| Dendrobium,belonging to Epidendreae of Orchidaceae,is an important germplasm resource of flowers because of its unique shape,rich color and sweet-smelling perfume.At present,in the cultivated hybrid Dendrobium,there is a lack of varieties with pure bright yellow flowers and fragrant aroma.And in the natural species,Dendrobium chrysotoxum flowers have pure bright yellow color with fragrant aroma.Therefore,in this study,the color of D.chrysotoxum in each flowering period was identified by Royal Horticultural Society of Britain color card and spectrophotometer;the qualitative and quantitative analysis of carotenoids was carried out by using convergence chromatography triple quadrupole mass spectrometer(UPC-MS/MS)and the qualitative and quantitative analysis of flavonoids and anthocyanins was carried out by ultra performance liquid chromatography-electrospray ion mass spectrometry(UPLC-ESI-MSn);the volatile components of flowers and their contents were determined by olid-phase microextraction(SPEM)and gas chromatography coupled with mass spectrometry(GC-MS);the RNA extraction method of D.chrysotoxum tissue was optimized and improved.The transcriptome sequencing for whole flower of D.chrysotoxum at different developmental stages was performed by Illumina sequencing technology,and the expression patterns of different genes in the synthetic route of carotenoids and terpenes in D.chrysotoxum were analyzed by qRT-PCR technology.So the molecular regulation mechanism of flower color and fragrance were able to be studied.This will provide a theoretical basis for the improvement of Dendrobium cultivars in the future.The main results are as follows:(1)the petals of D.chrysotoxum during the buds stage are green;the petals are yellow green during the translucent period;and at the opening period are bright yellow.The brightness and saturation of D.chrysotoxum rise first and then decrease during the petals opening process,reaching the highest at about the 3rd to 5th day.The content of flavonoids and anthocyanins in the opening period of D.chrysotoxm was detected.The results showed that the flavonoids and anthocyanins were not detected in the translucent period and opening period,and it was preliminarily determined that anthocyanins and flavonoids were not related to the yellow flowers.The content of lutein in the flower petals of D.chrysotoxum was 125.62 μg/g,antherxanthin was 18.35 μg/g and zeaxanthin was 59.34 μg/g,and the total content of carotenoid in the flower petals increased rapidly in the opening period,and the content of lutein,antherxanthin and zeaxanthin increased by 40.47 μg/g,89.85 μg/g and 397.06 μg/g,respectively.Therefore,lutein,antherxanthin and zeaxanthin are the main metabolites of yellow flowers of D.chrysotoxum.(2)From 12 to 14 PM,the aroma components of D.chrysotoxum flowers were measured,and 33 kinds of volatile components were identified.The aroma content was only 5.2 n mol ·flower-1,and only 3 kinds of aroma substances at translucent period,mainly terpenes,which increased to 13.48 n mol · flower-1 in the initial opening period.The six kinds of aroma substances were esters and terpenes,which accounted for 44.2%and 55.8%of the total aroma release respectively,and rise rapidly to 167.48 n mol · flower-1 in the opening period.31 aromatic substances were detected,mainly esters and terpenes,accounting for 53.6%and 30.2%of the total aroma,and then which would down to 10.36 n mol ·flower-1 in decay period,and 93.3%of which were terpenes.31 kinds of volatile components were identified in the flowers of D.chrysotoxum at different sampling time in one day.The total release of aroma was 99.08 n mol · flower-1 at 8:00.The 14 aroma substances were esters,terpenes and aldehydes,and the release amount accounted for 53%and 32.2%of the total aroma components,respectively.At 11:00,the total release amount increased to 122.86 n mol · flower-1 and the aroma substances increased to 23.The maximum 167.48 n mol· flower-1 was reached at 14:00,and the release amount of the 31 aroma substances reached the maximum at this time,and the esters and terpenes accounted for 53.6%and 30.2%of the total aroma components respectively.At 17:00,the content of alcohol and terpenes decreased significantly,and the release amount was reduced to 56.3 n mol · flower-1,11.8%and 5%respectively.At 20:00 it was only 25.07 n mol · flower-1.There were only 6 aroma substances,accounting for 80.1%of the total aroma components.By measuring the aroma value(content/sense threshold)of the volatile substances,the main aroma components of D.chrysotoxum were monoterpenes such as β-alenene,α-pinene,β-pinene and D-limonene.(3)Transcriptome sequencing of the whole flower of D.chrysotoxum during translucent period,half opening period,opening period and decay period was performed,and 18817483680 nt data were performed.60711 Unigene were assembled using de novo assembly technology.Then,through gene function annotation and differential expression analysis,13 genes related to carotenoid synthesis were obtained,including PSY、PDS、ZDS、CRTISO、ZISO、LCYB、CHYB、VDE、ZEP、LCYE、CYP450、NCED and CCD1;the four genes GPPS，DXR,DXS and TPS related to the synthesis of terpenes.