CN104357479B - Interference xanthein expression application of the overexpression lycopene in red petal brassica plant is prepared simultaneously - Google Patents

Abstract

The invention discloses interference xanthein expression application of the overexpression lycopene in red petal brassica plant is prepared simultaneously,Specifically disturb LCYB,LCYE,MYB12 and MYB111 gene expressions are while overexpression CRTISO genes,Also disclose interference LCYB,LCYE,The plant expression vector of MYB12 and MYB111 gene expression carotenoid isomerases CRTISO and the preparation method of the plant expression vector,By disturbing LCYB,LCYE,The biosynthesis of MYB12 and MYB111 gene expression inhibition yellow carotenoid and flavonols,Overexpression carotenoid isomerase CRTISO promotes lycopene accumulation,Thus make the aobvious red of petal,It is successfully prepared the brassica plant that petal shows red,Enrich the pattern of brassica plant.

Description

Overexpression lycopene is preparing red petal rue to the expression of interference xanthein simultaneously Application in a kind of sedge platymiscium

Technical field

The invention belongs to genetic engineering field, and in particular to interference LCYB, LCYE, MYB12 and MYB111 gene expression is same When application of the overexpression CRTISO genes in red petal brassica plant kind is prepared.

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 is by Brassica genus) three things Plant and 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.Research find, pattern mainly by flavonoids (flavonoids) and class recklessly Radish element (carotenoids) two major class pigment is determined.Flavonoids is the anthocyanidin most seen, contribution yellow, orange, red to purple A series of colour systems of color.Flavonoids is water-soluble substances, with a C15 skeleton, 9 classes is broadly divided into by structure：Chalcone (chalcones), aurones (aurones), isoflavones (isoflavonoids), flavones (flavones), flavonols (flavonols), flavandiols (flavandiols), anthocyanin (anthocyanins), condensed tannin (condensed Tannins, i.e. OPC, proanthocyanins) and tan acid anhydride (phlobaphenes), plant is coloured in them is contributed Maximum is anthocyanin, flavonols, chalcone and aurones.Wherein anthocyanin is maximum flavone material, be flower or its It organizes contribution magenta, the tone such as red, purple, blueness, accumulates in the vacuole or chromatophore of cell, yellow tone then by Chalcone, aurones, flavonols, flavones are provided.

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 (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 LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression are while overexpression carotenoid isomerase CRTISO Application of the gene in the brassica plant kind that petal takes on a red color is prepared；The second object of the present invention is to provide interference rape Beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression in platymiscium petal are while excess table Up to the plant expression vector of carotenoid isomerase CRTISO genes；The third object of the present invention is to provide above-mentioned plant table Up to the preparation method of carrier；The fourth object of the present invention is to provide to prepare what petal took on a red color using the plant expression vector The method of brassica plant kind.

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 interference brassica plant petal Overexpression carotenoid isomerase CRTISO genes are preparing the brassica plant kind that petal takes on a red color simultaneously for gene expression In application.

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 as shown in SEQ ID NO.2, the overexpression carotenoid isomerase The sequence of CRTISO genes is as shown in SEQ ID NO.3.

It is furthermore preferred that 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 Shown in the nucleotide sequence SEQ ID NO.1 the 267th to the 1044th of promoter PAtAP3.

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

2nd, beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 in interference brassica plant petal The plant expression vector of gene expression overexpression carotenoid isomerase CRTISO genes simultaneously, the plant vector includes Expression beta cyclase gene LCYB, ε mediated by petal specific promoter-cyclase gene LCYE, MYB12 and MYB111 gene The expression cassette of RNA interference sequences and the table by constitutive promoter mediation overexpression carotenoid isomerase CRTISO genes Up to frame.

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 Reverse complementary sequence and NOS terminator composition shown in the sequence shown, intervening sequence, SEQ ID NO.2.

