CN104480128B - Suppress xanthein accumulation and accumulate lycopene and anthocyanin simultaneously in the application for preparing petal and taking on a red color in brassica plant - Google Patents

Abstract

The invention discloses suppress xanthein accumulation to accumulate lycopene and anthocyanin simultaneously in the application for preparing petal and taking on a red color in brassica plant,Specially disturb LCYB,LCYE,MYB12 and MYB111 gene expressions while ectopic expression TT8 genes,Also disclose interference LCYB,LCYE,MYB12 and MYB111 gene expressions while the preparation method for preparing expression vector and carrier of ectopic expression TT8 genes,By disturbing LCYB,LCYE,MYB12 and MYB111 gene expression inhibition xanthein carotenoid and flavonols,And ectopic expression TT8 genes can promote red tomatoes red pigment and anthocyanin to accumulate,So that petal takes on a red color,It is successfully prepared the brassica plant that petal shows red,Enrich the pattern of brassica plant.

Description

Suppressing xanthein accumulation, accumulating lycopene and anthocyanin is preparing petal simultaneously The application to take on a red color in brassica plant

Technical field

The invention belongs to genetic engineering field, and in particular to suppress xanthein accumulation and accumulate lycopene and cyanine simultaneously Plain glycosides is in the application for preparing petal and taking on a red color in brassica plant.

Background technology

Rape is important oil crops, and cultivation history is long, economic value is high, widely used, strong adaptability, is China The first big oil crops, and global important oil crops.In species taxonomy, rape by Brassica genus three species Form, 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) evolved by double diplodization after natural interspecific hybridization and a kind of aggregate species come, although cultivation history only has the centuries, by Strong in its growth potential, yielding ability is high, the Yi Zhan worlds and China's 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 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 that its original origin and differentiation center, planting range spread all over The world.

With the sustainable development of economic level and the continuous lifting of social modernization's degree, the popular pursuit to life taste Also growing day by day, not only the tourism of 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.Tens well-known rape flowers have been formed in China and have viewed and admired area, rape is 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, the petal of cabbage mustard (B.oleracea var.alboglabra) is removed It is beyond milky, the petal of brassica plant is commonly Yellow series, and business rape variety is yellow petal entirely, excessively singly Adjust.With the fast development of rape pattern villatic zoology sightseeing industry, there is an urgent need to colourful pattern character, it is allowed to may be used also To output red, blueness etc., do not influenceing outside the oil yield of rape vegetable seed and other conventional uses, increasing its value of admiring the beauty of flowers, Boosting Ecological sightseeing is traveled.In addition, the selected marker character that specific pattern or rapeseed breeding circle are thirsted for, and pattern is non- Yellowing, which additionally aids, reduces the insect pests such as nitidulid.Therefore, it is necessary to parse the molecule mechanism of rape pattern character, and new flower is carried out 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 and the distance of Brassica genus core species are small 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 character Comparative studies, be not only the needs of these biological pattern character 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 mainly by flavonoids (flavonoids) and carotenoid (carotenoids) two major class pigments determine.

Flavonoids is the anthocyanidin most seen, contribution yellow, a series of orange, red colour systems to purple.Flavonoids is water Soluble substance, there is a C15 skeleton, 9 classes are 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), colour contribution maximum to plant in them is anthocyanin, flavonols, chalcone and aurones.Pattern Glycosides is maximum a kind of flavonoid substances, is flower or the tone such as other tissue contribution magentas, red, purple, blueness, 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 A variety of 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 using chalcone as substrate, through being hydroxylated, reducing, aoxidizing, 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 is more inclined to blueness.Under normal circumstances, chalcone and anthocyanidin Be further embellished, such as glycosylate (glycosyl transferase, GT), methylate (transmethylase, MT), be acylated (acyltransferase, AT), then transhipment (glutathione S-transferase, GST) is stored into 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.In addition, flavones and flavones Alcohol also plays certain contribution to pattern, and they can promote blue anthocyanin to be colourless or light yellow by the so-called effect of color altogether Formed and stably.Flavones and flavonols are using flavanones and flavanonol as substrate, in flavone synthase (FNS) and flavones respectively Synthesized under the catalysis of alcohol synthase (FLS).In addition to anthocyanidin, other flavonoid substances such as flavonols, chalcone may also 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, there is TT8, GL3, EGL3 etc.) and WD40 repeats what the transcription factor complex that (WDR, there is TTG1 etc.) is formed was regulated and controled, and the ternary complex has regulated and controled decorative flower Toad's-mouth, petunia, morning glory, African Chrysanthemum and flower of Radix Gentianae petal in the process that colours of anthocyanin, regulation and control behavior is by organ, group 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 The obvious division of labor occurs.

