Identification And Function Analysis Of MYB Transcription Factor Interacted With Soybean SGF14l

MYB transcription factor family,as an important transcription factor family,plays a vital role in the growth,development and metabolic regulation of soybean(Glycine max(Linn.)Merr.).R2R3 MYB transcription factor,as a major member of MYB transcription factor family in plants,is widely involved in plant stress response,hormone signal transduction,and the regulation of other life activities in plants.14-3-3 protein is widely found in eukaryotes which structure is highly conserved.It mainly functions as scaffold proteins by forming homologous or heterologous dimers with other proteins.Previous studies indicated that 14-3-3 protein could play its function by interacting with MYB transcription factor.In this study,MYB transcription factor interacting with SGF14 l was screened based on the preliminary work,and its bioinformatics analysis and preliminary functional analysis were conducted.The main research results are as follows:1.We identified 5 candidate MYB transcription factor genes and found that GmMYB81 could interact with SGF14 l through yeast two-hybrid experiment,while the other 4 transcription factors could not.The interaction was further verified by the bi-molecule fluorescence complementary experiment in tobacco,and the interaction was found to occur in the nucleus.At the same time,we also found that GmMYB81 could not only form homologous dimer,but also form heterodimer with GmMYB176,and this heterodimer was also formed in the nucleus.2.The expression patterns of GmMYB81 in different tissues and different abiotic stresses and hormone treatments were analyzed by qRT-PCR.It was found that,for the expression patterns in various tissues,GmMYB81 had the highest expression level in nodules,but lower expression level in roots and flowers.In seeds of different period,the expression level was the highest in seeds 60 days after flowering,and the expression level gradually increased with the maturity of seeds.Under different abiotic stress treatments,the response degree of GmMYB81 was the most obvious in salt treatment,which showed a trend of slight decrease at first and then continuous increase.The response modes of the cold treatment and the drought treatment were first increased,then decreased and finally remained stable.Hormone treatment: GmMYB81 showed no obvious response trend to ABA and MeJA treatment.The expression of GmMYB81 could be induced by SA.3.The 1512 bp GmMYB81 promoter sequence was cloned from soybean,and the cis-acting elements contained in the sequence were analyzed by PlantCARE website.There were multiple cis-acting elements such as light response element,drought response element and auxin response element existed in the promoter.The promoter fragments were cloned and constructed on the pMDC162 vector through the Gateway technology and transformed into Arabidopsis thaliana by floral dip.After T2 generation,GUS staining was used to analyze the expression patterns of GUS gene driven by this promoter in different tissues,during germination and mannite simulated drought stress.4.GmMYB81 was overexpressed in Arabidopsis thaliana to observe the phenotype of transgenic lines.We found that GmMYB81 overexpressed lines had the characteristics of small overall plant type,increased branching and decreased apical dominance.And the bolting time was later than wild type but the number of rosette leaves was similar with wild type.The total life span of rosette was longer than the wild type.RNA-seq results showed that the differentially expressed genes were mainly concentrated in plant hormone regulation pathways,and the expression levels of AtSMXL8 and AtIAA29 were decreased through qRT-PCR verification,while the expression levels of 15 WRKY transcription factors were up-regulated.5.Through the experiments of wild type and GmMYB81 overexpressed lines to salt stress and drought stress treatment,we found the germination rate and the rate of green seedlings of GmMYB81 overexpressed lines were higher than the wild type under salt stress,but its root elongation in seedling stage was not obviously difference,It explained over-expression of GmMYB81 for the improvement of salt resistance of the transgenic plants in the germination stage has obvious effect;Under the conditions of drought stress,transgenic lines also had higher tolerance to drought stress at the germination stage of seeds,GmMYB81 over-expression improved the tolerance of plants to drought stress at the germination period of seeds.6.The upstream 2000 bp promoter region sequences of AtWRKY28,AtWRKY53,AtWRKY60,AtWRKY70,AtSMXL8 and AtIAA29 was analyzed,it was found that the upstream 2000 bp promoter regions of the six genes contained the predicted binding sites of MYB transcription factor,mainly MRE binding sites and MBS binding sites.However,it was found that GmMYB81 only interacted with the fragment of WRKY28pro-1 through yeast one-hybrid experiment,which preliminarily indicated that GmMYB81 could directly bind to the promoter region of AtWRKY28.