Cloning And Characterization Of MYB Transcription Factor Genes From Soybean

Transcription factors play an important role in regulation of plant growth andphysiological metabolism by regulation of gene expression at the transcription level.MYB transcription factor gene is one of the largest families in plants, which involved inregulation of secondary metabolism, responding to hormones and environmental factors,also regulation of cell differentiation, cell cycles and the formation of leaves and otherorgans. Soybean is not only an important oil crop and a source of proteins, but also themost important source of isoflavonoids. Many MYB transcription factors were reported tohave ability to regulate the metabolism of flavonoids. In this paper, some novel genesencoding MYB transcription factors were isolated and characterized from soybean cultivars.The main results of this study are as follows:1. A pair of degenerate primers was designed according to the conserved regions whichencoding the MYB DNA binding domains in plant MYB genes. Fourteen fragments wereamplified from leaves of soybean cultivars ZhongDou-27, JiLin-3, HuaiDou-1 andZhangJiaKou Black Soybean using RT-PCR. Four novel genes encoding MYBtranscription factors, GmMYBZ1, GmMYBZ2, GmMYBJ6 and GmMYBJ7 were isolated byRACE-PCR. Comparison of deduced amino acid sequences showed that they are typicalR2R3MYB transcription factors because of including two MYB domains respectively.2. The expression pattern of the four members in different organs was studied usingsemi-quantitative RT-PCR. The results showed that GmMYBZ2 was expressed in roots,stems, leaves and immature seeds of soybean, the expression of GmMYBZ1 and GmMYBJ7was detected in the stems and leaves, whereas the expression of GmMYBJ6 was detectedonly in the leaves. Expression of GmMYBJ6 could be increased under UV-B radiation,drought and high-salt treatment, while the expression of GmMYBJ6 and GmMYBJ6 wouldbe decreased under the same treatments.3. The full-length DNA sequences of GmMYBZ1,GmMYBZ2,GmMYBJ6 andGmMYBJ7 were also amplified using genomic DNA as templates. The results indicatedthat GmMYBZ2 contains one intron, GmMYBJ6 and GmMYBJ7 contain two intronsrespectively, whereas the gene GmMYBZ1 lacks intron. 4. The recombinant vectors pBridge-GmMYBZ1, pBridge-GmMYBZ2, pBridge-GmMYBJ6 and pBridge-GmMYBJ7 were constructed and transformed into the yeast stainsAH109, then the positive yeast transformants were selected using the SD/-Trp selectivemedium and the SD/-Trp/-His selective medium. The transcriptional activation ofGmMYBZ1, GmMYBZ2, GmMYBJ6 and GmMYBJ7 proteins was confirmed by theyeast system, and itsβ-galactosidase activity was detected as 3.59U, 10.39U, 28.48U and24.31U, respectively. The results of the colony-lift filters assay indicated that the yeasttransformants of GmMYBZ2, GmMYBJ6 and GmMYBJ7 showed a clear blue, whereas theyeast transformants of GmMYBZ1 didn't.5. The green fluorescent protein expression vectors p163-GFP-GmMYBZ2,p163-GFP-GmMYBJ6 and p163-GFP-GmMYBJ7 were constructed and transformed into the epidermalcells of onion via particle bombardrnental method. The results of instantaneous expressionshowed that GmMYBZ2,GmMYBJ6 and GmMYBJ7 proteins all were localized in cellnucleus, which was consistent with the prediction of the subcellular localization.6. GrnMYBZ2,GmMYBJ6 and GmMYBJ7 were transformed into tobacco NC89 withAgrobacterium LBA4404 containing the plant expression vectors pCAMBIA2301-GmMYBZ2, pCAMBIA2301-GmMYBJ6 and pCAMBIA2301-GmMYBJ7, respectively.The positive tobacco transformants were selected using MS medium containing kanamycin,Gus activity assay and RT-PCR. Semi-quantitative RT-PCR analysis indicated thatGmMYBJ6 could improve the expression of some flavonoid biosynthetic genes, such asPAL (Phenylalanine ammonia lyase), C4H (cinnamate-4-hydroxylase), 4CL (4-coumaroyl-CoA ligase), CHS (Chalcone Synthase), CHI (chalcone isomerase), F3H (flavanone3-hydroxylase), and FLS (flavonol synthase), resulting the increase of the total flavonoidlevels. Whereas, over exprssion of GmMYBZ2 and GmMYBJ7 could decrease the totalflavonoid level in the transgenic tobacco plants due to the repression of the genes inflavonoid biosynthesis. Results of anti-stress experiments showed that the expression ofGmMYBJ6 could improve resistance to UV-B radiation and drought of transgenic tobaccoobviously.