Cloning Of MfERF070 Gene From Medicago Falcata And Preliminary Study On Its Anti-stress Function

Medicago falcata L.has the characteristics strong adaptability,drought resistance,cold resistance,salt and alkali resistance.It is more resistant than Medicago sativa and is an important gene pool for alfalfa resistance breeding.Based on the transcriptome data analysis of the earlier stage of the research group,a 954 bp-long M.falcata gene was cloned by RT-PCR technology,which was named MfERF070(Gene Bank No.ON567209)after phylogenetic analysis of the gene sequence.MfERF070 has a conserved AP2 structural domain and belongs to the ERF subfamily.The results of subcellular localization experiments indicated that MfERF070 was localized in the nucleus.The expression levels of the MfERF070 gene was analyzed by semi-quantitative RT-PCR under cold(4℃),salt(200 m M Na Cl),drought(300 m M mannitol)and ABA(100 μM)stress for 0 h,2 h,4 h,8 h,24 h and 48 h,and the results showed that the MfERF070 gene responded more significantly to cold stress and salt stress.A 2180 bp promoter fragment of MfERF070 gene was successfully cloned using chromosome stepping technique.The analysis revealed that this promoter not only contained the core promoter region of TATA-box and CAAT-box,but also a variety of abiotic stress-related cis-acting elements.Expression vectors for MfERF070-p1307-OE and MfERF070-p1307-RNAi were constructed and transformed into Medicago sativa L.cv.‘Zhongmu No.1’ by Agrobacterium-mediated leaf disc transformation.13 overexpressed MfERF070 transgenic strains and10 interfered MfERF070 transgenic strains were obtained.To further verify the effect of MfERF070 on the cold stress tolerance of the transgenic strains,six overexpression MfERF070 transgenic strains and six wild-type strains with similar growth conditions were randomly selected.The overground survival rate of MfERF070 transgenic strains and wild-type strains were calculated after 12 h of freezing stress and 3 d of incubation at room temperature.The results showed that the overexpression MfERF070 transgenic strains had better cold tolerance compared to the wild-type strains,MfERF070 was a positive regulator in the cold-resistant regulatory pathway.Q-PCR results showed that the expression level of the cold stress response gene COR413 in MfERF070 overexpressed strains was higher than that in control strains,suggesting that MfERF070 may regulate plant tolerance to low temperature through positive regulation of COR413 expression.This study provides preliminary that overexpression of MfERF070 transcription factor can improve cold tolerance in M.sativa,providing a theoretical basis and candidate genes for furture study on the mechanism of alfalfa resistance and breeding of new alfalfa resistant varieties.