Functional Analysis Of Transcription Factor PagMYB88 On Cadmium Tolerance Of Poplar

The heavy metal pollution situation of soil is very serious.Drought is also one of the important limiting factors to the sustainable development of agroforestry.In arid and semi-arid areas,the combined stress of drought and cadmium is often formed due to industrial pollution and sewage irrigation.The MYB transcription factor family has a large scale and plays an important regulatory role in plant growth,development,physiological metabolism and other processes,and is involved in plant response to abiotic stress such as drought and heavy metals.Studies have shown that MYB88 gene can improve drought resistance in plants through ABA pathway,and it also plays an important role in stomatal development and root geotropism,which are related to Cd stress tolerance.Based on these reasons,combining with bioinformatics,physiology and molecular biology,MYB88 gene as research gene and poplar as research tree species,the PagMYB88 gene of 84K poplar(Populus alba×P.glandulosa)was cloned.Tissue specific expression analysis was performed on poplar under cadmium stress and drought-cadmium combined stress,and transgenic function verification was carried out on poplar.PagMYB88 overexpressed transgenic positive plants were obtained,and positive plants were stressed on potted seedlings.Physiological indexes such as plant height diameter,relative water content,biomass,photosynthetic parameters and cadmium content were determined,and the function of PagMYB88gene in poplar coping with cadmium stress and drought-cadmium combined stress was further explored and analyzed,so as to provide research basis for the improvement of forest resistance.The main research results are as follows:(1)The sequence of Pt MYB88 gene was obtained through the published gene database of NCBI.The basic physicochemical properties,signal peptide and transmembrane domain analysis of Pt MYB88 gene showed that Pt MYB88 gene was an unstable basic hydrophilic protein.Conservative motif analysis showed that MYB88gene had two same motifs,which were evolutionarily conserved among five dicotyledonous plants including Populus trichocarpa,Arabidopsis thaliana,Malus domestica,Populus deltoides and Salix purpurea.Promoter analysis showed that Pt MYB88 gene had unique SA and JA response elements compared with homologous gene Pt MYB124,and the number of ABA and Me JA response elements was also higher than Pt MYB124.(2)Tissue-specific expression analysis of poplar potted seedlings under without stress,cadmium stress and drought-cadmium combined stress showed that Pa BMYB88 gene expression level was highest in stem,followed by root system and lowest in leaf under no stress condition.The expression of Pa BMYB88 gene was also the highest in stem under cadmium stress and drought-cadmium combined stress,but lower in roots and leaves,and there was no significant difference in gene expression levels between roots and leaves.(3)Pt MYB88 sequence was obtained from NCBI database,and primers were designed.PagMYB88 gene was successfully cloned by PCR and sequencing.MYB88gene cloning analysis of 84K poplar showed that the open reading frame of PagMYB88 was 972bp and consisted of 324 amino acid codes.After DNA and protein sequence alignment,the results showed that the consistency of the PagMYB88DNA sequence of 84K and Pt MYB88 sequence was 98.35%and the amino acid similarity was 97.83%.The sequence consistency of PagMYB88 and Pa BMYB88 was99.38%and the amino acid similarity was 99.07%.The amino acid sequence of the functional domain of Pa BMYB88 and Pt MYB88 is completely consistent with that of PagMYB88.Agrobacterium tumefaciens infection solution was prepared to infect leaves,and transgenic plants were obtained.After DNA and RNA level identification,PagMYB88 overexpression positive transgenic lines was obtained.(4)Transgenic lines with PagMYB88 overexpression positive were transplanted and treated with cadmium stress and drought-cadmium combined stress to analyze the physiological index data.It was found that the transgenic lines had lower branch height,fewer leaf litter,more leaf survival number and better leaf state than the wild type under the combined stress.Compared with cadmium stress,drought-cadmium combined stress decreased the relative water content of stem,biomass,stomatal conductance(g_s)photosynthetic rate(A)and water use efficiency(WUE_i)of poplar stems,but increased cadmium accumulation in roots and stems.Overexpression of PagMYB88 gene could reduce the inhibition effect of cadmium stress on plant diameter,increase the relative water content of stem and root,but inhibit stem biomass.PagMYB88 gene could increase plant photosynthetic rate and water use efficiency and decrease plant transpiration rate(E)under cadmium stress.The PagMYB88 gene can reduce the accumulation of cadmium in leaves,above-ground parts and whole plants under complex stress,and reduce the transport capacity of cadmium in plants under complex stress.In conclusion,PagMYB88 transgenic plants had better resistance to cadmium stress and drought-cadmium combined stress than the wild type.PagMYB88transgenic plants could improve the photosynthesis and water use efficiency of plants under cadmium stress,so as to enhance the plant tolerance to cadmium stress.PagMYB88 transgenic plants can reduce the absorption and transport of cadmium under drought-cadmium combined stress,so as to improve the tolerance of plants to drought-cadmium combined stress.