CN104531754A - Application of gene for interfering expression of LCYB and LCYE and simultaneously over-expressing PAP in preparation of brassica plant having red petals - Google Patents

Abstract

The invention discloses application of a gene for interfering expression of LCYB and LCYE genes and simultaneously over-expressing a PAP gene in preparation of a brassica plant having red petals. The invention further discloses an expression vector for interfering expression of the LCYB and LCYE genes and simultaneously over-expressing the PAP gene and a preparation method of the expression vector. Yellow carotenoid is inhibited and red lycopene is accumulated by interfering expression of the LCYB and LCYE genes; accumulation of cyanin can be promoted through over-expression of the PAP gene; therefore, the petals are red; the brassica plant having red petals is successfully prepared; and colours of the brassica plants are enriched.

Description

Interference LCYB, LCYE express the application that simultaneously overexpression PAP is preparing petal and takes on a red color in brassica plant

Technical field

The invention belongs to biological technical field, be specifically related to disturb LCYB, LCYE genetic expression overexpression PAP gene application preparing petal and take on a red color in brassica plant simultaneously.

Background technology

Rape is important oil crops, and cultivation history is long, economic worth is high, of many uses, strong adaptability, is the first oil crops of China, is also global important oil crops.In species taxonomy, rape is made up of three species of Brassica genus, be respectively swede type rape (Brassica napus, 2n=38, AACC), turnip type rape (Brassica rapa ssp.oleiferasyn.B.campestris, 2n=20, AA), mustard type rape (Brassica juncea, 2n=36, AABB).Swede type rape rape is by Chinese cabbage (Brassica rapa) and wild cabbage (Brassica oleracea, 2n=18, CC) evolved and next a kind of aggregate species by diploidization two after natural species hybridization, although cultivation history only has the centuries, but because its growth potential is strong, yielding ability is high, the Yi Zhan world and China's rapeseed cultivation area more than 90%.Turnip type rape be Brassica genus the earliest by one of the species of taming, the Chinese cabbage originating in NORTHWEST CHINA area develops, and has distribution area and long cultivation history widely in world wide.Mustard type rape is that China is its original origin and differentiation center, planting range throughout world by the double diploid aggregate species that formed of Natural double again after Chinese cabbage and black mustard (Brassica nigra, 2n=16, BB) natural hybrization.

Along with the sustainable development of economic level and the continuous lifting of social modernization's degree, the popular pursuit to life taste also grows with each passing day, and 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 very rapid.Rape big area produces the upper ocean forming golden yellow flower, is more and more important villatic zoology tourist resources.Rape flower time of viewing and admiring at the bottom of annual December to summer in the coming year.Each place is because the time of plantation is slightly different, and the florescence has some difference.Formed tens well-known rape flowers in China and viewed and admired area, rape is when the flowers are in blossom, competitively in full bloom, shine with glittering gold and colorful decorations, be continuous tens of in, like the ocean that gold wave is torrential.

Although nature flower color is of a great variety, except the petal of cabbage mustard (B.oleracea var.alboglabra) is except oyster white, the petal of brassica plant is commonly Yellow series, and business rape variety is yellow petal entirely, too dull.Along with the fast development of rape pattern villatic zoology sightseeing industry, in the urgent need to colourful pattern proterties, make it to output redness, blueness etc., outside the oil yield not affecting rape vegetable seed and other conventional use, increase its value of admiring the beauty of flowers, boosting Ecological sightseeing is traveled.In addition, the selective marker proterties that specific pattern or rapeseed breeding circle are thirsted for, and also the non-yellowization of pattern also contributes to reducing the insect pests such as nitidulid.Therefore, need the molecule mechanism of resolving rape pattern proterties, and carry out the molecular breeding of new pattern.

Radish (Raphanus sativus) belongs to Rhaphanus, molecular Evidence in recent years show radish and Chinese cabbage, wild cabbage, swede type rape distance even much smaller than black mustard and their distance, mean that the distance of radish and Brassica genus core species is less than the kind spacing of Brassica genus inside.But then, radish can output the pattern of red colour system, and Brassica genus and numerous relative genus (as sinapsis alba belongs to) then can only output the flower of yellow system.This is a strange phenomenon, therefore carries out the comparative studies of Brassica genus and radish pattern proterties, and be not only the needs of these biological pattern proterties fundamental researchs, the genetic improvement that also can be the brassica plant pattern proterties such as rape provides guidance.

The pattern molecule mechanism of important decorative flower and molecular breeding obtain remarkable break-throughs, provide important references for studying other flower color mechanism and carrying out molecular breeding.Pattern determines primarily of flavonoid (flavonoids) and the large class pigment of carotenoid (carotenoids) two.

Flavonoid is the cyanidin(e) seen most, contributes yellow, orange, red a series of colour systems to purple.Flavonoid is water-soluble substances, there is a C15 skeleton, 9 classes are mainly divided into: cinnamophenone (chalcones) by structure, aurones (aurones), isoflavones (isoflavonoids), flavones (flavones), flavonol (flavonols), flavandiols (flavandiols), anthocyanogen (anthocyanins), condensed tannin (condensed tannins, i.e. pycnogenols, and acid anhydride of tanning (phlobaphenes) proanthocyanins), in them to the painted contribution of plant maximum be anthocyanogen, flavonol, cinnamophenone and aurones.Anthocyanogen is a maximum class flavonoid substances, for flower or tone such as the contribution of other tissue magenta, redness, purple, blueness etc., accumulate in the vacuole or pigment cell of cell, pelargonin (pelargonidin), Cyanidin (cyanidin), delphinidin (delphinidin) are modal three class anthocyanogen; Yellow tone is then provided by cinnamophenone, aurones, flavonol, flavones.

Flavonoid biosynthesis pathway in the plants such as petunia, corn, Common Snapdragon, Arabidopis thaliana is fully resolved.It is an important branch approach in common body propane biosynthetic pathway downstream, and phenylpropyl alcohol alkane approach is by multiple secondary substances such as other branch approach synthesis xylogen, stilbene class, tonka bean camphor, plant protecting chemicals.The first step of flavonoid path forms lurid 4,2 ', 4 ', 6 '-tetrahydroxy cinnamophenone by looking into ketone synthase (CHS) catalysis 1 molecule p-coumaric acyl-CoA with 3 molecule malonyl--CoA condensations.Anthocyanogen aglucon such as pelargonin, Cyanidin, delphinidin etc. take cinnamophenone as substrate, through a few step enzymes such as hydroxylation, reduction, oxidation, side base modification catalyzed reaction and formed, relate to CHI (enzyme, namely chalcone isomerase), F3H (flavanone 3-hydroxylase), F3 ' H (flavonoid 3 '-hydroxylase), F3 ' 5 ' H (flavonoid 3 ', 5 '-hydroxylase), DFR/FNR (dihydroflavonol 4-reductase/flavanone 4-reductase enzyme), ANS (anthocyanidin synthetic enzyme) etc. successively.Hydroxyl on the B-ring of the anthocyanidin (anthocyanidins) determined by F3 ' H and F3 ' 5 ' H is more, then color is more partial to blueness.Under normal circumstances; cinnamophenone and anthocyanidin are modified further; as glycosylation (glycosyltransferase; GT), methylate (methyltransgerase; MT), acylations (acyltransferase; AT), then transport (glutathione S-transferase, GST) to store in vacuole.In the vacuole of the orfe showy flowers of herbaceous plants, cinnamophenone 4 '-O-glucoside is transformed into jonquilleous aureusidin (6-O-glucoside) by aureusidin synthetic enzyme (AS).Gorgeous paulownia grass waits the orange to red flower of plant, and flavanone synthesizes 3-deoxidation anthocyanogen through a few step reaction.In addition, flavones and flavonol also play certain contribution to pattern, and they are colourless or light yellow, be total to look be used for promoting the formation of blue anthocyanogen and stablize by what is called.Flavones and flavonol are with flavanone and flavanonol for substrate respectively, synthesize under the catalysis of flavone synthase (FNS) and flavonol synthase (FLS).Except anthocyanidin, other flavonoid substances such as flavonol, cinnamophenone also may relate to the modifications such as glycosylation.

