression in stems. Meanwhile, real-time fluorescent quantitative PCR was performed to identify the different experssion level under the treatment of woud and plant hormones GA and ABA. The results showed that only CsMYB4-7had more than2-fold expression by GA, ABA and wond stimulation.4. Recombinant plasmids pET32a+-CsMYB4-5and pET32a+-CsMYB4-6were constructed then transformed into expression host E.coli rosetta (DE3). The target proteins were expressed by IPTG induction.5. Recombinant plasmids pCB2004-CsMYB4-5, pCB2004-CsMYB4-6and pCB2004-CsMYB4-7were constructed then transformed into Agrobacterium tumefaciens C58C1and EHA105by electroporation. PAP1mutant of Arabidopsis and tobacco were successfully transformed.