CN104404079B - Simultaneously overexpression GL3 takes on a red color the application in brassica plant preparing petal for interference LCYB, LCYE expression - Google Patents

Abstract

The invention discloses interference LCYB, LCYE expression simultaneously overexpression GL3 takes on a red color the application in brassica plant preparing petal, interference LCYB, LCYE gene expression is also disclosed while the preparation method of the expression vector and the expression vector of overexpression GL3 genes, by disturbing LCYB, LCYE gene expression inhibition yellow carotenoid and accumulating red tomatoes red pigment, and overexpression GL3 genes can promote anthocyanin to accumulate, so that petal takes on a red color, the aobvious red brassica plant of petal is successfully prepared, the pattern of brassica plant is enriched.

Description

Simultaneously overexpression GL3 takes on a red color Caulis et Folium Brassicae campestriss preparing petal for interference LCYB, LCYE expression Application in platymiscium

Technical field

The invention belongs to biological technical field, and in particular to interference LCYB, LCYE gene expression is while overexpression GL3 bases Because taking on a red color the application in brassica plant preparing petal.

Background technology

Brassica campestris L is important oil crop, and cultivation history is long, economic worth is high, of many uses, strong adaptability, is China The first big oil crop, be also global important oil crop.In species taxonomy, three species of the Brassica campestris L by Brassica genus Constitute, respectively cabbage type rape (Brassica napus, 2n=38, AACC), turnip type rape (Brassica rapa Ssp.oleifera syn.B.campestris, 2n=20, AA), mustard type rape (Brassica juncea, 2n=36, AABB).Cabbage type rape Brassica campestris L be by Chinese cabbage (Brassica rapa) and Caulis et Folium Brassicae capitatae (Brassica oleracea, 2n=18, CC) after natural intervarietal hybridization double diploidization evolve and come a kind of aggregate species, although cultivation history only has the centuries, but by Strong in its growth potential, yielding ability is high, the Yi Zhan worlds and China rapeseed cultivation area more than 90%.Turnip type rape is Brassica genus One of species that is tamed earliest, the Chinese cabbage for originating in NORTHWEST CHINA area develop, and have extensive point in world wide Cloth area and long cultivation history.Mustard type rape is natural by Chinese cabbage and black mustard (Brassica nigra, 2n=16, BB) The amphidiploid aggregate species that Natural double is formed again after hybridization, China is its original origin and differentiation center, and planting range spreads all over The world.

With the sustainable development and the continuous lifting of social modernization's degree of economic level, the popular pursuit to life taste Also grow with each passing day, the tourism of not only traditional famous mountains and great rivers formula is more and more fiery, the development of rural area recreational tourism industry is also rapidly. The ocean of golden yellow dyeing defect is formed in the production of Brassica campestris L large area, is more and more important villatic zoology tourist resources.Rape flower is viewed and admired Time be to summer in the coming year at the bottom of annual December.Because the time of plantation is slightly different, the florescence has a bit in each place Difference.Tens well-known rape flowers have been formed in China and have viewed and admired area, Brassica campestris L has been when the flowers are in blossom, competitively in full bloom, shine with glittering gold and colorful decorations, be continuous number In ten, like the ocean that golden wave is torrential.

Although nature flower color species is various, except the petal of cabbage mustard (B.oleracea var.alboglabra) It is that the petal of brassica plant is commonly Yellow series, and business rape variety is yellow petal entirely, excessively singly beyond milky Adjust.With the fast development of Brassica campestris L pattern villatic zoology sightseeing industry, in the urgent need to colourful pattern character, it is allowed to may be used also To output redness, blueness etc., outside the oil yield and other conventional uses for not affecting Brassica campestris L Semen Allii Tuberosi, increase its value of admiring the beauty of flowers, Boosting Ecological sightseeing is traveled.Additionally, the selected marker character that specific pattern or rapeseed breeding circle are thirsted for, and pattern is non- Yellowing additionally aids the insect pests such as minimizing nitidulid.Accordingly, it would be desirable to parse the molecule mechanism of Brassica campestris L pattern character, and carry out new flower The molecular breeding of color.

Radix Raphani (Raphanus sativus) belongs to Rhaphanuss, and molecular Evidence in recent years shows Radix Raphani and Chinese cabbage, Caulis et Folium Brassicae capitatae, sweet The distance of blue type Brassica campestris L even much smaller than black mustard and their distance, means that Radix Raphani is little with the distance of Brassica genus core species Inter-species distance inside Brassica genus.But then, Radix Raphani can output the pattern of red colour system, and Brassica genus and numerous relative genus The flower of yellow class can only be outputed if (as Caulis et Folium Sinapis albee belongs to).This is a strange phenomenon, therefore carries out Brassica genus and Radix Raphani pattern character Comparative study, be not only the needs of these biological pattern character basic research, alternatively the brassica plant pattern such as Brassica campestris L The genetic improvement of shape provides guidance.

The pattern molecule mechanism of important decorative flower and molecular breeding, achieved with remarkable break-throughs, are to study other flower colors Mechanism and development molecular breeding provide important references.Pattern is mainly by flavonoid (flavonoids) and carotenoid (carotenoids) two big class pigments determine.

Flavonoid is the anthocyanidin that most sees, contribution yellow, a series of orange, red colour systems to purple.Flavonoid is water Soluble substance, with a C15 skeleton, is broadly divided into 9 classes by structure：Chalcone (chalcones), aurones (aurones), different Flavone (isoflavonoids), flavone (flavones), flavonol (flavonols), flavandiolss (flavandiols), flower Color glycosides (anthocyanins), condensed tannin (condensed tannins, i.e. procyanidin, proanthocyanins) and tan Acid anhydride (phlobaphenes), colour contribution maximum to plant in them is anthocyanin, flavonol, chalcone and aurones.Pattern Glycosides is maximum class flavonoid substances, is the tone such as flower or other tissue contribution magenta, redness, purple, bluenesss, accumulate in In the vacuole of cell or pigment cell, pelargonin (pelargonidin), cyanidin (cyanidin), delphinidin (delphinidin) it is modal three classes anthocyanin；And yellow tone is then provided by chalcone, aurones, flavonol, flavone.

Flavonoid biosynthesis pathway in the plants such as petunia, Semen Maydiss, Antirrhinum majus L., arabidopsiss is fully parsed.It It is an important branch approach in common body propane biosynthesis pathway downstream, phenylpropyl alcohol alkane approach is by other branch's approach synthesis Multiple secondary substances such as lignin, stilbene class, coumarin, protective plant protecting agent.The first step of flavonoid path is urged by looking into ketone synthase (CHS) Change 1 molecule p- coumaric acyls-CoA and 3 molecule malonyl-CoA to be condensed to form lurid 4,2 ', 4 ', 6 '-tetrahydroxy chalcone. Pattern aglucon such as pelargonin, cyanidin, delphinidin etc. are with chalcone as substrate, through hydroxylation, reduction, oxidation, side Base modification etc. the catalytic reaction of a few step enzymes and formed, be related to successively CHI (enzyme, namely chalcone isomerase), F3H (flavanone 3-hydroxylase), F3 ' H (3 '-hydroxylase of flavonoid), 5 ' H of F3 ' (3 ', 5 '-hydroxylase of flavonoid), DFR/FNR (dihydroflavonol 4-reductase/ Flavanone 4- reductases), ANS (anthocyanidin synzyme) etc..The anthocyanidin determined by 5 ' H of F3 ' H and F3 ' (anthocyanidins) the hydroxyl on B- rings is more, then color is more partial to blue.Under normal circumstances, chalcone and anthocyanidin Be further embellished, such as glycosylation (glycosyl transferase, GT), methylate (transmethylase, MT), be acylated (acyltransferase, AT), then transhipment (glutathione S-transferase, GST) is stored in vacuole.In the vacuole of orfe showy flowers of herbaceous plants, chalcone 4 '-O- glucosides are transformed into jonquilleous aureusidin (6-O- glucosides) by aureusidin synzyme (AS).Gorgeous TONGCAO In orange to red flower Deng plant, flavanone reacts through several steps and synthesizes 3- deoxidation anthocyanin.Additionally, flavone and flavone Alcohol also plays certain contribution to pattern, and they are colourless or light yellow, can promote blue anthocyanin by the effect of so-called color altogether Formed and stable.Flavone and flavonol are with flavanone and flavanonol as substrate, in flavone synthase (FNS) and flavone respectively Synthesis under the catalysis of alcohol synthase (FLS).In addition to anthocyanidin, other flavonoid substances such as flavonol, chalcone are likely to be related to Glycosylation etc. is modified.

