| Oriental melon (Cucumis melo var. makuwa Makino) is famous for excellent taste and unique aroma and it is important melon varieties resources in China. Pant lipoxygenase (LOXs) comprise a large gene family, different LOX isoforms may play different physiological roles in plant development, senescence, aroma sythesis and stress responses, aroma compounds of oriental sweet melon is an important quality indicator. LOXs in plants play an important role in the biosynthesis of aroma compounds, and related research has become a hot ponit in recent years. In this study, oriental sweet melon was used to identify the LOX gene family members and study the bioinformatic analysis. The gene expression analys is revealed that the 18 CmLOX genes exhibited a complex pattern. Three different types of LOX genes were cloned and the patterns of transcript abundance during postharvest (ethylene, 1-MCP, low temperature, and ethanol) storage were measured. Heterologous expression in yeast cells and transgenic techniques were applied to study biochemical characteristics and functions of CmLOX18. The main results are as followings:1. From the melon genome, this study identified eighteen candidate LOX genes and named CmLOX01-CmLOX18. Of the eighteen melon LOX sequences identified, eleven LOXs (CmLOX03-10, CmLOX12-13 and CmLOX18) encoded full-or nearly full-length functional proteins, the remaining seven LOX sequences (CmLOX01-02, CmLOX11 and CmLOX14-17) might encode truncated proteins, and found all LOX sequences indicating the importance of the functional domains (His-(X)4-His-(X)4-His-(X)17-His-(X)8-His) of LOXs. Among the eighteen melon LOX genes, CmLOX 12 and CmLOX 13 had the highest amino acid sequence identity, which was 80%, while CmLOX01 and CmLOX 16 shared the lowest sequence identity, which was 14%. The phylogenetic tree showed the CmLOX genes were divided into two types, type I CmLOX01-07 and CmLOX09, type II CmLOX08, CmLOX10-16 and CmLOX18; according to specific oxygen binding sites, only CmLOX07 and CmLOX09 clustered as 9-LOX, while the remaining LOXs, with the exception of CmLOX17, clustered as 13-LOXs.2. This research investigated the expressions of CmLOX members by RT-sqPCR and qPCR using extracted RNA from root, stem, young leaves, flowers, fruits (eight developing and mature fruits were harvested at 5,10,15,20,25,30,35 and 40 day after pollination), and seeds. Melon LOX gene expression profiles during melon growth and development show: Seventeen genes of the CmLOXs were detected in both vegetative and reproductive tissues of the oriental sweet melon cultivar "Yumeiren, only CmLOX07 was expressed in neither vegetative nor reproductive tissues. Three CmLOXs (CmLOX03, CmLOXOS and CmLOX06) had similar patterns of expression in all investigated tissues but were undetectable in young leaves. CmLOX09 and CmLOX18 expression were observed in all tissues except female flowers. CmLOX15 was not found in vegetative tissues, but expressed in all other tissues. For the remaining five genes, CmLOX11, CmLOX12, CmLOX14, CmLOX16, and OmLOX17 were undetectable in stems. Additionally, the CmLOX12 expression was not found in the flowers either. Neither of CmLOX14 and CmLOX17 was detected in young leaves. CmLOX01, CmLOX03 and CmLOX18 were stably expressed throughout fruit development, the transcript levels of these genes increased during fruit ripening. The transcript level of CmLOX06 increased relatively slowly from 5 DAP to 10 DAP and then decreased during fruit development. However, CmLOX02, CmLOX04, CmLOX08, CmLOX09, CmLOX10, CmLOX11, CmLOX13, CmLOX14, CmLOX15 and CmLOX16 had relatively higher expression levels at 5 DAP and then decreased dramatically during fruit development. Theses results suggested that different LOX genes family may play different roles during melon development, ripening and aroma synthesis.3. In order to investigate the effects of different postharvest treatments on the expression of LOX genes in fruit, three LOX genes (CmLOX03, CmLOX09, and CmLOX18) were cloned and the patterns of transcript abundance during postharvest storage were measured. Phylogenetic analyses indicated that CmLOX03, CmLOX09, and CmLOX18 represented three major LOX groupings:type1 13-LOXs, typel 9-LOXs, and type2 13-LOXs, respectively. The transcriptional regulation of CmLOX03, CmLOX09, and CmLOX18 that occur during postharvest melon fruit ripening were characterized. Quantitative real-time PCR (qPCR) showed that the expressions of CmLOX03 and CmLOX18 were up-regulated by ethylene treatment. By contrast, CmLOX09 transcript was negatively associated with the physiological and biochemical changes that occur during postharvest ripening. Treatments of 1-methylcyclopropene (1-MCP), ethanol, and low temperatures decelerated the ripening process and down-regulated the expression of both CmLOX03 and CmLOX18, while CmLOX09 had no such effects under the three treatments. In summary, these imply that CmLOX03 and CmLOX18 might be major LOX-encoding genes involved in melon fruit ripening, and that individual CmLOX isoforms are differentially regulated and have distinct functions during the postharvest ripening of melons.4. Melon CmLOX 18 were transformed into the Saccharomyces cerevisiae cell line INVScl for expression of recombinant protein; the purified recombinant protein was used to study enzymatic characteristics; chromatogram analysis was used to study the catalytic product of recombinant protein; subcellular localization analysis by trreaient expression of CmLOX18 in Arabidopsis mesophyll protoplasts was used to examine the localization of CmLOX18 in vivo. The results showed:in the buffer of pH 3.0-pH 9.0, recombinant CmLOX18 protein exhibited its highest activity at pH 4.5; to determine the optimum temperature for recombinant CmLOX 18, enzyme activity was measured over a range of temperatures (20℃-45℃), Maximal activities for recombinant protein were observed at 30 ℃; CmLOX18 showed 3.12-fold higher Km value for linoleic acid (LA) than for linolenic acid (LnA); comparison of the Vmax values showed that CmLOX 18 oxidized (LA approximately 4 times faster than LnA did. These results indicate that LA is clearly the preferred substrate for the recombinant CmLOX18; CmLOX18 was a 13-position-specific lipoxygenase, the stereochemistry of the reaction products was analyzed by chiral-phase HPLC; CmLOX18 was located in non-chloroplast organelle.5. By Gateway technology, CmLOX18 was transferred into the vector; CmLOX18 was introduced into "Zhongshu 6" tomato genome using the Agrobacterium-mediated method; PCR, Southern blot, western blot and vivo fluorescence imager were used to identify TO transgenic plants; detecting the expression level of CmLOX18, LeHPL and tomato LOXs in T1 transgenic fruits through real-time fluorescence quantitative techniques (Qpcr); GC-MS was used to exmain T1 transgenic fruit volatiles. The expression levels of CmLOX18, LeHPL, and the tomato LOX gene family in transgenic and "Zhongshu 6" fruits, were examined by real-time PCR; The expression of the exogenous CmLOX18 gene in the tomato can improve the expression levels of LeHPL, but there was no change in tomato TomloxA, TomloxB, TomloxC, TomloxD, TomloxE, and TomloxF; Transgenic tomato fruits exhibited increase in the biosynthesis of compounds hexanal, (Z)-3-hexanal and (Z)-3-hexen-1-ol; Moreover the compounds of 1-pentanol and 1-penten-3-one were not changed. These results indicate that biosynthesis of C6 volatiles is dependent upon CmLOX18-hydroperoxide lyase (HPL) pathway. |
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