Identification And Molecular Mechanism Analysis Of FtADH1 And FtPMEI13 Involved In Drought Resistance Of Fagopyrum Tataricum

In recent years,as a high-quality small miscellaneous crop,tartary buckwheat has significantly increased its planting area and yield.With the passage of time,the drought and desertification of the global land has become a limiting factor for crop production.Drought stress is an important factor in reducing the yield of tartary buckwheat in arid and semi-arid areas.Therefore,improving the tolerance of tartary buckwheat to drought stress is an urgent need for the development of tartary buckwheat production.In this experiment,290 resources were subjected to drought treatment during the germination period,and the relative germination rate,germination energy,germination index and seedling length were analyzed to evaluate the drought resistance levels of different materials.The total membership function and drought resistance index were used to conduct genome-wide association analysis.Significant independent single nucleotide polymorphisms(SNPs)associated with drought resistance were located,and two drought resistance candidate genes were identified according to SNPs.In this study,FtADH1 and FtPMEI13,were cloned from Pinku No.1 c DNA,and the p CAMBIA1302 overexpression vector was constructed.Through genetic transformation of tartary buckwheat hairy roots and heterologous expression of Arabidopsis thaliana,gene expression characteristics and detection under stress were analyzed.Physiological indicators related to drought resistance and identification of drought resistance-related phenotypes,evaluation of the drought resistance of transgenic lines,preliminary analysis of the molecular mechanism of FtADH1(Alcohol dehydrogenase)and FtPMEI13(Pectin methylesterase inhibitor)involved in drought resistance of tartary buckwheat.The main results are as follows:1.The membership function values of 290 materials showed positive distribution,and different materials showed different drought resistance ability,which were divided into five drought resistance levels,among which 29 materials with extreme drought resistance were selected.2.The genome-wide association analysis was conducted through the drought resistance index and the total membership function,we found a candidate locus on chromosome 8 which was significantly related to drought tolerance.The analysis of the upstream and downstream 400 kb segments revealed a total of 28 candidate genes.The candidate genes were induced by PEG6000,and 11 up-regulated genes were screened.It was found that FtADH1 and FtPMEI13 showed the same change trend after ABA induction as after PEG6000 induction.At the same time,we detected that FtADH1 has 3 SNP sites upstream of the gene.There is a SNP site in the exon,which makes Ala mutate to Gly,causing non-synonymous mutations,and the gene expression level of this gene is high in materials with high drought resistance level,and the gene expression level is low in materials with low drought resistance level.FtPMEI13 has two SNP sites in the exons,but they are all synonymous mutations.An insertion of base A was detected upstream of the gene.which destroyed the core element CAAT.At the same time,it was found that the expression of FtPMEI13 in different drought resistant materials also showed great differences.3.The FtADH1 gene was cloned and identified: Phylogenetic analysis shows that it is closely related to Fagopyrum esculentum,Beta vulgaris,Arabidopsis thaliana and Glycine max.The results of q RT-PCR analysis of tartary buckwheat during the whole growth period showed that FtADH1 was mainly expressed in roots and stems.Through the construction of p CAMBIA1302-FtADH1 overexpression vector,genetic transformation of tartary buckwheat hairy roots and Arabidopsis thaliana,PEG6000 treatment of hairy roots showed that the expression of FtADH1 gene was up-regulated after PEG6000 treatment for 2 and 6 days,and the expression of FtADH1 gene in the overexpressed roots was significantly higher than that in the control.The activity of CAT,POD and SOD were increased and the content of MDA was decreased under PEG6000 stress.Under mannitol treatment,the germination rate and root length of FtADH1 overexpression lines in Arabidopsis were higher than those of the control,and the water loss rate of the detached leaves was lower than that of the control.Through repeated drought,it was found that the transgenic line was more droughtresistant than the 1302 empty line.After drought,compared with the control,the transgenic line had higher antioxidant enzyme activity and reduced ROS content.The results of disease resistance test showed that FtADH1 gene was involved in the disease resistance pathway of tartary buckwheat,which may play a positive regulatory role.4.The FtPMEI13 gene was cloned and identified: Phylogenetic analysis shows that it is closely related to Fagopyrum esculentum,Beta vulgaris and Arabidopsis thaliana.The results of q RT-PCR analysis of tartary buckwheat during the whole growth period showed that FtPMEI13 was mainly expressed in stems and leaves.Through the construction of p CAMBIA1302-FtPMEI13 overexpression vector,genetic transformation of tartary buckwheat hairy roots and Arabidopsis thaliana,PEG6000 treatment showed that the expression of FtPMEI13 gene was significantly up-regulated in transgenic roots,and the expression of FtPMEI13 gene in the overexpressed roots was significantly higher than that in the control.After PEG6000 treatment,the activities of cat,pod and SOD in transgenic roots increased,while the content of MDA decreased.Under mannitol treatment,the germination rate and root length of FtPMEI13 overexpression lines in Arabidopsis were higher than those of the control,and the water loss rate of the detached leaves was lower than that of the control.Through repeated drought,it was found that the control line wilted seriously,while the transgenic line was more drought resistant.After drought,compared with the control,the transgenic line had higher antioxidant enzyme activity and reduced ROS content.The results of disease resistance test showed that FtPMEI13 gene was involved in the disease resistance pathway of tartary buckwheat,which may play a negative regulatory role.In conclusion,290 materials of tartary buckwheat were analyzed by genome-wide association analysis,and two candidate genes for drought resistance were found.These two candidate genes were transformed into hairy root of tartary buckwheat and heterologous expression of Arabidopsis thaliana for preliminary functional verification,which laid a foundation for further understanding of the molecular mechanism of tartary buckwheat drought resistance.