The research of model animals shows, the expression of flavonoid path structure gene (is had PAP by one by R2R3-MYB, PFG, TT2 etc.), basic helix-loop-helix (basic helix-loop-helix, bHLH, there is TT8, GL3, EGL3 etc.) and WD40 repetition (WDR, having TTG1 etc.) transcription factor complex that formed regulates and controls, this ternary complex has regulated and controled decorative flower Common Snapdragon, petunia, morning glory, the process that in the petal of African chrysanthemum and flower of Radix Gentianae, anthocyanogen is painted, regulation and control behavior is by organ, signal and light in the bodies such as tissue, the external signals such as ultraviolet affected.The research of Arabidopis thaliana shows, the expression of flavonol (glycosides) biosynthetic pathway of yellow hue is is then regulated and controled by PFG gene specific, and wherein the obviously division of labor occurs member MYB11, MYB12, MYB111 in organ specificity.

Carotenoid (carotenoids) is fat-soluble redness, orange and xanthein, is entrenched in the film of chloroplast(id) and chromoplastid.Carotenoid is C40-tetraterpenes compound, and synthesized by C5-isoprene basis unit, plant, fungi, algea and bacteria all can synthesize, though animal can not be synthesized, can take in and use the precursor of imitating element and vitamin A from food.Carotenoid is that many patterns contribute to yellow colour system, and for the petal of some plants such as rose, chrysanthemum contribute to orange/red, tawny and shade of brown separately or together with anthocyanogen.So far, almost whole key genes of Carotenoid in Plants biosynthetic pathway have been cloned and have identified, whole approach originates in the Isoprenoid (isopentenylpyrophosphate of the C5 in plastid, IPP) unit, form mixture between the Some Related Enzymes that it is believed that this approach and be incorporated on plastid film, 4 molecule I PP condensations are the GGPP (GGPP) of 1 molecule C20, under the catalysis of phytoene synthase (PSY), 2 molecule GGPP are coupled to the colourless phytoene of C40 head to head, it is first carotenoid.Subsequently, phytoene desaturase (PDS) and sigma carotene desaturase (ZDS) order in molecule introduces conjugated double bond, successively forms colourless phytofluene, lurid sigma carotene, orange-yellow neurosporene, red Lyeopene.Along with the increase of conjugated double bond number, absorbing wavelength moves to long wave direction.Some cisoid conformations produced in desaturation process are alltrans conformation by carotenoid isomerase (CRTISO, Z-ISO) catalyzed transitions.Lyeopene can by lycopene beta cyclase (LCYB) or Lyeopene ε-cyclase (LCYE) cyclisation, and this is a tapping point of this approach.Except the plants such as half head lettuce, in most plant, LCYE can only add ε-ring to Lyeopene, the alpha-carotene containing 1 β-ring and 1 ε-ring of synthesizing yellow and its derivative.Further hydroxylation can occur for β-and alpha-carotene or epoxidation is modified, and produces many new textures.The oxygen product that adds of carotene is called xanthophyll, and the dihydroxylation process of β-ring and ε-ring is completed by β-ring hydroxylase (CHYB) and ε-ring hydroxylase (CHYE) respectively.There is flower Idiotype and fruit differential type in PSY, GGPS and LCYB, the carotenogenesis approach that display existence chromoplastid is special.There is C5 in zeaxanthin epoxidase (ZEP) catalysis zeaxanthin, and 6 and C5 ', the epoxidation of 6 ' position, form yellow zeaxanthin epoxide and zeaxanthin diepoxide, it can be transformed into Neoxanthine again under the catalysis of Neoxanthine synthetic enzyme (NSY).9-cis-zeaxanthin diepoxide and 9-cis-Neoxanthine also can be used for synthesis dormin (ABA).

The regulatory gene clone of carotenogenesis approach need to strengthen, but research shows that the adjustment of this approach mainly occurs on transcriptional level, in acceptor and such environmental effects, PSY is important rate-limiting enzyme check point, photo-signal channel participates in the regulation and control to class radish element approach, and carotenoid cleavage dioxygenases (CCD)/NCED (9-cis-epoxy carotenoid dioxygenase) participates in important regulative from the angle of decomposing.In addition, VDE (Analysis of Violaxanthin De-Epoxidase) catalysis zeaxanthin diepoxide and zeaxanthin epoxide are converted into zeaxanthin.Arabidopis thaliana AtRAP2.2 has weak promoter action to the carotenoid accumulation in particular organization, but the hypoxia tolerance survival ability of the recently research function mainly organization of regulation control that shows it.Tomato DDB1 and DET1 suppresses carotenoid approach by the negative regulation of light reflex signal pathway, and wild cabbage OR (Orange) and tomato HSP21 is then the accumulation being promoted carotenoid by the formation of regulation and control chromoplastid.

In addition, also there is the 3rd class plant pigments, i.e. betanidin (betalains), it is the water-soluble alkaloid that a class is present in vacuole, the betaxanthin (betaxanthins) of the Betacyanins of purple colour system (betacyanins) and yellow colour system can be divided into, but in the plant that betanidin is only found in 10 sections of Caryophyllales and minority higher fungi, also do not find in other plant, and never find betanidin and anthocyanogen and be stored in same plant.

Because the pattern of the important decorative flower of majority is incomplete, there is the major defect on pattern, therefore the metabolic engineering of pattern molecular breeding has important application prospect.But comprise in the whole Brassica genus of rape, have no the report of the metabolic engineering rape pattern utilizing molecular breeding.

Summary of the invention

In view of this, an object of the present invention is to provide beta cyclase gene LCYB, ε-cyclase gene LCYE in interference brassica plant petal to express overexpression PAP gene simultaneously to prepare the application in the brassica plant kind that petal takes on a red color; Two of object of the present invention is to provide beta cyclase gene LCYB, ε-cyclase gene LCYE in interference brassica plant petal to express the plant expression vector of overexpression PAP gene simultaneously; Three of object of the present invention is the preparation method providing above-mentioned plant expression vector; Four of object of the present invention is to provide the method utilizing above-mentioned plant expression vector to prepare the brassica plant kind that petal takes on a red color.