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 is that many patterns contribute to yellow colour system, 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 identified, the Isoprenoid for the C5 that whole approach originates 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 1 molecule C20 GGPP (GGPP), are closed in phytoene 2 molecule GGPP are coupled to C40 colourless phytoene head to head under the catalysis of enzyme (PSY), and it is first class Hu Radish element.Then, phytoene desaturase (PDS) and sigma carotene desaturase (ZDS) are sequentially introduced into molecule Conjugate double bonds, successively form colourless phytofluene, lurid sigma carotene, orange-yellow neurosporene, red Lycopene.With the increase of conjugate double bonds number, absorbing wavelength moves to long wave direction.Some are suitable caused by during desaturation 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 can only give lycopene to add ε-ring in most plants, synthesizing yellow containing 1 β-ring and The alpha-carotene of 1 ε-ring and its derivative.Further hydroxylation or epoxidation modification can occur for β-and alpha-carotene, 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, the antheraxanthin and violaxanthin of yellow are formed, it again can be under neoxathin synzyme (NSY) catalysis It is transformed into neoxathin.9- is cis-violaxanthin and 9- it is cis-neoxathin can also be used to synthesize 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 photo-signal channel participates in pair The regulation and control of carotenoids approach, and carotenoid cleavage dioxygenases (CCD)/NCED (9- is cis-and epoxy carotenoid is double Oxygenase) from the angle of decomposition participate in important regulative.In addition, VDE (Analysis of Violaxanthin De-Epoxidase) catalysis violaxanthins 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 passes through anti-to light The negative regulation of induction signal approach and suppress carotenoid approach, and wild cabbage OR (Orange) and tomato HSP21 are then to pass through regulation and control The formation of chromoplast and promote the accumulation of carotenoid.

In addition, the 3rd class phytochrome, i.e. betanidin (betalains) also be present, be it is a kind of be present in it is water-soluble in vacuole Property alkaloid, can be divided into the Betacyanins (betacyanins) of purple colour system and the betaxanthin of yellow colour system (betaxanthins), but betanidin is only found in the plant and a small number of higher funguses of 10 sections of Caryophyllales, other plants 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, the major defect on pattern be present, therefore pattern molecule is educated The metabolic engineering of kind has important application prospect.But in 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 in the beta cyclase gene in interference brassica plant petal is provided Ectopic expression TT8 genes are preparing petal in red simultaneously for LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression Application in the brassica plant kind of color；The second object of the present invention is in β-cyclisation in interference brassica plant petal is provided Enzyme gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression while the plant table of ectopic expression TT8 genes Up to carrier；The third object of the present invention is the preparation method for providing above-mentioned plant expression vector；The fourth object of the present invention exists The method for preparing the brassica plant kind that petal takes on a red color using above-mentioned plant expression vector in offer.

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

1st, beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 in brassica plant petal are disturbed Gene expression application of the ectopic expression TT8 genes in the brassica plant kind that petal takes on a red color is prepared simultaneously.

Preferably, it is described interference brassica plant petal in beta cyclase gene LCYB, ε-cyclase gene LCYE, The sequence of MYB12 and MYB111 gene expressions is as shown in SEQ ID NO.2, the sequence such as SEQ ID NO.3 institutes of the TT8 genes Show.

Preferably, it is described interference brassica plant petal in beta cyclase gene LCYB, ε-cyclase gene LCYE, The sequence of MYB12 and MYB111 gene expressions is mediated by arabidopsis petal specific promoter PAtAP3 and expressed, and the petal is special Promoter PAtAP3 nucleotide sequence is 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, MYB12 and MYB111 in brassica plant petal are disturbed Gene expression while the plant expression vector of ectopic expression TT8 genes, the plant vector include being situated between by petal specific promoter The expression for expression beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 the gene RNA interference sequence led Frame and the expression cassette that TT8 genes are mediated by constitutive promoter.

Preferably, petal specific promoter mediation expression beta cyclase gene LCYB, ε-cyclase gene LCYE, The expression cassette of MYB12 and MYB111 gene RNA interference sequences is successively by petal specific promoter PAtAP3, SEQ ID NO.2 institutes The sequence shown, intervening sequence, reverse complementary sequence and NOS terminator composition shown in SEQ ID NO.2.

It is furthermore preferred that described mediate the expression cassette of TT8 genes successively by CaMV35S promoters, SEQ by constitutive promoter Sequence shown in ID NO.3 and NOS terminator composition.