It is furthermore preferred that the table by constitutive promoter mediation overexpression carotenoid isomerase CRTISO genes Up to frame successively by CaMV35S promoters, 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-B4RNAia, then sequence shown in SEQ ID NO.2 is reversely connected into intermediate carrier pFGC5941CEPE- Between the intervening sequence and OCS terminators of B4RNAia, pFGC5941CEPE-B4RNAi carriers are obtained, finally by SEQ ID NO.3 Shown sequence is connected into through the pFGC5941CEPE-B4RNAi carriers of same digestion by NcoI and AscI restriction enzyme sites, must be disturbed Beta cyclase gene LCYB, ε-cyclase gene LCYE, MYB12 and MYB111 gene expression are simultaneously super in brassica plant petal The plant expression vector of amount expression carotenoid isomerase CRTISO 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 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：The present invention by studying the Brassica genus core species such as rape and radish pattern mechanism, Specify that chrysanthemum, spend in vain, the key differences expressing gene site between safflower, thus the present invention utilize petal specific promoter The key gene expression of carotenoid and flavonoid path synthesis is disturbed in petal, so as to suppress xanthein in petal Accumulation, by the key gene on overexpression lycopene route of synthesis, makes lycopene excess accumulation, will press down in petal Xanthein processed simultaneously obtains the Brassica genus that take on a red color of petal after accumulating the plant expression vector conversion brassica plant of lycopene New variety of plant, by succeeding first for carotenoid approach metabolic engineering modified plant brassica 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 that (A is DFR in Brassica genus petal to the differential expression of DFR, TT19, TT8 between Brassica genus petal and radish petal With the differential expression between radish petal；B is differential expressions of the TT19 between Brassica genus petal and radish petal；C is TT8 in Brassica genus Differential expression between 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：Turnip type rape chrysanthemum valve；BoYPe：Collard chrysanthemum valve；BoWPe：Cabbage mustard Spend valve in vain；RsRPe：The pale reddish brown valve of radish red).

Fig. 3 is that (A is OR in Brassica genus petal and radish to the differential expression of OR, CCD1 between Brassica genus petal and radish petal Differential expression between petal；B is differential expressions of the CCD1 between Brassica genus petal and radish petal；BnYPe：Cabbage type rape is yellow Petal；BnWPe：Cabbage type rape spends valve in vain；BjYPe：Mustard type rape chrysanthemum valve；BrYPe：Turnip type rape chrysanthemum valve； BoYPe：Collard chrysanthemum valve；BoWPe：Cabbage mustard spends valve in vain；RsRPe：The pale reddish brown valve of radish red).

Fig. 4 is NOS-PAtAP3, B4RNAi and BnCRTISO1PCR amplification (A：NOS-PAtAP3；B：B4RNAi；C： BnCRTISO1)。

Digestion result (A during Fig. 5 pFGC5941CEPE and pBLycoRF1 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-B4RNAia；H：NcoI and AscI double digestion pMD19-T-BnCRTISO1).

Fig. 6 is pBLycoRF1 conversion cabbage type rape results (A：Wild type cabbage type rape；B：Transgenic positive is planted Strain).

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 (in double No. 9 kinds), cabbage type rape breast Petal, turnip type rape chrysanthemum valve (6Y733 strains), mustard type rape chrysanthemum valve (CNG12011 strains), collard Valve (R9057 product are spent in (B.oleracea var.acephala ftricolor, K10-3 strain) chrysanthemum valve, cabbage mustard mutation in vain System), radish red petal is taken from Chongqing City of Southwest University rape Engineering Technical Research Centre and has a rest the routine test plant in horse base. Arabidopsis (Arabidopsis thaliana, Columbia wild type) seed is purchased from international arabidopsis center, plants in interior Phjytotron.

The reagent and kit that embodiment is used are as follows：PrimeScript^TM RT reagent Kit with gDNA Eraser(Perfect Real Time)、 Premix Ex Taq^TM II(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 pFGC5941M plasmids used in the present invention are by cabbage type rape (Brassica napus) PAP2 genes the 2nd Novel spacers containing sub (BnPAP2I2) instead of original PhChsA spacer regions of pFGC5941, and in new spacer region and startup (its specific transformation process is referring to the beautiful duckweed in Ma Lijuan Feng Yu rivers to have increased a point of contact of Aat II at MCS between son newly Quick bavins friend is flourish for Shen；The transformation of pFGC5941 and Brassica genus transparent testa 1 gene (TT1) family rna interference vector build agriculturals Biotechnology journal, volume 18, the 6th phase, page 1189~1196 in 2010).

