Study On Cloning Of The Two Key Enzyme Genes In Ethylene Biosynthesis Pathway Of Olea Europaea L. And Prokaryotic Expression Of The ACO In E.coli

Olive(Olea europaea L.),an evergreen tree of Olea Genus,Oleaceae Family,and origin from Asia minor,was one of the four famous woody oil crops in the world.The olive oil,extracted from olive fruit,had a very high nutritional value and was honored as "Queen Of Vegetable Oil" and "Liquid Gold",because it was rich in unsaturated fatty acids and bioactive substances.Consequencely,it was recognized as one of the most healthy cooking oil in the world.Ethylene,known as a vital gaseous plant hormone,had an important influence on aging and loss of plant flower and fruit,make response of plant adversity stress,and played many important physiological function in plant lifecycle.Thus,ethylene seriously affected the quality and yield of olive fruit,and as a result,it had a great impact on the nutrition value and economic value of olive.Nowadays,the ethylene biosynthesis pathway had been clearly cognized in higher plants.It was well known that 1-aminocyclopropane-l-carboxylic acid synthase(ACS)'and 1-aminocyclopropane-1-carboxylic acid oxidase(ACO)were two key enzymes in this pathway,and they seriously affected the production of plant endogenous ethylene.The DNA and cDNA sequences of one olive ACS gene and three olive ACO genes were obtained from olive fruit genome DNA and total RNA by using RT-PCR,RACE,FPNI-PCR and an olive transcriptome database.And the four genes,designated as OeACS,OeACO1,OeACO2,OeACO3,respectively,were analyzed by using bioinformatics technology.In addition,the full ORF of three OeACOs were integrated into the expression vector pET30b(+),and successfully 'heterogeneous expressed in E.coli BL21(DE3)induced by IPTG.Besides,the enzyme function verification of the three recombinant proteins were also conducted.The main research results were as follows:1.The length of obtained OeACS DNA and cDNA were 1882bp and 1423bp respectively,containing four exons and three introns,encoding 423 amino acids in the ORF.Related bioinformatics analysis suggested that this OeACS protein fragment belonged to unstable protein,with a molecular weight of 47871.55,pI of 6.32,molecular formula of C2125H3339N583O638S19,and without signal peptide and transmembrane regions.The OeACS protein fragment was composed of alpha helix of 42.08%,beta turn of 11.82%and random coil of 26.24%.The amino acids sequences IQMGLAENQ and VYSLSKDLGLPGFRVG were highly conserved in the polypeptide chain,and N185,Y216,D213of them,N1 of PLP could form hydrogen bonding with O3'of the pyridine ring.Y128could stable the pyridine ring of PLP,K252 could bind with PLP and form schiff's base with PLP.The Y128,A103,T104,S249,S25 and R260 of the protein active site participated in enzymatic reaction,converting SAM to ACC.2.The length of the three OeACOs DNA were 1463bp,1481bp and 1414bp respectively,and all composed of four exons and three introns.The length of the genes cDNA were 1169bp,1150bp and 1155bp respectively,and the length of their ORF were 954bp,951bp and 954bp,encoding 317,316 and 317 amino acids,respectively.And the ORF sequences of these three genes shared a very high similar,above 90%,according homologous comparison.Related bioinformatics analysis suggested that:(1)OeACO1 was composed of alpha helix of 45.11%,beta turn of 11.36%and random coil of 24.92%,and it belonged to stable protein,with a molecular weight of 36051.3,pI of 5.34,molecular formula of C1610H2510N422O479S19.(2)OeACO2 was composed of alpha helix of 45.89%,beta turn of 11.08%and random coil of 25.63%,and it belonged to stable protein,with a molecular weight of 35859.1,pI of 5.48,molecular formula of C,611H2514N422O474S,5.(3)OeACO3 was composed of alpha helix of 45.74%,beta turn of 11.67%and random coil of 24.92%,and it belonged to stable protein,with a molecular weight of 36067.4,pI of 5.54,molecular formula of C1618H2530N424O477S16.In addition,all of these three proteins did not contain signal peptide and transmembrane regions.And all of them contained Fe2+ binding sites H177,D179 and H234 and substrate ACC binding site R244.3.The three ORF sequences of OeACOs were successfully integrated into the expression vector pET30b(+),then transfered into E.coli BL21(DE3),and successfully implemented prokaryotic expression induced.Electrophoresis detection of SDS-PAGE suggested that the molecular weight of the three recombinant proteins were all around 40kDa,consistent with theoretical size.The enzyme activity of the three recombinant proteins were determined by using GC-MS technology and the result showed that the three enzymatic activity of OeACO1,OeACO2 and OeACO3 were 10.94U,13.15U and 16.03U respectively,suggested that the recombinant enzyme proteins had the enzyme activity of plant ACO.