The Mechanism Of MAPK6 Regulating MYB77 Involved In Salt Acclimation Of Arabidopsis

Soil salinization is one of the important factors that restrict the growth and development of plants and affect the yield of food crops.Plants have formed a complex adaptive mechanism to salt stress during the long-term evolution.Studies have found that non-halophyte plants can gain tolerance to high salt stress through physiological adaptation and molecular and cellular responses at non-lethal salt concentrations.The study on the molecular mechanism behind salt acclimation will greatly promote the detection of the mechanism of plant salt tolerance.In our preliminary work,a set of plant salt acclimation system was established and it was found that the mapk6 mutant shows defect in salt acclimation,indicating that MAPK6 plays an important regulatory role in the process of salt acclimation.By mining the transcriptome data of mapk6 mutants and wild-type Arabidopsis during salt acclimation,we have screened out several MAPK6-regulated genes,one of which is MYB77(AT3G50060).In order to further study whether MYB77 is involved in salt acclimation in Arabidopsis and how MAPK6 regulates MYB77 participating in salt acclimation in Arabidopsis,the thesis work has been carried out from the following aspects:(1)First,the phylogenetic tree of the Arabidopsis R2R3 MYB subfamily was constructed by bioinformatics methods,and the evolutionary relationship among them was analyzed based on their genetic relationship.Then,the MYB77 was analyzed for the DNA binding elements and the sequence characteristics of its target genes.The prediction of the possible downstream target genes of MYB77 transcription factor,such as RAB18,ERF109,etc.,will provide ideas and reference for elucidating the function of MYB77 transcription factor.(2)Using the CRISPR/Cas9 gene editing system to edit the MYB77 gene in wild-type Arabidopsis(Col-0),two myb77 single mutant lines(myb77-12,myb77-15)were obtained,and the plant hybridization technique was used for cross between myb77 mutant and the mapk6 mutant to obtain the mapk6-2 myb77-12 double mutant.(3)The salt acclimation phenotype was analysed on wild-type Arabidopsis and myb77 mutants,and it was found that when they were acclimated on low-salt(50 m M Na Cl)medium for 48 h and then transferred to high-salt(200 m M Na Cl)medium,the wild-type seedlings can maintain basic growth,while the myb77 mutant seedlings appear albino death.When the seedlings were directly transferred to the high-salt medium without being acclimated by low-salt(50 m M Na Cl),all seedlings,both Arabidopsis wild type and myb77 mutant,showed albino death.The experimental results showed that MYB77 gene is involved in the process of plant salt acclimation.(4)Based on the experiment result that myb77 mutant showed the same salt acclimation deletion phenotype as the mapk6 mutant,combined with the information provided by the previous transcriptome data that MAPK6 regulates the expression of MYB77 during salt acclimation,we speculated that MYB77 might participate in the salt acclimation pathway mediated by MAPK6.In order to explore the relationship between MYB77 and MAPK6,we used yeast two-hybrid and luciferase complementation assay to detect whether the interaction between MAPK6 and MYB77 protein occurred.The results indicate that there is an interaction between MAPK6 and MYB77 protein.The mapk6-2 myb77-12 double mutant obtained by hybridization also showed the salt acclimation deletion phenotype,Those proved that MAPK6 participates in salt domestication by regulating MYB77.(5)In order to detect whether MAPK6 influenced the function of MYB77 via phosphorylation,we constructed the prokaryotic expression vector p GEX-4T-1-MAPK6 and p GEX-4T-1-MYB77 and optimized the condition for MYB77 protein expression in E.Coli DE3(BL21).We find that the optimum condition for MYB77 expression is at 30℃ for 4 h;We also constructed 1307-HA-Flag-MYB77 vectors and have screened resistant transgenic lines.These works provided research materials for the in vitro phosphorylation of MYB77 by MAPK6 and whether the phosphorylation of MYB77 by MAPK6 affects the regulation of MYB77 in the process of salt acclimation.(6)The p CAMBIA-3301H-MYB77 vector was constructed and the overexpression transgenic homozygous line(MYB77-OE)was screened.The subsequent salt acclimation experiment on the overexpression line is underway,hoping to provide more evidence for the involvement of MYB77 gene in the salt acclimation process.(7)The complementary vector of MYB77 gene was constructed in order to obtain MYB77 complementary transgenic lines,and experimental work is underway.