The research of model organism shows, the expression of flavonoid path structural gene be by one by R2R3-MYB (have PAP, PFG, TT2 etc.), basic helix-loop-helix (basic helix-loop-helix, bHLH have TT8, GL3, EGL3 etc.) and The transcription factor complex that WD40 repetitions (WDR has TTG1 etc.) is formed is regulated and controled, and the ternary complex has regulated and controled decorative flower The process of anthocyanin coloring in the petal of Antirrhinum majus L., petunia, morning glory, African Chrysanthemum and flower of Radix Gentianae, regulation and control behavior receive organ, group The external signal such as internal signal and light, ultraviolet such as knitting is affected.The research of arabidopsiss shows that the flavonol (glycosides) of yellow hue is raw The expression of thing route of synthesis is then regulated and controled by PFG gene specifics, and wherein member MYB11, MYB12, MYB111 is in organ specificity Occur substantially to divide the work.

Carotenoid (carotenoids) is fat-soluble red, orange and xanthein, is entrenched in chloroplast and has In the film of colour solid.Carotenoid is C40- tetraterpenes compounds, synthesize by C5- isoprene basis unit, plant, very Bacterium, algae and antibacterial can synthesize, though animal can not synthesize, can take in from food and with imitating element and vitamin A before Body thing.Carotenoid contribute to yellow colour system for many patterns, and individually or together with anthocyanin for Flos Rosae Rugosae, Flos Chrysanthemi etc. some The petal of plant contribute to orange/red, yellowish-brown and shade of brown.So far, Carotenoid in Plants biosynthesis pathway is almost complete Portion's key gene has been cloned and has identified, whole approach originates in the Isoprenoid of the C5 in plastid (isopentenyl pyrophosphate, IPP) unit, it is believed that form complex between the relevant enzyme of the approach and combine On plastid film, 4 molecule IPP condensations are the GGPP (GGPP) of 1 molecule C20, close in phytoene Under the catalysis of enzyme (PSY), 2 molecule GGPP are coupled to the colourless phytoene of C40 head to head, and it is first class Hu Radix Raphani element.Subsequently, phytoene desaturase (PDS) and sigma carotene desaturase (ZDS) are sequentially introduced in molecule Conjugate double bonds, successively forms colourless phytofluene, lurid sigma carotene, orange-yellow neurosporene, red Lycopene.With the increase of conjugate double bonds number, absorbing wavelength is moved to long wave direction.Some produced during desaturation are suitable Formula conformation is alltranses conformation by carotenoid isomerase (CRTISO, Z-ISO) catalyzed transitions.Lycopene can be by tomato red Plain beta cyclase (LCYB) or lycopene ε-cyclase (LCYE) cyclisation, this is a branch point of the approach.Remove half balling Outside the plants such as Caulis et Folium Lactucae sativae, in most plants, LCYE adds ε-ring can only to lycopene, synthesizing yellow containing 1 β-ring and The alpha-carotene of 1 ε-ring and its derivant.β-and alpha-carotene can occur further hydroxylation or epoxidation modification, produce Many new constructions.The oxygenation product of carotene is referred to as the dihydroxylation process of xanthophyll, β-ring and ε-ring respectively by β-ring hydroxylase (CHYB) complete with ε-ring hydroxylase (CHYE).There is flower Idiotype and fruit differential type in PSY, GGPS and LCYB, show and exist The special carotenogenesis approach of one chromoplast.Zeaxanthin epoxidase (ZEP) catalysis zeaxanthin occur C5,6 and C5 ', the epoxidation of 6 ' positions form antheraxanthin and the violaxanthin of yellow, and it again can be under the catalysis of neoxathin synzyme (NSY) It is transformed into neoxathin.9- is cis-violaxanthin and 9- cis-neoxathin can be additionally used in synthesizing abscisic acid (ABA).

The controlling gene clone of carotenogenesis approach is also to be strengthened, but research shows that the regulation of the approach is main Occur on transcriptional level, by internal and such environmental effects, PSY is important rate-limiting enzyme check point, and it is right that photo-signal channel is participated in The regulation and control of carotenoids approach, and carotenoid cleavage dioxygenases (CCD)/NCED (9- is cis-and epoxy carotenoid is double Oxygenase) important regulative is participated in from the angle that decomposes.Additionally, VDE (Analysis of Violaxanthin De-Epoxidase) catalysis violaxanthins and flower Medicine Huang matter is converted into cryptoxanthin.Arabidopsiss AtRAP2.2 to particular organization in carotenoid accumulation have weak facilitation, But recent research indicate that its function is mainly the anoxia enduring survival ability of organization of regulation control.Fructus Lycopersici esculenti DDB1 and DET1 are by anti-to light The negative regulation of induction signal approach and suppress carotenoid approach, and Caulis et Folium Brassicae capitatae OR (Orange) and Fructus Lycopersici esculenti HSP21 be then by regulation and control The formation of chromoplast and promote the accumulation of carotenoid.

Additionally, also there is the 3rd class plant pigment, i.e. betanidin (betalains), it is water-soluble in vacuole to be that a class is present in Property alkaloid, can be divided into Betacyanins (betacyanins) and the betaxanthin of yellow colour system of purple colour system , but betanidin is only found in the plant and minority higher funguses of 10 sections of Caryophyllales, other plants (betaxanthins) In it has not been found that, and never find betanidin and anthocyanin and be stored in same plant.

As the pattern of most important decorative flowers is not complete, there is the major defect on pattern, therefore pattern molecule is educated The metabolic engineering that plants has important application prospect.But in including the whole Brassica genus including Brassica campestris L, have no and utilize molecular breeding Metabolic engineering Brassica campestris L pattern report.

Content of the invention

In view of this, an object of the present invention is to provide beta cyclase gene in interference brassica plant petal Overexpression GL3 genes are preparing the brassica plant kind that petal takes on a red color simultaneously for LCYB, ε-cyclase gene LCYE expression In application；The second object of the present invention is to provide beta cyclase gene LCYB, ε-cyclisation in interference brassica plant petal The expression of enzyme gene LCYE is while the plant expression vector of overexpression GL3 genes；The third object of the present invention is to provide above-mentioned The preparation method of plant expression vector；The fourth object of the present invention is that offer prepares petal using above-mentioned plant expression vector and is in The method of red brassica plant kind.

For achieving the above object, the present invention provides following technical scheme：

1st, in interference brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expresses excess table simultaneously Reach application of the GL3 genes in the brassica plant kind that petal takes on a red color is prepared.