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

1, disturb beta cyclase gene LCYB, ε-cyclase gene LCYE in brassica plant petal to express overexpression PAP gene simultaneously and prepare the application in the brassica plant kind that petal takes on a red color.

Preferably, the sequence that in described interference brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expresses is as shown in SEQ ID NO.2, and the sequence of described overexpression PAP gene is as shown in SEQ ID NO.3.

Preferably, the sequence that beta cyclase gene LCYB, ε in described interference brassica plant petal-cyclase gene LCYE expresses is mediated by Arabidopis thaliana petal specific promoter PAtAP3 and expresses, and the nucleotide sequence of described petal specific promoter PAtAP3 is as shown in SEQ ID NO.1 the 267th to the 1044th.

Most preferred, described brassica plant is swede type rape (Brassica napus).

2, disturb beta cyclase gene LCYB, ε-cyclase gene LCYE in brassica plant petal to express the plant expression vector of overexpression PAP gene simultaneously, described plant vector comprises the expression cassette of expression beta cyclase gene LCYB and the ε-cyclase gene LCYE RNA interference sequence mediated by petal specific promoter and is mediated the expression cassette of expressing PAP gene by constitutive promoter.

Preferably, the expression cassette that beta cyclase gene LCYB, ε-cyclase gene LCYE RNA interference sequence is expressed in described petal specific promoter mediation is made up of the reverse complementary sequence shown in the sequence shown in petal specific promoter PAtAP3, SEQ ID NO.2, intervening sequence, SEQID NO.2 and NOS terminator successively.

Preferred, described by constitutive promoter mediate express PAP gene expression cassette successively by CaMV35S promotor, sequence shown in SEQ ID NO.3 and NOS terminator composition.

3, the preparation method of described plant expression vector, comprise the steps: sequence shown in SEQ ID NO.1 after AscI and SwaI double digestion, be connected into the pFGC5941M plasmid cut through same enzyme, obtain pFGC5941CEPE plasmid, then the sequence shown in SEQ IDNO.2 is connected between the PAtAP3 promotor of pFGC5941CEPE carrier and intervening sequence, form intermediate carrier pFGC5941CEPE-B2RNAia, again sequence shown in SEQ ID NO.2 is oppositely connected between the intervening sequence of intermediate carrier pFGC5941CEPE-B2RNAia and OCS terminator, obtain pFGC5941CEPE-B2RNAi carrier, finally sequence shown in SEQ ID NO.3 is connected into the pFGC5941CEPE-B2RNAi carrier cut through same enzyme by NcoI and AscI restriction enzyme site, beta cyclase gene LCYB in brassica plant petal must be disturbed, ε-cyclase gene LCYE expresses the plant expression vector of overexpression PAP gene simultaneously.

Preferably, described intervening sequence is swede type rape PAP2 gene intron 2.

4, utilize described plant expression vector to prepare the method for the brassica plant kind that petal takes on a red color, comprise the steps:

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

B. step a gained engineering bacteria is transformed the hypocotyl of brassica plant aseptic seedling, after Dual culture, differentiation-inducing acquisition regrowth under Basta Herbicid resistant, screens positive seedling transgenic seedling and obtains the brassica plant kind that petal takes on a red color.

Beneficial effect of the present invention is: the invention provides the Brassica genus core species such as rape and radish pattern mechanism, specify that chrysanthemum, spend in vain, key differences expressing gene site between safflower, the present invention clones petal specific promoter and a kind of novel plant expression platform carrier being suitable for flower color gene engineering of transformation acquisition thus, then genes involved or gene fragment is cloned, build to obtain and a kind ofly in petal, suppress yellow carotenoid and accumulate the plant expression vector of Lyeopene and Kuromanine, the new type resource material of reddish oil cauliflower phenotype is createed after transforming rape, this technical tactic is initiating in the Brassica genus flower color gene engineerings such as rape, also be the first by carotenoid and flavonoid path associating metabolic engineering modified plant pattern.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 is the microscopic findings (A: swede type rape (BnY) of Brassica genus and radish petal; B: swede 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 the differential expression of DFR between Brassica genus petal and radish petal to DFR, TT8, GL3, EGL3 differential expression between Brassica genus petal and radish petal; B is the differential expression of TT8 between Brassica genus petal and radish petal; C is the differential expression of GL3 between Brassica genus petal and radish petal; D is the differential expression of EGL3 between Brassica genus petal and radish petal; BnYPe: swede type rape chrysanthemum lobe; BnWPe: swede type rape spends lobe in vain; BjYPe: mustard type rape chrysanthemum lobe; BrYPe: turnip type rape chrysanthemum lobe; BoYPe: kale chrysanthemum lobe; BoWPe: lobe spent in vain by cabbage mustard; RsRPe: the pale reddish brown lobe of radish red).

Fig. 3 is OR, CCD1 differential expression between Brassica genus petal and radish petal (BnYPe: swede type rape chrysanthemum lobe; BnWPe: swede type rape spends lobe in vain; BjYPe: mustard type rape chrysanthemum lobe; BrYPe: turnip type rape chrysanthemum lobe; BoYPe: kale chrysanthemum lobe; BoWPe: lobe spent in vain by cabbage mustard; RsRPe: the pale reddish brown lobe of radish red).

Fig. 4 is NOS-PAtAP3, B2RNAi and BnPAP1PCR amplification (A:NOS-PAtAP3; B:B2RNAi; C:BnPAP1).

In Fig. 5 pFGC5941CEPE and pBLycoRF7 vector construction process, enzyme cuts result (A:AscI and SwaI double digestion pFGC5941M; B:AscI and SwaI double digestion pMD19-T-NOS-PAtAP3; C:SwaI and AatI double digestion pFGC5941CEPE; D:SwaI and AatII double digestion pMD19-T-B2RNAi; E:BamHI and XbaI double digestion pMD19-T-B2RNAi; F:BamHI and XbaI double digestion pFGC5941CEPE-B2RNAia; G:NcoI and AscI double digestion pFGC5941CEPE-B2RNAi; H:NcoI and AscI double digestion pMD19-T-BnPAP1).

Fig. 6 is the petal color (A: control group that pBLycoRF7 transforms rapeseed plants; B: transgene rape).

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.

The vegetable material that the embodiment of the present invention adopts: swede type rape chrysanthemum lobe and grow seed in mid-term (in two No. 9 kinds), lobe (R9057 strain) is spent in swede type rape breast petal, turnip type rape chrysanthemum lobe (6Y733 strain), mustard type rape chrysanthemum lobe (CNG12011 strain), kale (B.oleracea var.acephala ftricolor, K10-3 strain) chrysanthemum lobe, cabbage mustard mutation in vain, radish red petal is all taken from Chongqing City of Southwestern University rape Engineering Technical Research Centre and to be had a rest the routine test plant in horse base.Arabidopis thaliana (Arabidopsis thaliana, Columbia wild-type) seed, purchased from international Arabidopis thaliana center, is planted in indoors artificial climatic chamber.

