CN104404080B - Overexpression EGL3 is preparing the application that petal takes on a red color in brassica plant simultaneously for interference LCYB, LCYE expression - Google Patents

Abstract

The invention discloses interference LCYB, LCYE expression, overexpression EGL3 is preparing the application that petal takes on a red color in brassica plant simultaneously, interference LCYB, LCYE gene expression is also disclosed while the expression vector and the preparation method of expression vector of overexpression EGL3 genes, by disturbing LCYB, LCYE gene expression inhibition yellow carotenoid and accumulating red tomatoes red pigment, and overexpression EGL3 genes can promote anthocyanin to accumulate, so that petal takes on a red color, the brassica plant that petal shows red is successfully prepared, the pattern of brassica plant is enriched.

Description

Overexpression EGL3 takes on a red color rue preparing petal simultaneously for interference LCYB, LCYE expression Application in a kind of sedge platymiscium

Technical field

The invention belongs to genetic engineering field, and in particular to interference LCYB, LCYE expression is while overexpression EGL3 genes Preparing the application that petal takes on a red color in brassica plant.

Background technology

Rape is important oil crops, and cultivation history is long, economic worth is high, widely used, strong adaptability, is China The first big oil crops, be also global important oil crops.In species taxonomy, rape by Brassica genus three species 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 rape be by Chinese cabbage (Brassica rapa) and wild cabbage (Brassica oleracea, 2n=18, CC) by double diplodization after natural interspecific hybridization 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 tamed earliest, the Chinese cabbage for originating in NORTHWEST CHINA area develops, and has 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 growing day by day, the tourism of not only traditional famous mountains and great rivers formula is more and more fiery, and the development of rural area recreational tourism industry is also rapidly. The ocean of golden yellow dyeing defect is formed in the production of rape 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.Formed tens well-known rape flowers in China and viewed and admired area, rape has been when the flowers are in blossom, it is 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 beyond milky, the petal of brassica plant is commonly Yellow series, and business rape variety is full yellow petal, excessively singly Adjust.With the fast development of rape pattern villatic zoology sightseeing industry, in the urgent need to colourful pattern proterties, it is allowed to may be used also To output red, blueness etc., outside the oil yield and other conventional uses for not influenceing rape vegetable seed, increase its value of admiring the beauty of flowers, Boosting Ecological sightseeing is traveled.Additionally, the selected marker proterties that specific pattern or rapeseed breeding circle are thirsted for, and pattern is non- Yellowing additionally aids the insect pests such as reduction nitidulid.Accordingly, it would be desirable to parse the molecule mechanism of rape pattern proterties, and carry out new flower The molecular breeding of color.

Radish (Raphanus sativus) belongs to Rhaphanus, and molecular Evidence in recent years shows radish and Chinese cabbage, wild cabbage, sweet The distance of blue type rape even much smaller than black mustard and their distance, means that radish is small with the distance of Brassica genus core species Inter-species distance inside Brassica genus.But then, radish 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 (such as sinapsis alba category).This is a strange phenomenon, therefore carries out Brassica genus and radish pattern proterties Comparative studies, the need for being not only these biological pattern proterties basic research, or the brassica plant pattern such as rape The genetic improvement of shape provides guidance.

The pattern molecule mechanism and molecular breeding of important decorative flower have obtained remarkable break-throughs, to study other flower colors Mechanism and development molecular breeding provide important references.Pattern is main by flavonoids (flavonoids) and carotenoid (carotenoids) two major class pigments are determined.

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

Flavonoid biosynthesis pathway in the plants such as petunia, corn, toad's-mouth, arabidopsis is fully parsed.It It is an important branch approach in common body propane biosynthesis pathway downstream, phenylpropyl alcohol alkane approach is synthesized by other branch's approach Various secondary substances such as lignin, stilbene class, cumarin, protective plant protecting agent.The first step of flavonoid path is urged by looking into ketone synthase (CHS) Change 1 molecule p- coumaric acyl-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 (flavonoids 3 '-hydroxylase), the H of F3 ' 5 ' (3 ', 5 '-hydroxylase of flavonoids), DFR/FNR (dihydroflavonol 4-reductase/ Flavanones 4- reductases), ANS (anthocyanidin synzyme) etc..The anthocyanidin determined by the H of F3 ' H and F3 ' 5 ' (anthocyanidins) the hydroxyl on B- rings is more, then color gets over deflection blueness.Under normal circumstances, chalcone and anthocyanidin Be further embellished, such as glycosylation (glycosyl transferase, GT), methylate (transmethylase, MT), be acylated (acyltransferase, AT), then transport (glutathione S-transferase, GST) to storage in vacuole.In the vacuole of the orfe showy flowers of herbaceous plants, chalcone 4 '-O- glucosides are transformed into the aureusidin (6-O- glucosides) of glassy yellow by aureusidin synzyme (AS).Gorgeous paulownia grass In orange to red flower Deng plant, flavanones reacts by several steps and synthesizes 3- deoxidation anthocyanin.Additionally, flavones and flavones Alcohol also plays certain contribution to pattern, and they are colourless or light yellow, can act on promoting blue anthocyanin by so-called color altogether Formed and stablized.Flavones and flavonols are respectively with flavanones and flavanonol as substrate, in flavone synthase (FNS) and flavones Synthesize under the catalysis of alcohol synthase (FLS).In addition to anthocyanidin, other flavonoid substances such as flavonols, chalcone are likely to be related to The modification such as glycosylation.

