Research On The Mechanism Of MIR156 Regulated Plant Stress Tolerance And Its Application

The use of phytoremediation of soil pollution is a new environmental biotechnology.The research is based on the discovery and application of hyperaccumulators.However,hyperaccumulators have the disadvantages of relatively small biomass and insufficient root development.To large-scale production practice.As a significant regulator of plant growth and development,MIR156-SPLs has a broad application prospect,such as plant growth cycle,biomass and root development.In this study,we used overexpression of MIR156 Arabidopsis thaliana(35S::MIR156A)and MIR156 knockout Arabidopsis thaliana(Ubi10::MIM156),and their phenotype and mechanism of Cd stress were studied.In addition,overexpression and knockout of MIR156 were carried out on the callus of Sedum plumbizincicola to explore the differences in plant-related cadmium tolerance and enrichment under the pathways of MIR156-SPLs.The MIR156-SPLs signal pathway used to improve Sedum plumbizincicola accumulating characteristics is possible。The main conclusions as follows:(1)The transgenic plants and the wild type plants showed different phenotypes after the true leaves of Arabidopsis plants grew.35S::MIR156A leaves growth rate were significantly faster than WT,and the abaxial trichome germination was significantly later than WT.Ubi10::MIM156 showed a delayed Phase change phenotapes,the number of leaves less than 35S::MIR156A and WT,leaves possessed serrated lealf margins..(2)In order to study the role of MIR156 in response to cadmium stress in A.thaliana,we observed the phenotypic and injury levels of A.thaliana under cadmium stress.The results showed that compared with WT,overexpression of 35S::MIR156A was relieved after cadmium stress,while Ubi10::MIM156 was susceptible to severe cadmium stress.(3)The results of the determination of Cd content in the ground and underground of Arabidopsis thaliana showed that the absorption of cadmium at root was 35S::MIR156A>Ubi10::MIM156>WT,While the ground part of the cadmium content is different with underground parts and the absorption of cadmium at root was Ubi10::MIM156>WT>35S::MIR156A.Although Ubi10::MIM156 had the lowest cadmium uptake,the 35S::MIR156A plant was the highest,but the cadmium content on the aerial part was opposite to that of the subsurface.This indicates that Ubi10::MIM156 cadmium transport efficiency is significantly higher than WT and 35S::MIR156A.The results of this study show that MIR156 can participate in the regulation of cadmium uptake and transport.(4)In order to elucidate the molecular mechanism of Cd in Arabidopsis overexpression of MIR156 and mutant Ubi10::MIM156,We measured the expression levels of several Cd transporter genes,such as ATP-binding cassette transporters,HAM(heavy metal ATPases)and ZIP(Zrt/Irt-like Protein).The results showed that the expression of Cd in the roots of the heavy metal transporter ZIP 1-4 and the expression of Cd in the root cells into the vacuole ABCC1 gene in 35S::MIR156A were significantly higher than those in WT,While in Ubi10::MIM156 was significantly lower than WT.The expression level of HMA4 gene responsible for transporting Cd to the aerial part of the stem is Ubi10::MIM156>35S::AfMIR156A>WT.Based on this,combined with the accumulation of cadmium on the ground part of the three materials,it is speculated that the cadmium absorbed into the root of the 35S::MIR156A material,and a large amount of cadmium was transported and fixed in the cell vacuole,and the upward transport capacity of the stem decreased Root accumulation of cadmium.(5)MIR156 plays an important role in plant development.Under normal growth conditions and the 5μM cadmium sulfate solutions,35S::MIR156A biomass was significantly higher than that of WT,Ubi10:,MIM156 was significantly lower than that of WT.The number of lateral roots of 35S::MIRS::plant was significantly higher than that of WT and Ubi10::MIM156 plant,while the root of the 35S::MIR156A is widest.The principal root of Ubi10::MIM156 is significantly longer than that of WT and 35S::MIR156A.This indicates that the Ubi10::MIM156 plants are more conducive to the repair of deep soil.(6)The regeneration medium was established by MS medium supplemented with 300 mg/L hydrolyzed casein,and the recipient material was provided for the genetic transformation of S.plumbizincicola.The results showed that the best medium for callus induction was 2,4-D lmg/L + 6-BA 0.3mg/L,the induction rate was 92.01%;The optimal differentiation medium was 2,4-D 0.1 mg/L + 6-BA 1 mg/L,the differentiation rate was 27.44%;The suitable bud growth medium was:2,4-D 0.1mg/L+ 6-BA 0.5mg/L;suitable rooting medium:IBA 1-2mg/L.The experiment of antibiotic susceptibility of S.pliumbizincicola showed that:the type of antibiotics suitable for the genetic transformation of G418,the critical concentration was 40mg/L for 20 d.(7)Theplant expression vector PBI121 containing the NPTII(aminoglycoside-3-phosphate transferase)resistance marker gene was used as a backbone.The MIR156a overexpression vector(MIR156PBI)and the target gene Mimicry156 expression vector(MIMPBI)were constructed.The PBI121,MIR156PBI,MIMPBI carriers were used to bombard the callus and resistant calli were obtained by G418 multi-generation screening.Three resistant calli were cultured in 0μM and 1000pMCdSO4 medium.The results showed that the growth of callus of MIR156PBI vector was the fastest and the empty vector was the second,and the MIMPBI vector was the slowest.(8)Treated with Op.M and 1000μMCd,the ratio of fresh weight growth rate after cadmium treatment was used to measure the effect of Cd stress on the callus.The ratios of pBI121,MIR156PBI and MIMPBI were 0.49,0.66,and 0.31.The quality of callus transformed to MIR156PBI vector under the condition of 1000pM Cd was better than that of MIM156 callus.These results indicated that the MIR156 callus showed stronger tolerance on Cd stress.At the cellular level,MIR156 could enhance the tolerance of S.plumbizincicola to Cd stress.