The reagent that the embodiment of the present invention adopts and test kit: PrimeScript ^tMrT reagent Kit with gDNAEraser (Perfect Real Time), premix Ex Taq ^tMiI (Tli RNaseH Plus) ROX plus, DNALigation 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 precious biological (TaKaRa) biotechnology company limited in Dalian; RNAprep Pure plant total RNA extraction reagent box is TIANGEN Biotech's product; Glue reclaims test kit, in a small amount method plasmid extraction test kit purchased from Shanghai Hua Shun Bioisystech Co., Ltd; Restriction enzyme is purchased from MBI Fermentas company of Lithuania; MS (Murashige & Skoog medium, including vitamins) substratum is Dutch Duchefa Products; The reagent such as DL-2000plus, Easy-Taq enzyme, dNTPs are purchased from Beijing full formula gold (Transgen) Bioisystech Co., Ltd; Other biochemistry and molecular biology reagent such as X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid), Rifampin (Rif), Streptomycin sulphate (Str), kantlex (Kan), penbritin (Amp), agarose, the saturated phenol of Tris, CTAB, Tris (pH=8.0), Tryptone, Yeast Extract, X-gal, IPTG, CTAB is purchased 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 adopts: Veriti ^tMmultiple temperature control PCR instrument purchased from American Applied Biosystems company; CFX96 quantitative real time PCR Instrument purchased from American Bio-Rad company; And molecular biology and engineered miscellaneous equipment facility.

Preferred embodiment PCR primer used synthesis and order-checking are completed by Shanghai English fine horse/Invitrogen Corp., the raw company trade such as work, Beijing six directions Hua Da in Shanghai.

The microscopic examination of embodiment 1, Brassica genus and radish petal colour developing subcellular organelle

Take fresh swede type rape chrysanthemum lobe (BnY) respectively; Swede type rape breast petal (BnW); Mustard type rape chrysanthemum lobe (BjY); Turnip type rape chrysanthemum lobe (BrY); Kale chrysanthemum lobe (BoY); Lobe (BoW) spent in vain by cabbage mustard; Radish red petal (RsR), be placed on the cold fresh preservation in ice face and transport laboratory back, then free-hand section, observes rapid screening with low power lens, and select qualified section and carry out examining and photograph, result as shown in Figure 1.Result shows, in the chrysanthemum lobe of the several species of Brassica genus, the material of displaing yellow is rendered as numerous fine granularity in a cell, is distributed in cell unevenly, is expressed to adherent position by vacuole, illustrate that in yellow petal, phaeochrome cell device is chromoplastid, its pigment is yellow carotenoid.Also have some cells to have light yellow chromoplastid in swede type rape breast petal, but total number is few and iuntercellular is inconsistent, cabbage mustard is spent in vain in lobe and almost be can't see chromoplastid, and explanation is that the minimizing of chromoplastid or disappearance cause its yellow shoal or disappear.Radish petal mediumpurple pigment is uniformly distributed in the middle position of each cell, and central authorities are denseer, and more offset from center is lighter, and illustrate that the colour developing subcellular organelle of radish petal is vacuole, substance that show color is Kuromanine.

Embodiment 2, detection Brassica genus and radish flower petal pigment

Just open swede type rape chrysanthemum lobe (BnY) is got respectively morning at full-bloom stage; Swede type rape breast petal (BnW); Mustard type rape chrysanthemum lobe (BjY); Turnip type rape chrysanthemum lobe (BrY); Kale chrysanthemum lobe (BoY); Lobe (BoW) spent in vain by cabbage mustard; Radish red petal (RsR), shady and coolly immediately fresh-keepingly transports laboratory back, 50 ~ 60 DEG C of oven dry, crosses 60 mesh sieves, lucifuge kept dry after grinding powder.

1, sherwood oil, hydrochloric acid and ammoniacal liquor test result

Take the petal powder 0.100g of preservation, put into respectively and compile number tool plug test tube, add sherwood oil, 10% hydrochloric acid, each about 10mL of 30% ammoniacal liquor respectively, mix gently, filter, observe colour-change, result is as follows:

(1) sherwood oil reaction: swede type rape, turnip type rape, mustard type rape all show glassy yellow, show that the content of carotenoids content is high; Kale shows light yellow, illustrates that it contains a small amount of carotenoid; And cabbage mustard and radish flower show colourless, show not containing carotenoid.

(2) hydrochloric acid test: only have radish red petal to show pink, illustrate containing Kuromanine, and swede type rape, turnip type rape, mustard type rape petal show yellow in various degree, illustrate containing flavones (alcohol) not containing anthocyanidin.Cabbage mustard is white and kale performance is closely colourless, illustrate not containing anthocyanidin, and flavones (alcohol) is also little.

(3) ammoniacal liquor test: each material of Brassica genus all shows yellow in various degree, illustrate that it contains flavones (alcohol) more or less, and the yellow-green colour that radish flower shows, this color is that the yellow that the blueness that presented by anthocyanogen and flavonoid present mixes, but all material does not all show orange red or red, illustrate not containing aurones.

2, the color reaction of flavonoid

Go bail for the petal powder 0.100g deposited, and uses methanol extraction 24h, filters, be settled to 50mL, respectively get 2mL extracting solution, then carry out following color reaction, observes colour-change.

(1) concentrated hydrochloric acid-magnesium powder reaction: add a small amount of magnesium powder, then after add concentrated hydrochloric acid 5, shake up gently, leave standstill 1h.Result shows, and the display of swede type rape, cabbage mustard is colourless, may contain cinnamophenone, aurones; Swede type rape, turnip type rape, mustard type rape display pole lavender blush and micro-purplish red, illustrates and does not contain cinnamophenone, aurones and catechin, may contain flavones, flavonol, flavanonol, flavanone; Radish flower shows pink, illustrates containing anthocyanidin.

(2) concentrated hydrochloric acid-zinc powder reaction: add a small amount of zinc powder, then add concentrated hydrochloric acid 10, shake up gently, leave standstill 1h.Result shows, and radish flower presents pink, illustrates containing Kuromanine.All the other are all colourless or yellow, illustrate not containing Kuromanine.

(3) plumbic acetate reaction: add 1.0% plumbic acetate 2mL, shake up gently, leaves standstill 2h.Result shows, and yellow mercury oxide in various degree all appears in all Brassica genus materials, illustrates that flavonoid possesses phenolic hydroxyl group and not containing phenyl styryl ketone and aurones, may have adjacent two phenolic hydroxyl groups or have 4-ketone group, 3-OH or 4-ketone group, 5-OH structure concurrently; There is green precipitate in radish flower, illustrates containing Kuromanine.

(4) ferric chloride reaction: add 5.0% iron trichloride 2ml, shake up gently.Result shows, and all material all occurs yellow, and not phenolic hydroxy group is described in pigment molecular.

(5) aluminum chloride reaction: add 1.0% aluminum chloride methanol solution l ml.Result shows, and all material all presents the different yellow of degree, illustrates containing flavonoid substances.

