| Orchidaceae is a huge family with wide distribution and high ornamental,medicinal and economic value.Volatile organic compounds are the special language for plants to communicate with the surroundings.They help plants attract pollinators and resist all kinds of stress.Orchids are known for their deceptive pollination,attracting pollinators by releasing VOCs to mimic other odour of flowers containing nectar or sex hormones of female insects.In fact,VOCs greatly promote the effective pollination and reproductive development of orchids.The Orchidaceae germplasm resources in Jiangxi Province are extremely rich,with 153 orchid species in 60 genera,but there are relatively few reports on the orchid volatiles in Jiangxi Province.In this paper,C.ensifolium was selected as experimental materials.By exploring the influence of adsorption method,extraction temperature and sample state on the detection,the sample pretreatment and GC-MS technology suitable for orchid VOCs analysis were optimized and established.Based on the established method,the chemical composition of VOCs emitted by seven Cymbidiums were analyzed systematically in Jiangxi Province,including C.floribundum,C.kanran,C.faberi,C.lancifolium,C.ensifolium,C.goeringii and C.sinense,and the data were analyzed by chemometrics.Finally,the diurnal variation of VOCs fragrance and related gene expression in C.ensifolium were studied.The results provide a data base for further research on biosynthetic pathways,regulatory mechanisms,and pollination biology of orchids.The main results are as follows.(1)The method suitable for VOCs collection and analysis of orchid samples was established.In this paper,C.ensifolium was used as the experimental material to comparatively analyze the effects of solid-phase microextraction and dynamic headspace adsorption on VOCs detection of fresh samples and powder samples under extraction temperatures of 30?,45? and 60?.The results showed that SPME collected 33 kinds of C.ensifolium powder VOCs in 7 categories,31 kinds of C.ensifolium fresh samples VOCs in 8 categories,while DHS collected 24 compounds in 6 categories at room temperature.SPME is simple,fast,with sample purification,accurate analysis,low organic solvent consumption,mainly to collect esters,aldehydes and terpenes,of which 72 compounds were detected;Dynamic headspace can directly detect fresh samples,which can reflect the true state of plants,mainly to collect esters,alkanes and aldehydes,of which 24 compounds were detected.The results show that the SPME method can collect the volatile components of C.ensifolium flowers more effectively at 60?.From the analysis of extraction temperature,the types and composition of volatile components are gradually enriched with the increase of extraction temperature.From 30? to 60?,the VOCs of C.ensifolium fresh samples increased from 6 to 31and the chemical composition increased from 3 to 7 categories,while the VOCs of C.ensifolium powder samples increased from 8 to 33 categories,and the chemical composition increased from 5 categories to 8 categories.The results showed that C.ensifolium VOCs extracted at high temperature of 60? had the most varieties and the most abundant composition.From the analysis of the sample state,when extracting at a low temperature of 30?,the powder samples were detected 8 compounds in 5 categories,while the fresh samples were detected 6 compounds in 3 categories.When extracting at 45?,25 compounds in 8 categories were detected in the powder samples,while 17 compounds in 5 categories were detected in the fresh samples.Under the condition of low temperature extraction,the VOCs composition of the powder samples were significantly richer than that of the fresh samples..At 60?high temperature extraction,the volatile components of fresh samples and powder samples are the same,which are mainly aldehydes,followed by terpenes and other substances.But,considering that fresh samples can reflect the real state of plants better than powder samples,fresh samples are selected for the next experiment.(2)Using the established method,7 species of Cymbidium species in Jiangxi province such as C.floribundum,C.kanran,C.faberi,C,lancifolium,C.ensifolium,C.goeringii and C.sinense were used as experimental materials to identify floral VOCs by GC-MS.The identified compounds were clustered by PCA and CA.The results showed that the number of volatile components emitted by C.floribundum,C.kanran,C.faberi,C.lancifolium,C.ensifolium,C.goeringii and C.sinense 7 was 31,26,24,23,22,19 and 19 respectively.The VOCs of C.faberi Cymbidium,C.floribundum,C.ensifolium were mainly aldehydes and the highest relative contents were 2-ethylbutanal,phenylacetaldehyde and 2-pentylfuran respectively;The VOCs of C.goeringii and C.lancifolium were mainly alcohols,and the compounds with the highest relative contents were benzyl alcohol and nerolidol respectively.The terpenes in the volatile components of C.kanran were the highest,especially(E)-?-Bergamotene.The odor of C.sinense were mainly ketones,and ?-ionone accounts for 76.07%of the total.In the PCA,the cumulative contribution rate of PC1,PC2 and PC3 reached 73.5%.In CA,when the critical value was 10,the characteristic values from C.faberi and C.lancifolium were close,and there were 5 common compounds,such as(E)-2-heptenal,2-pentylfuran and 3-ethyl-2-methyl-1,3-hexadiene,while C.sinense and C.goeringii both emitted(E)-2-heptenal,6-methyl-5-Hepten-2-one,octanal and other 10 volatiles.When the critical value was 4,C.kanran and C.floribundum got together,and they possessed 7 compounds such as 1-Octen-3-ol,?-Bisabolene and nonanal,and then formed a large group with C ensifolium at a critical value of 8.In summary,the 7 species of orchids are rich in volatile components,with different main components such as phenylacetaldehyde,2-ethylbutyraldehyde and 2-n-pentylfuran.A variety of VOCs such as 2-n-amylfuran and nonanal have been detected in 7 species of Cymbidium,which are the important components of VOCs emitted by orchids in Jiangxi Province.(3)The diurnal variation rule of C.ensifolium VOCs release was clarified,and related gene expression patterns were explored.In this chapter,fresh samples and powder samples of C.ensifolium flowers were collected and analyzed at 4:00,8:00,12:00,16:00,20:00 and 24:00.The results show that the release of VOCs in C.ensifolium had o an obvious daily variation rule.From 4:00 am to 24:00 pm,the types of VOCs showed a trend of increasing first and then decreasing with time.At 12:00,the number and composition of VOCs were the most abundant.Methyl jasmonate and its isomers its isomers[1-?,2-?(Z)]-Cyclopentaneacetic acid,3-oxo-2-(2-pentenyl)-,methyl ester were the main VOCs that make up the fragrance emitted by C.ensifolium,and they first increased and then decreased with the change of time,reaching the highest relative content at 12:00.In order to clarify the expression rules of their related synthetic genes,qRT-PCR was used to detect the expression levels of the key synthetic genes CeLOX,CeAOS,CeAOC and CeJMT.The results showed that the relative expression levels of all genes reached the highest at 12:00 and the lowest at 20:00.The expression patterns of these key synthetic genes are similar to the volatiles,which further regulates the synthesis of the volatiles.The study systematically explored the volatile components of Jiangxi orchid flowers for the first time,enriched the data resources of orchid fragrance research in Jiangxi Province,laid a foundation for the study of the biosynthesis pathway,regulation mechanism and pollination biology of orchid fragrance in the future,which is helpful for the conservation and protection of orchids. |
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