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 What the transcription factor complex that WD40 repeats (WDR has TTG1 etc.) formation was regulated and controled, the ternary complex has regulated and controled decorative flower Organ, group are received in the process of anthocyanin coloring in the petal of toad's-mouth, petunia, morning glory, African Chrysanthemum and flower of Radix Gentianae, regulation and control behavior The external signal such as internal signal and light, ultraviolet such as knitting is influenceed.The research of arabidopsis shows that the flavonols (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 There is the substantially division of labor.

Carotenoid (carotenoids) is fat-soluble red, orange and xanthein, is entrenched in chloroplaset and has In the film of colour solid.Carotenoid is C40- tetraterpenes compounds, by C5- isoprene basis unit synthesize, plant, very Bacterium, algae and bacterium can synthesize, though animal can not synthesize, can be taken in from food and with before imitating element and vitamin A Body thing.Carotenoid contribute to yellow colour system for many patterns, and individually or together with anthocyanin for rose, chrysanthemum 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 compound is formed between the relevant enzyme of the approach and is combined In on plastid film, 4 molecule IPP condensations are the GGPP (GGPP) of 1 molecule C20, are closed in phytoene 2 molecule GGPP are coupled to the colourless phytoene of C40 head to head under the catalysis of enzyme (PSY), it be first class recklessly Radish element.Then, phytoene desaturase (PDS) and sigma carotene desaturase (ZDS) in molecule to being sequentially introduced 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 alltrans 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.Except half balling Outside the plants such as lettuce, LCYE adds ε-ring can only to lycopene in most plants, synthesizing yellow containing 1 β-ring and 1 alpha-carotene of ε-ring and its derivative.β-and alpha-carotene can occur further hydroxylation or epoxidation modification, produce Many new constructions.The oxygenation product of carrotene is referred to as xanthophyll, and the dihydroxylation process of β-ring and ε-ring is respectively by β-ring hydroxylase (CHYB) completed with ε-ring hydroxylase (CHYE).There is flower Idiotype and fruit differential type in PSY, GGPS and LCYB, display is present The special carotenogenesis approach of one chromoplast.Luteole epoxidase (ZEP) catalysis luteole occur C5,6 and C5 ', the epoxidation of 6 ' positions forms the antheraxanthin and 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- it is cis-neoxathin can be additionally used in synthesis 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) participate in important regulative from the angle decomposed.Additionally, VDE (Analysis of Violaxanthin De-Epoxidase) catalysis violaxanthin and flower Medicine Huang matter is converted into zeaxanthin.Arabidopsis AtRAP2.2 has weak facilitation to the carotenoid accumulation in particular organization, But recent research indicate that its function is mainly the resist oxygen lack survival ability of organization of regulation control.Tomato DDB1 and DET1 is by anti-to light The negative regulation of induction signal approach and suppress carotenoid approach, and wild cabbage OR (Orange) and tomato HSP21 be then by regulation and control The formation of chromoplast and promote the accumulation of carotenoid.

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

Because 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 planted has important application prospect.But in including the whole Brassica genus including rape, have no and utilize molecular breeding Metabolic engineering rape pattern report.

The 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 EGL3 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 Enzyme gene LCYE expression is while the plant expression vector of overexpression EGL3 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 to provide to prepare petal using above-mentioned plant expression vector and be in The method of red brassica plant kind.

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

1st, beta cyclase gene LCYB, ε-cyclase gene LCYE express excess table simultaneously in interference brassica plant petal Up to application of the EGL3 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 EGL3 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 arabidopsis petal specific promoter PAtAP3 mediate express, the nucleotides sequence of the petal specific promoter PAtAP3 Row are as shown in SEQ ID NO.1 the 267th to the 1044th.

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

2nd, beta cyclase gene LCYB, ε-cyclase gene LCYE express excess table simultaneously in interference brassica plant petal Up to the plant expression vector of EGL3 genes, the plant vector includes the expression beta cyclase base mediated by petal specific promoter Because of the expression cassette of the RNA interference sequences of LCYB and ε-cyclase gene LCYE and by constitutive promoter mediation overexpression EGL3 The expression cassette of gene.

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

It is furthermore preferred that the expression cassette by constitutive promoter mediation overexpression EGL3 genes is successively by CaMV35S Promoter, sequence shown in SEQ ID NO.3 and NOS terminator are constituted.