(6) strong sulfuric acid response: add the dense H of 1.5mL ₂sO ₄, shake up gently, then put boiling water bath 5min.All Brassica genus materials all present yellow in various degree, illustrate that in boiling water, 5min color does not change containing flavones (alcohol), illustrate not containing cinnamophenone, aurones, may not contain flavanone, possible isoflavone-containing and isoflavanone.Radish flower occurs orange-yellow, illustrates containing anthocyanidin.

(7) tetrahydro boron sodium reaction: add tetrahydro boron sodium 8mg, then add 1.0% hydrochloric acid 2mL, shake up gently, leaves standstill 2h.All Brassica genus materials all present the different yellow of degree, illustrate not containing flavanone and flavanonol.Radish flower presents extremely rose pink, illustrates containing flavanone and/or flavanonol.

(8) alkaline reagents reaction: add 5%Na ₂cO ₃3ml, shakes up gently, airtight standing 30min, blowing air 10min.All material all presents the different yellow of degree, and after blowing air, color is constant, illustrates not containing flavanonol.

(9) ammonia cesium chloride reaction: get methyl alcohol 10ml, add ammoniacal liquor and be settled to 25ml, become by the water saturated methanol solution of ammonia.In sample liquid, add 0.01mol/L strontium chloride methanol solution 10, then add by the water saturated methanol solution of ammonia 10, even with have gentle hands jog, leave standstill lh.Radish flower presents precipitation, has illustrated that 3 ', 4 '-dihydroxyl replaces.

(10) acid reaction: add 1.0% boric acid 10, then add 2.0%H ₃bO ₃3ml, cabbage mustard is spent lobe in vain and is presented colourless, illustrates that cabbage mustard is spent lobe flavonoid in vain and may not be contained C ₅-OH.

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

(1) chlorophyll: the petal 0.100g taking preservation, rapidly by liquid nitrogen grinding to powder, adopt volume fraction be the acetone of 90%: ethanol (4:l, V/V) 24h is extracted, filter, be settled to 25ml, adopt ultraviolet-visible spectrophotometer in the interscan of 200 ~ 700nm scope.Result shows, all samples, illustrates all not containing chlorophyll all without absorption peak at 662nm and 644nm place.

(2) carotenoid: the petal 0.100g taking preservation, rapidly by liquid nitrogen grinding to powder, add sherwood oil: acetone (1:1, V/V) 24h is extracted, filter, be settled to 25ml, adopt ultraviolet-visible spectrophotometer in the interscan of 200 ~ 700nm scope.Result shows, swede type rape, turnip type rape, mustard type rape, kale have absorption peak in 440 and about 470nm, illustrate containing carotenoid, the content that quantitative analysis measures is respectively 6.824,6.712,5.548,1.248mg/g.Cabbage mustard and radish petal then do not have charateristic avsorption band, illustrate not containing carotenoid.

(3) flavonoid: the petal 0.100g taking preservation, rapid liquid nitrogen grinding, to powder, adds acidified methyl alcohol (pH=3) 2ml and is placed in 4 DEG C of refrigerators and extracts 24h, filters, be settled to 25ml, with ultraviolet-visible spectrophotometer in the interscan of 220 ~ 600nm scope.Result shows, the extracting solution of swede type rape, turnip type rape, mustard type rape, kale, cabbage mustard, radish petal all has absorption peak in 330 and 270nm, illustrate that they contain flavonoids, the content that quantitative analysis measures be respectively 7.483,7.651,7.001,1.391,1.003,8.373mg/g.

(4) Kuromanine: the petal 0.100g taking preservation, rapidly by liquid nitrogen grinding to powder, add methanol extraction 24h, filter, be settled to 50ml, with ultraviolet-visible spectrophotometer in the interscan of 200 ~ 700nm scope.Result shows, radish flower petal pigment has obvious anthocyanogen charateristic avsorption band at about 532nm, is anthocyanogen band I absorption peak, and with the peak of a not too strong band II in 260-270nm region, the content that quantitative analysis measures is 237.27mg/100g.All Brassica genus sample standard deviations, without anthocyanogen absorption peak, namely do not contain anthocyanogen.

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

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

For the molecule mechanism of difference appears in research Brassica genus and radish flower petal pigment, the RT-PCR of design Brassica genus and radish flavonoid pathway gene detects primer, and using 5SrRNA as internal reference, specifically as shown in table 1:

Table 1, flavonoid path RT-PCR detect primer

Just open swede type rape chrysanthemum lobe (BnY) is got respectively morning at full-bloom stage; Swede type rape breast petal (BnW); Mustard type rape chrysanthemum lobe (BjY); Turnip type rape chrysanthemum lobe (BrY); Kale chrysanthemum lobe (BoY); Lobe (BoW) spent in vain by cabbage mustard; Radish red petal (RsR), liquid nitrogen freezing transports, and-80 DEG C of Refrigerator stores are for subsequent use.Then RNAprep Pure plant total RNA extraction reagent box extracted total RNA is used, through electrophoretic analysis and spectrophotometry qualified after, the DNA impurity in total serum IgE is removed with RNase-freeDNase I, remove genomic dna (gDNA) further with RT Reagent Kit With gDNA Eraser (Perfect Real Time) test kit and carry out reverse transcription, obtaining total cDNA first chain.Pcr amplification is carried out with a pair conserved regions primer of CHS gene family, target area span intron, electrophoresis showed all samples only expands and predicts cDNA band of the same size and do not have gDNA band, detect with internal standard gene 25SrRNA further, amplified band between each sample is special and luminance difference is little, thoroughly eliminate the gDNA in total serum IgE before reverse transcription is described, all samples reverse transcription success and total cDNA concentration are more or less the same, and may be used for the experiments such as quantitative gene expression PCR.

Then adopt premix Ex Taq ^tMiI (Tli RNaseH Plus) kit is at CFX96 ^tMreal-Time System carries out fluorescence real-time quantitative PCR, and the method according to specification sheets performs PCR program.Reaction system is that 20 μ L system quantitative RT-PCRs contain reverse transcription product 0.1 μ L, SYBR Premix Ex Taq ^tMiI (2 ×) 10 μ L, each primer (10 μMs) 0.4 μ L, all the other volumes ddH ₂o polishing.PCR loop parameter is: 95 DEG C of denaturation 3min; 50 amplification cycles (95 DEG C of sex change 10s, corresponding temperature annealing 30s, 72 DEG C of extension 30s); 65 DEG C of 5s; 95 DEG C of 5s.Setup Experiments 2 RNA extraction-reverse transcriptions repeat (biology repetitions), and each reverse transcription repeats 2 PCR (detecting repetition), 4 results averaged.And by the specificity of melting curve analysis confirmatory reaction, relative expression quantity is according to 2 ^{-Δ Ct}calculate, result as shown in Figure 2.

