Cloning And Functional Verification Of Medicago Falcata L. Grazing Tolerance Candidate Genes

Medicago falcata L.,also known as medicago falcate and wild alfalfa,is a perennial alfalfa leguminous herbage,widely distributed in Russia,northeast China and other cold regions.It is an important wild forage.M falcata.has strong adaptability,and has excellent characteristics such as grazing tolerance,drought resistance,cold resistance,salt and alkali resistance,and nitrogen fixation root system.M falcata.has low grazing cost and high nutritional value,but the existing alfalfa varieties have poor tramping resistance and are susceptible to the stress of biological and abiotic factors.Most alfalfa varieties have low grazing resistance and decreased grass persistence under continuous grazing.Alfalfa with strong grazing tolerance is urgently needed for the improvement of degraded grassland and the establishment of artificial grassland.Therefore,it is of great significance to improve grazing tolerance and breed M falcata..Based on the sequence information of differentially expressed genes in the transcriptome sequencing of grazing tolerance traits in our research group,upregulated genes were screened,and specific primers were designed for gene cloning,and 164 genes of MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 obtained.Bioinformatics analysis was carried out on these four genes,and the expression characteristics of these four genes were studied by q RT-PCR technology,and they were transformed into Arabidopsis thaliana by Agrobacterium-mediated method for grazing tolerance function verification,which laid a foundation for further research on the functional characteristics of grazing tolerance related genes.Specific research results are as follows:1.The 4 genes MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 of M falcata.were cloned.The cDNA length of MfMYB30 transcription factor gene was 1060 bp,the open reading frame（ORF）was 957 bp,encoding 318 amino acids.The similarity between this gene and transcription factor Mt MYB30 was 92%.The cDNA length of MfMYBR1transcription factor was 1345 bp,the open reading frame（ORF）was 1230 bp,encoding409 amino acids.The similarity between the DNA and transcription factor Mt MYBR1 of Medicago truncatula was 96%.The cDNA length of MfCAB1 gene was 893 bp,the open reading frame（ORF）was 801 bp,encoding 266 amino acids.The similarity between the DNA sequence of MfCAB1 gene and that of Mt CAB1 gene was 99%.The cDNA length of MfCOL16 gene was 1338 bp,the open reading frame（ORF）was 1251 bp,encoding 416amino acids.The similarity between the DNA sequence of MfCOL16 gene and that of Mt COL16 gene of Medicago truncatula was 97%.2.qRT-PCR was used to analyze the expression characteristics of MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 genes under mowing（simulated grazing）conditions,and the results showed that the relative expression of MfMYB30 gene showed a gradual increase with the increase of mowing days and reached the peak after 7 d of mowing;the relative expression of MfMYBR1 gene showed a gradual decrease with the increase of mowing days and reached the highest expression after 3 d of mowing and started to decrease after 5 d of mowing.The relative expression of MfMYBR1 gene showed a decreasing trend with the increase of mowing days,with the highest expression after 3 d of mowing and the decreasing expression after 5 d of mowing;the relative expression of MfCAB1 gene showed a decreasing trend with the increase of mowing days,with the highest expression after 3 d of mowing and the decreasing expression after 5 d of mowing,but the increasing expression after 7 d of mowing.Under mowing stress,the relative expression of MfCOL16 gene showed a gradual increase with the increase of mowing days,and reached the peak expression after 7 days of mowing.This indicates that mowing treatment had a significant effect on the expression of these four genes,and the MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 genes may have certain regulatory functions in the face of mowing or grazing stress in M falcata..3.Using A.thaliana protoplast transfection technology,transient expression vectors were constructed and subcellular localization analysis was performed.Fluorescence signals were observed using laser confocal microscopy,which further confirmed that MfMYB30,MfMYBR1 and MfCOL16 proteins were subcellularly localized in the nucleus and MfCAB1 protein was localized in the chloroplast.The transcriptional activation repressor activity of MfMYB30 and MfMYBR1 was also detected,and the GUS activity results showed that MfMYB30 and MfMYBR1 transcription factors were transcriptional repressors.4.The plant expression vectors of four genes MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 were successfully constructed,namely 1300-MfMYB30-c YFP,1300-MfMYBR1-c YFP,1300-MfCAB1-c YFP and 1300-MfCOL16-c YFP vectors.A.thaliana was transformed using Agrobacterium tumefaciens for functional verification,and after screening and identification,T₃generation transgenic pure seeds were obtained.The phenotypic observation of the T₂generation transgenic strain showed that compared with the wild type,the A.thaliana strain overexpressing the MfMYB30 gene grew vigorously and significantly faster than the wild type,with significantly more rosette leaves and stem leaves than the wild type;overexpression of MfMYBR1 gene suppressed the early growth of A.thaliana;overexpression of MfCAB1 gene promoted the speed of A.thaliana and thus the growth and development of A.thaliana;overexpression of MfCOL16 gene delayed the speed of A.thaliana and the growth and development of the plant.5.The transgenic A.thaliana homozygous lines were mowed（simulated grazing）,and the phenotype and gene expression were observed 3,5 and 7 days after mowing.The results showed as follows:The growth and development of A.thaliana plants overexpressing MfMYB30 gene were faster than those of wild type,and the relative expression of MfMYB30 gene increased gradually after mowing and reached a peak value 7days after mowing.The growth and development of A.thaliana plants overexpressing MfMYBR1 gene were similar to that of the wild type,and the expression level was 2.3times higher than that of the wild type after the 3rd,5th and 7th days of mowing,and the expression level was not significantly up-regulated or down-regulated with the increase of days after mowing.With the increase of mowing days,the expression level of MfCAB1overexpressed strains gradually increased,and the expression level reached a peak on the7th day after mowing,which was about 5.7 times that of the wild type,indicating that the MfCAB1 gene continued to be highly expressed in A.thaliana under mowing conditions.With the increase of cutting days,overexpression of MfCOL16 gene promoted the growth and development of A.thaliana,indicating that MfMYB30,MfMYBR1,MfCAB1 and MfCOL16 genes had an effect on the growth and development of A.thaliana,and it was speculated that these four genes were involved in the regulation of grazing tolerance mechanism of M falcata.6.Chlorophyll content of transgenic MfCOL16 and MfCAB1 A.thaliana was measured.The results showed that the content of chlorophyll a in transgenic MfCAB1 A.thaliana was slightly higher than that of wild type,and the content of chlorophyll b was slightly lower than that of wild type.The content of chlorophyll a and b was significantly higher than that of wild type on the 21 st day.The content of chlorophyll a,chlorophyll b and total chlorophyll of transgenic A.thaliana with MfCOL16 gene was slightly lower than that of wild type at 7 d,and the content of chlorophyll a,chlorophyll b and total chlorophyll at 21 d were significantly higher than that of wild type.It shows that MfCOL16and MfCAB1 can regulate the biosynthesis of chlorophyll in A.thaliana,improve the total content of chlorophyll in transgenic plants,and then improve the photosynthesis of A.thaliana to promote its growth and development.This study explores and analyzes the expression characteristics and functions of these important genes,which will help to carry out in-depth research on the grazing tolerance of M falcata.,and also lay a foundation for breeding M falcata.varieties with strong grazing tolerance by molecular biological means.