Pectin Methylesterase Inhibitor MePMEI1 Functional Analysis And Screening Of Interacting Roteins

Pectin methylesterase inhibitors(PMEIs)are a family of polygenes,which can regulate the activity of pectin methylesterase(PMEs).PMEI was first found in kiwifruit to inhibit the activity of plant-derived pectin methylesterase(PMEs).At present,PMEI has been reported in many species,such as A.thaliana,rice(O.sativa),cotton(G.hirsutum),etc.It indicates that PMEI has a variety of biological functions: such as affecting plant growth,fruit development and ripening,cell and organ size,plant stress resistance and pathogen defense.At present,there are few reports about PMEI in cassava,and its physiological function and regulation mechanism need to be analyzed.A new cassava germplasm(RYG-1)with resistant to postharvest physiological deterioration was obtained in the early of our laboratory,it was found that the gene expression of a plant pectin methylesterase inhibitor was significantly increased,which might be related to the stress resistance process of cassava,and named as Me PMEI1.In this study,the gene was cloned and overexpressed in A.thaliana to explore the gene functions in salt and drought stresses,exogenous ABA response.In order to explore the interaction network of Me PMEI1,a mixed c DNA library of cassava roots,stems,leaves,flowers,fruits and seeds was constructed.The interaction proteins of Me PMEI1 were screened by yeast two-hybrid,the authenticity of protein interaction was verified by rotation verification and bimolecular fluorescent complimentary(Bifc)technique.The transcriptome data of SC8 and RYG-1 were used to analyze the expression of Me PMEI1,Gly2 and VDAC2 genes in fresh root tubers and root tubers placed for 3 weeks.The results provide a new way for us to further understand the interaction network of cassava Me PMEI1 protein and its regulation mechanism in the process of stress resistance.The main experimental results are as follows:1.A gene Me PMEI1 encoding pectin methylesterase inhibitor was cloned from cassava.The gene CDS was 609 bp,without intron and encoded 203 amino acids.At PMEI13 was the highest homology protein with Me PMEI1 in A.thaliana.Bioinformatics analysis showed that Me PMEI1 contained a signal peptide and a conserved PMEI domain.2.The fusion protein of Me PMEI1 and GFP was expressed in tobacco leaves instantaneously.The results showed that the fusion protein was located in the extracellular space,thus it was speculated that Me PMEI1 is a secretory protein.3.The results of phenotype observation and stress treatment of transgenic A.thaliana showed that: under the same culture conditions,the growth trend of over expression of Me PMEI1 was significantly better than that of wild-type A.thaliana.Me PMEI1 overexpressing plants still survived,whereas the WT plants was died under 100 mmol/L Na Cl treatment;Under the treatment of mannitol and ABA,the root system was more developed than the control.In summary,Me PMEI1 might participate in salt stress,drought stress and ABA response in transgentic A.thaliana,and improves the resistance of A.thaliana seedlings.4.p GBKT7-Me PMEI1 bait carrier was constructed successfully,and toxicity test and self activation test were carried out.The point-to-point interaction of Me PMEI1 and three pectin methylesterase Me PME1～3 was carried out by yeast two hybrid technology.The results showed that Me PME1～3 protein did not directly interact with Me PMEI1.5.The yeast two-hybrid c DNA library was successfully constructed from the root,stem,leaf,flower,fruit and seed of South China 8 cassava.were obtained,co-transformation of p GBKT7-Me PMEI bait plasmid and c DNA library into ah109 yeast cells.37 proteins with complete ORF were screened by yeast two-hybrid system,which includes Glyoxalase(Glyoxalase-2),voltage-dependent anion channel protein 2(VDAC2),nitrogen-regulated protein,xylose-glucose transferase(TCH4)and etc.6.12 candidate proteins full length sequence of gene CDS were successfully cloned from the c DNA of South China 8 cassava,include LHCA4、DUF4409、DUF3007、PSAK、PUB、VDAC2、P-II、Glyoxalase2、RPS20、P2C、CCR4、PPCTI,and p GADT7 prey vector was constructed.The results showed that VDAC2 and Gly2 could interact with Me PMEI protein by verification of point-point rotation.We successfully constructed Bimolecular fluorescence expression vectors: p NC-Me PMEI1-Ecc,p NC-VDAC2-Enc and p NC-Gly2-Enc.The results showed that VDAC2 and Gly2 proteins interacted with Me PMEI1 protein.7.Transcriptome analysis results showed that Me PMEI1,VDAC2 and Gly2 genes were expressed in 11 tissues of cassava,including fibrous root,tuber root,root tip,stem,stem tip,leaf,petiole,axillary bud,midvein,somatic embryo and brittle callus,in which the expression level of VDAC2 gene was the highest,and Me PMEI1 was lowest.VDAC2 had a high expression in tissues or organs with vigorous cell division of root tip,shoot tip,axillary bud and somatic embryo;while the expression of Gly2 gene was higher only in root tip,and the expression of Me PMEI1 gene was very low in all 11 tissues.Based on the analyses of the transcriptome data of fresh tuber roots and storage for 3-week tuber roots of SC8 and RYG-1,it was found that the expression of VDAC2 gene was higher in SC8 and significantly decreased in RYG-1 3-week stored tuber roots,while the expressions of Gly2 and Me PMEI1 genes were significantly up-regulated in RYG-13-week stored tuber roots,and the expression levels were lower in fresh tuber roots of SC8 and RYG-1,and SC8 3-week stored tuber roots.It was speculated that there may be negative regulation between VDAC2 and Me PMEI1 genes,while there is positive regulation between Gly2 and Me PMEI1 in delaying postharvest deterioration in cassava tuber roots.