Transcriptome Analysis On Freezing Tolerance And Cloning And Functional Characterization Of MsNCX And MsCML In Alfalfa

Medicago sativa L.?alfalfa?‘Zhaodong',a perennial forage crop is widely cultivated in alfalfa plantation areas of China.Whether alfalfa survives during harsh winter depends largely on the amount and revival ability of crown buds.To investigate the molecular mechanisms of frost tolerance in alfalfa,RNA-seq technology was used to analyze field-grown alfalfa crown buds during winter.A large of genes on transcript levels in alfalfa responsed to frozen stress were enriched.In this study,MsNCX and Ms CML genes were cloned,and analyzed their function on abiotic stresses in transgenic plant.The summary of the main results are as follows:1.Transcriptome analysis the cold resistance mechanisms of Medicago sativa L.De novo transcriptome assembly of the field-grown alfalfa crown buds using Illumina platform provided an overview of the dynamic transcript levels of genes in alfalfa under frozen stress.A total of 5,605 differentially expressed genes?DEGs?identified under freezing stress including 1,900 up-regulated and 3,705 down-regulated DEGs.The results of GO analysis showed that most DEGs were enriched in subset of biologe process,organic cyclic compound metabolic process,oxidation-reduction process and single-organism metabolic process.The function of DEGs were centralized in subset of cell component,cell wall and membrane-enclosed lumen.The combination of molecular function and nucleic acid binding,binding and catalytic activity.The DEGs were identified numerous potential freeze-sensing TF genes and various key signal transduction components at transcript level.There were 350 unique genes were identified,these TF families included the AP2/EREBP,Hsfs,MYB,bHLH,WRKY,NAC,bZIP,and MADS family.The key genes enriched from calcium signaling pathway?such as cation transporter gene and calmodulin like genes?,plant hormone signaling pathway,and ubiquitination protein pathways were up-regulated expression.The genes enriched from metabolic pathway were down-regulated expression.Using the replicates of qPCR,we analyzed the expression profiles of the selected genes during the overwintering period and compared them with those of the RNA-seq assay.The expression pattern of functional proteins was induced immediately in the cold acclimation stage and was further up-regulated under freezing stress.During the mid-winter stage,the functional proteins were depressed.The regulatory proteins were induced significantly from the cold acclimation to mid-winter stage and had higher expression in the mid-winter stage than in the freezing stress stage.Compared to the freezing stress stage,the DEGs were not all induced in the deacclimation stage.Moreover,qPCR showed a high correlation of fold change between RNA sequencing analysis and qPCR.The expression patterns in qPCR assay were generally in agreement with the results of the RNA-seq.The genes expression pattern in alfalfa during overwintering stage of two consecutive years was analysised using qPCR.The results showed that the expression pattern of genes relatived to calcium signal and antioxidase were the same in two consecutive years of alfalfa overwintering period.During cold acclimation and freezing stress period,the genes related to plant hormone signal transduction expressed pattern was similar to the second year,and the different expression pattern was in deacclimation stage.These results suggested that the genes relatived to calcium signal play important role in alfalfa response to freezing stress.2.Cloning and molecular function analysis of MsNCXMs NCX gene contains a pair of Na⁺/Ca²⁺exchangers domain was cloned by PCR,encoded a polypeptide of 597 amino acids.MsNCX protein was homology with MtCAX.The expression of MsNCX was the highest in roots,followed by the stems and leaves.MsNCX was rapidly induced in leaves under low temperature treatment,and was the highest induced in roots under salt stress.The expression of MsNCX in roots reached the highest level under drought and saline-alkali treatment with 6 h,and ABA treatment with 12 h.The plant expression vector pCBM-MsNCX was constructed,and studied on the genetic transformation of tobacco.Four lines of higher expression level were used for subsequent functional analysis by PPT resistance identification,PCR and qRT-PCR analysis.Over-expression of MsNCX significantly increased freezing stress tolerance in tobacco,as indicated by the higher survival rate,lower MDA after freezing treatment,and higher activity of antioxidant enzymes,contents of proline,solute protein and sugar.Moreover,lower O₂^-and H₂O₂ contents and higher chlorophyll content were found in the transgenic lines compared to wild-type plants when response to salt and drought stresses.The increased activity of CAT,and more protective substances were accumulated improved the tolerance of tobacco,especially proline.Compared to wild-type plants,the lower Na⁺and higher K⁺contents were accumulated in leaves of transgenic lines under salt stress.The expression of stress responsive genes in transgenic lines were higher than wild type tobacco,these results suggested that MsNCX gene can enhance the ability of tabacco to response to abiotic stresses.3.Cloning and molecular function analysis of MsCMLMs CML gene contains a EF-hand domain was cloned by PCR,encoded a polypeptide of 196 amino acids,MsCML protein was homology with CaCML45.The expression of MsCML was the highest in stems,followed by the roots and leaves.And MsCML in leaves was rapidly responsed to abiotic stresses,the results were as follow:MsCML was rapidly induced and highly induced under low temperature and salt stresses with 1 h.The expression of MsCML in leaves reached the highest level under saline-alkali and ABA treatment with 3 h,and mannitol treatment with 24 h.These results suggested that MsCML gene played important role in the early response to abiotic stresses.The over-expression plant vector pCBM-MsCML was constructed,and studied the genetic transformation on tobacco.Three lines of higher expression level were used for subsequent functional analysis by PPT resistance identification,PCR and qRT-PCR analysis.Over-expression of MsCML significantly increased freezing stress tolerance in tobacco,as indicated by the higher survival rate,lower MDA after freezing treatment,and higher proline,solute protein and sugar,and ROS scavenging genes after cold stress.Moreover,higher O₂^-and H₂O₂ contents and lower chlorophyll content were found in the wild-type plants compared to transgenic lines when response to salt and drought stresses.The increased activity of antioxidant enzymes,and more proline were accumulated reduced the damage of tobacco under abiotic stress.Compared to wild-type plants,the lower Na⁺and higher K⁺contents were accumulated in leaves of transgenic lines under salt stress.The expression of responsive genes in overexpression MsCML tobacco were much higher than wild type lines,these results suggested that MsCML gene can enhance the resistance tolerance of tabacco under abiotic stresses.