Preferably, beta cyclase gene LCYB, ε-cyclase gene LCYE expression in the interference brassica plant petal Sequence as shown in SEQ ID NO.2, the sequence of the overexpression GL3 genes is as shown in SEQ ID NO.3.

Preferably, beta cyclase gene LCYB, ε-cyclase gene LCYE expression in the interference brassica plant petal Sequence by arabidopsiss petal specific promoter PAtAP3 mediation expression, the nucleotides sequence of the petal specific promoter PAtAP3 Row are as shown in SEQ ID NO.1 the 267th are to the 1044th.

Most preferably, the brassica plant is cabbage type rape (Brassica napus).

2nd, in interference brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expresses excess table simultaneously Up to the plant expression vector of GL3 genes, the plant vector includes the expression beta cyclase base mediated by petal specific promoter Expression GL3 genes are mediated because of the expression cassette of LCYB and ε-cyclase gene LCYE RNA interference sequences and by constitutive promoter Expression cassette.

Preferably, the petal specific promoter mediation expression beta cyclase gene LCYB, ε-cyclase gene LCYE The expression cassette of RNA interference sequences sequence successively shown in petal specific promoter PAtAP3, SEQ ID NO.2, intervening sequence, Reverse complementary sequence shown in SEQ ID NO.2 and NOS terminator composition.

It is furthermore preferred that described mediate the expression cassette of expression GL3 genes to be started by CaMV35S successively by constitutive promoter Son, sequence shown in SEQ ID NO.3 and NOS terminator composition.

3rd, the preparation method of the plant expression vector, comprises the steps：By sequence shown in SEQ ID NO.1 through AscI PFGC5941M plasmids with being connected into after SwaI double digestions through same enzyme action, obtain pFGC5941CEPE plasmids, then by SEQ ID Sequence shown in NO.2 is connected between the PAtAP3 promoteres of pFGC5941CEPE carriers and intervening sequence, forms intermediate carrier PFGC5941CEPE-B2RNAia, then sequence shown in SEQ ID NO.2 is reversely connected into intermediate carrier pFGC5941CEPE- Between the intervening sequence of B2RNAia and OCS terminators, pFGC5941CEPE-B2RNAi carriers are obtained, finally by SEQ ID NO.3 Shown sequence is connected into the pFGC5941CEPE-B2RNAi carriers through same enzyme action by NcoI and AscI restriction enzyme sites, must disturb In brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expression is while the plant of overexpression GL3 genes Thing expression vector.

Preferably, the intervening sequence is cabbage type rape PAP2 gene intron 2s.

4th, the method for preparing the brassica plant kind that petal takes on a red color using the plant expression vector, including following step Suddenly：

A. by plant expression vector conversion Agrobacterium described in any one of claim 5-7, engineering bacteria is obtained；

B., step a gained engineering bacteria is converted the hypocotyls of brassica plant aseptic seedling, after co-cultivation, in Basta weedings Under agent resistance, induction differentiation obtains regrowth, and the positive Seedling transgenic seedling of screening obtains the brassica plant kind that petal takes on a red color.

The beneficial effects of the present invention is：The invention provides the Brassica genus core species such as Brassica campestris L and Radix Raphani pattern mechanism, bright Really Hemerocallis citrina Baroni, spend in vain, the key differences expressing gene site between Flos Carthami, thus present invention clone's petal specific promoter is simultaneously Transformation obtains a kind of novel plant expression platform carrier for being suitable for flower color gene engineering, then clone's related gene or gene piece Section, the plant expression for building a kind of suppression yellow carotenoid in petal of acquisition and accumulating lycopene and anthocyanin are carried Body, creates the new type resource material of red rape flower phenotype after converting Brassica campestris L, the technical tactic is the Brassica genus pattern such as Brassica campestris L Pioneering in genetic engineering, and the first for combining metabolic engineering modified plant pattern by carotenoid and flavonoid path.

Description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：

Fig. 1 is the microscopic findings (A of Brassica genus and Radix Raphani petal：Cabbage type rape (BnY)；B：Cabbage type rape (BnW)；C：Mustard type rape (BjY)；D：Turnip type rape (BrY)；E：Caulis et Folium Brassicae capitatae (BoY)；F：Cabbage mustard (BoW)；G：Radix Raphani (RsR)).

Fig. 2 is that (A is DFR in Brassica genus to differential expression between Brassica genus petal and Radix Raphani petal of DFR, TT8, GL3, EGL3 Differential expression between petal and Radix Raphani petal；B is differential expressions of the TT8 between Brassica genus petal and Radix Raphani petal；C be GL3 in rue A kind of sedge belongs to the differential expression between petal and Radix Raphani petal；D is differential expressions of the EGL3 between Brassica genus petal and Radix Raphani petal；BnYPe： Cabbage type rape Hemerocallis citrina Baroni lobe；BnWPe：Cabbage type rape spends lobe in vain；BjYPe：Mustard type rape Hemerocallis citrina Baroni lobe；BrYPe：Chinese cabbage type oil Dish Hemerocallis citrina Baroni lobe；BoYPe：Brassica Oleracea Var.Acephala Hemerocallis citrina Baroni lobe；BoWPe：Lobe spent in vain by cabbage mustard；RsRPe：The pale reddish brown lobe of radish red).

Fig. 3 is the differential expression (BnYPe of OR, CCD1 between Brassica genus petal and Radix Raphani petal：Cabbage type rape Hemerocallis citrina Baroni lobe； BnWPe：Cabbage type rape spends lobe in vain；BjYPe：Mustard type rape Hemerocallis citrina Baroni lobe；BrYPe：Turnip type rape Hemerocallis citrina Baroni lobe；BoYPe：Plumage Clothing Caulis et Folium Brassicae capitatae Hemerocallis citrina Baroni lobe；BoWPe：Lobe spent in vain by cabbage mustard；RsRPe：The pale reddish brown lobe of radish red).

Fig. 4 is NOS-PAtAP3, B2RNAi and BnGL3-1PCR amplification (A：NOS-PAtAP3；B：B2RNAi；C： BnGL3-1).

Enzyme action result (A during Fig. 5 pFGC5941CEPE and pBLycoRF5 vector constructions：AscI and SwaI double digestions pFGC5941M；B：AscI and SwaI double digestion pMD19-T-NOS-PAtAP3；C：SwaI and AatI double digestions pFGC5941CEPE；D：SwaI and AatII double digestion pMD19-T-B2RNAi；E：BamHI and XbaI double digestion pMD19-T- B2RNAi；F：BamHI and XbaI double digestion pFGC5941CEPE-B2RNAia；G：NcoI and AscI double digestions pFGC5941CEPE-B2RNAi；H：NcoI and AscI double digestion pMD19-T-BnGL3-1).

Fig. 6 is the petal color (A that pBLycoRF5 converts rapeseed plants：Matched group；B：Transgene rape).

Specific embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted concrete in embodiment The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. write) Described in condition, or according to the condition proposed by manufacturer.

The vegetable material that the embodiment of the present invention is adopted：Cabbage type rape Hemerocallis citrina Baroni lobe and development mid-term seed (in double No. 9 product Kind), cabbage type rape breast petal, turnip type rape Hemerocallis citrina Baroni lobe (6Y733 strains), mustard type rape Hemerocallis citrina Baroni lobe (CNG12011 product System), Brassica Oleracea Var.Acephala (B.oleracea var.acephala ftricolor, K10-3 strains) Hemerocallis citrina Baroni lobe, cabbage mustard mutation spend in vain Lobe (R9057 strains), radish red petal are taken from Chongqing City of Southwestern University Brassica campestris L Engineering Technical Research Centre and have a rest the routine in horse base Test plant.Arabidopsiss (Arabidopsis thaliana, Columbia wild type) seed is planted purchased from international arabidopsiss center Plant in indoors artificial climatic chamber.

