Isolation And Characterization Of MYB Transcription Factors, GmMYBJ1and GmMYBJ2, From Soybean (Glycine Max L.)

Soybean is a significant crop providing humans and animals with abundantproteins and vegetable oil. However, with the rapid development of the society andthe global climate change, its growth and development were increasingly affected bydiverse abiotic stresses, such as drought, cold, soil salinization and disease. Thesestresses may severely affect their growth and development, causing great financiallosses. Recent studies show that MYB transcription factors are involved in responseto biotic and abiotic stresses. In this study, two novel MYB transcription factors,designated as GmMYBJ1and GmMYBJ2respectively, were isolated from soybeanJilin32and made further research for their roles. This study also enriches the generesource of soybean MYB. The main experiment results are as follows:1. Two new MYB genes, GmMYBJ1and GmMYBJ2, were isolated fromsoybean Jilin32using RT-PCR method. GmMYBJ1has an ORF of816bp whichencodes a putative protein of271amino acids, GmMYBJ2has an ORF of960bpwhich encodes a putative protein of319amino acids. Both GmMYBJ1andGmMYBJ2predicted proteins contained two conserved MYB domain and belong totypical R2R3MYB transcription factors.2. The green fluorescent protein fused expression vectors of GmMYBJ1andGmMYBJ2were constructed by SOE-PCR, and then were introduced intoAgrobacterium tumefaciens strain EHA105via Agrobacterium-mediated method fortransient transformation of onion epidermal cells. The results indicated GmMYBJ1and GmMYBJ2were both localized in nucleus and are correspond with their role astranscription factors.3. pGBKT7-GmMYBJ1and pGBKT7-GmMYBJ2were constructed and were expressed in yeast to analyze their transcriptional activation. The results showed thatGmMYBJ1had no or weak transcriptional activation ability in yeast, on the contrary,GmMYBJ2had transcriptional activation ability in yeast which suggested it is atranscription activator.4. The expression patterns of GmMYBJ2and GmMYBJ2were analyzed usingquantitative RT-PCR. The results indicated that GmMYBJ1and GmMYBJ2responded differently to abiotic stresses such as drought, cold, high salt and ABA.5. The plant expression vector pCB35SR1R2-GFP-GmMYBJ1was constructedusing GATEWAY clone technology. Then the construct was electroporated intoAgrobacterium tumefaciens EHA105and transformed into ArabidopsisCol-0usingfloral dipping method. The seed germination rates of GmMYBJ1-transgenicArabidopsis were significantly higher than that of wild type Arabidopsis. Fordehydration treatment in soil, WT and transgenic plants were subjected to droughtstress for16days by withholding water and then were re-watered for10days, thetransgenic lines showed normal growth compared with WT. Meanwhile,the plantsurvival and height were counted and compared.The results indicated that the plantheight and survival rates of most transgenic lines were remarkably higher comparedwith the WT plants. The MDA contents increased in WT and transgenic plant after24h PEG treatment.However, less MDA contents were observed in transgenic plantthan those in WT. The water loss rate of transgenic plants was lower than that of WTplants within12h. Soil-grown seedlings were placed at4oC for7d, then normalconditions were provided for recovery, After recovery for15days, the WT plantsexhibited nearly all etiolated and withered. On the contrary, the transgenic linesshowed pretty better growth. Subsequently, the plant height and survival weremeasured and compared, the results indicated that the transgenic plant showedsignificant difference in height and survival compared to WT.The soluble sugar wasmeasured and the results showed that the level of soluble sugar was markedly higherin transgenic lines than that in WT. Semi-quantitative RT-PCR was used to examinethe level of downstream genes. The results showed that some stress-responsive geneswere altered in transgenic plants. The expressions of RD29B, COR47, COR78, COR15a and P5CS were up-regulated in overexpressing-GmMYBJ1plants. However,the expression level of DREB2A was slightly down-regulated in transgenic plantscompared with WT plants. All these results showed that overexpression ofGmMYBJ1enhanced the tolerance of transgenic plants to cold and drought.6. The plant expression vector pCB35SR1R2-GFP-GmMYBJ12wasconstructed using GATEWAY clone technology. Then the construct waselectroporated into Agrobacterium tumefaciens EHA105and transformed intoArabidopsisCol-0using floral dipping method. The seed germination rates ofGmMYBJ2-transgenic Arabidopsis were significantly higher than that of wild typeArabidopsis.The transgenic plants exhibited better growth compared with WT after19days by withholding water. The soluble sugar and excised-leaf water loss ratewere measured， the results demonstrated the content of soluble sugar wassignificantly higher in transgenic lines than that in WT and the water loss rate waslower compared with WT within10h. In addition, some stress-responsive geneswere up-regulated in transgenic plant. These results to some extent showedoverexpression of GmMYBJ2could enhance the tolerance of transgenic plants todrought.