Cloning And Functional Verification Of Two MYB Transcription Factors In Tea Plant [Camellia Sinensis (L.)]

Phenylpropanoid metabolism is an important pathway involved in the synthesis of multiple secondary metabolites like lignin, coumaric acid esters and flavonoids. Some members of MYB proteins,a large and functionally diverse superfamily of regulatory transcription factors, participate in the regulation of phenylpropanoid metabolism.This study reports that two R2R3-MYB transcriptor factors in subgroup 4 were isolated from tea plant. The differential expressions of the two genes were analyzed by Real-time fluorescent quantitative PCR; and the functions of the two genes were studied by using tobacco and arabidopsis genetic transformation system.The main results are as follows:1. Firstly, by homology search, the candidate ESTs of R2R3-MYB transcription factors probably involved in the regulation of phenylpropanoid metabolism were screened from NCBI database and Anhui Agricultural University transcriptome of Camellia sinensis. The ORFs of two candidate R2R3-MYB transcription factors were cloned by RACE.2. Based on bioinformatics analysis,the two R2R3-MYB transcription factors, predicted to have the regulation function on the biosynthesis of polyphenols, were in sg4 among the 22 R2R3-MYB subgroups (C2 repressor motif clade), and named CsMYB4-5 and CsMYB4-6, respectively. The comparison of amino acid sequences by DNAMAN showed that CsMYB4-5 displayed 48.45% and 44.79% sequence identity with AmMYB330 and AtMYB3 respectively while CsMYB4-6 69.80% to AmMYB308 and 62.41% to AtMYB4.3. A Real-time quantitative PCR approach was performed to study the tempospatial differential expressions of the two genes in tea plants under different induction conditions. The results showed, CsMYB4-5 and CsMYB4-6 were both expressed low in the stem and high in roots, but with the development of leaves, the expression of two genes did not follow the same trend; the expressions of CsMYB4-5 and CsMYB4-6 were slightly influenced when treated by exogenous plant hormones GA and ABA or wound4. Genetic transformation system of tobacco was applied to verify the functions of CsMYB4-5 and CsMYB4-6. The results showed that compared with the wild-type untransformed tobacco, the growth of tobaccos expressing with CsMYB4-5 and CsMYB4-6 were inhibited different degrees, and over-expression of CsMYB4-6 exhibitted a stronger inhibition effect.Different tobacco lines transformed with CsMYB4-6 appeared more significant phenotype with the rise of CsMYB4-6 expression levels. The main phenotype were as follows:plant dwarf; austerity and uneven leaf veins; white spots in mature and senescent leaves (isolated dead cells); lowly developed roots and unobvious taproot; the flower color of transformative tobacco lines was decreased with the increasing expression level of CsMYB4-6.In the regeneration process, the tobacco callus of transformation CsMYB4-6 turned yellow and growth was significantly slower than that of wild type; besides, its second generation seed planting appeared distinctly short, yellowing, short roots compared with the wild type tobacco seedlings II.Microdissection observation displayed that glandular hairs of tobacco leaves transformation with CsMYB4-6 turned shorter and decreased; cells filled between the upper and lower leaf epidermal were not full with most part empty, by hloroglucinol staining, red color of stem cross-section faded which indicated that the lignin content decreased.The characterization of polyphenols content showed that lignin content in tobacco leaf transformation with CsMYB4-6 reduced by 36.1%-44.4% compared to the control group and cellulose reduced by up to 15.1%; anthocyanin content of high expressed tobacco lines decreased by 47.7% while increased 38.3% in low expressed tobacco lines.To analyze the expression difference of the leaves transformation with CsMYB4-6 of tobacco by Real-time quantitative PCR, the expression of genes COMT、 CCR、CAD、C4H、4CL involved in lignin biosynthesis pathway were all down-regulated, among which 4CL was most remarkable; genes PAL、C4H、CHS、 CHI、DFR、LAR、ANR、ANS concerned with flavonoid biosynthetic pathway in tobacco flowers were all down-regulated while up-regulated in tobacco lines with lower CsMYB4-6 expression.5. Arabidopsis genetic transformation system was used to verify the functions of CsMYB4-5 and CsMYB4-6. The results showed that the growth of papl seedlings transformed with CsMYB4-6 was significantly inhibited; the leaves turned dark green; red and purple color increased both in leaf and stem; the content of anthocyanin went up by 115.0%.