3rd, the preparation method of the plant expression vector, comprises the following steps：By sequence shown in SEQ ID NO.1 through AscI With the pFGC5941M plasmids through same digestion are connected into after SwaI double digestions, 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-B4RNAia, then sequence shown in SEQ ID NO.2 is reversely connected into intermediate carrier pFGC5941CEPE- Between B4RNAia intervening sequence and OCS terminators, pFGC5941CEPE-B4RNAi carriers are obtained, finally by SEQ ID NO.3 Shown sequence is connected into the pFGC5941CEPE-B4RNAi carriers through same digestion by NcoI and AscI restriction enzyme sites, must disturb Beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression are simultaneously different in brassica plant petal The plant expression vector of position expression TT8 genes.

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. the plant expression vector is converted into Agrobacterium, engineering bacteria is made；

B., engineering bacteria obtained by step a is converted to the hypocotyl of brassica plant aseptic seedling, after co-cultivation, in Basta weedings Induction differentiation obtains regrowth under agent resistance, screens positive seedling transgenic seedling and 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 key differences expressing gene site between chrysanthemum, white flower, 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, structure obtain a kind of suppression xanthein in petal and accumulate the plant expression vector of lycopene and anthocyanin, turn The new type resource material of red rape flower phenotype is createed after carburetion dish, this be the Brassica genus flower color gene such as rape engineering first Success, and succeeding first by carotenoid approach metabolic engineering modified plant pattern.

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 the differential expression (BnYPe of DFR, TT19, TT8 between Brassica genus petal and radish petal：Cabbage type rape is yellow Petal；BnWPe：Cabbage type rape white flower valve；BjYPe：Mustard type rape chrysanthemum valve；BrYPe：Turnip type rape chrysanthemum valve； BoYPe：Collard chrysanthemum valve；BoWPe：Cabbage mustard white flower valve；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 white flower valve；BjYPe：Mustard type rape chrysanthemum valve；BrYPe：Turnip type rape chrysanthemum valve；BoYPe：Plumage Clothing wild cabbage chrysanthemum valve；BoWPe：Cabbage mustard white flower valve；RsRPe：The pale reddish brown valve of radish red).

Fig. 4 is NOS-PAtAP3, B4RNAi and BnTT8-1PCR amplification (A：NOS-PAtAP3；B：B4RNAi；C： BnTT8-1)。

Digestion result (A during Fig. 5 pFGC5941CEPE and pBLycoRF2 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-B4RNAi；E：BamHI and XbaI double digestions pMD19-T- B4RNAi；F：BamHI and XbaI double digestions pFGC5941CEPE-B4RNAia；G：NcoI and AscI double digestions pFGC5941CEPE-B4RNAi；H：NcoI and AscI double digestion pMD19-T-BnTT8-1).

Fig. 6 is the petal color (A that pBLycoRF2 converts rapeseed plants：Control group；B：Transgene rape).

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 method of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (work such as the third edition, J. Pehanorm Brookers) Described in condition, or according to the condition proposed by manufacturer.

The vegetable material that the embodiment of the present invention uses：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 white flower Valve (R9057 strains), radish red petal are taken from Chongqing City of Southwest University rape Engineering Technical Research Centre and had a rest the routine in horse base Test plant.Arabidopsis (Arabidopsis thaliana, Columbia wild type) seed is purchased from international arabidopsis center, kind Plant in indoors artificial climatic chamber.

The reagent and kit that the embodiment of the present invention uses：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 limited public affairs of precious biological (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 kits 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-2000 plus, Easy-Taq enzymes, dNTPs are raw purchased from Beijing full formula golden (Transgen) 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 reagent purchases such as Tryptone, Yeast Extract, X-gal, IPTG, CTAB 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 uses：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 in preferred embodiment and sequencing give birth to work, Beijing by Shanghai English fine horse/Invitrogen Corp., Shanghai The company trades such as six directions Hua Da are completed.

The microexamination of embodiment 1, Brassica genus and 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 white flower valve (BoW)；Radish safflower Valve (RsR), it is placed on the cold fresh preservation of ice face and transports laboratory back, then free-hand section, with low power sem observation rapid screening, it is qualified to select Section is examined and taken a picture, as a result as shown in Figure 1.As a result show, 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, illustrate that phaeochrome cell device is chromoplast in yellow petal, 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, is almost seen in cabbage mustard white flower valve Less than chromoplast, explanation is that the reduction of chromoplast or disappearance 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

Take just open cabbage type rape chrysanthemum valve (BnY) 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, 50~60 DEG C of drying, 60 mesh are crossed 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 compile number tool plug test tube in, be separately added into petroleum ether, Each about 10mL of 10% hydrochloric acid, 30% ammoniacal liquor, gently mixes, filters, and observes color change, as a result as follows：

(1) petroleum ether reacts：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 to be free of carotenoid.

(2) hydrochloric acid is tested：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 seldom.