Reagent and test kit that the embodiment of the present invention is adopted：PrimeScript^TMRT reagent Kit with gDNA Eraser(Perfect Real Time)、Premix Ex Taq^TMII(Tli RNaseH Plus)ROX plus、 DNA Ligation Kit, pMD19-T, Taq archaeal dna polymerases, DNase I (RNase-free) and buffer, RNase Inhibitor, DL-2000 and λ-HindIII DNA Marker are purchased from the limited public affairs of precious biology (TaKaRa) biological engineering in Dalian Department；RNAprep Pure plant total RNA extraction reagents box is TIANGEN Biotech's product；Glue reclaim reagent Box, little mensuration plasmid extraction test kit are purchased from Shanghai Hua Shun Bioisystech Co., Ltd；Restricted enzyme is purchased from Lithuania MBI Fermentas companies；MS (Murashige＆Skoog medium, including vitamins) culture medium is Holland Duchefa Products；The reagents such as DL-2000plus, Easy-Taq enzyme, dNTPs are raw purchased from full formula gold (Transgen) in Beijing Thing Technology Co., Ltd.；X-Gluc (5-bromo-4-chloro-3-indolyl- β-D-glucuronic acid), rifampicin (Rif), streptomycin (Str), kanamycin (Kan), ampicillin (Amp), agarose, Tris, CTAB, Tris saturated phenol (pH=8.0), other biochemistry and molecular biology reagent purchases such as Tryptone, Yeast Extract, X-gal, IPTG, CTAB From Shanghai Sangon Biological Engineering Technology And Service Co., Ltd；Phytohormone sows the companies such as rich garden supplies purchased from Shanghai.

The key instrument that the embodiment of the present invention is adopted：Veriti^TMMultiple temperature control PCR instrument is purchased from U.S. Applied Biosystems companies；CFX96 quantitative real time PCR Instruments are purchased from Bio-Rad companies of the U.S.；And molecular biology and genetic engineering Miscellaneous equipment facility.

PCR primer synthesis used by preferred embodiment and sequencing are by Shanghai English fine horse/Invitrogen Corp., Shanghai life work, Beijing The company trades such as six directions Hua Da are completed.

Embodiment 1, Brassica genus and the microexamination of Radix Raphani petal colour developing subcellular organelle

Fresh cabbage type rape Hemerocallis citrina Baroni lobe (BnY) is taken respectively；Cabbage type rape breast petal (BnW)；Mustard type rape Hemerocallis citrina Baroni lobe (BjY)；Turnip type rape Hemerocallis citrina Baroni lobe (BrY)；Brassica Oleracea Var.Acephala Hemerocallis citrina Baroni lobe (BoY)；Lobe (BoW) spent in vain by cabbage mustard；Radix Raphani Flos Carthami Lobe (RsR), is placed on cold fresh preservation of ice face and transports laboratory back, then freehand section, and with low power sem observation rapid screening, it is qualified to select Section is examined and photograph, as a result as shown in Figure 1.As a result show, displaing yellow in the Hemerocallis citrina Baroni lobe of the several species of Brassica genus Material be rendered as numerous fine granularities in a cell, be non-uniformly distributed in intracellular, be expressed to by vacuole adherent Position, illustrates that phaeochrome cell device is chromoplast in yellow petal, and its pigment is yellow carotenoid.Cabbage type rape breast petal In also have some cells to have light yellow chromoplast, but total number is few and iuntercellular is inconsistent, and cabbage mustard is spent in vain in lobe and almost seen Less than chromoplast, explanation is that the minimizing of chromoplast or disappearance cause its yellow to shoal or disappear.In Radix Raphani petal, aubergine pigment is equal The even middle position for being distributed in each cell, central authorities are denseer, and more offset from center is lighter, illustrate that the colour developing of Radix Raphani petal is sub- thin Born of the same parents' device is vacuole, and substance that show color is anthocyanin.

Embodiment 2, detection Brassica genus and radish flower petal pigment

Firm open cabbage type rape Hemerocallis citrina Baroni lobe (BnY) is taken respectively in full-bloom stage morning；Cabbage type rape breast petal (BnW)；Mustard type rape Hemerocallis citrina Baroni lobe (BjY)；Turnip type rape Hemerocallis citrina Baroni lobe (BrY)；Brassica Oleracea Var.Acephala Hemerocallis citrina Baroni lobe (BoY)；Cabbage mustard is white Petal (BoW)；Radish red petal (RsR), shady and cool immediately fresh-keeping transport laboratory back, 50～60 DEG C drying, after smashing cross 60 mesh Sieve, lucifuge kept dry.

1st, petroleum ether, hydrochloric acid and ammonia test result

Weigh the petal powder 0.100g of preservation, be respectively put into volume number tool plug test tube in, be separately added into petroleum ether, The each about 10mL of 10% hydrochloric acid, 30% ammonia, gently mixes, filters, and observes color change, as a result as follows：

(1) petroleum ether reaction：Cabbage type rape, turnip type rape, mustard type rape all show glassy yellow, show class recklessly The content of Radix Raphani content is high；Brassica Oleracea Var.Acephala shows light yellow, illustrates which contains a small amount of carotenoid；And cabbage mustard and radish flower Show colourless, show without carotenoid.

(2) hydrochloric acid test：Only radish red petal shows pink, illustrates containing anthocyanin, and cabbage type rape, Turnip type rape, mustard type rape petal show different degrees of yellow, illustrate containing flavone (alcohol) and do not contain anthocyanidin.Cabbage mustard The performance of white and Brassica Oleracea Var.Acephala is closely colourless, illustrates not containing anthocyanidin, and flavone (alcohol) is also little.

(3) ammonia test：The each material of Brassica genus shows different degrees of yellow, illustrates which contains more or less Huang Ketone (alcohol), and the yellow green that radish flower shows, the color are the yellow mixing of the blueness and flavonoid presentation presented by anthocyanin Form, but all material does not show orange red or red, illustrates without aurones.

2nd, the chromogenic reaction of flavonoid

Go bail for the petal powder 0.100g for depositing, and uses methanol extraction 24h, filters, is settled to 50mL, respectively takes 2mL extracting solution, so After carry out following color reaction, observe color change.

(1) concentrated hydrochloric acid-magnesium powder reaction：A small amount of magnesium powder is added, then adds concentrated hydrochloric acid 5 to drip afterwards, gently shaken up, stand 1h.Knot Fruit shows that cabbage type rape, cabbage mustard show colourless, may contain chalcone, aurones；Cabbage type rape, turnip type rape, Caulis et Folium Brassicae junceae Type Brassica campestris L shows pole lavender blush and micro- purplish red, illustrate without chalcone, aurones and catechin, may containing flavone, flavonol, two Hydrogen flavonol, flavanone；Radish flower shows pink, illustrates containing anthocyanidin.

(2) concentrated hydrochloric acid-zinc powder reaction：A small amount of zinc powder is added, concentrated hydrochloric acid 10 is added and is dripped, gently shake up, stand 1h.As a result Show, radish flower assumes pink, illustrates containing anthocyanin.Remaining colourless or yellow, illustrates without anthocyanin.