3rd, the preparation method of the plant expression vector, comprises the following steps：By sequence shown in SEQ ID NO.1 through AscI Be connected into after SwaI double digestions through the pFGC5941M plasmids of same digestion, pFGC5941CEPE plasmids are obtained, then by SEQ ID Sequence shown in NO.2 is connected between the PAtAP3 promoters 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 and OCS terminators of B2RNAia, pFGC5941CEPE-B2RNAi carriers are obtained, finally by SEQ ID NO.3 Shown sequence is connected into through the pFGC5941CEPE-B2RNAi carriers of same digestion by NcoI and AscI restriction enzyme sites, must be disturbed Beta cyclase gene LCYB, ε-cyclase gene LCYE expression are while the plant of overexpression EGL3 genes in brassica plant petal Thing expression vector.

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

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

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

B., step a gained engineering bacterias are converted the hypocotyl of brassica plant aseptic seedling, after co-cultivation, in Basta weedings Differentiation is induced to obtain regrowth under agent resistance, 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 are：It is bright the invention provides the Brassica genus core species such as rape and radish pattern mechanism Really chrysanthemum, spend in vain, the key differences expressing gene site between safflower, 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 clones 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 is carried Body, creates the new type resource material of red rape flower phenotype after conversion rape, the technical tactic is the Brassica genus pattern such as rape It is pioneering in genetic engineering, it is also that the pioneering of metabolic engineering modified plant pattern is combined by carotenoid and flavonoid path.

Brief 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 radish petal：Cabbage type rape (BnY)；B：Cabbage type rape (BnW)；C：Mustard type rape (BjY)；D：Turnip type rape (BrY)；E：Wild cabbage (BoY)；F：Cabbage mustard (BoW)；G：Radish (RsR))。

Fig. 2 is that (A is DFR in Brassica genus to the differential expression of DFR, TT8, GL3, EGL3 between Brassica genus petal and radish petal Differential expression between petal and radish petal；B is differential expressions of the TT8 between Brassica genus petal and radish petal；C is GL3 in rue A kind of sedge belongs to the differential expression between petal and radish petal；D is differential expressions of the EGL3 between Brassica genus petal and radish petal；BnYPe： Cabbage type rape chrysanthemum valve；BnWPe：Cabbage type rape spends valve in vain；BjYPe：Mustard type rape chrysanthemum valve；BrYPe：Chinese cabbage type oil Dish chrysanthemum valve；BoYPe：Collard chrysanthemum valve；BoWPe：Cabbage mustard spends valve in vain；RsRPe：The pale reddish brown valve of radish red).

Fig. 3 is the differential expression (BnYPe of OR, CCD1 between Brassica genus petal and radish petal：Cabbage type rape chrysanthemum valve； BnWPe：Cabbage type rape spends valve in vain；BjYPe：Mustard type rape chrysanthemum valve；BrYPe：Turnip type rape chrysanthemum valve；BoYPe：Plumage Clothing wild cabbage chrysanthemum valve；BoWPe：Cabbage mustard spends valve in vain；RsRPe：The pale reddish brown valve of radish red).

Fig. 4 is NOS-PAtAP3, B2RNAi and BnEGL3-1PCR amplification (A：NOS-PAtAP3；B：B2RNAi；C： BnEGL3-1)。

Digestion result (A during Fig. 5 pFGC5941CEPE and pBLycoRF6 vector constructions：AscI and SwaI double digestions pFGC5941M；B：AscI and SwaI double digestions pMD19-T-NOS-PAtAP3；C：SwaI and AatI double digestions pFGC5941CEPE；D：SwaI and AatII double digestions pMD19-T-B2RNAi；E：BamHI and XbaI double digestions pMD19-T- B2RNAi；F：BamHI and XbaI double digestions pFGC5941CEPE-B2RNAia；G：NcoI and AscI double digestions pFGC5941CEPE-B2RNAi；H：NcoI and AscI double digestion pMD19-T-BnEGL3-1).

Fig. 6 is the petal color (A that pBLycoRF6 converts rapeseed plants：Control group；B：Transgene rape).

Specific embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific 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 used：Cabbage type rape chrysanthemum valve and development mid-term seed (in double No. 9 product Kind), cabbage type rape breast petal, turnip type rape chrysanthemum valve (6Y733 strains), mustard type rape chrysanthemum valve (CNG12011 product System), collard (B.oleracea var.acephala ftricolor, K10-3 strain) chrysanthemum valve, cabbage mustard mutation spend in vain Valve (R9057 strains), radish red petal are taken from Chongqing City of Southwest University rape Engineering Technical Research Centre and have a rest the routine in horse base Experiment plant.Arabidopsis (Arabidopsis thaliana, Columbia wild type) seed is purchased from international arabidopsis center, plants Plant in indoors artificial climatic chamber.

Reagent and kit that the embodiment of the present invention is used：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 polymerase, DNase I (RNase-free) and buffer, RNase Inhibitor, DL-2000 and λ-HindIII DNA Marker are purchased from the precious biology limited public affairs of (TaKaRa) bioengineering in Dalian Department；RNAprep Pure plant total RNA extraction reagents box is TIANGEN Biotech's product；Glue reclaim reagent Box, a small amount of method plasmid extraction kit are purchased from Shanghai Hua Shun Bioisystech Co., Ltd；Restriction 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), rifampin (Rif), streptomysin (Str), kanamycins (Kan), ampicillin (Amp), agarose, Tris, CTAB, Tris saturated phenol (pH=8.0), other biochemistry and molecular biology reagents such as Tryptone, Yeast Extract, X-gal, IPTG, CTAB purchase From Shanghai Sangon Biological Engineering Technology And Service Co., Ltd；Plant hormone sows the companies such as rich garden supplies purchased from Shanghai.

