Cloning And Characterization Of Two MYB Transcription Factor Genes From Soybean(Glycine Max[L.]Merr.)

Soybean can provide abundant protein and oil, it also act as the main source of varies isofalvone compounds. Isoflavones are considered to play diverse roles in plant-microbe interaction and also have great potential to human nutrition and health. Soybean isoflavones are a class of secondary metabolites of the phenylpropanoid pathway which exists throughout the whole plant system. Many researches have approved that lots of MYB transcription factors could regulate the biosynthesis of isofalvones in plants. MYB transcription factors are the largest transcription factor family in plant, their functions in secondary metabolism, environmental stress, cell differentiation, cell cycle and morphogenesis of organs are all well known now. To further confirm the function of MYB transcription factors in the regulation of isofalvone biosynthesis, we cloned and characterized two MYB transcription factors in soybean.In this study, we cloned a novel MYB transcription factor from soybean (Jilin 32) and named GmMYB12B2 (GenBank accession number:JF510467). We also cloned the GmMYB12a. The function of them in regulation of isoflavone biosynthesis was discussed. The main results are as follow:1. We cloned the GmMYB12B2 transcription factor using Reverse Transcript-PCR method from soybean (Jilin32). It contains 783 nucleotides and encodes 260 amino acids, it contains two MYB domains according to sequence alignment and belongs to typical R2R3-MYB transcription factor. We also cloned the GmMYB12a.2. We constructed the pET28-GmMYB12a and pET28-GmMYB12B2 plasmids and transformed them into E. coli Rosetta (DE3). The proteins were induced in 28℃for 6 hours by 1.2 mMIPTG..3. We analysised the expression levels of GmMYB12a and Gm MYB12B2 under salt, low temperature, drought, ABA and UV radiation treatments using semi-quantitative RT-PCR. The expression of GmMYB12a and Gm MYB12B2 are all induced by UV radiation and salt treatment dramatically, especially GmMYB12B2. They are not induced by low temperature, drought and ABA treatment. 4. We constructed yeast effect plasmids pGBK7-GmMYB12a and pGBK7-GmMYB12B2, and then they were transformed into the yeast strain AH 109. The positive transformants were selected using nutrient-deficient medium SD/-Trp/-Ade /-His with 3-AT. The result showed that GmMYB12B2 have transcriptional activation, GmMYB12a have no or weak transcriptional activation.5. We constructed pBI121-GFP-GmMYB12a and pBI121-GFP-GmMYB12B2 plasmids, and then they were transformed into onion epidermal cells using Agrobacterium-mediated method. The subcellular location showed that they are all located in the nucleus.6. Real-time RT-PCR was carrying out to analyze the tissue-specific expression of GmMYB12B2 and GmMYB12a in soybean. The GmMYB12B2 transcripts were higher in root and mature seed than other organs. GmMYB12a have the same expression pattern as GmMYB12B2, but its expression levels were lower than GmMYB12B2.7. The contents of isoflavones were detected in different tissues using HPLC method. The results showed that the isoflavones contents in following organs were:soybean seed> 60d embryo> 50d embryo> 40d embryo> flower> pod> 20d embryo> 30d embryo> leaf> root> stem. The accumulation of isoflavones in immature embryos increased during plant growth. This result is consistent with the genes expression levels.8. pCAMBIA1301-CHS8P. PPZP-GmMYB12a and PPZP-GmMYB12B2 plasmids were constructed and transformed into soybean callus cells. CHS8 is the key enzyme in plant flavonoid biosynthesis, the cotransformation of MYBs and CHS8 will confirm the interaction of them. The results showed that the cells that cotransformed of pCAMBIA1301-CHS8P and PPZP-GmMYB12B2 have the highest GUS flurescence. It proved that GmMYB12B2 could regulate CHS8 to produces more fluorescence.9. We constructed PPZP-GmMYB12a and PPZP-GmMYB12B2 plasmids, and then they were transformed into Arabidopsis thaliana. The expression levels of some key enzymes in flavonid biosynthesis were dectected in T3 progeny. The results showed that:the expression levels of PALI, CHS and FLS were significantly higher than wild-type and vector in GmMYB12B2 transgenic lines, while the expression levels of CHI. F3H and F3'H is same in all lines. The expression level of DFR is lower than WT and vector in GmMYB12B2 transgenic lines. GmMYB12a have the same expression pattern as GmMYB12B2 in GmMYB12a transgenic lines, but its expression levels were lower than GmMYB12B2. These results proved that GmMYB12B2 and GmMYB12a are all regulate the flavonid biosynthesis. The transgenic lines of them have more tolerance during salt and UV radiation treatment.