Cloning And Functional Analysis Of GmMYB46 Gene From Soybean Under Drought Stress

Soybean[Glycine max(L.)Merr.]is an important crop rich in plant protein and oil.However,soybeans are inevitably affected by abiotic stress in the progress of growth and development,reducing yield and causing seriously economic losses.Transcription factors regulate plant growth and metabolism by regulating the expression of target genes at the transcriptional level.MYB transcription factor,as one of the largest family of transcription factors in plants,plays an important role in the response to abiotic stress.Among various abiotic stresses,drought is a major factor limiting global crop yeild.Therefore,a new MYB transcription factor gene,GmMYB46,was cloned from soybean cultivar ’Jindou 21’ in this study.The function responsed to drought stress were studied.The main results of this study are as follows:1.Induced expression analysis of soybean GmMYB genesUsing AtMYB44(AT5G67300.1)TaODORANT1(KY013614.1),OsMYB2(LOC_Os03g20090),GmMYBJ1(Glyma.05G051700.1),FtMYB13(FtPinG0005410000)as probes,BLAST alignment was performed in soybean database.The GmMYB genes with a score≥ 100 were selected to construct a phylogenetic tree,discarding the GmMYB genes with distant relationship.The difference in transcriptional level of these GmMYB genes after drought treatment was analyzed by real-time PCR.The results showed that the expression difference of GmMYB46 after drought treatment reached an extremely significant level.2.Cloning of GmMYB46The specific primer was designed and the GmMYB46 gene was cloned from the soybean cultivar ’Jindou 21’.The result showed that the CDS of the gene was 1 005 bp,encoding 334 amino acid,the relative molecular weight of the protein was 82.53 KDa,and the isoelectric point was 4.88.3.Bioinformatics analysis of GmMYB46Amino acid multiple sequence alignment of GmMYB46 and MYB proteins from other species showed that the GmMYB46 encoding product had 99%、77%、74%、71%、69%、60%sequence similarities with Glycine soja,Cajanus cajan,Vigna angularis,Phaseolus vulgaris,Vigna radiata var.radiate,Medicago truncatula,respectively.Phylogenetic tree analysis indicated that the protein encoded by GmMYB46 was closely related to wild soybean GsMYB46 protein.Promoter element analysis indicated that GmMYB46 contains stress response elements such as MBS、ABRE、GARE、TCA and LTR.4.Expression pattern of GmMYB46 under different processing and Tissue specific expressionReal-time fluorescence quantitative analysis showed that the transcriptional level of GmMYB46 was upregulated under the drought,salt(NaCl,200 mmol·L-1)、cold(4℃)、ABA(200 μmol·L-1)、SA(500 μmol·L-1)、GA(100 μmol·L-1)treatments.Tissue expression analysis showed that the relative expression of GmMYB46 in stems was higher,while the expression was lower in cotyledons,followed in roots and leaves and the expression levels were 4 times and 3 times that of the control cotyledon,respectively.5.Subcellular localization of GmMYB46Domain analysis revealed that the amino acid sequence contained two highly conserved SANT domains.The full-length of GmMYB46(aal-334),the portion containing only the MYB conserved domain(M1,aal-123)and the part removing the MYB conserved domain(M2,aa124-334)were constructed on the pBinGFP4 vector.Subcellular localization analysis was performed by gene gun transformation of onion epidermal cell transient expression.The results showed that the green fluorescence of pBinGFP4::GmMYB46Ml and pBinGFP4::GmMYB46 were concentrated in the nucleus,while the pBinGFP4::GmMYB46M2 fusion protein was distributed in the nucleus.It was demonstrated that the MYB domain of GmMYB46 is required for nuclear localization signals.6.GmMYB46 enhanced the drought resistanceThe overexpression vector pTF101.1::GmMYB46 that contained GmMYB46 gene was constructed,and over-expressing transgenic soybean plants were obtained by Agrobacterium transformation.Both wild-type plants and over-expressed plants were exposured under drought stress treatment,the results showed that compared with wild type,over-expressing lines showed higher survival rate,lower malondialdehyde(MDA)content and higher proline(Pro)content under drought stress conditions.These results indicate that GmMYB46 plays a positive regulatory role in drought stress.