The key instrument that the embodiment of the present invention is used：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 radish petal colour developing subcellular organelle

Fresh cabbage type rape chrysanthemum valve (BnY) is taken respectively；Cabbage type rape breast petal (BnW)；Mustard type rape Chrysanthemum valve (BjY)；Turnip type rape chrysanthemum valve (BrY)；Collard chrysanthemum valve (BoY)；Cabbage mustard spends valve (BoW) in vain；Radish safflower Valve (RsR), is placed on the cold fresh preservation of ice face and transports laboratory back, and then free-hand section, with low power sem observation rapid screening, selects qualified Section examined with photograph, as a result as shown in Figure 1.Result shows, displaing yellow in the chrysanthemum valve of the several species of Brassica genus Material numerous fine granularities are rendered as in a cell, be non-uniformly distributed in intracellular, be expressed to by vacuole adherent Position, phaeochrome cell device is chromoplast in illustrating yellow petal, and its pigment is yellow carotenoid.Cabbage type rape breast petal In also there are some cells to possess light yellow chromoplast, but total number is few and iuntercellular is inconsistent, and cabbage mustard is almost seen in spending valve in vain Less than chromoplast, explanation is that the reduction or disappearance of chromoplast cause its yellow to shoal or disappear.Aubergine pigment is equal in radish petal The even middle position for being distributed in each cell, center is denseer, and more offset from center is lighter, illustrates that the colour developing of radish 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 chrysanthemum valve (BnY) is taken respectively in full-bloom stage morning；Cabbage type rape breast petal (BnW)；Mustard type rape chrysanthemum valve (BjY)；Turnip type rape chrysanthemum valve (BrY)；Collard chrysanthemum valve (BoY)；Cabbage mustard is white Petal (BoW)；Radish red petal (RsR), it is shady and cool immediately it is fresh-keeping transport laboratory back, 60 mesh are crossed in 50~60 DEG C of drying after smashing Sieve, lucifuge kept dry.

1st, petroleum ether, hydrochloric acid and ammoniacal liquor 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, Each about 10mL of 10% hydrochloric acid, 30% ammoniacal liquor, gently mixes, and 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 radish content is high；Collard shows light yellow, illustrates that it 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 to be free of anthocyanidin containing flavones (alcohol).Cabbage mustard The performance of white and collard is near colourless, illustrates not containing anthocyanidin, and flavones (alcohol) is also little.

(3) ammoniacal liquor test：Each material of Brassica genus shows different degrees of yellow, illustrates that it contains more or less Huang Ketone (alcohol), and the yellow green that radish flower shows, the color are that the yellow that the blue and flavonoids presented by anthocyanin is presented mixes Form, but all material does not show orange red or red, illustrates without aurones.

2nd, the chromogenic reaction of flavonoids

Gone bail for the petal powder 0.100g for depositing, and 24h is extracted with methyl alcohol, and filtering is settled to 50mL, respectively takes 2mL extract solutions, 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, leaf mustard Type rape shows pole lavender blush and micro- purplish red, illustrate to be free of chalcone, aurones and catechin, may contain flavones, flavonols, two Hydrogen flavonols, flavanone；Radish flower shows pink, illustrates to contain 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 shaken up, stand 1h.As a result It has been shown that, radish flower is presented pink, illustrates to contain anthocyanin.Remaining colourless or yellow, illustrates without anthocyanin.

(3) lead acetate reaction：Plus 1.0% lead acetate 2mL, gently shake up, stand 2h.Result shows, all Brassica genus materials There is different degrees of yellow mercury oxide in material, illustrates that flavonoids possesses phenolic hydroxyl group and without chalcone 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 to contain flower Blue or green element glycosides.

(4) ferric chloride reaction：Plus 5.0% ferric trichloride 2ml, gently shake up.Result shows that Huang all occurs in all material Color, illustrates in pigment molecular not phenolic hydroxy group.

(5) alchlor reaction：Plus 1.0% alchlor methanol solution l ml.Result shows that all material is all presented 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 Different degrees of yellow is presented, (alcohol) containing flavones is illustrated, 5min colors do not change in boiling water, illustrate without chalcone, aurones, can Flavanone, possible isoflavone-containing and isoflavanone can be free of.Radish flower appearance is orange-yellow, illustrates to contain anthocyanidin.

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

(8) alkaline reagent reaction：Plus 5%Na₂CO₃3ml, gently shakes up, closed standing 30min, blowing air 10min.It is all Material is presented the different yellow of degree, and color is constant after blowing air, illustrates without flavanonol.

