Cloning And Expression Analysis Of Lipoxygenase Gene CsLOX2 In Cucumis Sativus(Cucumber)

To investigate the mechanism of cucumber lipoxygenase gene participating in the formation of fruit aldehydes aroma,a full length cDNA named CsLOX2 was cloned from the Northern china cultivar No.26.Analysing the bioinformatics and constructed the optimum expression system of prokaryotic expression.Transient expression analysis showed that CsLOX2 was a cytoplasm protein.The expression profiling and the lipoxygenase activity of cucumber fruit during fruit development were studied.The expression profiling of CsLOX2 gene was measured in the different tissues during the cucumber plant development.The main results were as follows:1.Obtaining the full cDNA fragment of cucumber lipoxygenase gene CsLOX2.A full length cDNA named CsLOX2 was cloned in cucumis sativus belonging to NO.26 cucumber breed by RT-PCR and 3’RACE.Sequence analysis showed that the full cDNA fragment was 2878bp,open reading frame encompassed 2640bp and encoding a polypeptide of 879 amino acids.2.Bioinformatics ananlysis of cucumber lipoxygenase gene CsLOX2Sequence alignment analysis showed that the cDNA fragment shared 94%,81%,94%identity with cucumber LOX-1(accession No.U25058)、cucumber CrLOX1(ccession No.U36339)and Cucumis melo CmLOX2(ccession No.GQ386815.1)respectively.The bioinformatics analysis showed that CsLOX2 encoded an 879 polypeptide,the calculated protein molecular mass was 99.39kD and isoelectric point was 6.28.The amino acids sequence of CsLOX2 shared three conserved regions and six highly conserved histidines with other plant LOXs.Cluster analysis revealed that the CsLOX2 protein belonged to type I LOX and shared 13-LOX activity.3.Construturing the prokaryotic expression vector P4t-LOX2,and determined the optimum expression system of the fusion CsLOX2 proteinBy means of constructing a recombined expression plasmid p4t-LOX2 and transformed the recombined plasmid into competent cell,obtained a 115kDa CsLOX2/GST protein induced by IPTG.The fusion protein was expressed in inclusion body,the molecular weight of it was 115 kD,remove the 26kD GST protein induced by pGEX-4t-1,the CsLOX2 gene encoded a 89kD protein.Further assay showed that the optimum induction condition is temperature 37℃,IPTG concentration 0.8 mM and induced for 10.5h.4.To determine the subcellular localization of CsLOX2 protein,CsLOX2-GFP fusion protein was transformed into onion epidermis cell by bombardment.The result showed that the green fluorescent signal of CsLOX2 was detected in the cytoplasm,which indicated CsLOX2 was a cytoplasm protein.5.Difinted theespression profile of CsLOX2 geneThe RT-PCR analysis showed that CsLOX2 gene was expressed during the fruit developing period,the highest expression was at the 3 d after pollination,and then downregulated gradually and lowest at the 15d after pollination.The CsLOX2 gene was expressed in different tissues during the plant development.6.Study the total LOX activity during during the cucumber fruit developing periodThe total LOX activity rose from the 3 d to 12d after pollination,and it was up to the highest activity at 12 d after pollination.The results showed that this enzyme may take part in the fruit enlargment during the cucumber fruit development.