Molecular Regulation Mechansim And Key Gene Cloning Expression Of Floral Color Formation In Cymbidium Ensifolium

Cymbidium ensifolium is the key one of Chinese orchids,and is on the ever-increasing demand by the modern society.The color is one of the key factors for evaluating its quality,and it is also a key research direction for ornamental value and breeding.However,the color research of this speceis has no major breakthrough.In this study,the physiological and biochemical characteristics of the multicolor of petal and the molecular mechanism of color formation were explored for the complex-colored C.ensifolium cultivars.The results are as follows:1.The yellow-green and red petals tissue of the six plant were used in this study.The material basis and ingredient of flower color formation was explored in detection analysis for the cultivation of new varieties.The phenotypes and digital color matching was detected,and the content of petals protein and p H value were also measured in this research.Finally,The pigment composition in two flowers tissue was detected by UV-Vis spectrophotometer and high performance liquid chromatography-mass spectrometry(LC-MS).The results are as follows:(1)The yellow-green petals in brightness value(L*,70.11±1.7)was About two times more than that in red petals.The red petals in Red value(a*,51.20±1.9)was four times more than that of yellow-green petals.The yellow-green petals in yellow value(b*,35.21±1.5)was about three times more than that of red petals.The result of above showed conspicuous chromaticity difference between two petals;(2)the red petals in protein content(3.435±1.7)was about 2 times more than that in yellow-green petals;(3)12 flavonol glycosides and 8anthocyanin were inspected in these flowers.The former mainly includes three types of Quercetin glycogen,Kaempferol glycosides and Isorhamnetin glycosides.The latter mainly includes four types of Centaurin,Delphindin glycogen,Geranium glycogen and a spot of Peonidin;(4)Isorhamnetin glycosides is located only in yellow-green petals.And some of which indwell only in the red petals,such as Geranium-diglucoside,Delphinium glucoside,Kaempferol rhamnoside,Cornflower rutin glycoside,Kaempferol rutinoside and Kaempferol Rutinoside Galactoside.2.In order to explore the molecular mechanism of the color formation of Cymbidium flower,in this study,high-throughput sequencing technique was used to construct transcriptome library with the yellow-green and red petals of one Cymbidium plant as experimental materials.And we explored the biosynthesis metabolism pathway of color related ingredients,even the transcriptional activity of key regulatory genes.The results showed that:(1)A total of 131 110030 Clean Reads and 106 479 Unigenes were got by transcriptome sequencing,and 29 748 Unigenes with annotated information was obtained by comparing with NR,GO,KEGG and other public databases;(2)Compared with yellow-green petals and FPKM data analysis,776 genes expression increased and 589 genes expression decreased in red petals;(3)the above genes were identified to 93 metabolic pathways by KEGG database,and involved 6pathways of flavonoid biosynthesis and total 20 DEGs;(4)Among them,the up regulation gene expression of chorismate mutase(CM),phenylalanine ammonia-lyase(PAL),cinnamate 4-hydroxylase(C4H)and 4-coumarate: Co A ligase(4CL)are in favor of flavonoid precursor accumulation;(5)Chalcone synthase(CHS),dihydroflavonol4-reductase(DFR)and anthocyanidin synthase(ANS)were hardly expressed in yellow-green petals,but significantly increased in the red petals,which could be related to the floral color formation of Cymbidium.3.A key gene from anthocyanidin synthesis pathways was cloned and named as Ce CHS1.And we reseached its nucleotide and amino acid sequences.The results displayed:(1)Ce CHS1 is 1185 bp in length and encodes 394 Amino Acid chains,and is rich in Leucine.The protein has a typical conserved catalytic center of chalcone synthase family(Cys164-His303-Asn336)and a C-terminal conserved GFGPG and other Sequence components.The amino acid sequence was found to have a high degree of similarity to that of other Orchidaceae plants;(2)After recombination with the plasmid vector of Pet28 a and expression in the E.coli(BL21,DE3),the molecular weight of the fusion protein was discoverd to be about 43 Kda;(3)After optimization study of the induction conditions,the result showed that the p ET28-Ce CHS1 in the prokaryotic expression was efficiently induced by1 m M IPTG,at 37°C and for 6 h.This paper firstly systematically studied the physiological influence factors,material composition,gene transcriptomes and key regulatory genes sequence analysis about flower color of C.ensifolium.Through the results of this paper,it provides the technological basis for the molecular mechanism of floral color formation of C.ensifolium,cultivation of new floral color varieties and gardening application.