Studies On Genetic Transformation And Droung-resistance Of Alfalfa HD-Zip Transcription Factor Gene Mfhb-1 In Arabidopsis And Alfalfa

Mfhb-1 gene was obtained by subtractive hybridization in the early stage of Alfalfa somatic embryogenesis.Bioinformatics analysis showed that the gene belonged to class I transcription factor gene of homeodomain leuzine zipper protein（Homeodomain-leuzine zipper protein,HD zip）.At present,the research on transcription factors is becoming more and more comprehensive and in-depth.More and more HD zip class I transcription factors have been found to be involved in plant drought resistance response,embryonic development and photomorphogenesis.In order to comprehensively study the biological function of Mfhb-1 gene,using wild-type Arabidopsis thaliana as the starting material,the Mfhb-1 gene was transferred into Arabidopsis thaliana through agrobacterium tumefaciens mediated genetic transformation system.After molecular biological identification,the transgenic Arabidopsis plants were obtained.Then take the Mfhb-1 transgenic Arabidopsis and wild-type Arabidopsis as the research object,use the method of stopping watering to simulate drought stress,observe the apparent morphology of Mfhb-1 transgenic Arabidopsis and wild-type Arabidopsis,detect the expression level of Mfhb-1 transgenic Arabidopsis,detect the physiological and biochemical indexes related to drought resistance in Mfhb-1 transgenic Arabidopsis and wild-type Arabidopsis,and evaluate the drought resistance of the two groups of Arabidopsis.The main research results are as follows:（1）Complete the genetic transformation of Mfhb-1 gene in Arabidopsis and obtain the transformed plants of Mfhb-1 gene in Arabidopsis.（2）The transgenic plants of Arabidopsis thaliana with Mfhb-1 gene were screened by molecular biology,and the T₃ generation homozygous Arabidopsis thaliana strains were obtained.（3）The total DNA of T₃ generation homozygous Arabidopsis was extracted for PCR reaction and agarose gel electrophoresis was carried out.The results showed that the target band of 1300 bp,which preliminarily proved that Mfhb-1 gene had been successfully integrated into the genome of Arabidopsis thaliana.（4）Agarose gel electrophoresis and quality detection were carried out on the T₃ generation homozygous transgenic Mfhb-1 gene Arabidopsis RNA.c DNA was obtained by reverse transcription.PCR amplification was carried out with c DNA as template.The product was detected by agarose gel electrophoresis.The result showed that the target band was 250 bp,which further proved that Mfhb-1 gene had been successfully transferred into Arabidopsis and expressed in Arabidopsis.（5）The method of stopping watering was used to carry out drought stress on T₃ generation homozygous Mfhb-1 transgenic Arabidopsis and wild-type Arabidopsis.The real-time fluorescence quantitative expression of T₃ generation homozygous Mfhb-1 transgenic Arabidopsis was detected in different periods of drought.The results showed that the expression level of Mfhb-1 gene increased significantly with the extension of drought time.（6）The apparent morphology of the two groups of Arabidopsis were observed in different periods of drought,and the physiological and biochemical indexes related to drought resistance of the two groups of Arabidopsis were measured.The results showed that the growth state and drought resistance indexes of the transgenic group were better than those of the wild group.（7）The transgenic alfalfa plants with Mfhb-1 gene were obtained by Agrobacterium tumefaciens mediated genetic transformation.In this experiment,the best infection solution OD value of alfalfa genetic transformation system was 0.55,the best infection time was 10 min,and the best screening concentration of hygromycin was 35 mg / L.（8）Twelve alfalfa plants with hygromycin resistance were screened by applying hygromycin to the transgenic alfalfa plants with Mfhb-1 gene.The PCR results showed that the total DNA of Alfalfa Strain mhb-1 and Alfalfa Strain mhb-1 were amplified successfully,and the PCR results of Alfalfa Strain mhb-1 and Alfalfa Strain mhb-1 were positive.It is proved that Mfhb-1 gene has been successfully integrated into the genome of alfalfa and has been successfully expressed in alfalfa.The results showed that under normal growth conditions,there was no significant difference in apparent morphology between transgenic and wild-type Arabidopsis,and there was no significant difference in physiological and Biochemical Resistance Indexes between the two groups.However,drought induced the expression of Mfhb-1 gene in Arabidopsis,and the Mfhb-1 transcription factor involved in regulating the response of Arabidopsis to drought,thus increasing the synthesis and accumulation of osmoregulatory substances in Arabidopsis,improving the water retention capacity of Arabidopsis,ensuring the synthesis of chlorophyllin and delaying the degradation of chlorophyll.At the same time,the expression of Mfhb-1 gene increased the enzyme activity of antioxidant enzymes in Arabidopsis,enhanced the ability of Arabidopsis to scavenge reactive oxygen species,and reduced the damage of reactive oxygen species to plasma membrane.These physiological and biochemical differences make the growth status of Arabidopsis in the transgenic group better than that in the control group.In conclusion,transgenic Arabidopsis with Mfhb-1 gene showed better adaptability to drought stress.It is speculated that the expression of Mfhb-1 gene improved the drought resistance of Arabidopsis.This provides a theoretical basis and clue for a comprehensive and in-depth study of the biological function of Mfhb-1 gene and the regulatory mechanism of its function.In addition,the Mfhb-1 gene has been successfully integrated into Alfalfa by Agrobacterium mediated method,which provides experimental materials for the quality improvement of alfalfa.