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

(10) acid reaction：Plus 1.0% boric acid 10 drip, then add 2.0%H₃BO₃3ml, cabbage mustard spends valve in vain and shows colourless, says Bright cabbage mustard spends valve flavonoids in vain and may be free of 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, use volume fraction for 90% acetone:Ethanol (4:L, V/V) 24h is extracted, filtering is settled to 25ml, using ultraviolet-visible spectrophotometer 200 Scanned in the range of~700nm.Result shows that all samples, without absworption peak, illustrate 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, filtering is settled to 25ml, using ultraviolet-visible spectrophotometer in the range of 200~700nm Scanning.Result shows that cabbage type rape, turnip type rape, mustard type rape, collard have suction in 440 and 470nm or so Receive peak, illustrate containing carotenoid, the content of quantitative analysis measure is respectively 6.824,6.712,5.548,1.248mg/g.And Cabbage mustard and radish petal then without characteristic absorption peak, illustrate not containing carotenoid.

(3) flavonoids：The petal 0.100g of preservation is weighed, rapidly with liquid nitrogen grinding to powder, acidified methyl alcohol (pH is added =3) 2ml be placed in 4 DEG C of refrigerators and extract 24h, filter, 25ml is settled to, with ultraviolet-visible spectrophotometer in 220~600nm In the range of scan.Result shows, cabbage type rape, turnip type rape, mustard type rape, collard, cabbage mustard, radish petal Extract solution has absworption peak in 330 and 270nm, illustrates that they contain flavonoids, the content point that quantitative analysis is determined 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, adds methyl alcohol to extract 24h, filtering, is settled to 50ml, is scanned in the range of 200~700nm with ultraviolet-visible spectrophotometer.Result shows, radish 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 analysis is determined is 237.27mg/100g.All Brassica genus sample standard deviations without Anthocyanin absworption peak, i.e., without anthocyanin.

The Molecular Identification of embodiment 3, Brassica genus and radish flower petal pigment biosynthesis pathway

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

For the molecule mechanism of difference occur in research Brassica genus and radish flower petal pigment, Brassica genus and radish flavonoid path are designed The RT-PCR detection primers of gene, and using 5SrRNA as internal reference, it is specific as shown in table 1：

Table 1, flavonoid path RT-PCR detection primers

Firm open cabbage type rape chrysanthemum valve (BnY) is taken respectively in full-bloom stage morning；Cabbage type rape breast petal (BnW)；Mustard type rape chrysanthemum valve (BjY)；Turnip type rape chrysanthemum valve (BrY)；Collard chrysanthemum valve (BoY)；Cabbage mustard is white Petal (BoW)；Radish red petal (RsR), liquid nitrogen frozen transport, -80 DEG C of Refrigerator stores are standby.Then planted with RNAprep Pure Thing total RNA extraction reagent box extracted total RNA, through electrophoretic analysis and spectrophotometry it is 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) kit is further removed genomic DNA (gDNA) and is carried out reverse transcription, obtains the chains of total cDNA first.Use CHS genes A pair of conserved region primers of family enter performing PCR amplification, target area span introne, and electrophoresis showed all samples only expand big with prediction Small consistent cDNA bands are further detected, the amplification between each sample without gDNA bands with internal standard gene 25SrRNA Band is special and luminance difference less, illustrate the gDNA thoroughly eliminated in total serum IgE before reverse transcription, 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 use Premix Ex Taq^TMII (Tli RNaseH Plus) kit is in CFX96^TM Real- Fluorescence real-time quantitative PCR is carried out on Time System, the method according to specification performs PCR programs.Reaction system is 20 μ L bodies It is that quantitative RT-PCR contains the μ L of reverse transcription product 0.1, SYBR Premix Ex Taq^TMThe μ L of II (2 ×) 10, each primer (10 μM) 0.4 μ L, remaining volume ddH₂O polishings.PCR cycle parameter is：95 DEG C of predegeneration 3min；50 amplification cycles (95 DEG C of denaturation 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 (biology repetition), each reverse transcription is repeated 2 times PCR (detection is repeated), 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.

Result shows, CHS, CHI, F3H, TTG1, PAP gene family is vigorous in all material or moderate table 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 without significantly expression or Expression is extremely low, but expression quantity is very high in radish.Illustrate flavonols synthesis be in Brassica genus and radish petal it is vigorous Or more vigorous working condition, the synthesis of anthocyanin only in radish petal vigorous work but in Brassica genus petal in closing Closed state.

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

For further the molecule mechanism of difference occur in research Brassica genus and radish flower petal pigment, design Brassica genus and radish kind are recklessly Radish element 17 RT-PCR primers of functional site gene family of approach, and using 25SrRNA as internal reference, the specific primer such as institute of table 2 Show.

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

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.

Result shows that all detection gene locis of carotenoid approach are expressed in 7 petals of species material, But there is also some differences, one be Brassica genus it is white/newborn petal in CCD1 expressions it is substantially higher than in chrysanthemum valve, two is radish flower , in closed mode without expression, OR expressions are also without in tetraploid aggregate species in Brassica genus dliploid elementary species for OR in valve It is high.The missing of carotenoid in radish petal is illustrated because OR is closed and can not be formed with colour solid, Brassica genus are white/newborn petal in The reduction of carotenoid is because the decomposition of carotenoid is too strong.