(3) ammoniacal liquor is tested：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 is shown, the color are that the yellow of the blueness and flavonoids presentation presented by anthocyanin mixes Form, but all material does not show orange red or red, illustrates to be free of aurones.

2nd, the chromogenic reaction of flavonoids

Go bail for the petal powder 0.100g deposited, extracts 24h with methanol, 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 afterwards and drips, gently shakes up, stands 1h.Knot Fruit shows, 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 containing anthocyanidin.

(2) concentrated hydrochloric acid-zinc powder reaction：A small amount of zinc powder is added, concentrated hydrochloric acid 10 is added and drips, gently shake up, stands 1h.As a result It has been shown that, radish flower are presented pink, illustrated containing anthocyanin.Remaining colourless or yellow, illustrate to be free of anthocyanin.

(3) lead acetate reacts：Add 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 flavonoids possesses phenolic hydroxyl group and is free of chalcone and aurones, may have Adjacent two phenolic hydroxyl groups either 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 plain glycosides.

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

(5) alchlor reacts：Add 1.0% alchlor methanol solution l ml.As a result show, journey is all presented in all material Different yellow is spent, illustrates to contain flavonoid substances.

(6) strong sulfuric acid response：Add 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, illustrates (alcohol) containing flavones, 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 containing anthocyanidin.

(7) tetrahydro boron sodium reacts：Add tetrahydro boron sodium 8mg, then add 1.0% hydrochloric acid 2mL, gently shake up, stand 2h.All rues The different yellow of degree is presented in a kind of sedge category material, illustrates to be free of flavanone and flavanonol.Pole rose pink is presented in radish flower Color, illustrate containing flavanone and/or flavanonol.

(8) alkaline reagent reacts：Add 5%Na₂CO₃3ml, gently shake up, closed standing 30min, blowing air 10min.Institute There is material that the different yellow of degree is presented, color is constant after blowing air, illustrates to be free of flavanonol.

(9) ammonia cesium chloride reacts：Methanol 10ml is taken, ammonification water is settled to 25ml, turns into molten by the water saturated methanol of ammonia Liquid.0.01mol/L strontium chlorides methanol solution 10 is added into sample liquid to drip, then is added 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, illustrates there is the substitution of 3 ', 4 '-dihydroxy.

(10) acid reaction：Add 1.0% boric acid 10 to drip, then add 2.0%H₃BO₃3ml, cabbage mustard white flower valve show it is colourless, Illustrate that cabbage mustard white flower valve flavonoids may be free of C₅-OH。

3rd, the ultraviolet-visible spectrum analysis 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) extraction 24h, filtering, 25ml is settled to, using ultraviolet-visible spectrophotometer 200 Scanned in the range of~700nm.As a result show, 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, adds petroleum ether:Third Ketone (1:1, V/V) 24h is extracted, filtering, 25ml is settled to, using ultraviolet-visible spectrophotometer in the range of 200~700nm Scanning.As a result show, 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 that quantitative analysis determines is respectively 6.824,6.712,5.548,1.248mg/g.And Cabbage mustard and radish petal do not have characteristic absorption peak then, illustrate not containing carotenoid.

(3) flavonoids：The petal 0.100g of preservation is weighed, rapidly with liquid nitrogen grinding to powder, adds acidified methanol (pH =3) 2ml is placed in 4 DEG C of refrigerators and extracts 24h, filtering, 25ml is settled to, 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, collard, cabbage mustard, radish petal Extract solution has absworption peak in 330 and 270nm, illustrates that they contain flavonoids, the content point of quantitative analysis measure 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 methanol extraction 24h, filtering, is settled to 50ml, is scanned with ultraviolet-visible spectrophotometer in the range of 200~700nm.As a result show, radish Petal pigment has obvious anthocyanin characteristic absorption peak in 532nm or so, is anthocyanin band I absworption peaks, in 260-270nm areas With the one less strong peak with II in domain, the content of quantitative analysis measure 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

There is the molecule mechanism of difference for research Brassica genus and radish flower petal pigment, design Brassica genus and radish flavonoid path 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

Take just open cabbage type rape chrysanthemum valve (BnY) 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 refrigerators save backup.Then planted with RNAprep Pure Thing total RNA extraction reagent box extracted total RNA, after electrophoretic analysis and spectrophotometry are qualified, uses 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 carries out reverse transcription, obtains total chains of cDNA first.With 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 is little, illustrates thoroughly to have eliminated the gDNA in total serum IgE before reverse transcription, all samples reverse transcription into Work(and total cDNA concentration is more or less the same, it can be used for the experiment such as quantitative gene expression PCR.

