Functional Characterization Of Saline-alkali Resistance Of MsbZIP11 Gene In Alfalfa

Alfalfa(Medicago sativa L.)is a perennial leguminous forage with high protein content.The yield and quality of alfalfa were seriously affected by soil salinization in northern China.Medicago sativa cv.Zhaodong,as a local variety of alfalfa,it could grow in the soil environment with severe condition of moderate saline-alkali,which contained rich genes of saline alkali resistance.In order to explore the molecular mechanism of saline-alkali resistance of alfalfa,Zhaodong alfalfa was used as research material,and the b ZIP(basic leucine zipper)transcription factor encoding gene MsbZIP11 was cloned based on the transcriptome data of alfalfa under saline-alkali stress in the early stage of laboratory.The function of MsbZIP11 was predicted by bioinformatics,and its molecular mechanism of saline-alkali resistance was explored through the analysis of its expression pattern,identification of interaction protein and upstream and downstream genes.Under saline-alkali treatment,the phenotypes of transgenic Arabidopsis,WT(Wild-Type)Arabidopsis and Arabidopsis mutant bzip11 were observed,and the related indexes of three types of Arabidopsis were measured and analyzed.The main results are as follows:1.Cloning and molecular function analysis of MsbZIP11The MsbZIP11 gene was cloned from alfalfa by RT-PCR(Reverse Transcription-Polymerase Chain Reaction),which contained a BRLZ domain and encodes 156 amino acids.Phylogenetic analysis showed that the MsbZIP11 protein of alfalfa was closely related to the members of b ZIP transcription factor family of Leguminosae,and had the highest similarity with Mtb ZIP11(Medtr4g070860).The expression of MsbZIP11 was similar in alfalfa root and leaf,and the highest in stem.The MsbZIP11 in the shoot of alfalfa responded to cold,drought,salt,saline-alkali and ABA(Abscisic Acid)treatments,and reached the peak value at 1 h or 3 h;the MsbZIP11 in the root of alfalfa was up-regulated after salt,saline-alkali and ABA treatments.It indicated that MsbZIP11 plays a key role in the early response of abiotic stresses.The MsbZIP11 was located in nucleus.The interaction protein MsbZIP42 of MsbZIP11 was screened by yeast two hybrid assay.The intensity of transcriptional self activation activity of MsbZIP11 and MsbZIP42 was verified.The results showed that MsbZIP11 had transcriptional self activation activity,while MsbZIP42 had no transcriptional self activation activity.After adding MsbZIP42,the transcriptional activity of MsbZIP11 was significantly increased.The results of Bi FC(Bimolecular Fluorescence Complementary)assay showed that the yellow fluorescence signal produced by the interaction between MsbZIP11 and MsbZIP42 were specifically present in the nucleus.The interaction between MsbZIP11 and MsbZIP42 was verified by in vitro pull-down and in vivo Co-IP(Co-immunoprecipitation)assay.The results showed that the two proteins were interacted and located in the nucleus.2.Cloning and functional analysis of MsbZIP11 upstream and downstream geneThe promoter of MsbZIP11 gene was cloned from alfalfa,which contains the cis-acting element ABRE(Abscisic Acid Response Element).The upstream gene Ms ABF2(ABRE Binding Factor 2)of MsbZIP11 was screened by yeast one hybrid assay.Ms ABF2 could activate the transcription of MsbZIP11 by binding to the cis-acting element ABRE in the promoter region of MsbZIP11.The expression product of MsbZIP11 from alfalfa belonged to GBF(G-box Binding Factor)transcription factor,which had the ability to bind G-box elements in the promoter region of its downstream gene.Yeast one hybrid assay showed that MsbZIP11 could bind to Ms NCED3(9-Cis-Epoxycarotenoid Dioxygenase 3),a key gene in ABA synthesis pathway.The G-box element in the promoter region indicated that Ms NCDE3 was located downstream of MsbZIP11.In the presence of MsbZIP42,the binding efficiency of MsbZIP11 to the G-box element in the promoter region of Ms NCED3 was higher,which indicated that the protein complex formed by the interaction of MsbZIP11 and MsbZIP42 regulated the transcription level of Ms NCED3 and promotes the expression of Ms NCED3.3.Analysis of saline-alkali resistance of transgenic Arabidopsis with MsbZIP11The plant overexpression vector p CAMBIA1302-MsbZIP11-GFP was constructed and transformed into Arabidopsis.Through resistance screening and PCR identification,q RT-PCR and Western blotting were selected to detect the high level expression lines for subsequent gene function analysis.Under saline-alkali stress,compared with WT,transgenic Arabidopsis with MsbZIP11 had longer root length,higher survival rate,higher chlorophyll content,lower ion leakage and MDA(Malondialdehyde)content;however,Arabidopsis T-DNA insertion mutant bzip11 reduced the tolerance of plants to saline-alkali stress due to deletion of MsbZIP11 homologous endogenous gene.The growth and development of seedlings were seriously restricted,resulting in shorter root length,lower survival rate,a larger decline in chlorophyll content,and a significant increase in ion leakage and malondialdehyde content.The endogenous ABA content in transgenic Arabidopsis,WT and bzip11 after saline-alkali treatment was determined by plant abscisic acid ELISA(Enzyme-Linked Immunosorbent Assay)kit.The endogenous ABA content in transgenic Arabidopsis was significantly higher than that in WT,and the ABA content in bzip11 was the lowest.