PCR primer synthesis used by embodiment and sequencing are by Shanghai English fine horse/Invitrogen Corp., Shanghai life work, Beijing six directions The company trades such as 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 1ml.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.Institute There is material that the different yellow of degree is presented, color is constant after blowing air, illustrate 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 spend in vain valve show it is colourless, Illustrate that 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, 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

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 tetravalence RNA interference fragments B4RNAi

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 Be 504bp), with primer combine FBMYB12i and RBMYB12i expand MYB12 gene families RNA interference fragments (417bp, plus It is 441bp after joint), combine the RNA interference fragments that FBMYB111i and RBMYB111i expands MYB111 gene families with primer (524bp is 550bp after enzyme-added enzyme site and joint), is separately recovered.Then will make after 4 kinds of recovery product mixed in equal amounts of PCR It is masterplate, combining FBLCYBi and RBMYB111i with primer expands into 1808bp fragments, the fragment series connection CYBi, CYE, MYB12 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 is connected to obtain with pMD19-T Bacillus DH5 α competent cells, the transformant monoclonal to Amp resistance LB plate screenings combines FBLCYBi+ using primer RBMYB111i enters performing PCR detection, send positive colony to be sequenced, and sequencing result is as shown in SEQ ID No.2.

(4) plant expression vector pFGC5941CEPE-B4RNAi builds

Extracting pMD19-T-B4RNAi plasmids, with SwaI and AatII double digestions (SwaI first cuts, then adds AatII), reclaim B4RNAi 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 B4RNAi fragments of recovery are connected into pFGC5941CEPE plant expression vectors is opened Between mover and introns BnPAP2I2, intermediate carrier pFGC5941CEPE-B4RNAia 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 RBnMYB111i and RBnPAPI2 enters performing PCR detection, and PCR positive clone molecules are standby.

Extracting pMD19-T-B4RNAi plasmids, with BamHI and XbaI double digestions, reclaim the sense fragment of B4RNAi B4RNAi, as shown in E in Fig. 5.PFGC5941CEPE-B4RNAia plasmids are extracted simultaneously, with BamHI and XbaI double digestions, are reclaimed 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 carries out PCR identifications to primer combination, and positive clone molecule sequencing chooses clone of the sequence without mutation standby.

(5) clone of the cDNA code areas of cabbage type rape C RTISO1 genes

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 FBnCRTISO1 and RBnCRTISO1 amplification BnCRTISO1 genes cDNA code areas 1770bp (be after enzyme-added enzyme site 1778bp), as shown in C in Fig. 4.By amplified fragments glue reclaim, pMD19-T-BnCRTISO1 is connected into pMD19-T, conversion is big Enterobacteria DH5 α competent cells, with the monoclonal transformant of Amp resistance LB plate screenings, are then combined using primer FBnCRTISO1 and RBnCRTISO1 enter performing PCR detection, send multiple positive clone molecules to be sequenced, BnCRTISO1 sequences such as SEQ ID Shown in No.3.