Then usePremix Ex Taq^TMII (Tli RNaseH Plus) kit is in CFX96^TM Real- Fluorescence real-time quantitative PCR is carried out on Time System, PCR programs are performed according to the method for specification.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^TMμ 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 pre-degeneration 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 extractions-reverse transcription weights of Setup Experiments Multiple (biology repetition), each reverse transcription are repeated 2 times PCR (detection repeats), 4 results averageds.It is and 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 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, EGL3 without significantly expression or are expressed in each material of Brassica genus It is extremely low, but expression quantity is very high in radish.GL3 is not detected by its expression in Brassica genus and radish material.Illustrate flavonols Synthesis vigorous or more vigorous working condition is in Brassica genus and radish petal, the synthesis of anthocyanin is only in radish flower Vigorous work is but closed in Brassica genus petal in valve.

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

There is the molecule mechanism of difference for further research Brassica genus and radish flower petal pigment, design Brassica genus and radish kind recklessly The RT-PCR primer of radish element 17 functional site gene families 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 in the present invention of table 2

Then RT-PCR is carried out using method same as described above, and passes through 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 showing, all detection gene locis of carotenoid approach are expressed in the petal of 7 species materials, But there is also some differences, first, CCD1 expressions are substantially higher than in chrysanthemum valve in white/newborn petal of Brassica genus, second, radish flower OR is in closed mode without expressing in valve, also there is no in tetraploid aggregate species OR expressions in Brassica genus diploid elementary species It is high.The missing for illustrating carotenoid in radish petal is can not be formed with colour solid, in white/newborn petal of Brassica genus because OR is closed The reduction of carotenoid is because the decomposition of carotenoid is too strong.

The structure of embodiment 4, plant expression platform carrier pFGC5941CEPE

It is poor according to gene expression difference on Brassica genus and radish petal carotenoid and flavonoids route of synthesis, design clone The primer of different expressing gene and construction of expression vector, specific primer are 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

Using pCAMBIA2301 plasmids as pcr template, expanded with primer combination FTNOS and RTNOS and reclaim 286bp piece Section, obtains the fragment containing NOS terminator, 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 with primer combination FPAtAP3 and RPAtAP3 and reclaims 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 positions.Then pcr template is used as after two kinds of recovery products are mixed, with primer combine FTNOS with RPAtAP3 is expanded, and the fusion fragment NOS-PAtAP3 that clip size is 1044bp is obtained, 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 of acquisition is converted into Escherichia coli DH5 α competent cells, the transformant monoclonal of Amp resistance LB plate screenings is entered using primer combination FTNOS and RPAtAP3 Performing PCR is detected, and sequencing result is shown, fusion fragment NOS-PAtAP3 sequences are as shown in SEQ ID No.1.

(2) pFGC5941CEPE structure

PMD19-T-NOS-PAtAP3 recombinant plasmids are extracted, then carry out double digestion with AscI and SwaI, reclaim NOS- PAtAP3 fragments, see B in Fig. 5；PFGC5941M plasmids are extracted simultaneously, carrier framework are reclaimed after AscI and SwaI double digestions, such as In Fig. 5 shown in A.11241bp is obtained after NOS-PAtAP3 fragments are connected with pFGC5941M plasmids is suitable for flower color gene work The plant expression vector pFGC5941CEPE of journey, pFGC5941CEPE is converted into bacillus coli DH 5 alpha competent cell, is coated on Concentration containing Kan is that monoclonal transformant is screened on 50mg/L LB flat boards, will screen the monoclonal transformant primer sets of acquisition Close FTNOS and RPAtAP3 and F35S3N and ROCST5N and enter performing PCR detection, screening positive clone sequencing, choose sequence without prominent The clone of change is standby.In its pFGC5941CEPE recombinant plasmid obtained, contain Bar expression casettes in T-DNA sections and (carry For the resistance to herbicide basta) and 2 expression cassettes that can be used for insertion foreign gene, exogenous gene expression box 1 contains composition Promoter CaMV 35S and NOS terminator, justice or antisense expression available for target gene；Exogenous gene expression box 2 contains petal Specific promoter PAtAP3, introns BnPAP2I2 (cabbage type rape (Brassica napus) PAP2 genes intron 2) With OCS PolyA terminators, RNA interference, justice or antisense expression available for target gene.

