Cloning And Drought Resistance Function Analysis Of The MsTHI1 Gene Of Alfalfa

The frequency and intensity of extreme climate events,such as drought caused by global warming,increase significantly.The conomic losses caused by drought account for more than 50%of meteorological disasters.Alfalfa is a global growing feed crop with high biomass production and nutritional value.It is an excellent leguminous forage cultivated in the north of China.Improving the drought resistance of alfalfa is related to the improvement of alfalfa productivity in arid and semi-arid areas.The THI1 gene is involved in the synthesis of thiamine,which relieves the stress of external stress on organism and plays a vital role in plant growth and development.In this study,the THI1 gene of Medicago truncatula(Medicago truncatula L.,Medtr4g081130.1)with known function was selected as the reference sequence,and the homologous Ms THI1 gene of alfalfa was cloned and verified.The main results of this study were as follows:(1)Analysis of the expression of Ms THI1 gene in alfalfaThe expression of Ms THI1 gene showed different trends in alfalfa under different stresses.Under drought stress,the expression of Ms THI1 gene in stem of alfalfa was less than that in roots and leaves.And Ms THI1 gene had the highest expression in leaves after 3 h of stress treatment,which was 3.85 times higher than that of control treatment.Low temperature stress can inhibit Ms THI1 gene expression in alfalfa significantly.Under 4°C stress,Ms THI1 gene relative expression in different parts of alfalfa were lower than that of control treatment.The relative expression of Ms THI1 gene in alfalfa stem was the highest under 150 mmol/L Na Cl stress for 12 h,which was 70.85 times higher than that of control treatment.Under 150 mmol/L Na HCO3 stress for6 h,the expression of Ms THI1 gene in alfalfa root was the highest,which was 3.65 times higher than that of control treatment.(2)Cloning and sequence analysis of alfalfa Ms THI1 geneIn this study,THI1 gene of Medicago truncatula was used as a reference sequence,to gain Ms THI1 gene of alfalfa(Gene Accession: MH206189)by homologous cloning.Bioinformatics analysis showed that the gene contained an open reading frame of 1052 bp,encoding 350 amino acids,a molecular weight of 36.90 k D,and a theoretical isoelectric point of 5.68.Sequence comparison showed that the similarity with the Medicago truncatula sequence was as high as98.9%.In the protein sequence,?-helix accounted for 32.86%,extended chain accounted for18.57%,?-turn angle accounted for 8.29%,and irregular curl accounted for 40.29%.The transientexpression of Ms THI1 gene in tobacco showed that Ms THI1 was expressed in chloroplast and cell membrane of tobacco,indicating that the gene may be involved in photosynthesis,transmembrane transport and osmotic regulation of cells.(3)Transformation of Ms THI1 gene in alfalfa and tobaccoIn this experiment,we used p CAMBIA1300 as vector skeleton,Bam HI and Pst I as restriction sited to construct tobacco overexpression vector of Ms THI1 gene.Agrobacterium tumefaciens were transformed in tobacco K326.19 tobacco plants overexpression of Ms THI1 gene were identified by PCR.The fruitful seeds were screened and planted in germination medium containing20ug/ml hygromycin,and obtain three T1 transgenic tobaccos for physiological indicators.The alfalfa overexpression vector of Ms THI1 gene was constructed by using p MDC123 as vector skeleton,Spe I and Xba I as restriction sited.Agrobacterium tumefaciens were transformed in alfalfa(Longmu 801).Nine alfalfa plants overexpression of Ms THI1 gene were identified by PCR.Alfalfa cuttings were used to provide plant materials for further study of Ms THI1 gene.(4)Changes of physiological indicators of transgenic tobacco with Ms THI1 gene under drought stressThe VB1 contents of overexpressing plants at each time point were higher than that of wild type plant,which prove that Ms THI1 gene can improve the contents of VB1 in tobacco.Overexpression of Ms THI1 gene can increase SPAD value in leave of plant under long-term drought stress.SPAD value of overexpressing plants were higher than those of wild type plant at the third day and the fifth day of stress treatment.The Fv/Fm of wild type tobacco,plant No.3 and plant No.7 showed a decreasing trend with the increase of stress time,but the change was not significant.At the same time under drought stress,the contents of MDA in wild type plant were the highest at the three time points of control treatment,stress treatment for 1 day and the third day.The contents of O2-in wild type plant were significantly higher than that in overexpressing plants.The contents of O2-in plant No.7 were significantly different from that in plant No.2 and plant No.3.The contents of SOD in wild plant,overexpressing plants showed an increasing trend with the increase of stress time,SOD contents in wild type plant were the highest at the same time point under drought stress.The contents of POD in wild plant were significantly lower than that in overexpressing plants,and the overexpression of Ms THI1 gene increased the contents of POD.With the increase of stress treatment time,the contents of Pro were the highest in the wild type plant after the third day of stress treatment,followed by the decrease of Pro contents,while the Pro contents of plant overexpressing plants showed an increasing trend,indicated that Ms THI1 gene overexpression improve the drought resistance of tobacco.