(4)qRT-PCR was perfomed for the genes related to the synthesis of 13 carotenoids obtained in the young buds,buds,transcoloration,half opening,opening and decay periods of D.chrysotoxum flowers.The results showed that PSY continued to be expressed in the young buds,buds,translucent and half opening periods of developing flower,and the expression amount did not change basically,which may be related to the continuous synthesis of carotenoids in the process of flower development.LCYE and CYP450 were expressed continuously during the buds and opening periods in the development,and the change was little.This may be the reason for ensuring the continuous synthesis of lutein in petals.The expression of LCYB in the flower development process is generally higher,and the expression amount is maximum at the opening period,and the increase of the expression of LCYB may be the reason to cause the increased lutein biosynthesis during development.The expression of PDS,ZDS,CRTISO and ZISO increased rapidly during the opening period of D.chrysotoxum flower,which was in accordance with the rapid increase of carotenoid content during the coloring process.Therefore,the increase of these genes was one of the reasons for the rapid increase of carotenoid content;the expression of LCYB,CHYB and VDE reached the peak in the opening period of the flower.The three genes are all related to the synthesis of zeaxanthin and antheraxanthin,so these three genes may be the main factors for the significant increase in the flowering period of zeaxanthin and antheraxanthin.NCED can decompose antheraxanthin,and its product is the precursor of abscisic acid(ABA)synthesis.Its expression increases continuously at all stages of the flowering period,and reaches the highest value in the decay period.Therefore,NCED may play a leading role in the degradation of β-carotene and throughout the whole flowering period.It also participates in the aging process of flowers.CCD1 is also involved in the decomposition of carotenoids,and its expression reached the highest in the half-flowering period,indicating that it is mainly involved in the decomposition of carotenoids in the half-flowering period.qRT-PCR was also performed of 13 genes in the flower petals of D.catenatum and D.chrysotoxum.The results showed that the expression of PSY,PDS,ZDS,LCYE,ZEP,NCED and CYP450 in the D.chrysotoxum petals was lower than that of these genes in D.catenatum.The expression levels of ZISO,CRTISO,LCYB,CHYB and VDE in the petal of D.chrysotoxum were higher than those in D.catenatum.Therefore,the large expression of ZISO,CRTISO,LCYB,CHYB and VDE in the petals of D.chrysotoxum may be the cause of the difference in the color of the flowers of the D.catenatum,and which mau be the reasons for the more carotenoids in D.chrysotoxum.(5)The 4 structural genes involved in the main chain of terpenes and the CCD1 gene involved in the decomposition of carotenoid were detected by qRT-PCR during the translucent,half opening,opening and delay periods of D.chrysotoxum and the opening period of D.catenatum.The results showed that the expression of these 5 genes in the petal of D.chrysotoxum during flowering stage was much higher than that in D.catenatum.At the same time,there was a rapid increase in the expression of GPPS,DXS,DXR and TPS between the translucent and half opening periods of petals,and reached the highest in the half opening period,and then decreased with the expression.This is consistent with the yield variation of terpene volatile compounds in D.chrysotoxum.Therefore,the four genes of GPPS,DXS,DXR and TPS may be the main cause of the production of terpene volatile compounds from D.chrysotoxum.The trend of the expression of CCD1 in D.chrysotoxum is similar to that of GPPS,DXS,DXR and TPS,and the expression of CCD1 in the opening period of D.chrysotoxum is much higher than that in D.catenatum.CCD1 can catalyze the decomposition of carotenoids,and some of the decomposition products can participate in the synthesis of some volatile substances.Therefore,the carotenoid decomposition products obtained through it may be involved in the synthesis of the flower fragrance of D.chrysotoxum.In this study,the method of extracting RNA from the flower tissue of D.chrysotoxum was established.The volatile components and the content of the flower color of Dendrobium was clarified.Through transcriptome analysis and qRT-PCR technology,candidate coding genes of key enzymes involved in the biosynthesis of D.chrysotoxum and terpenes were selected.Among the flowers of D.chrysotoxum,isopentenyl pyrophosphate(IPP)is a synthetic precursor of carotenoids and an important intermediate for the biosynthesis of terpenoids.At the same time,the large expression of D.chrysotoxum CCD1 in the petals may cause partial carotenoid cleavage.The cleavage product may be a synthetic precursor of volatile substances,which helps to increase the yield of volatile anthraquinones in Dendrobium nobile.Therefore,both the IPP metabolic pathway and the CCD1 gene in D.chrysotoxum can affect carotenoids and terpenoid synthesis pathways.Through the regulation of IPP metabolic pathway and CCD1,the purpose of co-regulation of the flower buds of D.chrysotoxum can be achieved.These results will provide a theoretical basis for the improvement of Dendrobium varieties through genetic engineering in the future. |
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