(3) series connection tetravalence RNA interference fragments B4RNAi clone

The total chains of cDNA first of cabbage type rape petal for using above-mentioned reverse transcription to obtain are combined for template using primer The RNA interference fragments of FBLCYBi and RBLCYBi amplification LCYB gene families (387bp, are after enzyme-added enzyme site and joint 411bp), the RNA interference fragments (480bp, after adjunction head that FBLCYEi and RBLCYEi expand LCYE gene families is combined with primer For 504bp), the RNA interference fragments that FBMYB12i and RBMYB12i amplification MYB12 gene families are combined with primer (417bp, add It is 441bp after joint), with the RNA interference fragments of primer combination FBMYB111i and RBMYB111i amplification MYB111 gene families (524bp, enzyme-added enzyme site and joint then be 550bp), is separately recovered.Then will make after 4 kinds of PCR recovery product mixed in equal amounts For masterplate, FBLCYBi and RBMYB111i amplifications are combined into 1808bp fragments, the fragment series connection CYBi, CYE, MYB12 with primer With the RNA interference fragments of tetra- genes of MYB111, B4RNAia is named as, as a result (is after enzyme-added enzyme site as shown in B in Fig. 4 1834bp).Then B4RNAia is reclaimed, pMD19-T-B4RNAi, pMD19-T-B4RNAi conversion large intestines are connected to obtain with pMD19-T Bacillus DH5 α competent cells, FBLCYBi+ is combined using primer to the transformant monoclonal of Amp resistance LB plate screenings RBMYB111i enters performing PCR detection, send positive colony to be sequenced, sequencing result is as shown in SEQ ID No.2.

(4) plant expression vector pFGC5941CEPE-B4RNAi is built

PMD19-T-B4RNAi plasmids are extracted, with SwaI and AatII double digestions (SwaI is first cut, then adds AatII), recovery B4RNAi fragments, as shown in D in Fig. 5；PFGC5941CEPE plasmids are extracted simultaneously, after SwaI and AatII double digestions, recovery carries Body skeleton, as shown in C in Fig. 5.The PAtAP3 that the B4RNAi fragments of recovery are connected into pFGC5941CEPE plant expression vectors is opened Between mover and introns BnPAP2I2, intermediate carrier pFGC5941CEPE-B4RNAia is formed, converts 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 RBnMYB111i and RBnPAPI2 enters performing PCR detection, and PCR positive clone molecules are standby.

PMD19-T-B4RNAi plasmids are extracted, with BamHI and XbaI double digestions, reclaim B4RNAi sense fragment B4RNAi, as shown in E in Fig. 5.PFGC5941CEPE-B4RNAia plasmids are extracted simultaneously, with BamHI and XbaI double digestions, recovery Carrier framework, as shown in F in Fig. 5.By B4RNAi be connected into pFGC5941CEPE-B4RNAia plasmids (introns BnPAP2I2 with Between OCS terminators), plant expression vector pFGC5941CEPE-B4RNAi is formed, by plant expression vector PFGC5941CEPE-B4RNAi 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, RBnMYB111i and RBnPAPI2, FBnPAPI2 and RBnMYB111i 4 combines to primer carries out PCR identifications, positive clone molecule sequencing, it is standby to choose clone of the sequence without mutation.

(5) clone of the cDNA code areas of cabbage type rape TT8-1 (BnTT8-1) gene

We show that cabbage type rape TT8-1 (BnTT8-1) gene is mainly expressed in kind of skin in the research of the past, activation The synthesis of flavonoids-OPC, also expressed in the cauline leaf that aging turns red, activate the synthesis of flavonoids-anthocyanin.Cause This, the present invention clones BnTT8-1 cDNA code areas from the seed of cabbage type rape development, is subsequently used for building just carrier And convert the rape ectopic expression in petal later.The total serum IgE of cabbage type rape development mid-term seed is extracted, such as preceding method removes gDNA Reverse transcription obtains the first chain cDNA afterwards, then using the first chain cDNA as template, is combined and expanded using FBnTT8-1 and RBnTT8-1 primers Increase BnTT8-1 genes, obtain BnTT8-1 genes 1566bp code area (being 1576bp after enzyme-added enzyme site), electrophoresis result such as 4 Shown in middle C, by its glue reclaim, pMD19-T-BnTT8-1 is connected into pMD19-T, converts bacillus coli DH 5 alpha competent cell, Performing PCR detection is entered using primer combination FBnTT8-1 and RBnTT8-1 to the transformant monoclonal of Amp resistance LB plate screenings, sent Multiple positive clone molecule sequencings, sequencing result show BnTT8-1 sequences as shown in SEQ ID No.3, choose sequence without mutation Clone is standby.