(6) plant expression vector pBLycoRF1 builds

Extracting pMD19-T-BnCRTISO1 plasmids, with NcoI and AscI double digestions (AscI first cuts, then adds NcoI), reclaim Genetic fragment BnCRTISO1, as shown in E in Fig. 5；PFGC5941CEPE-B4RNAi plasmids are extracted simultaneously, it is double with NcoI and AscI Digestion, reclaims carrier framework, as shown in F in Fig. 5.BnCRTISO1 is connected with pFGC5941CEPE-B4RNAi skeleton carriers, The pentavalent plant expression vector of the tetravalence that must connect RNAi and monovalence overexpression, is named as pFGC5941CEPE-B4RNAi- BnCRTISO1ox (referred to as pBLycoRF1), bacillus coli DH 5 alpha competent cell is converted by pBLycoRF1, uses Kan resistances The monoclonal transformant of LB plate screenings, then respectively with FPAtAP3 and FBLCYBi, FBLCYBi and ROCST5N, RBnMYB111i and RBnPAPI2, FBnPAPI2 and RBnMYB111i, F35S3N and RCRTISO1, FCRTISO1 and RTNOS 6 PCR identifications are carried out to primer combination, positive clone molecule converts agrobacterium tumefaciens lba4404 using freeze-thaw method, using containing Kan, Str With the monoclonal transformant of the LB plate screenings of the triple resistances of Rif, the transformant for obtaining will be screened with 6 kinds of primers as implied above Combine into performing PCR detection, positive clone molecule is preserved as engineered strain, for Plant Transformation.

Chrysanthemum valve is modified to red by embodiment 5, pBLycoRF1 conversion 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.By the ethanol surface sterilization that seed volume fraction double No. 10 in cabbage type rape typical case's chrysanthemum kind is 75% Aseptic water washing being used after 1min 3 times, then soaking 20min with the sodium hypochlorite that mass fraction is 5%, sterilized water repeatedly rinses dry Only, MS solid mediums (MS powder 4.41g/L+Phytagel 2.6g/L+ sucrose 30.0g/L, pH5.8, sterilizing are then inoculated in Pot moist heat sterilization；It is not added with Phytagel as fluid nutrient mediums), then cultivated in 25 DEG C, 2000Lux illumination, 16h/d photoperiods (condition of culture is identical with this in addition to especially person is indicated between tissue culture below).Cut the hypocotyl of seedling age 8d or so aseptic seedling The segment for being about 0.5~1.0cm is cut into, culture medium MSp (MS culture medium+1.0mg/L 6-BA+1.0mg/L2,4-D) is inoculated into Upper preculture 3d.

By the obtained LBA4404- engineered strains containing pBLycoRF1 added with 100mg/L Kan+20mg/L Str+ In 28 DEG C, 250r/min shaken cultivations to logarithmic phase in the LB fluid nutrient mediums of 40mg/L Rif, switching is cultivated once, Ran Hou Thalline is collected by centrifugation under the conditions of 5000rpm, 10min, with dip-dye culture medium MSm (MS fluid nutrient mediums+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, as 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 Unnecessary bacterium solution is blotted on sterilizing paper, common training culture medium MSc (MS solid medium+2.0mg/L 6-BA+0.5mg/ are inoculated into LNAA in), 23.5 DEG C of light culture 48h.With sterilizing liquid culture medium MSk (MS fluid nutrient medium+1.0mg/L 2,4-D+1.0mg/ L 6-BA+500mg/L Cef) washing by soaking 3 × 10min of explant, surface liquid is blotted with sterilizing paper, it is transferred to induction screening Culture medium MSi (MS solid medium+1.0mg/L 6-BA+1.0mg/L 2,4-D+500mg/L Cef+12.5mg/L Basta+ 6mg/L AgNO₃) culture, about 2 weeks subcultures 1 time to growing macroscopic kanamycin-resistant callus tissue, then are transferred to differential medium MSd (MS solid medium+4.0mg/L 6-BA+2.0mg/L ZT+5.0mg/L AgNO₃+500mg/LCef+12.5mg/L Basta culture more than 14d in), evoked callus differentiate budlet, then are transferred to stem differential medium MSs (MS solid cultures Base+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+12.5mg/L Basta) culture is to growing small stem, then be transferred to Cultivated extremely in long shoot culture medium MSe (MS solid medium+0.05mg/L 6-BA+500mg/L Cef+12.5mg/L Basta) The complete unrooted seedling of length, then root media MSr [MS solid medium+2mg/L NAA] cultures are transferred to growing flourishing root System, after seedling after taking root is through domestication, is transplanted to containing sterilize perlite, vermiculite, (mass ratio is 1 to turf earth mixtures:1:1) Basin alms bowl in, be managed by greenhouse pot culture.

