Prokaryotic Expression, Bochemical Characteristic And Promoter Analysis Of CmLOX10 And CmLOX13 In Oriental Melon (Cucumis Melo Var. Makuwa Makino)

The oriental melon (Cucumis melo var. makuwa Makino) is an important agricultural commodity and widely grown in China and eastern Asian countries. In comparison with muskmelons, the oriental melon plants are weak in stress and disease resistance. Previous studies have shown that the jasmonates and green leaf volatiles from LOX pathway generally are regarded as the key factors which involve in the expression of the genes relating with resistance in plants. The study of LOX genes can provide theoretical basis to improve the stress resistance of the oriental melon by gene engineering. A lot of LOXs have been found in plants in recent decades, but there is relatively little known about oriental melon LOXs. With the completeness of melon genomic sequencing project which provides an ideal way for melon gene cloning and functional analysis,18 candidate LOX genes have been identified in the melon genome database and named as CmLOX01 to CmLOX18. The phylogenetic tree including the putative amino acid sequences of the oriental melon CmLOX10, CmLOX13 and other plant LOXs of which the functions have been characterized were constructed. CmLOX10 and CmLOX13 get together closely in the phylogenetic tree and share 58.11% identity with each other by sequence alignments of the predicted amino acid sequences. In order to identify the functions of CmLOX10 and CmLOX13 genes, the oriental melon cultivar ’Yumeiren’ were used as experimental material to study. The main results are described as followings:1. The full-length cDNAs of the oriental melon CmLOX10 and CmLOX13 were acquired by a combined strategy using RT-PCR and RACE-PCR. The CmLOX10 (GenBank: KT613843) harbours a 2709-bp open reading frame (ORF) flanked by a 47 bp 5’-untranslated region (UTR) and 118 bp 3’-UTR. The deduced CmLOX10 protein sequence consists of 902 amino acid residues with a calculated molecular mass of 102.301 kDa and a theoretical isoelectric point (pI) of 6.0. The CmLOX13 (GenBank:KT613844) harbours a 2721-bp ORF encoding a predicted protein of 906 amino acid residues with a calculated molecular mass of 102.312 kDa and a theoretical pI of 6.34. The ORF of CmLOX13 is flanked by the 5’-UTR of 48 bp and 3’-UTR of 21 bp. Two sequence alignments of the nucleotides and deduced amino acid sequences of CmLOX10 isolated from oriental melon (Cucumis melo var. makuwa Makino) and from the melon (Cucumis melon L.) genome database were done and seven different nucleotides and three different amino acids are found. For CmLOX13, there are five different nucleotides and one different amino acid2. By analyzing the deduced amino acid sequences of CmLOX10 and CmLOX13 using some bioinformatics software, the PLAT and LOX domains are found and the length of the chloroplast transit peptide of the N-terminus of the deduced CmLOX10 and CmLOX13 proteins are 37 and 23 respectively. Several highly conserved regions of the predicted CmLOX10 and CmLOX13 proteins are identified by amino acid sequence alignments and it is predicted that they are 13-LOXs.3. CmLOX10 and CmLOX13 promoter fragments which are 2155 and 2097 bp respectively were achieved by PCR strategy from the genomic DNA of the oriental melon. Two sequence alignments of the CmLOX13 promoter sequences achieved above and GeLOX13 from the melon (Cucumis melon L.) genome database were done and they share 98.72% identity with each other. CmLOX10 and GeLOX10 promoter sequences only share 93.19% identity with each other. The putative cis-regulatory elements of CmLOX10 and CmLOX13 promoter were analyzed by PlantCARE and PLACE databases and several elements responding to hormone and abiotic were found, such as TCA-element responding to SA, TGA-element responding to auxin, GAREAT responding to GA and WUN-motif responding to wounding.4. After CmLOX10 and CmLOX13 recombinant proteins were acquired by prokaryotic expression followed by purifying protein, identifying with SDS-PAGE and Western-blot technique and concentrating protein, the purified proteins were used to analyze the enzyme activity. The result shows that the recombinant CmLOX10 and CmLOX13 proteins display the highest catalytic activity at pH 5.0 and 5.5 and the optimum temperature for CmLOX10 and CmLOX13 are observed at 45℃ and 35℃, respectively. By enzymatic kinetics and HPLC analysis, the result shows that linoleic acid is the preferred substrate for the recombinant CmLOX10 and CmLOX13 and the two proteins are 13-LOXs.5. The overexpression vectors of CmLOX10 and CmLOX13 were constructed by gateway method followed by transferring into agrobacterium. The subcellular localization of CmLOX10 and CmLOX13 were examined by transient expression of the two proteins in tobacco leaves. The result shows that CmLOX10 protein is located in plasma membrane which is not in accord with the result of the bioinformatics analysis and CmLOX13 protein is located in chloroplasts.6. The CmLOX10 and CmLOX13 promoter sequences were analyzed by PlantCARE and PLACE databases. Then 5’deletion expression vectors of the CmLOX10 and CmLOX13 promoters were constructed. After the 5’deletion expression vectors of the CmLOX13 promoters transiently expressed in tobacco leaves, the leaves were treated by hormone and environmental stress. The results from GUS histochemical staining and fluorimetric assay show that the GUS activity of pl21GUS:13-5 deletion structure is significantly higher than the other deletion structures and CmLOX13 promoter is involved in responding to all treatments including ABA, SA, GA, IAA, MeJA, wounding, cold and heat except ethylene.7. The overexpression vectors of CmLOX10 and CmLOX13 were transformed into Arabidopsis thaliana by floral-dip method. T1 transgenic plants were screened by spraying 0.0005% Basta and RT-PCR identification and provided to study the gene functions of the oriental melon CmLOX10 and CmLOX13.