(6) plant expression vector pBLycoRF2 is built

PMD19-T-BnTT8-1 plasmids are extracted, with NcoI and AscI double digestions (AscI is first cut, then adds NcoI), recovery BnTT8 genetic fragments, as shown in H in Fig. 5；PFGC5941CEPE-B4RNAi plasmids are extracted simultaneously, with the double enzymes of NcoI and AscI Cut, carrier framework is reclaimed, as shown in G in Fig. 5.By the BnTT8 genetic fragments of recovery and pFGC5941CEPE-B4RNAi carrier bones Frame connects, and forms the pentavalent plant expression vector of series connection tetravalence RNAi and monovalence ectopic expression, is named as pFGC5941CEPE- B4RNAi-BnTT8-1ox, referred to as pBLycoRF2.Then pBLycoRF2 is converted into bacillus coli DH 5 alpha competent cell, it is right The transformant monoclonal of Kan resistance LB plate screenings respectively with FPAtAP3 and FBLCYBi, FBLCYBi and ROCST5N, RBnMYB111i and RBnPAPI2, FBnPAPI2 and RBnMYB111i, F35S3N and RTT8-1, FTT8-1 and RTNOS 6 are to drawing Whether carrier is successfully constructed respectively for thing combination, and freeze-thaw method conversion Agrobacterium tumefaciems is used after positive clone molecule then is extracted into plasmid LBA4404, then with the monoclonal transformant of the resistance LB plate screenings containing Kan, Str and Rif, and with above-mentioned 6 pairs of primer sets Close and detected into performing PCR, screening positive clone preserves as engineered strain, for Plant Transformation.

Embodiment 5, pBLycoRF2 conversion rapes

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 handled.Double No. 10 seeds is rushed with after 75% ethanol surface sterilization 1min with sterilized water in cabbage type rape typical case's chrysanthemum kind Wash 3 times, then soak 20min with 5% sodium hypochlorite, (MS powder is inoculated 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 liquid to be not added with Phytagel Culture medium), cultivated in 25 DEG C, 2000Lux illumination, 16h/d photoperiods (between tissue culture below condition of culture except especially indicate person Outside, 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- Train (MS culture medium+1.0mg/L 6-BA+1.0mg/L 2,4-D) preculture 3d on culture medium MSp.

By the LBA4404 engineered strains containing pBLycoRF2 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 Str and 40mg/L Rif LB fluid nutrient mediums, Agrobacterium is set to grow to pair 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 liquid for shell.

Hypocotyl section after preculture is immersed into 5~10min in dip dyeing liquid for shell, 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, each 10min, then blots surface with sterilizing paper Liquid, it 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) in culture more than 14d, evoked callus 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) 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, the seedling after taking root is transplanted to containing sterilizing perlite, vermiculite, turf earth mixtures after domestication (mass ratio 1:1:1) in basin alms bowl, it is managed by greenhouse pot culture.

Finally, pBLycoRF2, which is converted, obtains 22 plants of regeneration plants after double No. 10 in cabbage type rape；To regeneration plant blade 200mg/L Basta solution detection resistance is added dropwise, and extract blade genome DNA be respectively adopted primer combination F35S3N and RBnTT8-1, FBLCYBi and ROCST5N enter performing PCR detection, the results showed that obtain 13 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 obtained grows substantially not to be changed with background character, but petal becomes red, and petal cell is micro- Observation shows that red material is existing from chromoplast, also has and also indicates that new generation from vacuole, biochemical composition detection 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 go out the homozygous excellent strain of transgene rape, and its petal color is redder than transgenosis present age plant, illustrates to turn It is more preferable than the transgene traits of contemporary (heterozygosis) individual plant that gene character can stablize hereditary and homozygous offspring's strain.

Other explanations

Finally illustrate, above example is only to illustrate technical scheme, but be 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 declare, the following change on application form also all necessarily belongs to the present invention's Spirit and scope are covered：

1st, TT8-1, LCYB, LCYE, MYB12, MYB111 gene/gene fragment in the present invention, except institute in sequence table Beyond the nucleotide sequence of row, in addition to come from the corresponding gene order in parent species Chinese cabbage or wild cabbage, also include coming from The sequence of the same other members of functional site gene family in these species, although them and nucleotides listed in sequence table Sequence may have small difference.

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

3rd, in the present invention LCYB, LCYE, MYB12, MYB111 site RNA interference fragments, except listed in sequence table Beyond nucleotide sequence, also include the fragment for coming from same gene other positions.

4th, the suppression in the present invention to the expression of LCYB, LCYE, MYB12, MYB111 site, except being lifted in preferred embodiment RNA perturbation techniques beyond, can also using the technologies such as antisense RNA, genome editor (ZFN, TALEN, CRISPR-Cas) come Reach same or analogous purpose.

5th, the gene and its fragment in the present invention, except being carried as the transformation using pFGC5941 lifted in preferred embodiments Beyond body is built, plant expression vector construction can also be carried out using other carriers, including using other promoters and Terminator, to reach same or analogous purpose；Vector construct in the present invention, except being adopted as what is lifted in preferred embodiments Carried out beyond genetic transformation, can also be entered using other methods with the Agrobacterium tumefaciens strain LBA4404 improvement leaf disk methods mediated Row Genetic Transformation in Higher Plants.

