Cloning,Prokaryotic Expression And Subcellular Localization Of Pectin Methylesterase Gene In Onion

Onion(Allium cepa L.)belongs to the genus Allium L.,subfamily allioideae,family Amaryllidaceae,order Asparagales vegetable crops in the taxonomy(APGIII system),which is widely cultivated around the world.Due to its huge genomic(15290 Mbp/C),the research on the field of molecular biology is relatively weak,which is mainly on the molecular markers of fertility and color related genes,and little research on onion gene function.Pectin esterase [pectin methylesterase,PME],is a kind of cell wall proteins that catalyzes the D-galactose acid units methylated in pectin complexes,according to its conserved domain,the enzyme forms a large gene family,and it plays a important role in different developmental stages of plant growth and physiological processes by acting on the cell wall.Previous studies had found that pectin esterase was involved in a variety of physiological processes,such as fruit maturation,organ abscission,microspore development and pollen tube elongation,seed germination,disease resistance,cambium cell differentiation and so on.In the research of onion fertility differential gene expression,we found that a 350 bp fragment is always expressed in fertile materials,and cloned its coding sequence.The results of the protein sequence alignment showed that it has pectin methyl-esterase domain,belonging to a member of the pectin methyl-esterase superfamily.In this study,the onion fertility restorer line 12-10(S,MsMs)was used as research materials,and the methods of gene cloning,sequence analysis,prokaryotic protein expression,biological activity analysis and gene gun transformation of onion epidermis were used,it was preliminarily proved that the onion AcPME gene is a member of the PME protein superfamily from the molecular biology experiment,which was located in the cell wall or cell membrane with PME protein activity during onion pollen development.The results of this study were as follows:(1)The sequence of Ac PME gene obtained through rapid amplification of cDNA ends(RACE)based on the sample of flower bud in onion.The analysis of amino acid deduced from the cDNA exhibited a higher identitiy between AcPME and PME of homologous monocotyledons such as rice,maize and sorghum.Bioinformatics analysis showed that AcPME protein had significant signal peptide and Transmembrane domain(SP/TM),PMEI and PME domains belongs to type I with a long N terminal PRO region in the PME families on the basis of analysis of secondary structure.AcPME tertiary structure showed an obvious shallow cleft,4 amino acid residues(T423,Q453,R565 and W567)binding to substrate and 2 residues(D476 and D497)involved in catalysis.(2)The pGEX4T-1-PME and pET24a-PMEI prokaryotic expression vectors were successfully constructed and optimized for expression conditions in E.coli BL21(DE3)strain.The expression of pGEX4T-1-PME was determined to be 17 oC,0.3 mM IPTG induced for 4 h;the expression of pET24a-PMEI was determined to be 37 oC,0.1 mM IPTG induced for 4 h.SDS-PAGE electrophoresis indicated the presence of recombinant proteins,the prokaryotic expression protein pGEX4T-1-PME was a soluble protein,while the pET24a-PMEI protein was in the form of inclusion bodies.The corresponding molecular weights of the expressed proteins were about 61.73 kD and 24.67 k D respectively,which were consistent with the expected sizes of the target proteins.(3)The target protein was purified from the p GEX4T-1-PME expression product by GST-tag purification column,the target protein pET24a-PMEI was also purified by His-tag purification column.(4)The biological activity analysis showed that pGEX 4T-1-PME(PME)had significant PME activity;PET24a-PMEI(PMEI)was able to inhibit the activity of its own protein PME but not to inhibit the activity of exogenous protein P5400.(5)The pUC19-35S-AcPME-AcGFP subcellular localization vector was successfully constructed by pUC19 vector and AcGFP(Clontech)green fluorescent protein gene,the pUC19-35S-AcGFP vector was used as a positive control.Under the fluorescence microscope,it was found that the AcPME fusion protein was localized in the cell membrane or cell wall,which proved that the protein was a kind of action on the cell membrane or cell wall protein.