Transcriptional Analysis Of Alfalfa During Cold Stress And Functional Identification Of MsMYB144 Gene

Alfalfa is the most widely cultivated leguminous forage,which is known as the“queen of forage crops”because of higher nutritional value.However,abiotic stress seriously hinders the survival and yield of alfalfa.Due to the incompleteness of genomic information and the lack of expression profile data,the excavation of alfalfa resistance genes has just started compared with traditional crops.In order to comprehensively reveal the changes of gene expression in alfalfa in response to cold stress,this study used high-throughput sequencing BGISEQ-500 technology combined with full-length transcriptome data to compare the transcriptome dynamics of alfalfa under 4℃ cold stress.The MYB gene family plays an important role in plant response to abiotic stress,but has not been systematically analyzed in alfalfa.Based on the previous transcriptome experiments,the members of MsMYB gene family were recognized and identified and the expression patterns of different members under abiotic stresses such as cold stress were analyzed,and the gene functions of MsMYB 144 were identified in Arabidopsis thaliana and alfalfa by genetic transformation.The main research results are:1.The change characteristics of eight physiological indexes of alfalfa under cold stress were analyzed.As a result,increase patterns in the electrolyte leakage level,CAT and POD activity,and GSH,H2O2,MDA and PRO content were observed under cold stress compared with the control.However,the chlorophyll content was lower in the three groups(C2,C3,and C4)under cold treatment compared with normal temperature(P<0.05).2.The response of alfalfa to cold stress was analyzed at full-length transcriptome level.(1)The transcriptome changes in alfalfa under cold stress at 4℃ for 2,6,24 and 48 h(three replicates for each time point)were analysed using the high-throughput sequencing platform BGISEQ-500,resulting in the identification of 50,809 annotated unigenes and 5,283 differentially expressed genes;(2)Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism,photosynthesis,plant hormone signal transduction and the biosynthesis of amino acids;(3)The physiological changes of glutathione and proline content,catalase and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes;(4)Some transcription factors maybe play important roles in the alfalfa response to cold stress as determined by the expression pattern of the related genes during 48 h cold stress treatment,including MYB,AP2,NAC,and WRKY transcription factors3.The response of MsMYB gene family to abiotic stresses such as cold stress was studied at genome-wide level,and the predicted interaction genes were verified by yeast two-hybrid test.(1)A total of 265 MsMYB proteins were obtained in the present study,including 50 R1-MYB,186 R2R3-MYB,26 R1R2R3-MYB,and three atypical-MYB proteins.These predicted MsMYB proteins were divided into 12 subgroups by phylogenetic analysis;(2)The expression profiles and quantitative real-time PCR analysis indicated that some MsMYB genes might play a crucial role in the response to abiotic stresses;(3)A total of 170 and 914 predicted protein-protein and protein-DNA interactions were obtained,respectively.The interactions between MsMYB043 and MSAD320162,MsMYB253 and MSAD320162,and MsMYB253 and MSAD308489 were confirmed by a yeast two-hybrid system4.The gene function of alfalfa MsMYB144 was identified.(1)MsMYB144,an R2R3-MYB transcription factor,comprised three exons and two introns.The yeast cells harboring pLexA-MsMYB144 turn blue on SD/-U-H+X-gal induction medium,which indicates that MsMYB 144 possesses transcriptional activity in yeast and proves that it is a transcription factor.MsMYB144 was notably induced by ABA,cold,drought,and salt treatments;(2)There was no significant difference between the transgenic lines and wild-type Arabidopsis under cold stress at 4℃.However,Overexpression of MsMYB144 in Arabidopsis seedlings increased the number of lateral roots and survival rate in response to salt stress(P<0.05),and decreased sensitivity to ABA;(3)A total of 54 strains of MsMYB144 transgenic alfalfa positive plants were obtained by transgenic means,with positive rates of 56%.Compared with the control,overexpression of MsMYB144 in alfalfa seedlings also increased salt tolerance;(4)MsMYB144-overexpressing alfalfa and Arabidopsis seedlings both increased flavonoid accumulation in response to salt stress(P<0.05);(5)14 MsMYB144 RNAi transgenic alfalfa lines were obtained,and the expression level of MsMYB144 gene was significantly lower than that of wild type.These results suggest that MsMYB144 gene can enhance the salt tolerance of alfalfa and Arabidopsis,but its potential mechanism still needs further study.