The structure of embodiment 4, plant expression platform carrier pFGC5941CEPE

According to gene expression difference on Brassica genus and radish petal carotenoid and flavonoids route of synthesis, clone is poor for design The primer of different expressing gene and construction of expression vector, specific primer is as shown in table 3.

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

(1) acquisition of NOS-PAtAP3 fragments

With pCAMBIA2301 plasmids as pcr template, combine FTNOS and RTNOS with primer and expand and reclaim the piece of 286bp Section, obtains the fragment containing NOS terminator, and its nucleotide sequence is as shown in SEQ ID No.1 1-286.Extracted using CTAB methods The genome DNA of Arabidopsis leaf is pcr template, and combining FPAtAP3 and RPAtAP3 with primer expands and reclaim 778bp's The PAtAP3 of specific promoter containing petal fragments (GenBank accession number:U30729), its nucleotide sequence such as SEQ Shown in ID No.1 267-1044.Then as pcr template after two kinds of recovery products are mixed, with primer combine FTNOS and RPAtAP3 is expanded, and it is the fusion fragment NOS-PAtAP3 of 1044bp to obtain clip size, 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, the recombinant vector that will be obtained converts Escherichia coli DH5 α competent cells, the transformant monoclonal to Amp resistance LB plate screenings is entered using primer combination FTNOS and RPAtAP3 Performing PCR detects that sequencing result shows, fusion fragment NOS-PAtAP3 sequences are as shown in SEQ ID No.1.

(2) structure of pFGC5941CEPE

Extracting pMD19-T-NOS-PAtAP3 recombinant plasmids, then carry out double digestion with AscI and SwaI, reclaim NOS- PAtAP3 fragments, as shown in B in Fig. 5；PFGC5941M plasmids are extracted simultaneously, and carrier bone is reclaimed after AscI and SwaI double digestions Frame, as shown in A in Fig. 5.11241bp is obtained after NOS-PAtAP3 fragments and pFGC5941M plasmids are connected is suitable for pattern The plant expression vector pFGC5941CEPE of genetic engineering, bacillus coli DH 5 alpha competent cell is converted by pFGC5941CEPE, Coat and screen monoclonal transformant on the LB flat boards that concentration containing Kan is 50mg/L, the monoclonal transformant for obtaining will be screened and used Primer combines FTNOS and RPAtAP3 and F35S3N and ROCST5N and enters performing PCR detection, and sequence is chosen in screening positive clone sequencing Arrange the clone without mutation standby.It is interval containing Bar gene tables in T-DNA in its pFGC5941CEPE recombinant plasmid for obtaining Up to box (providing the resistance to herbicide basta) and 2 expression cassettes that can be used to insert foreign gene, exogenous gene expression box 1 CaMV35S containing composition promoter and NOS terminator, can be used for the justice of genes of interest or antisense expression；Exogenous gene expression The PAtAP3 of specific promoter containing petal of box 2, introns BnPAP2I2 (cabbage type rape (Brassica napus) PAP2 genes 2 intrones) and OCS PolyA terminators, can be used for RNA interference, justice or the antisense expression of genes of interest.

(3) clone of series connection divalence RNA interference fragments B2RNAi

The total chains of cDNA first of cabbage type rape petal for using above-mentioned reverse transcription to obtain are template, are combined using primer (387bp is the RNA interference fragments of FBLCYBi and RBLCYBi amplification LCYB gene families after enzyme-added enzyme site and joint 411bp), the RNA interference fragments (480bp, after adjunction head that FBLCYEi and RBLCYEI expands LCYE gene families is combined with primer It is 506bp), it is separately recovered.Then, as masterplate, FBLCYBi will be combined with primer after 2 kinds of recovery product mixed in equal amounts 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 and detects 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 adds AatII), reclaim B2RNAi fragments, as shown in D in Fig. 5；PFGC5941CEPE plasmids are extracted simultaneously, after SwaI and AatII double digestions, are reclaimed and are carried 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 By 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 clone molecules 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.PFGC5941CEPE-B2RNAia plasmids are extracted simultaneously, with BamHI and XbaI double digestions, are reclaimed 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 clone molecule sequencing chooses clone of the sequence without mutation standby.