Result shows, and CHS, CHI, F3H, TTG1, PAP gene family is all vigorous or moderate expression in all material, and storeroom difference is little.F3 ' H, F3H, FLS, PFG (MYB12, MYBL2) all have expression in each material, and only only storeroom has height difference.DFR, ANS, TT19, TT8, GL3, EGL3 all significantly do not express or express extremely low in each material of Brassica genus, but expression amount is very high in radish.Illustrate that the synthesis of flavonol is all in vigorous or more vigorous working order in Brassica genus and radish petal, the synthesis of Kuromanine only in radish petal vigorous work but in Brassica genus petal, be in closing condition.

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

Occur the molecule mechanism of difference for studying Brassica genus and radish flower petal pigment further, the RT-PCR primer of design Brassica genus and radish kind carotene approach 17 functional site gene families, and using 25SrRNA as internal reference, concrete primer is as shown in table 2.

The carotenoid approach RT-PCR detection primer that table 2 the present invention is used

Then utilize method same as described above to carry out RT-PCR, and pass through the specificity of melting curve analysis confirmatory reaction, relative expression quantity is according to 2 ^{-Δ Ct}calculate, result as shown in Figure 3.

Result shows, all detection gene locuss of carotenoid approach are all expressed in the petal of 7 species material, but also there are some differences, one be Brassica genus white/newborn petal in CCD1 expression level obviously than high in chrysanthemum lobe, two is that in radish petal, OR is closing condition and does not express, and in Brassica genus diploid elementary species, OR expression level does not have in tetraploid aggregate species high yet.Illustrate that the disappearance of carotenoid in radish petal can not be formed with colour solid because OR closes, Brassica genus is white/newborn petal in the minimizing of carotenoid be because the decomposition of carotenoid is excessively strong.

The structure of embodiment 4, expression of plants platform carrier pFGC5941CEPE

According to gene expression difference on Brassica genus and radish petal carotenoid and flavonoid route of synthesis, the primer of design clonal bias expressing gene and construction of expression vector, concrete primer is as shown in table 3.

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

(1) acquisition of NOS-PAtAP3 fragment

With pCAMBIA2301 plasmid for pcr template, increase with combination of primers FTNOS and RTNOS and reclaim the fragment of 286bp, must containing the fragment of NOS terminator, its nucleotide sequence is as shown in SEQ ID No.1 1-286 position.The genome DNA adopting CTAB method to extract Arabidopsis leaf is pcr template, increase with combination of primers FPAtAP3 and RPAtAP3 and reclaim 778bp containing petal specific promoter PAtAP3 fragment (GenBank accession number:U30729), its nucleotide sequence is as shown in SEQ ID No.1 267-1044 position.Then reclaim two kinds as pcr template after products mixing, with combination of primers FTNOS and RPAtAP3 amplification, obtaining clip size is the fusion fragment NOS-PAtAP3 of 1044bp, and result is as shown in A in Fig. 4.Then cut glue to reclaim, the pMD19-T-NOS-PAtAP3 recombinant vectors be connected with pMD19-T, by the recombinant vectors transformation of E. coli DH5 α competent cell obtained, combination of primers FTNOS and RPAtAP3 is adopted to carry out PCR detection to the transformant mono-clonal of Amp resistance LB plate screening, sequencing result shows, and merges fragment NOS-PAtAP3 sequence as shown in SEQ ID No.1.

(2) structure of pFGC5941CEPE

Extracting pMD19-T-NOS-PAtAP3 recombinant plasmid, then carries out double digestion with AscI and SwaI, reclaims NOS-PAtAP3 fragment, to see in Fig. 5 shown in B; The pFGC5941M of extracting simultaneously plasmid, reclaims carrier framework, as shown in A in Fig. 5 after AscI and SwaI double digestion.The plant expression vector pFGC5941CEPE being suitable for flower color gene engineering of 11241bp is obtained after NOS-PAtAP3 fragment being connected with pFGC5941M plasmid, by pFGC5941CEPE transformation of E. coli DH5 α competent cell, coating containing Kan concentration is the LB dull and stereotyped top sieve selected monoclonal transformant of 50mg/L, the mono-clonal transformant combination of primers FTNOS and RPAtAP3 and F35S3N obtain screening and ROCST5N carries out PCR detection, the order-checking of screening positive clone, chooses sequence for subsequent use without the clone of sudden change.In its pFGC5941CEPE recombinant plasmid obtained, can be used for containing Bar expression casette (providing the resistance to herbicide basta) and 2 expression cassette inserting foreign gene in T-DNA interval, exogenous gene expression box 1, containing composition promoter CaMV 35S and NOS terminator, can be used for justice or the antisense expression of goal gene; Exogenous gene expression box 2, containing petal specific promoter PAtAP3, introns BnPAP2I2 (swede type rape (Brassica napus) PAP2 gene intron 2) and OCS PolyA terminator, can be used for the RNA interference of goal gene, justice or antisense expression.

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

Swede type rape petal total cDNA first chain adopting above-mentioned reverse transcription to obtain is template, adopt the RNA interference fragment (387bp of combination of primers FBLCYBi and RBLCYBi amplification LCYB gene family, be 411bp after adding restriction enzyme site and joint), with the RNA interference fragment (480bp of combination of primers FBLCYEi and RBLCYEI amplification LCYE gene family, be 506bp after adding joint), reclaim respectively.Then using after the recovery product balanced mix of 2 kinds of PCR as masterplate, 867bp fragment is increased into combination of primers FBLCYBi and RBLCYEI, the RNA interference fragment of this fragment series LC YB, LCYE two genes, called after B2RNAia, result is (being 893bp after adding restriction enzyme site) as shown in B in Fig. 4.Then B2RNAia is reclaimed, pMD19-T-B2RNAi is connected to obtain with pMD19-T, pMD19-T-B2RNAi transformation of E. coli DH5 α competent cell, combination of primers FBLCYBi+RBLCYEI is adopted to carry out PCR detection to the transformant mono-clonal of Amp resistance LB plate screening, send positive colony to check order, sequencing result is as shown in SEQ ID No.2.

(4) plant expression vector pFGC5941CEPE-B2RNAi is built

Extracting pMD19-T-B2RNAi plasmid, with SwaI and AatII double digestion (SwaI first cuts, then adds AatII), reclaims B2RNAi fragment, to see in Fig. 5 shown in D; Simultaneously extracting pFGC5941CEPE plasmid, after SwaI and AatII double digestion, reclaims carrier framework, to see in Fig. 5 shown in C.The B2RNAi fragment of recovery is connected between the PAtAP3 promotor of pFGC5941CEPE plant expression vector and introns BnPAP2I2, form intermediate carrier pFGC5941CEPE-B2RNAia, transformation of E. coli DH5 α competent cell, with the mono-clonal transformant of Kan resistance LB plate screening, then adopt combination of primers FPAtAP3 and FBLCYBi and RBLCYEI and RBnPAPI2 to carry out PCR detection respectively, PCR positive colony is for subsequent use.