(3) lead acetate reaction：Plus 1.0% lead acetate 2mL, gently shake up, stand 2h.As a result show, all Brassica genus materials There is different degrees of yellow mercury oxide in material, illustrates that flavonoid possesses phenolic hydroxyl group and without chalcone derivative and aurones, may have Adjacent two phenolic hydroxyl groups have 4- ketone groups, 3-OH or 4- ketone groups, 5-OH structures concurrently；There is green precipitate in radish flower, illustrates containing flower Blue or green element glycosides.

(4) ferric chloride reaction：Plus 5.0% ferric chloride 2ml, gently shake up.As a result show, Huang all occurs in all material Color, illustrates in pigment molecular not phenolic hydroxy group.

(5) aluminum chloride reaction：Plus 1.0% aluminum chloride methanol solution l ml.As a result show, all material all assumes journey The different yellow of degree, illustrates containing flavonoid substances.

(6) strong sulfuric acid response：Plus the dense H of 1.5mL₂SO₄, gently shake up, then put boiling water bath 5min.All Brassica genus materials are equal Assume different degrees of yellow, (alcohol) containing flavone is described, 5min colors do not change in boiling water, illustrate without chalcone, aurones, can Flavanone, possible isoflavone-containing and isoflavanone can not contained.Radish flower appearance is orange-yellow, illustrates containing anthocyanidin.

(7) tetrahydro boron sodium reaction：Plus tetrahydro boron sodium 8mg, then plus 1.0% hydrochloric acid 2mL, gently shake up, stand 2h.All rues A kind of sedge belongs to material and assumes the different yellow of degree, illustrates without flavanone and flavanonol.Radish flower assumes pole rose pink Color, illustrates containing flavanone and/or flavanonol.

(8) alkaline reagent reaction：Plus 5%Na₂CO₃3ml, gently shakes up, closed standing 30min, blowing air 10min.Institute There is material to assume the different yellow of degree, color is constant after blowing air, illustrate without flavanonol.

(9) ammonia cesium chloride reaction：Methanol 10ml is taken, ammonification water is settled to 25ml, become molten by the water saturated methanol of ammonia Liquid.To in sample liquid, add 0.01mol/L strontium chlorides methanol solution 10 to drip, then plus dripped by the water saturated methanol solution 10 of ammonia, light with handss Jog is even, stands lh.Radish flower presents precipitation, has illustrated that 3 ', 4 '-dihydroxy replaces.

(10) acid reaction：Plus 1.0% boric acid 10 drip, then plus 2.0%H₃BO₃3ml, cabbage mustard spend in vain lobe present colourless, Illustrate that cabbage mustard is spent lobe flavonoid in vain and may not contain C₅-OH.

3rd, the ultraviolet-visible light analysis of spectrum of petal pigment composition

(1) chlorophyll：Weigh the petal 0.100g of preservation, rapidly with liquid nitrogen grinding to powder, adopt volume fraction for 90% acetone:Ethanol (4:L, V/V) 24h is extracted, filter, be settled to 25ml, using ultraviolet-visible spectrophotometer 200 Scan in the range of～700nm.As a result show, all samples without absworption peak, are illustrated green without leaf at 662nm and 644nm Element.

(2) carotenoid：The petal 0.100g of preservation is weighed, rapidly with liquid nitrogen grinding to powder, petroleum ether is added:Third Ketone (1:1, V/V) 24h is extracted, filters, be settled to 25ml, using ultraviolet-visible spectrophotometer in the range of 200～700nm Scanning.As a result show, cabbage type rape, turnip type rape, mustard type rape, Brassica Oleracea Var.Acephala have suction in 440 and 470nm or so Peak is received, is illustrated containing carotenoid, the content that quantitative analyses are determined respectively 6.824,6.712,5.548,1.248mg/g.And Cabbage mustard and Radix Raphani petal illustrate not containing carotenoid then without characteristic absorption peak.

(3) flavonoid：The petal 0.100g of preservation is weighed, rapidly with liquid nitrogen grinding to powder, acidified methanol (pH is added =3) 2ml be placed in 4 DEG C of refrigerators extraction 24h, filter, be settled to 25ml, with ultraviolet-visible spectrophotometer in 220～600nm In the range of scan.As a result show, cabbage type rape, turnip type rape, mustard type rape, Brassica Oleracea Var.Acephala, cabbage mustard, Radix Raphani petal Extracting solution has absworption peak 330 and 270nm, illustrates that they contain flavonoidss, the content that quantitative analyses are determined point Not Wei 7.483,7.651,7.001,1.391,1.003,8.373mg/g.

(4) anthocyanin：The petal 0.100g of preservation is weighed, rapidly with liquid nitrogen grinding to powder, methanol extraction is added 24h, filters, is settled to 50ml, is scanned in the range of 200～700nm with ultraviolet-visible spectrophotometer.As a result show, Radix Raphani Petal pigment has obvious anthocyanin characteristic absorption peak in 532nm or so, is anthocyanin band I absworption peaks, in 260-270nm areas With a less strong peak with II in domain, the content that quantitative analyses are determined is 237.27mg/100g.All Brassica genus sample standard deviations without Anthocyanin absworption peak, that is, do not contain anthocyanin.

Embodiment 3, Brassica genus and the Molecular Identification of radish flower petal pigment biosynthesis pathway

(1) expression characteristic of Brassica genus and Radix Raphani petal flavonoid pathway gene

Occur the molecule mechanism of difference, design Brassica genus and Radix Raphani flavonoid path for studying Brassica genus and radish flower petal pigment The RT-PCR detection primers of gene, and using 5SrRNA as internal reference, concrete as shown in table 1：

Table 1, flavonoid path RT-PCR detection primers

Firm open cabbage type rape Hemerocallis citrina Baroni lobe (BnY) is taken respectively in full-bloom stage morning；Cabbage type rape breast petal (BnW)；Mustard type rape Hemerocallis citrina Baroni lobe (BjY)；Turnip type rape Hemerocallis citrina Baroni lobe (BrY)；Brassica Oleracea Var.Acephala Hemerocallis citrina Baroni lobe (BoY)；Cabbage mustard is white Petal (BoW)；Radish red petal (RsR), liquid nitrogen freezing are transported, and -80 DEG C of Refrigerator stores are standby.Then planted with RNAprep Pure Thing total RNA extraction reagent box extracted total RNA, through electrophoretic analysiss and spectrophotometry qualified after, use RNase-free DNase I remove the DNA impurity in total serum IgE, with RT Reagent Kit With gDNA Eraser (Perfect Real Time) test kit is further removed genomic DNA (gDNA) and is carried out reverse transcription, obtains the first chains of total cDNA.Use CHS genes A pair of conserved region primers of family enter performing PCR amplification, target area span intron, and electrophoresis showed all samples only expand big with prediction Little consistent cDNA bands are further detected with internal standard gene 25SrRNA, the amplification between each sample without gDNA bands Band is special and luminance difference less, thoroughly eliminated the gDNA in total serum IgE before reverse transcription is described, all samples reverse transcription into Work(and total cDNA concentration is more or less the same, can be used for the experiment such as quantitative gene expression PCR.

Then adoptPremix Ex Taq^TMII (Tli RNaseH Plus) kit is in CFX96^TMReal- Fluorescence real-time quantitative PCR is carried out on Time System, and PCR programs are executed according to the method for description.Reaction system is 20 μ L bodies It is that quantitative RT-PCR contains 0.1 μ L of reverse transcription product, SYBR Premix Ex Taq^TM10 μ L of II (2 ×), each primer (10 μM) 0.4 μ L, remaining volume ddH₂O polishings.PCR cycle parameter is：95 DEG C of denaturations 3min；50 amplification cycles (95 DEG C of degeneration 10s, corresponding temperature annealing 30s, 72 DEG C of extension 30s)；65℃5s；95℃5s.2 RNA extraction-reverse transcription weights of Setup Experiments Multiple (repetition biology), each reverse transcription are repeated 2 times PCR (detection repeats), 4 results averageds.And it is bent by melting The specificity of line analysis confirmatory reaction, relative expression quantity is according to 2^-ΔCtCalculate, as a result as shown in Figure 2.

