Preliminary Study On Identification Of Downstream Genes Regulated By Medicago Ruthenica MrDREB1 Transcription Factor And Its Mechanism

In the stress environment,DREB transcription factors have a very positive effect on plants,most of them are analyzed for their functions,and their regulatory mechanisms are rarely reported.Studies have shown that the exogenous MrDREB1 gene can affect the expression of the downstream stress response gene MrCAS15A.The 14 th amino acid(valine V)and 19 th amino acid(glutamate E)in its protein are important CRT components.The main results of this study are as follows:1.Medicago ruthenica were used as the material to extract the genomic DNA of the 4-week-old seedlings,and the DNA(Deoxyribonucleic Acid)sequence of MrCAS15A with a size of 1266 bp was cloned by PCR.The genomic sequence information obtained by amplification was then amplified using Genome Walking technology to obtain 1968 bp MrCAS15A promoter fragment.PlantCARE predictive analysis results show that the promoter sequence contains multiple functional elements related to stress resistance,of which the DRE element(ATGTCGGTA)is located at-(1643-1651)bp,and the promoter also contains abscisic acid and salicylic acid.Related components such as hormone response.2.The yeast one-hybrid experiment preliminarily verified the transcriptionalactivation of MrCAS15A by MrDREB1 protein in vivo.The results showed that on SD/-Ura/-Leu/AbA double-deficient medium,pGADT7-MrDREB1-pAbAi-3×DRE yeast strains grew single colonies,and The growth trend is basically the same as that of the positive control,and pGADT7-MrDREB1-pAbAi-3×DRE mutant has no single yeast colonies.The results show that MrDREB1 transcription factor regulates its downstream reporter gene MrCAS15A by combining DRE cis-acting elements.3.In order to further investigate whether MrDREB1 also has a regulatory effect under the resistance of lentil beans to low temperature,drought and high salt stress,this study used the hairy root transformation method to introduce foreign genes overexpressing(OE)and tasiRNA interference expression(tasi)into lfalfa Bean seedling root tissue.The seedlings with basically the same growth conditions were selected and subjected to stress treatment at-8?,300 mM Mannitol and 200 mM NaCl.After treatment,the total RNA of the roots of single alfalfa beans was extracted,and each sample was subjected to 3 biological replicates.After reviewing the literature,we found that ERD10,COR413,P5CS,and PP2C are regulated by DREB1 in plants such as chrysanthemum,Arabidopsis,and alfalfa.We tested whether they are also regulated in lentil beans.The results of qRT-PCR showed that the exogenous MrDREB1 gene positively regulated the expression of MrERD10 and MrCOR413,and negatively regulated the expression of MrP5CS.MrPP2C may not be directly regulated by MrDREB1.We tested the physiological and biochemical indicators of transgenic lentil bean plants.The results showed that MrDREB1 reduced the content of MDA and increased the enzyme activity of POD and SOD and the content of Pro.In summary,the preliminary verification that MrDREB1 regulates MrCAS15A is achieved by specifically binding to the DRE cis-acting element.MrDREB1 also has a regulatory effect on MrERD10,MrCOR413,MrP5CS and MrPP2C.Overexpression of MrDREB1 can also improve the tolerance of lentil beans under stress.