Extracting pMD19-T-B2RNAi plasmid, with BamHI and XbaI double digestion, reclaims the sense fragment B2RNAi of B2RNAi, to see in Fig. 5 shown in E.Simultaneously extracting pFGC5941CEPE-B2RNAia plasmid, with BamHI and XbaI double digestion, reclaims carrier framework, to see in Fig. 5 shown in F.B2RNAi is connected into pFGC5941CEPE-B2RNAia plasmid (between introns BnPAP2I2 and OCS terminator), form plant expression vector pFGC5941CEPE-B2RNAi, by plant expression vector pFGC5941CEPE-B2RNAi transformation of E. coli DH5 α competent cell, with Kan resistance LB plate screening mono-clonal transformant, with FPAtAP3 and FBLCYBi, FBLCYBi and ROCST5N, RBLCYEI and RBnPAPI2, FBnPAPI2 and RBLCYEI4, PCR qualification is carried out to combination of primers again, the order-checking of positive colony, chooses sequence for subsequent use without the clone of sudden change.

(5) clone of the cDNA coding region of swede type rape PAP1 (BnPAP1) gene

We show in the research of the past, and swede type rape PAP1 (BnPAP1) gene is mainly expressed in seed coat, activate the synthesis of flavonoid-pycnogenols, also express in the cauline leaf that aging turns red, activate the synthesis of flavonoid-Kuromanine.Therefore, the present invention clones the cDNA coding region of BnPAP1 from the seed that swede type rape is grown, then for building just carrier and transforming rape overexpression in petal later.Extract the total serum IgE that swede type rape grows seed in mid-term, as front method remove gDNA after reverse transcription obtain the first chain cDNA, then with the first chain cDNA for template, adopt FBnPAP1 and RBnPAP1 combination of primers amplification BnPAP1 gene, obtain the coding region (being 754bp after adding restriction enzyme site) of BnPAP1 gene 744bp, electrophoresis result is as shown in C in 4, its glue is reclaimed, pMD19-T-BnPAP1 is connected into pMD19-T, transformation of E. coli DH5 α competent cell, combination of primers FBnPAP1 and RBnPAP1 is adopted to carry out PCR detection to the transformant mono-clonal of Amp resistance LB plate screening, send the order-checking of multiple positive colony, sequencing result display BnPAP1 sequence is as shown in SEQ ID No.3, choose sequence for subsequent use without the clone of sudden change.

(6) plant expression vector pBLycoRF7 is built

Extracting pMD19-T-BnPAP1 plasmid, with NcoI and AscI double digestion (AscI first cuts, then adds NcoI), reclaims BnPAP1 gene fragment, to see in Fig. 5 shown in H; Simultaneously extracting pFGC5941CEPE-B2RNAi plasmid, with NcoI and AscI double digestion, reclaims carrier framework, to see in Fig. 5 shown in G.The BnPAP1 gene fragment reclaimed be connected with pFGC5941CEPE-B2RNAi carrier framework, form the trivalent plant expression vector of series connection divalence RNAi and monovalence overexpression, called after pFGC5941CEPE-B2RNAi-BnPAP1ox, referred to as pBLycoRF7.Then by pBLycoRF7 transformation of E. coli DH5 α competent cell, respectively FPAtAP3 and FBLCYBi is used to the transformant mono-clonal of Kan resistance LB plate screening, FBLCYBi and ROCST5N, RBLCYEI and RBnPAPI2, FBnPAPI2 and RBLCYEI, F35S3N and RBnPAP1, FBnPAP1 and RTNOS6 to combination of primers respectively carrier whether successfully construct, then freeze-thaw method transform Agrobacterium tumefaciens LBA4404 is adopted after positive colony being extracted plasmid, then with containing Kan, the mono-clonal transformant of the resistance LB plate screening of Str and Rif, and carry out PCR detection by above-mentioned 6 pairs of combination of primers, screening positive clone is preserved as engineering strain, for Plant Transformation.

Embodiment 5, pBLycoRF7 transform rape

All tissue culture operations are all carried out under the plant tissue culture condition of standard, and the clean rank between Bechtop, cultivation, between domestication is respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagent, material, vessel all carry out aseptically process by code.In swede type rape typical case chrysanthemum kind, the seed of two No. 10 uses aseptic water washing 3 times with after 75% ethanol surface sterilization 1min, then 20min is soaked with 5% clorox, (MS powder 4.41g/L+Phytagel2.6g/L+ sucrose 30.0g/L in MS solid medium is inoculated in after aseptic water washing is clean, pH5.8, Autoclave moist heat sterilization; Do not add Phytagel and be liquid nutrient medium), in 25 DEG C, 2000Lux illumination, 16h/d photoperiod cultivate (between group training below, culture condition is except indicating except person especially, identical all therewith).The hypocotyl cutting seedling age about 8d aseptic seedling is cut into the segment being about 0.5 ~ 1.0cm, is inoculated into (MS substratum+1.0mg/L6-BA+1.0mg/L2,4-D) preculture 3d on pre-training substratum MSp.

-80 DEG C are preserved containing the LBA4404 engineering strain of pBLycoRF7 in the LB liquid nutrient medium being added with 100mg/L Kan, 20mg/L Str and 40mg/L Rif in 28 DEG C, 250r/min shaking culture 1 ~ 2d, make Agrobacterium grow to logarithmic phase, switching is cultivated once.Then at 5000rpm, 10min room temperature collected by centrifugation thalline, regulate bacterial concentration to OD with dip-dye substratum MSm (MS liquid nutrient medium+1.0mg/L2,4-D+1.0mg/L6-BA+100 μm of ol/L AS) ₆₀₀about about 0.5, be dip-dyeing solution.

Hypocotyl section after preculture is immersed 5 ~ 10min in dip-dyeing solution, intermittence is swayed gently, then hypocotyl section is blotted unnecessary bacterium liquid on sterilizing paper, be inoculated in common training substratum MSc (MS solid medium+2.0mg/L6-BA+0.5mg/LNAA), in 23.5 DEG C of light culture 48h.With sterilizing liquid substratum MSk (MS liquid nutrient medium+1.0mg/L2 after light culture, 4-D+1.0mg/L 6-BA+500mg/L Cef) washing by soaking explant 3 times, each 10min, then surface liquid is blotted with sterilizing paper, be transferred to induction screening culture medium MSi (MS solid medium+1.0mg/L6-BA+1.0mg/L2,4-D+500mg/L Cef+12.5mg/L Basta+6mg/L AgNO ₃) cultivate, every 2 weeks subcultures 1 time, to growing macroscopic kanamycin-resistant callus tissue, then are transferred to division culture medium MSd (MS solid medium+4.0mg/L6-BA+2.0mg/L ZT+5.0mg/L AgNO ₃+ 500mg/L Cef+12.5mg/L Basta) middle cultivation more than 14d, evoked callus differentiates budlet, be transferred to stem division culture medium MSs (MS solid medium+3.0mg/L6-BA+2.0mg/L ZT+500mg/L Cef+12.5mg/L Basta) to be again cultured to and to grow little stem, be transferred to again in long shoot substratum MSe (MS solid medium+0.05mg/L6-BA+500mg/L Cef+12.5mg/L Basta) and be cultured to long complete unrooted seedling, be transferred to root media MSr (MS solid medium+2mg/L NAA) to be again cultured to and to grow flourishing root system, seedling after taking root is after domestication, be transplanted to containing sterilizing perlite, vermiculite, in the basin alms bowl of turfy soil mixture (mass ratio is 1:1:1), manage by greenhouse pot culture.