As a result show, CHS, CHI, F3H, TTG1, PAP gene family vigorous or moderate table in all material Reach, storeroom difference is little.F3 ' H, F3H, FLS, PFG (MYB12, MYBL2) have expression in each material, only only material Between have height difference.DFR, ANS, TT19, TT8, GL3, EGL3 in each material of Brassica genus significantly expression or Expression is extremely low, but expression is very high in Radix Raphani.Illustrate flavonol synthesis be in Brassica genus and Radix Raphani petal vigorous Or more vigorous working condition, the synthesis of anthocyanin only in Radix Raphani petal vigorous work but in Brassica genus petal in closing Closed state.

(2) expression characteristic of Brassica genus and Radix Raphani petal carotenoid approach

For the molecule mechanism that difference occur in further research Brassica genus and radish flower petal pigment, design Brassica genus and radish kind Hu The RT-PCR primer of Radix Raphani element 17 functional site gene families of approach, and using 25SrRNA as internal reference, concrete primer such as 2 institute of table Show.

Carotenoid approach RT-PCR detection primer used by 2 present invention of table

Then RT-PCR is carried out using method same as described above, and by the special of melting curve analysis confirmatory reaction Property, relative expression quantity is according to 2^-ΔCtCalculate, as a result as shown in Figure 3.

As a result show, all detection gene locis of carotenoid approach are expressed in the petal of 7 species materials, But there is also some differences, one be Brassica genus white/newborn petal in CCD1 expressions substantially high than in Hemerocallis citrina Baroni lobe, two is radish flower In lobe OR in closed mode and do not express, in Brassica genus diploid elementary species OR expressions also without tetraploid aggregate species in High.Illustrate that the disappearance of carotenoid in Radix Raphani petal is because that OR is closed and can not be formed chromoplast, Brassica genus are white/newborn petal in The minimizing of carotenoid is because that the decomposition of carotenoid is too strong.

Embodiment 4, plant expresses the structure of platform carrier pFGC5941CEPE

According to gene expression difference on Brassica genus and Radix Raphani petal carotenoid and flavonoid route of synthesis, clone is poor for design Different expressing gene and the primer of construction of expression vector, concrete primer are as shown in table 3.

Gene cloning, vector construction and detection the primer in 3 present invention of table

(1) acquisition of NOS-PAtAP3 fragments

With pCAMBIA2301 plasmids as pcr template, expanded and reclaimed the piece of 286bp with primer combination FTNOS and RTNOS Section, obtains the fragment containing NOS terminator, and its nucleotide sequence is as shown in SEQ ID No.1 1-286 positions.Extracted using CTAB methods The genome DNA of Arabidopsis leaf is pcr template, is expanded and reclaimed 778bp's with primer combination FPAtAP3 and RPAtAP3 PAtAP3 fragments (the GenBank accession number of specific promoter containing petal:U30729), its nucleotide sequence such as SEQ Shown in ID No.1 267-1044 positions.Then using after the mixing of two kinds of recovery products as pcr template, with primer combination FTNOS and RPAtAP3 is expanded, and obtains fusion fragment NOS-PAtAP3 of the clip size for 1044bp, as a result as shown in A in Fig. 4.Then cut Glue reclaim, the pMD19-T-NOS-PAtAP3 recombinant vectors being connected with pMD19-T, by the recombinant vector conversion escherichia coli for obtaining DH5 α competent cells, are entered using primer combination FTNOS and RPAtAP3 to the transformant monoclonal of Amp resistance LB plate screenings Performing PCR detects that sequencing result shows, fusion fragment NOS-PAtAP3 sequence is as shown in SEQ ID No.1.

(2) structure of pFGC5941CEPE

Extracting pMD19-T-NOS-PAtAP3 recombiant plasmid, then carries out double digestion with AscI and SwaI, reclaims NOS- PAtAP3 fragments, as shown in B in Fig. 5；Extract pFGC5941M plasmids simultaneously, carrier bone is reclaimed after AscI and SwaI double digestions Frame, as shown in A in Fig. 5.Obtain 11241bp after NOS-PAtAP3 fragments and pFGC5941M plasmids are connected is suitable for pattern PFGC5941CEPE is converted bacillus coli DH 5 alpha competent cell by engineered plant expression vector pFGC5941CEPE, Coating and monoclonal transformant is screened on the LB flat boards that concentration containing Kan is 50mg/L, the monoclonal transformant that screening is obtained is used Primer combination FTNOS and RPAtAP3 and F35S3N and ROCST5N enters performing PCR detection, and sequence is chosen in screening positive clone sequencing The clone arranged without mutation is standby.In its pFGC5941CEPE recombiant plasmid for obtaining, interval containing Bar gene tables in T-DNA Up to box (providing the resistance to herbicide basta) and 2 expression cassettes that can be used to insert exogenous gene, exogenous gene expression box 1 CaMV35S containing composition promoter and NOS terminator, can be used for justice or the antisense expression of genes of interest；Exogenous gene expression 2 PAtAP3 of specific promoter containing petal of box, introns BnPAP2I2 (cabbage type rape (Brassica napus) PAP2 genes 2 introns) and OCS PolyA terminators, can be used for RNA interference, justice or the antisense expression of genes of interest.

(3) clone of series connection bivalence RNA interference fragment B2RNAi

Total the first chains of cDNA of the cabbage type rape petal that adopts above-mentioned reverse transcription to obtain are combined using primer for template (387bp after enzyme-added enzyme site and joint is the RNA interference fragments of FBLCYBi and RBLCYBi amplification LCYB gene families 411bp), with the RNA interference fragments of primer combination FBLCYEi and RBLCYEI amplification LCYE gene families, (480bp, after adjunction head For 506bp), it is separately recovered.Then as masterplate, FBLCYBi will be combined with primer after the recovery product mixed in equal amounts of 2 kinds of PCR 867bp fragments are expanded into RBLCYEI, the RNA interference fragments of two genes of fragment series LC YB, LCYE are named as B2RNAia, as a result as shown in B in Fig. 4 (being 893bp after enzyme-added enzyme site).Then B2RNAia is reclaimed, is connected with pMD19-T PMD19-T-B2RNAi, pMD19-T-B2RNAi convert bacillus coli DH 5 alpha competent cell, to Amp resistance LB plate screenings Transformant monoclonal enters performing PCR detection using primer combination FBLCYBi+RBLCYEI, send positive colony to be sequenced, sequencing knot Fruit is as shown in SEQ ID No.2.

(4) plant expression vector pFGC5941CEPE-B2RNAi is built

Extracting pMD19-T-B2RNAi plasmids, with SwaI and AatII double digestions (SwaI first cuts, then plus AatII), reclaim B2RNAi fragments, as shown in D in Fig. 5；Simultaneously extract pFGC5941CEPE plasmids, with SwaI and AatII double digestions after, reclaim and carry Body skeleton, as shown in C in Fig. 5.The PAtAP3 that the B2RNAi fragments of recovery are connected into pFGC5941CEPE plant expression vectors is opened Between mover and introns BnPAP2I2, intermediate carrier pFGC5941CEPE-B2RNAia is formed, convert bacillus coli DH 5 alpha sense Receive state cell, with the monoclonal transformant of Kan resistance LB plate screenings, be then respectively adopted primer combination FPAtAP3 and FBLCYBi and RBLCYEI and RBnPAPI2 enters performing PCR detection, and PCR positive colony are standby.