(5) clone of the cDNA code areas of cabbage type rape EGL3-1 (BnEGL3-1) gene

We show that cabbage type rape EGL3-1 (BnEGL3-1) gene is mainly expressed in kind of skin, is swashed in the research of the past The synthesis of flavonoids-OPC living, also expresses in the cauline leaf that aging turns red, activates the synthesis of flavonoids-anthocyanin.Cause This, the present invention clones the cDNA code areas of BnEGL3-1 from the seed of cabbage type rape development, is subsequently used for building just load Body simultaneously converts the rape overexpression in petal later.The total serum IgE that cabbage type rape develops mid-term seed is extracted, such as preceding method is gone Reverse transcription obtains the first chain cDNA after gDNA, is then template with the first chain cDNA, using FBnEGL3-1 and RBnEGL3-1 primers Combination amplification BnEGL3-1 genes, obtain the code area (being 1831bp after enzyme-added enzyme site) of BnEGL3-1 genes 1821bp, electricity Swimming result, by its glue reclaim, pMD19-T-BnEGL3-1 is connected into pMD19-T as shown in C in 4, converts bacillus coli DH 5 alpha Competent cell, the transformant monoclonal to Amp resistance LB plate screenings combines FBnEGL3-1 and RBnEGL3-1 using primer Enter performing PCR detection, send multiple positive clone molecules to be sequenced, sequencing result shows that BnEGL3-1 sequences as shown in SEQ ID No.3, are selected Take clone of the sequence without mutation standby.

(6) plant expression vector pBLycoRF6 is built

Extracting pMD19-T-BnEGL3-1 plasmids, with NcoI and AscI double digestions (AscI first cuts, then adds NcoI), reclaim BnEGL3-1 genetic fragments, as shown in H in Fig. 5；PFGC5941CEPE-B2RNAi plasmids are extracted simultaneously, it is double with NcoI and AscI Digestion, reclaims carrier framework, as shown in G in Fig. 5.The BnEGL3-1 genetic fragments and pFGC5941CEPE-B2RNAi that will be reclaimed Carrier framework is connected, and forms the trivalent plant expression vector of series connection divalence RNAi and monovalence overexpression, is named as PFGC5941CEPE-B2RNAi-BnEGL3-1ox, referred to as pBLycoRF6.Then pBLycoRF6 is converted into bacillus coli DH 5 α competent cells, to 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 RBnEGL3-1, FBnEGL3-1 and RTNOS 6 combines whether carrier respectively successfully constructs to primer, is turned using freeze-thaw method after positive clone molecule then is extracted into plasmid Change agrobacterium tumefaciens lba4404, then with the monoclonal transformant of the resistance LB plate screenings containing Kan, Str and Rif, and use Stating 6 pairs of primer combinations carries out PCR detections, and screening positive clone is preserved as engineered strain, for Plant Transformation.

Embodiment 5, pBLycoRF6 converts rape

All tissue cultures operations are carried out under the conditions of the Plant Tissue Breeding 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.Double No. 10 seeds with sterilized water after 75% ethanol surface sterilization 1min with being rushed in cabbage type rape typical case's chrysanthemum kind Wash 3 times, then soak 20min with 5% sodium hypochlorite, (MS powder in MS solid mediums is inoculated in 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 as liquid Culture medium), cultivated in 25 DEG C, 2000Lux illumination, 16h/d photoperiods (between tissue culture below condition of culture except especially indicate person Outward, it is identical with this).The hypocotyl for cutting seedling age 8d or so aseptic seedling is cut into the segment for being about 0.5~1.0cm, is inoculated into pre- (MS culture medium+1.0mg/L 6-BA+1.0mg/L 2,4-D) preculture 3d on training culture medium MSp.

By -80 DEG C of LBA4404 engineered strains containing pBLycoRF6 of preservation added with 100mg/L Kan, 20mg/L In 28 DEG C, 250r/min 1~2d of shaken cultivation in the LB fluid nutrient 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) adjust bacterial concentration to OD₆₀₀About 0.5 or so, i.e., It is dip dyeing liquid for shell.

By 5~10min in the hypocotyl section immersion dip dyeing liquid for shell after preculture, intermittence is gently swayed, then by hypocotyl 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/ LNAA in), in 23.5 DEG C of light culture 48h.After light culture with sterilizing liquid culture medium MSk (MS fluid nutrient 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 and culturing 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, every 2 weeks subcultures 1 time, to growing macroscopic kanamycin-resistant callus tissue, then It is transferred to differential 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, evoked callus differentiate budlet, then are 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) is cultivated extremely Small 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 culture is to the complete unrooted seedling of length 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, is transplanted to and contains sterilizing perlite, vermiculite, turf earth mixtures (mass ratio is 1:1:1) in basin alms bowl, it is managed by greenhouse pot culture.

Finally, 16 plants of regeneration plants are obtained after double No. 10 in pBLycoRF6 conversions cabbage type rape；To regeneration plant blade Be added dropwise 200mg/L Basta solution detection resistance, and extract blade genome DNA be respectively adopted primer combination F35S3N and RBnEGL3-1, FBLCYBi and ROCST5N enter performing PCR detection, as a result show to obtain 9 plants of double positive transgenic plant.

Comprehensive biology and agronomy observation are carried out to transgenic positive plant, as a result as shown in Figure 6.Result shows, obtains The transfer-gen plant for obtaining grows and substantially do not change with background proterties, but petal becomes red, and petal cell is micro- Observation shows that red material is existing from chromoplast, also has from vacuole, and biochemical composition detection also indicates that new generation The existing lycopene of red material, also have anthocyanin.Show that xanthein and product can be suppressed by the method for the present invention Tired lycopene and anthocyanin can create red rape flower.

