Identification And Functional Analysis Of The MYB Transcription Factor GmMYBLJ Associated With Leaf Senescence In Soybean

Soybean(Glycine max(Linn.)Merr.)is a full-canopy photosynthetic plant with 90-95% of the yield from the products of photosynthesis.The most sensitive part of their sensory photosynthesis are leaves,and their growth development and senescence are closely related to crop yield.In agricultural production,crop yield reduction due to early leaf senescence is one of the major causes of crop yield reduction worldwide.In this study,a soybean leaf senescence-associated CCA1-like MYB transcription factor,GmMYBLJ was identified,and the function of the GmMYBLJ transcription factor was investigated using the Arabidopsis heterologous overexpression,promoterdriven GUS gene expression assay system,soybean hairy roots,and soybean genetic transformation.The main results are as follows.1.Tissue expression assays showed that the expression level of GmMYBLJ gene was higher in senescing leaves,and responded to dark treatment,SA treatment,and ABA treatment.The promoterdriven GUS expression assay also indicated that GmMYBLJ accumulated more in the leaves of senescing leaves and dark treatment.2.To investigate the function of GmMYBLJ,we constructed a p35S::GmMYBLJ-YFP fusion protein vector and performed subcellular localization experiments using tobacco.The result showed that GmMYBLJ was localized in the nucleus.3.Phenotypic analysis of GmMYBLJ transgenic Arabidopsis showed that the transgenic lines exhibited early leaf senescence,darker leaf color,shorter plant height and increased branch number relative to the wild type under normal conditions,and more ROS accumulated in the leaves of the transgenic strain after DAB staining.Further study indicated that GmMYBLJ overexpression accelerated the senescence process of Arabidopsis leaves after dark treatment and Pst DC3000 treatment.4.To explore the molecular mechanism of GmMYBLJ in leaf senescence of Arabidopsis,we detected the expression levels of senescence related marker genes SAG12,SAUR36,ORE1,SGR1 and several WRKY genes associated with leaf senescence in transgenic Arabidopsis.The results showed that these four senescence related marker genes and several WRKY genes such as WRKY75,WRKY45,and WRKY30 were up-regulated expression in transgenic Arabidopsis,suggesting that GmMYBLJ may control leaf senescence in Arabidopsis directly or indirectly by regulating the expression of these genes.5.To verify the function of GmMYBLJ in soybean hairy roots,we obtained an overexpressed vector with a separate promoter driving strong GFP expression and named it pK7 GM.Then overexpressed and RNAi soybean hairy roots were obtained by soybean hairy root transformation and analyzed by DAB staining,respectively.And the results showed that relative to the control,overexpressed hairy roots accumulated more ROS,while RNAi hairy roots accumulated the least amount of ROS,suggesting that GmMYBLJ may regulate senescence through the ROS pathway.6.To verify the biological function of GmMYBLJ in soybean,we constructed an overexpression vector for GmMYBLJ and obtained stable genetic GmMYBLJ transgenic soybean positive plants by Agrobacterium-mediated genetic transformation of soybean,and found that GmMYBLJ transgenic soybean grew faster than wild type,which provided a basis for further studies.