Extracting pMD19-T-B2RNAi plasmids, with BamHI and XbaI double digestions, reclaim the sense fragment of B2RNAi B2RNAi, as shown in E in Fig. 5.Extract pFGC5941CEPE-B2RNAia plasmids simultaneously, with BamHI and XbaI double digestions, reclaim Carrier framework, as shown in F in Fig. 5.By B2RNAi be connected into pFGC5941CEPE-B2RNAia plasmids (introns BnPAP2I2 with Between OCS terminators), plant expression vector pFGC5941CEPE-B2RNAi is formed, by plant expression vector PFGC5941CEPE-B2RNAi converts bacillus coli DH 5 alpha competent cell, is converted with Kan resistance LB plate screenings monoclonal Son, then with FPAtAP3 and FBLCYBi, FBLCYBi and ROCST5N, RBLCYEI and RBnPAPI2, FBnPAPI2 and RBLCYEI 4 pairs of primer combinations carry out PCR identifications, and positive colony sequencing chooses clone of the sequence without mutation standby.

(5) clone of the cDNA coding regions of cabbage type rape GL3-1 (BnGL3-1) gene

We show that cabbage type rape GL3-1 (BnGL3-1) gene is mainly expressed in kind of skin in the research of the past, activation The synthesis of flavonoid-procyanidin, also expresses in the stem and leaf that aging turns red, activates the synthesis of flavonoid-anthocyanin.Cause This, the present invention clones the cDNA coding regions of BnGL3-1 from the seed of cabbage type rape development, is subsequently used for building just carrier And convert Brassica campestris L overexpression in petal later.The total serum IgE that cabbage type rape develops mid-term seed is extracted, such as front method removes gDNA Reverse transcription obtains the first chain cDNA afterwards, then with the first chain cDNA as template, is expanded using the combination of FBnGL3-1 and RBnGL3-1 primers Increase BnGL3-1 genes, obtain the coding region (being 1900bp after enzyme-added enzyme site) of BnGL3-1 gene 1890bp, electrophoresis result such as 4 Shown in middle C, by its glue reclaim, pMD19-T-BnGL3-1 is connected into pMD19-T, converts bacillus coli DH 5 alpha competent cell, Performing PCR detection is entered using primer combination FBnGL3-1 and RBnGL3-1 to the transformant monoclonal of Amp resistance LB plate screenings, is sent Multiple positive colony sequencings, sequencing result show BnGL3-1 sequences as shown in SEQ ID No.3, choose sequence without mutation Clone is standby.

(6) plant expression vector pBLycoRF5 is built

Extracting pMD19-T-BnGL3-1 plasmids, with NcoI and AscI double digestions (AscI first cuts, then plus NcoI), reclaim BnGL3-1 genetic fragments, as shown in H in Fig. 5；PFGC5941CEPE-B2RNAi plasmid is extracted simultaneously, with the double enzymes of NcoI and AscI Cut, reclaim carrier framework, as shown in G in Fig. 5.By the BnGL3-1 genetic fragments for reclaiming and pFGC5941CEPE-B2RNAi carriers Skeleton connects, and forms the trivalent plant expression vector of series connection bivalence RNAi and monovalence overexpression, is named as pFGC5941CEPE- B2RNAi-BnGL3-1ox, referred to as pBLycoRF5.Then pBLycoRF5 is converted bacillus coli DH 5 alpha competent cell, right The transformant monoclonal of Kan resistance LB plate screenings respectively with FPAtAP3 and FBLCYBi, FBLCYBi and ROCST5N, RBLCYEI and RBnPAPI2, FBnPAPI2 and RBLCYEI, F35S3N and RBnGL3-1, FBnGL3-1 and RTNOS 6 are to primer Whether carrier successfully constructs respectively for combination, then extracts positive colony and convert Agrobacterium tumefaciems using freeze-thaw method after plasmid LBA4404, then with the monoclonal transformant of the resistance LB plate screening containing Kan, Str and Rif, and with above-mentioned 6 pairs of primer sets Close and detect into performing PCR, screening positive clone is preserved as engineered strain, for Plant Transformation.

Embodiment 5, pBLycoRF5 converts Brassica campestris L

All tissue culture's operations are carried out under the conditions of the plant tissue culture of standard, between superclean bench, culture, are tamed and dociled Clean rank between change is respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagent, material, vessel carry out nothing by code Bacterium is processed.In cabbage type rape typical case's Hemerocallis citrina Baroni kind, double No. 10 seeds is with being rushed with sterilized water after 75% ethanol surface sterilization 1min Wash 3 times, then 20min is soaked with 5% sodium hypochlorite, be inoculated in (MS powder in MS solid mediums after aseptic water washing is clean 4.41g/L+Phytagel 2.6g/L+ sucrose 30.0g/L, pH5.8, autoclave moist heat sterilization；It is not added with Phytagel and is liquid Culture medium), cultivate in 25 DEG C, 2000Lux illumination, 16h/d photoperiods (between tissue culture below condition of culture except especially indicate person Outward, identical with this).The hypocotyls for cutting seedling age 8d or so aseptic seedling are cut into the segment for being about 0.5～1.0cm, are inoculated into pre- (MS culture medium+1.0mg/L 6-BA+1.0mg/L 2,4-D) preculture 3d in training culture medium MSp.

By the LBA4404 engineered strains containing pBLycoRF5 of -80 DEG C of preservations added with 100mg/L Kan, 20mg/L In 28 DEG C, 250r/min 1～2d of shaken cultivation in the LB fluid mediums of Str and 40mg/L Rif, grow to Agrobacterium right The number phase, switching culture is once.Then thalline is collected by centrifugation in 5000rpm, 10min room temperature, with dip-dye culture medium MSm (MS liquid Culture medium+1.0mg/L 2,4-D+1.0mg/L 6-BA+100 μm ol/L AS) bacterial concentration is adjusted to OD₆₀₀About 0.5 or so, i.e., For dip-dyeing solution.

5～10min in hypocotyls section immersion dip-dyeing solution after by preculture, intermittence is gently swayed, then by hypocotyls Section blots unnecessary bacterium solution on sterilizing paper, is inoculated into common training culture medium MSc (MS solid medium+2.0mg/L 6-BA+0.5mg/ L NAA) in, in 23.5 DEG C of light culture 48h.After light culture with sterilizing liquid culture medium MSk (MS fluid medium+1.0mg/L2, 4-D+1.0mg/L 6-BA+500mg/L Cef) washing by soaking explant 3 times, then each 10min blots surface with sterilizing paper Liquid, is transferred to induction screening culture medium MSi (MS solid medium+1.0mg/L 6-BA+1.0mg/L 2,4-D+500mg/L Cef+12.5mg/L Basta+6mg/L AgNO₃) culture, per 2 weeks subcultures 1 time, to growing macroscopic kanamycin-resistant callus tissue, then It is transferred to division culture medium MSd (MS solid medium+4.0mg/L 6-BA+2.0mg/L ZT+5.0mg/L AgNO₃+500mg/ L Cef+12.5mg/L Basta) middle culture more than 14d, callus induction differentiates budlet, then is transferred to stem differentiation culture Base MSs (MS solid medium+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+12.5mg/L Basta) cultivate to Little stem is grown, then is transferred to long shoot culture medium MSe (MS solid medium+0.05mg/L6-BA+500mg/L Cef+12.5mg/L Basta unrooted seedling complete to length is cultivated in), then is transferred to root media MSr (MS solid medium+2mg/L NAA) trainings Support to flourishing root system is grown, after the seedling after taking root is through domestication, be transplanted to containing sterilizing perlite, Vermiculitum, turf earth mixtures (mass ratio is 1:1:1), in basin alms bowl, it is managed by greenhouse pot culture.

