Functional Analyses Of MfSRP54?MsRabD2a?MtARF37 And MtARF38

Low temperature is one of the major abiotic stresses that leads to decrease in the yield and quality of forage crops.Medicago sativa subsp.falcata(i.e.M.falcta)exhibits great tolerance to cold and drought.Medicago sativa subsp.alfalfa(i.e.M.sativa)is known as “the king of forage”,which has the advantages of abundant protein and high yield.Medicago truncatula(i.e.M.truncatula)is a model legume plant which has completed genome sequencing.Functions Mf SRP54 ?Ms Rab D2 a ?Mt ARF37 and Mt ARF38 were preliminary investigated in the present study.It is helpful to reveal the molecular mechanism of cold resistance,and provide some theoretical support for cultivating new cold-resistant varieties.MfSRP54 was cloned from M.falcata.The full length of the sequence was 1722 bp,and the ORF was 1662 bp,encoding 553 amino acids.We selected 6 samples after treated with low temperature in comparison to the room temperature control,the results of quantitative real-time PCR(q RT-PCR)showed the expression of Mf SRP54 was increased after 12 h treatment with low temerature,therefore Mf SRP54 was up-regulated induced by low-temperature.We constructed overexpression vector and RNAi vector to transform M.sativa.by Agrobacterium mediating.Finally we obtained 3 overexpressed-transgenic plants,all of the Mf SRP54-RNAi plants are in rooting stage.We analysed the expression of transgenic plants by q RT-PCR,selected line 1 and line 2 for cold-tolerance testing,which expression was significantly improved,the results of lethal temperature(LT₅₀)showed significant difference between wild type and transgenic plants whether before or after acclimation,indicating that overexpression of Mf SRP54 could improve the cold resistance of alfalfa.MsRabD2 a was cloned from M.sativa.The full length of the sequence was 1069 bp,and the ORF was 612 bp,encoding 203 amino acids,the GC content was 41.83%and the theoretical isoelectric point was 4.962.We selected 6 samples after treated with low temperature in comparison to the room temperature control,the results of quantitative q RT-PCR showed the expression of Ms Rab D2 a was increased after 12 h treatment with low temerature,therefore Ms Rab D2 a was up-regulated induced by low-temperature.We constructed overexpression vector and RNAi vector to transform M.sativa.by Agrobacterium mediating.All of the Ms Rab D2 a overespressed plants and Ms Rab D2a-RNAi plants are in rooting stage.MtARF37 was cloned from M.truncatula.The full length of the sequence was2700 bp,and the promoter was 2500 bp.At ARF6 was the most homologous gene to Mt ARF37 in Arabidopsis.we observed the phenotype of mtarf37,the height of NF16996 was 10% higher than wild type;We evaluated the cold tolerance by testing lethal temperature,the results showed that the mutants had stronger cold resistance than the wild type whether before or after acclimation.MtARF38 was cloned from M.truncatula.The full length of the sequence was2500 bp,and the promoter was 1800 bp.We found the height had no difference between mtarf38 mutant NF6366 and wild type,but the leaf was larger than wild type and the margin was wider.The mutant NF6366 had lower lethal temperature than wild type,so we concluded that the mutants had stronger cold resistance than wild type after acclimation.At ARF2 was the most homologous gene to Mt ARF38 in Arabidopsis.Compared to the wild type,the mutant was higher and had less twitching;The rosette leaves were larger and darker in color,and the fruit pods were larger;The flowering period was later than wild type and the seeds were larger.