6th, gene, genetic fragment and the vector construct in the present invention, except as being lifted in preferred embodiments for sweet Beyond blue type rape, other sibling specieses of its parent species Chinese cabbage, wild cabbage and Brassica genus can also be applied to, it is identical to reach Or similar purpose.

Claims (7)

1. disturb β-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 in brassica plant petal Application of the ectopic expression TT8 genes in the brassica plant kind that petal takes on a red color is prepared, its feature exist simultaneously for gene expression In：For disturbing β in brassica plant petal-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 The sequence of gene expression is as shown in SEQ ID NO.2, the sequence such as SEQ ID NO.3 institutes for ectopic expression TT8 genes Show.

2. the application according to claim 1, it is characterised in that：For disturbing β in brassica plant petal-cyclase base Because the sequence of LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression is by arabidopsis petal specific promoter PAtAP3 mediation expression, the nucleotide sequence such as SEQ ID NO.1 the 267th of the petal specific promoter PAtAP3 are extremely Shown in 1044th.

3. according to the application described in claim any one of 1-2, it is characterised in that：The brassica plant is cabbage type rape (Brassica napus)。

4. disturb β-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 in brassica plant petal Gene expression while the plant expression vector of ectopic expression TT8 genes, it is characterised in that：The plant vector is included by petal Expression β-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene of specific promoter mediation The expression cassette of RNA interference sequences and the expression cassette that TT8 genes are mediated by constitutive promoter；

Petal specific promoter mediation expression β-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and The expression cassette of MYB111 gene RNA interference sequences is successively as shown in petal specific promoter PAtAP3, SEQ ID NO.2 Sequence, intervening sequence, the reverse complementary sequence and OCS terminators composition of sequence shown in SEQ ID NO.2；

It is described that the expression cassette of TT8 genes is mediated successively by CaMV35S promoters, SEQ ID NO.3 institutes by constitutive promoter Show sequence and NOS terminators composition；

Its preparation method is as follows：Sequence shown in SEQ ID NO.1 is connected into through same digestion after AscI and SwaI double digestions PFGC5941M plasmids, obtain pFGC5941CEPE plasmids, be then connected into the sequence shown in SEQ ID NO.2 Between the PAtAP3 promoters and intervening sequence of pFGC5941CEPE carriers, intermediate carrier pFGC5941CEPE- is formed B4RNAia, then sequence shown in SEQ ID NO.2 is reversely connected into intermediate carrier pFGC5941CEPE-B4RNAia interval sequence Between row and OCS terminators, pFGC5941CEPE-B4RNAi carriers are obtained, finally pass through sequence shown in SEQ ID NO.3 NcoI and AscI restriction enzyme sites are connected into the pFGC5941CEPE-B4RNAi carriers through same digestion, obtain interference brassica plant β-cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression while ectopic expression in petal The plant expression vector of TT8 genes.

5. the preparation method of plant expression vector described in claim 4, it is characterised in that comprise the following steps：By SEQ ID Sequence shown in NO.1 is connected into the pFGC5941M plasmids through same digestion after AscI and SwaI double digestions, obtains PFGC5941CEPE plasmids, then the sequence shown in SEQ ID NO.2 is connected into the PAtAP3 of pFGC5941CEPE carriers Between promoter and intervening sequence, intermediate carrier pFGC5941CEPE-B4RNAia is formed, then by sequence shown in SEQ ID NO.2 Reversely it is connected between intermediate carrier pFGC5941CEPE-B4RNAia intervening sequence and OCS terminators, obtains PFGC5941CEPE-B4RNAi carriers, finally sequence shown in SEQ ID NO.3 is connected by NcoI and AscI restriction enzyme sites Enter the pFGC5941CEPE-B4RNAi carriers through same digestion, β-cyclase gene in brassica plant petal must be disturbed The expression of the plant of LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression while ectopic expression TT8 genes Carrier.

6. preparation method according to claim 5, it is characterised in that：The intervening sequence is cabbage type rape PAP2 bases Because of the 2nd introne.

7. preparing the method for the brassica plant kind that petal takes on a red color using plant expression vector described in claim 4, it is special Sign is, comprises the following steps：

A. plant expression vector described in claim 4 is converted into Agrobacterium, engineering bacteria is made；

B., engineering bacteria obtained by step a is converted to the hypocotyl of brassica plant aseptic seedling, after co-cultivation, in Basta herbicides Induction differentiation obtains regrowth under resistance, screens positive seedling transgenic seedling and obtains the brassica plant kind that petal takes on a red color.