Finally, double No. 10 in pBLycoRF5 conversions cabbage type rape after obtain 28 plants of regeneration plants；To regeneration plant blade Deca 200mg/L Basta solution detect resistance, and extract blade genome DNA be respectively adopted primer combination F35S3N and RBnGL3-1, FBLCYBi and ROCST5N enter performing PCR detection, as a result show to obtain 17 plants of double positive transgenic plant.

Comprehensive biology and agronomy observation is carried out to transgenic positive plant, as a result as shown in Figure 6.As a result show, obtain The transfer-gen plant growth promoter and background character for obtaining substantially does not change, but petal becomes red, and petal cell is micro- Observation shows that red material is existing from chromoplast, also has from vacuole, and biochemical component detection also indicates that new generation The existing lycopene of red material, also have anthocyanin.Show to suppress xanthein product by the method for the present invention Tired lycopene and anthocyanin can create red rape flower.

Self propagated is carried out to transgenic present age plant, using blade Deca Basta as transgenic present age plant Resistance detecting, Screening and Identification go out the excellent strain of transgene rape homozygosis, and its petal color is redder than transgenic present age plant, illustrates to turn Gene character can stablize heredity and homozygosis offspring strain is more preferable than the transgene traits of contemporary (heterozygosis) individual plant.

Finally illustrate, above example is only in order to illustrate technical scheme, but is not limited to this. Although by referring to the preferred embodiments of the present invention, invention has been described, and one of ordinary skill in the art should Work as understanding, various changes can be made to which in the form and details, be limited without departing from appended claims Fixed the spirit and scope of the present invention.Here especially declare, the following change on application form also all necessarily belongs to the present invention's Spirit and scope are covered：

1st, GL3-1, LCYB, LCYE gene/gene fragment in the present invention, except listed nucleotide sequence in sequence table In addition, also include coming from parent species Chinese cabbage or the corresponding gene order in Caulis et Folium Brassicae capitatae, also include coming from these species together The sequence of one functional site gene family other members, although they may have with listed nucleotide sequence in sequence table little Difference.

2nd, the gene in the present invention and its fragment, in addition to listed nucleotide sequence in sequence table, also include and it In continuous 80bp and above have more than 98.00% conforming any nucleotide sequence.

3rd, in the present invention LCYB, LCYE site RNA interference fragments, except listed nucleotide sequence in sequence table with Outward, also include the fragment for coming from same gene other positions.

4th, the suppression that LCYB, LCYE site is expressed in the present invention, except the RNA perturbation techniques that is lifted in preferred embodiment In addition, the technology such as antisense RNA, genome editor (ZFN, TALEN, CRISPR-Cas) can also be adopted same or similar to reach Purpose.

5th, the gene in the present invention and its fragment, except the transformation of the employing pFGC5941 as being lifted in preferred embodiments is carried Beyond body is built, can also using other carriers carrying out plant expression vector construction, including using other promoteres and Terminator, reaches same or analogous purpose；Vector construct in the present invention, except as adopting for being lifted in preferred embodiments Beyond the improvement leaf disk method mediated with Agrobacterium tumefaciens strain LBA4404 carries out genetic transformation, it would however also be possible to employ other methods are entered Row Genetic Transformation in Higher Plants.

6th, in the present invention gene, genetic fragment and vector construct, except as being lifted in preferred embodiments for sweet Beyond blue type Brassica campestris L, other sibling specieses of its parent species Chinese cabbage, Caulis et Folium Brassicae capitatae and Brassica genus can also be applied to, be reached identical Or similar purpose.

Claims (7)

1. beta cyclase gene in brassica plant petal is disturbedLCYB, ε-cyclase geneLCYEExpression is while overexpressionGL3Application of the gene in the brassica plant kind that petal takes on a red color is prepared, it is characterised in that：The interference brassica plant Beta cyclase gene in petalLCYB、ε-cyclase geneLCYEThe sequence of expression as shown in SEQ ID NO.2, the excess table ReachGL3The sequence of gene is as shown in SEQ ID NO.3.

2. application according to claim 1, it is characterised in that：Beta cyclase gene in the interference brassica plant petalLCYB, ε-cyclase geneLCYEThe sequence of expression is by arabidopsiss petal specific promoterPAtAP3Mediation expression, the petal Specific promoterPAtAP3Nucleotide sequence as shown in SEQ ID NO.1 the 267th are to the 1044th.

3. application according to claim 1 and 2, it is characterised in that：The brassica plant is cabbage type rape （Brassica napus）.

4. beta cyclase gene in brassica plant petal is disturbedLCYB, ε-cyclase geneLCYEExpression is while overexpressionGL3The plant expression vector of gene, it is characterised in that：The plant vector include the expression β that mediated by petal specific promoter- Cyclase geneLCYB, ε-cyclase geneLCYEThe expression cassette of RNA interference sequences and by constitutive promoter mediation expressionGL3 The expression cassette of gene；

The petal specific promoter mediation expression beta cyclase geneLCYB, ε-cyclase geneLCYERNA interference sequences Expression cassette sequence successively shown in petal specific promoter PAtAP3, SEQ ID NO.2, intervening sequence, SEQ ID NO.2 institutes The reverse complementary sequence for showing and NOS terminator composition；

Described mediation by constitutive promoter is expressedGL3The expression cassette of gene is successively by CaMV35S promoteres, SEQ ID NO.3 Shown sequence and NOS terminator composition.

5. the preparation method of plant expression vector described in claim 4, it is characterised in that comprise the steps：By SEQ ID Sequence shown in NO.1 is passed throughAsc WithSwa The pFGC5941M plasmids through same enzyme action are connected into after double digestion, obtain pFGC5941CEPE Plasmid, then the sequence shown in SEQ ID NO.2 is connected into PAtAP3 promoteres and the intervening sequence of pFGC5941CEPE carriers Between, intermediate carrier pFGC5941CEPE-B2RNAia is formed, then sequence shown in SEQ ID NO.2 is reversely connected into intermediate carrier Between the intervening sequence of pFGC5941CEPE-B2RNAia and OCS terminators, pFGC5941CEPE-B2RNAi carriers are obtained, finally Sequence shown in SEQ ID NO.3 is passed throughNco WithAsc Restriction enzyme site is connected into the pFGC5941CEPE- through same enzyme action B2RNAi carriers, must disturb beta cyclase gene in brassica plant petalLCYB, ε-cyclase geneLCYEGene expression is same When overexpressionGL3The plant expression vector of gene.

6. preparation method according to claim 5, it is characterised in that：The intervening sequence is cabbage type rapePAP2Gene Intron 2.

7. the method for preparing the brassica plant kind that petal takes on a red color using plant expression vector described in claim 4, its are special Levy and be, comprise the steps：

A. by plant expression vector conversion Agrobacterium described in claim 4, engineering bacteria is obtained；

B., step a gained engineering bacteria is converted the hypocotyls of brassica plant aseptic seedling, after co-cultivation, anti-in Basta herbicides Property under induction differentiation obtain regrowth, the positive Seedling transgenic seedling of screening obtains the brassica plant kind that petal takes on a red color.