Function Analysis Of WRKY Transcription Factor CmWRKY12 In Soybean (Glycine Max L.)

As the main plant source of animal feed protein and edible oil,soybean(Glycine max.L)is of great significance worldwide.However,soybean faced with drought,high salinity,extreme temperature and various stresses during their growth stage,resulting in reduced yield.Therefore,exploring how to use biotechnology and genetic engineering technology to improve soybean stress tolerance is important for soybean genetic breeding.As a member of the transcription factor superfamily,WRKY transcription factor plays an important role in plant growth and development,biological stress and abiotic stress.At present,multiple WRKY genes related to plant stress have been identified in soybean,and an intensive study of their mechanism is needed to improve the stress resistance of soybean.In this study,RNA-Seq was used to annotate and analyze GmWRKYs in response to drought and salt,and real-time fluorescence quantitative RCP analysis(RT-qPCR)was used to screen out GmWRKY12 gene that was induced by both drought and salt stress,as well as had a large up-regulation multiple.Function of GmWRKY12 was preliminarily studyed through biological analysis,expression pattern analysis,subcellular localization and tissue specific expression.Besides,Agrobacterium-mediated genetic transformation technology was used to transform GmWRKY12 into Arabidopsis thaliana and soybean,respectively,to carry out phenotypic identification experiments of drought and salt tolerance of over-expression lines.Main results in this study are as follows:1.The fragment of GmWRKY12 gene was amplified from soybean(Williams 82)genome by polymerase chain reaction(PCR).2.Biological analysis showed that GmWRKY12 had a molecular weight of 26.615 KD,CDS 714 bp,and encoded 237 amino acids,belonging to the WRKY family Group IIc.Multiple sequence alignment and evolutionary tree analysis showed that GmWRKY12 was very conservative in evolution and had the highest similarity with VrWRKY12(Vigna radiata).The subcellular localization indicated that GmWRKY12 was a nuclear protein.Both tissue specific expression and GUS staining showed that GmWRKY12 was expressed in the roots,stems and leaves of Arabidopsis.3.Over-expression of GmWRKY12 can improve the drought tolerance of transgenic Arabidopsis.Acording to the analysis of expression pattern,the expression level of GmWRKY12 increased greatly under drought stress.To further determine it's function under drought stress,GmWRKY12 was transformed into model plant Arabidopsis for tolerance identification.Results of germination and root growth of Arabidopsis shown that the root length of over-expression lines were longer than the control(Col-0)under PEG treatment.Besides,under the treatment of different concentrations PEG,the shoots of Col-0 showed yellowing phenomenon,while the over-expression plants growing in green color,which indicating that the over-expression of GmWRKY12 could improve the drought tolerance of Arabidopsis.4.Over-expression of GmWRKY12 can improve the salt tolerance of transgenic Arabidopsis.In order to further determine it's function response to salt stress,GmWRKY12 was transformed into Arabidopsis fo rtolerance identification.Results of germination and root length of over-expression Arabidopsis shown that the root length and root area of over-expression Arabidopsis were longer and larger than the control(Col-0)under NaCl treatment,respectively,which indicating that the over-expression of GmWRKY12 could improve the salt tolerance of Arabidopsis.5.Over-expression of GmWRKY12 improved the drought and salt tolerance of soybean.GmWRKY12 was transformed into soybean in vitro to further study.Compared with the control(Williams 82),over-expression of GmWRKY12 in soybean increased proline(Pro)content and decreased malondialdehyde(MDA)content under drought and salt treatment,which enhanced the drought and salt tolerance of soybean.The analysis of root traits after treatment showed that GmWRKY12 could improve the tolerance of soybean under drought and salt stress by promoting root growth.The current experiment results provide a theoretical support to future study GmWRKY transcription factors in soybean.