Finally, pBLycoRF7 transforms in swede type rape and obtains 28 strain regeneration plants after two No. 10; 200mg/L Basta solution is dripped to regeneration plant blade and detects resistance, and extract blade genome DNA and adopt combination of primers F35S3N and RBnPAP1, FBLCYBi and ROCST5N to carry out PCR detection respectively, result shows the two positive transgenic plant of acquisition 11 strain.

Carry out comprehensive biology and agronomy observation to transgenic positive plant, result as shown in Figure 6.Result shows, the transfer-gen plant obtained grows and background proterties does not obviously change, but petal all becomes red, petal cell microscopic examination shows the existing chromoplastid that derives from of red material, also to have and derive from vacuole, biochemical component detects and also shows the new existing Lyeopene of red material produced, and also has Kuromanine.Showing can by method of the present invention, suppresses xanthein and accumulates Lyeopene and Kuromanine can create reddish oil cauliflower.

Self propagated is carried out to transgenosis plant in the present age, the blade the same with transgenosis plant in the present age is adopted to drip Basta Resistance detecting, Screening and Identification goes out transgene rape and to isozygoty excellent strain, its petal color is redder than the contemporary plant of transgenosis, illustrate transgene traits can genetic stability and offspring's strain of isozygotying than the present age (heterozygosis) individual plant transgene traits better.

Finally illustrate, above embodiment only in order to illustrate technical scheme of the present invention, but is not limited to this.Although by referring to the preferred embodiments of the present invention, invention has been described, but those of ordinary skill in the art is to be understood that, various change can be made to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.Here declare especially, the following change on application form also all must belong to the spirit and scope of the present invention and covered:

1, PAP1, LCYB, LCYE gene/gene fragment in the present invention, except nucleotide sequence listed in sequence table, also comprise the corresponding gene order come from parent species Chinese cabbage or wild cabbage, also the sequence coming from same other member of functional site gene family in these species is comprised, although nucleotide sequence listed in they and sequence table may have little difference.

2, the gene in the present invention and its fragment, except nucleotide sequence listed in sequence table, also to comprise with them at continuous 80bp and have more than 98.00% conforming any nucleotide sequence above.

3, the RNA interference fragment in LCYB, LCYE site in the present invention, except nucleotide sequence listed in sequence table, also comprises the fragment coming from other position of same gene.

4, in the present invention to LCYB, LCYE site express suppression, except the RNA perturbation technique lifted in preferred embodiment, the technology such as sense-rna, genome editor (ZFN, TALEN, CRISPR-Cas) can also be adopted to reach same or analogous object.

5, the gene in the present invention and its fragment, except the transformation carrier resembling the employing pFGC5941 lifted in preferred embodiments builds, other carrier can also be adopted to carry out plant expression vector construction, comprise and adopt other promotor and terminator, reach same or analogous object; Vector construct in the present invention, except the improvement leaf disk method resembling the employing Agrobacterium tumefaciens strain LBA4404 mediation of lifting in preferred embodiments carries out except genetic transformation, also can adopt other method to carry out Genetic Transformation in Higher Plants.

6, the gene in the present invention, gene fragment and vector construct, except resemble lift in preferred embodiments for except swede type rape, other sibling species of its parent species Chinese cabbage, wild cabbage and Brassica genus can also be applied to, reach same or analogous object.

Claims (10)

1. disturb beta cyclase gene LCYB, ε-cyclase gene LCYE in brassica plant petal to express overexpression PAP gene simultaneously and prepare the application in the brassica plant kind that petal takes on a red color.

2. application according to claim 1, it is characterized in that: the sequence that in described interference brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expresses is as shown in SEQ ID NO.2, and the sequence of described overexpression PAP gene is as shown in SEQ ID NO.3.

3. application according to claim 1, it is characterized in that: the sequence that in described interference brassica plant petal, beta cyclase gene LCYB, ε-cyclase gene LCYE expresses is mediated by Arabidopis thaliana petal specific promoter PAtAP3 and expresses, and the nucleotide sequence of described petal specific promoter PAtAP3 is as shown in SEQ ID NO.1 the 267th to the 1044th.

4. the application according to any one of claim 1-3, is characterized in that: described brassica plant is swede type rape (Brassicanapus).

5. disturb beta cyclase gene LCYB, ε-cyclase gene LCYE in brassica plant petal to express the plant expression vector of overexpression PAP gene simultaneously, it is characterized in that: described plant vector comprises the expression cassette of expression beta cyclase gene LCYB, ε of being mediated by petal specific promoter-cyclase gene LCYE RNA interference sequence and mediated the expression cassette of expressing PAP gene by constitutive promoter.

6. plant expression vector according to claim 5, is characterized in that: the expression cassette that beta cyclase gene LCYB, ε-cyclase gene LCYE RNA interference sequence is expressed in described petal specific promoter mediation is made up of the reverse complementary sequence shown in the sequence shown in petal specific promoter PAtAP3, SEQ ID NO.2, intervening sequence, SEQ ID NO.2 and NOS terminator successively.

7. plant expression vector according to claim 5, is characterized in that: described by constitutive promoter mediate express PAP gene expression cassette successively by CaMV35S promotor, sequence shown in SEQ ID NO.3 and NOS terminator composition.

8. the preparation method of plant expression vector described in any one of claim 5-7, it is characterized in that, comprise the steps: sequence shown in SEQID NO.1 after AscI and SwaI double digestion, be connected into the pFGC5941M plasmid cut through same enzyme, obtain pFGC5941CEPE plasmid, then the sequence shown in SEQ ID NO.2 is connected between the PAtAP3 promotor of pFGC5941CEPE carrier and intervening sequence, form intermediate carrier pFGC5941CEPE-B2RNAia, again sequence shown in SEQ IDNO.2 is oppositely connected between the intervening sequence of intermediate carrier pFGC5941CEPE-B2RNAia and OCS terminator, obtain pFGC5941CEPE-B2RNAi carrier, finally sequence shown in SEQ ID NO.3 is connected into the pFGC5941CEPE-B2RNAi carrier cut through same enzyme by NcoI and AscI restriction enzyme site, beta cyclase gene LCYB in brassica plant petal must be disturbed, ε-cyclase gene LCYE expresses the plant expression vector of overexpression PAP gene simultaneously.

9. preparation method according to claim 8, is characterized in that: described intervening sequence is swede type rape PAP2 gene intron 2.

10. utilize plant expression vector described in any one of claim 5-7 to prepare the method for the brassica plant kind that petal takes on a red color, it is characterized in that, comprise the steps:

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

B. step a gained engineering bacteria is transformed the hypocotyl of brassica plant aseptic seedling, after Dual culture, differentiation-inducing acquisition regrowth under Basta Herbicid resistant, screens positive seedling transgenic seedling and obtains the brassica plant kind that petal takes on a red color.