| Medicago falcata L is an important legume forage,which is closely related to the Medicago sativa.Drought resistance gene resources make it strongly drought-resistant and the resources are also an important germplasm resource in stress resistance breeding of legume forage and a significant gene bank for alfalfa variety improvement.The arid climate in Xinjiang seriously affects the yield and quality of alfalfa,which is impossible to change.Therefore,the new alfalfa varieties with strong drought tolerance need to be cultivated to promote the development of alfalfa industry.In this experiment,Xinjiang M.falcata was used as experimental material.Under the simulated drought stress condition of 400 m M mannitol,the changes of morphology and physiology of M.falcata seedlings and the response mechanism of seedlings at different stress time(0 h、2 h、6 h、12 h、24 h、36 h and 48 h)were studied.The key drought resistant genes and modules in M.falcata were excavated through transcriptome analysis,trend analysis and WGCNA coexpression network analysis,the key ERF genes were screened for functional research.The main results are as follows:(1)With the increase of drought stress time,the wilting degree of M.falcata plants morphology increased.By measuring a variety of physiological indexes,it was found that the contents of malondialdehyde and proline increased in M.falcata and the activities of antioxidant enzymes such as SOD,POD and CAT increased,indicating that M.falcata was injured by osmotic stress to some extent.(2)The seedlings of M.falcata were under drought stress treatment at different time were sequenced by transcriptome sequencing.A total of 172,892 annotated genes and 16,304 differential genes were obtained.Compared with CK in the control group,1,219 differentially co expressed genes were screened including 88 transcription factor genes,which were divided into 16 families,b HLH、ERF、b ZIP、NAC and HD-Zip were more.Three modules closely related to drought response of M.falcata were identified by WGCNA analysis,and four ERF genes related to drought stress(MfERF053,MfERF9,MfERF034 and MfRAP2.1)were screened.(3)MfERF053,MfERF9,MfERF034 and MfRAP2.1 of full-length coding area were obtained by homologous cloning methods,sequence analysis showed that they all contained AP2 conserved domain,which had high homology with the ERF genes of Medicago truncatula.Through the analysis of expression profile data,it was found that the expression levels of four ERF homologous genes of alfalfa and M.truncatula increased significantly under drought,salt,ABA and cold stress.The expression level of MfERF053 gene in different tissue materials of M.falcata was analyzed,it was found that the gene was expressed in all tissues,and the expression level was the highest in roots,which may be related to the regulation of water transport and osmotic stress in roots.(4)In order to further explore the function of MfERF053 genes,transcriptional activity vector containing MfERF053 was constructed in this paper,transient expression results in tobacco leaves showed that MfERF053 protein had transcriptional activation activity.Subcellular localization showed that MfERF053 protein was mainly located in nucleus and plasma membrane.(5)In order to verify the function of MfERF053 in plants,MfERF053 was transferred into the model plant Arabidopsis by inflorescence infection method.A total of 25 homozygous lines with overexpression of Arabidopsis were obtained,and three lines with high expression were screened for stress resistance evaluation.The results showed that under drought,salt and ABA stress,the root length,number of lateral roots and fresh weight of Arabidopsis overexpression MfERF053 genes were significantly higher than those of wild type.q RT-PCR analysis showed that overexpression of MfERF053 gene could activate the expression of stress-related genes in Arabidopsis and resist abiotic stress.(6)A total of 72 transgenic alfalfa strains were obtained by Agrobacterium mediated leaf transformation.40 positive strains were detected by PCR,and 3 high expression strains were screened by q RT-PCR for subsequent stress resistance analysis.Phenotypic analysis showed that the plant height,branch number and growth rate of alfalfa overexpressing MfERF053 gene were higher than those of wild type.The expression of stress response genes ATPase and HSP23 in overexpressed alfalfa was significantly higher than that in wild-type alfalfa.In conclusion,four drought related ERF genes were screened and cloned by transcriptome analysis of M.falcata after drought treatment.It was found that one of the genes MfERF053 was specifically highly expressed in roots,and its expression level was induced by various stresses.Overexpression of MfERF053 gene in Arabidopsis can improve drought and salt resistance,and sensitivity of ABA by increasing root length and the number of lateral roots,and can inducing the expression level of stress resistance related genes.Overexpression of MfERF053 gene in alfalfa can increase plant height,branch number and the expression level of induced stress resistance related genes.In this paper,drought stress response genes were excavated from M.falcata,and the overexpression material of drought related gene MfERF053 was created,which provided theoretical basis and new materials for alfalfa drought resistance molecular breeding. |
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