Self propagated is carried out to transgenosis present age plant, Basta is added dropwise using the blade as transgenosis present age plant Resistance detecting, Screening and Identification goes out the excellent strain of transgene rape homozygosis, and its petal color is redder than transgenosis 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 used 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 it in the form and details, limited without departing from appended claims Fixed the spirit and scope of the present invention.Here especially statement, the following change on application form also all necessarily belongs to of the invention Spirit and scope are covered：

1st, EGL3-1, LCYB, LCYE gene/gene fragment in the present invention, except nucleotides sequence listed in sequence table Beyond row, also including coming from parent species Chinese cabbage or wild cabbage in corresponding gene order, also including coming from these species in The sequence of same functional site gene family other members, although they may have small with nucleotide sequence listed in sequence table Difference.

2nd, the present invention in gene and its fragment, in addition to nucleotide sequence listed in sequence table, also including with it Have any nucleotide sequence of more than 98.00% uniformity in continuous 80bp and the above.

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

4th, to the suppression of LCYB, LCYE site expression in the present invention, except the RNA perturbation techniques lifted in preferred embodiment In addition, can also be same or similar to reach using technologies such as antisense RNA, genome editors (ZFN, TALEN, CRISPR-Cas) Purpose.

5th, the gene and its fragment in the present invention, except the transformation of the use pFGC5941 as being lifted in preferred embodiments is carried Beyond body is built, plant expression vector construction can also be carried out using other carriers, including using other promoters and Terminator reaches same or analogous purpose；Vector construct in the present invention, except as adopting for being lifted in preferred embodiments The improvement leaf disk method mediated with Agrobacterium tumefaciens strain LBA4404 is carried out beyond 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 rape, other sibling specieses that can also be applied to its parent species Chinese cabbage, wild cabbage and Brassica genus are identical to reach Or similar purpose.

Claims (6)

1. beta cyclase gene in brassica plant petal is disturbedLCYB, ε-cyclase geneLCYEExpression is while overexpressionEGL3Application of the gene in the brassica plant kind that petal takes on a red color is prepared；β-ring in the interference brassica plant petal Change enzyme geneLCYB, ε-cyclase geneLCYEThe sequence of expression is by SEQ ID NO.2, intervening sequence and SEQ ID NO.2 Reverse complementary sequence is constituted, the overexpressionEGL3The sequence of gene is as shown in SEQ ID NO.3；The brassica plant is Cabbage type rape（Brassica napus）.

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 arabidopsis petal specific promoterPAtAP3Mediation expression, the petal Specific promoterPAtAP3Nucleotide sequence as shown in SEQ ID NO.1 the 267th to the 1044th.

3. beta cyclase gene in brassica plant petal is disturbedLCYB, ε-cyclase geneLCYEExpression is while overexpressionEGL3The plant expression vector of gene, it is characterised in that：The plant vector includes the expression mediated by petal specific promoter Beta cyclase geneLCYB, ε-cyclase geneLCYEThe expression cassette of RNA interference sequences and by constitutive promoter mediate expressEGL3The expression cassette of gene；

The petal specific promoter mediation expression beta cyclase geneLCYB, ε-cyclase geneLCYERNA interference sequences Expression cassette sequence, intervening sequence, SEQ ID NO.2 institutes successively as shown in petal specific promoter PAtAP3, SEQ ID NO.2 Show reverse complementary sequence and OCS the terminators composition of sequence；

It is described that overexpression is mediated by constitutive promoterEGL3The expression cassette of gene is successively by CaMV35S promoters, SEQ ID Sequence shown in NO.3 and NOS terminator are constituted.

4. the preparation method of plant expression vector described in claim 3, it is characterised in that comprise the following steps：By SEQ ID Sequence shown in NO.1 is passed throughAsc WithSwa It is connected into after double digestion through the pFGC5941M plasmids of same digestion, obtains pFGC5941CEPE Plasmid, then the sequence shown in SEQ ID NO.2 is connected into the PAtAP3 promoters and 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 and OCS terminators of pFGC5941CEPE-B2RNAia, 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 digestion B2RNAi carriers, must disturb beta cyclase gene in brassica plant petalLCYB, ε-cyclase geneLCYEExpression is simultaneously super Amount expressionEGL3The plant expression vector of gene.

5. preparation method according to claim 4, it is characterised in that：The intervening sequence is cabbage type rapePAP2Gene Intron 2.

6. the method that the cabbage type rape variety that petal takes on a red color is prepared using plant expression vector described in claim 3, it is special Levy and be, comprise the following steps：

A. by plant expression vector conversion Agrobacterium obtained in preparation method described in claim 3, engineering bacteria is obtained；

B., step a gained engineering bacterias are converted the hypocotyl of cabbage type rape aseptic seedling, it is anti-in Basta herbicides after co-cultivation Property under induce differentiation to obtain regrowth, the positive seedling transgenic seedling of screening obtains the cabbage type rape variety that petal takes on a red color.