Finally, 27 plants of regeneration plants are obtained after double No. 10 in pBLycoRF1 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 RBnCRTISO1 and FBLCYBi and ROCST5N enters performing PCR detection, as a result shows to obtain 14 plants of double positive transgenic plant.

Comprehensive biology and agronomy observation are carried out to transgenic positive plant, the above-mentioned kind of transfer-gen plant of carrier is not There is obvious side effect, grow and substantially do not change with background proterties, but petal becomes red (Fig. 6), petal Cell micro observation shows this red material from chromoplast, and biochemical composition detection also indicates that the red of this new generation Matter is lycopene.Show, suppress cabbage type rape xanthein and accumulate lycopene and 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.

Other explanations

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, CRTISO1, LCYB, LCYE, MYB12, MYB111 gene/gene fragment in the present invention, except in sequence table Beyond listed nucleotide sequence, also including coming from parent species Chinese cabbage or wild cabbage in corresponding gene order, also including come From the sequence of other members of same functional site gene family in these species, although them and nucleosides listed in sequence table Acid sequence may have small 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, MYB12, MYB111 site RNA interference fragments, except listed in sequence table Beyond nucleotide sequence, also including coming from the fragment of same gene other positions.

4th, to the suppression of LCYB, LCYE, MYB12, MYB111 site expression in the present invention, except as institute in preferred embodiments Beyond the RNA perturbation techniques of act, can also be using technologies such as antisense RNA, genome editors (ZFN, TALEN, CRISPR-Cas) To reach same or analogous 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 (5)

1. the method for preparing the brassica plant kind that petal takes on a red color, it is characterised in that：By disturbing brassica plant petal Middle beta cyclase geneLCYB, ε-cyclase geneLCYE、MYB12WithMYB111Gene expression is while overexpression carotenoids Plain isomeraseCRTISOGene, wherein for disturbing beta cyclase gene in brassica plant petalLCYB, ε-cyclase geneLCYE、MYB12WithMYB111The sequence of gene expression as shown in SEQ ID NO.2, for overexpression carotenoid isomeraseCRTISOThe sequence of gene is as shown in SEQ ID NO.3.

2. method according to claim 1, it is characterised in that：For disturbing beta cyclase gene in brassica plant petalLCYB, ε-cyclase geneLCYE、MYB12WithMYB111The sequence of gene expression is by arabidopsis petal specific promoterPAtAP3 Mediation expression, the petal specific promoterPAtAP3Nucleotide sequence such as the 267th to the 1044th institute of SEQ ID NO.1 Show.

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

4. beta cyclase gene in brassica plant petal is disturbedLCYB, ε-cyclase geneLCYE、MYB12WithMYB111Gene Expression is while overexpression carotenoid isomeraseCRTISOThe plant expression vector of gene, it is characterised in that：The plant carries Body includes the expression beta cyclase gene mediated by petal specific promoterLCYB, ε-cyclase geneLCYE、MYB12WithMYB111The expression cassette of gene RNA interference sequence and by constitutive promoter mediation expression carotenoid isomeraseCRTISOBase The expression cassette of cause；

The petal specific promoter mediation expression beta cyclase geneLCYB, ε-cyclase geneLCYE、MYB12WithMYB111 The expression cassette of gene RNA interference sequence sequence, interval successively as shown in petal specific promoter PAtAP3, SEQ ID NO.2 Sequence, the reverse complementary sequence of sequence shown in SEQ ID NO.2 and NOS terminator composition；It is described to be mediated by constitutive promoter Expression carotenoid isomeraseCRTISOThe expression cassette of gene is successively by CaMV35S promoters, sequence shown in SEQ ID NO.3 Constituted with NOS terminator.

5. the method that the brassica plant kind that petal takes on a red color is prepared using plant expression vector described in claim 4, it is special Levy and be, comprise the following steps：

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

B., step a gained engineering bacterias are converted the hypocotyl of brassica plant 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 brassica plant that petal takes on a red color.