CN106609268A - Use of cucumber gene CsMYB6 in regulation and control of plant epidermal fur and fruit bur development - Google Patents

Abstract

The invention relates to a use of a cucumber gene CsMYB6 in regulation and control of plant epidermal fur and fruit bur development. An overexpression vector of a gene CsMYB6 related to epidermal fur development is constructed and then is transferred into Arabidopsis thaliana wild type and Arabidopsis thaliana gene MYB6 mutants and it is proved that overexpression of heterogenetic CsMYB6 can cause great reduction of the number of Arabidopsis thaliana leaf epidermal fur and the number of epidermal fur branches and thus the gene CsMYB6 has a part of functions the same to that of the Arabidopsis thaliana gene MYB6, can inhibit epidermal fur branch formation, is different from Arabidopsis thaliana gene MYB6, and can inhibit formation of Arabidopsis thaliana epidermal fur and cucumber fruit bur. The invention first validates the function of the gene CsMYB6 of cucumber and plays an important role in improvement of the quality of cucumber.

Description

Fructus Cucumidis sativi CsMYB6 genes are in regulation and control plant epidermal hair and the developmental application of fruit thorniness

Technical field

The invention belongs to agricultural biological technical field, specifically, is related to Fructus Cucumidis sativi CsMYB6 bases Because in regulation and control plant epidermal hair and the developmental application of fruit thorniness.

Background technology

Fructus Cucumidis sativi (Cucumis sativus L.) is Cucurbitaceae (Cucurbitaceae) Cucumises (Cucumis) annual herb plant, Chromosome number is 2n=2x=14.Fructus Cucumidis sativi originates in temperature The Himalayas southern foot north India area of warm moistening, in existing more than the 2000 years cultivation of China History, and become the Secondary centers of origin of Fructus Cucumidis sativi.Fructus Cucumidis sativi is the big important vegetable in the world four One of, it is also the Main Vegetable Species Suitable For Culture of China's facility cultivation, occupy in the vegetable production of China Critical role.

The fruit of Fructus Cucumidis sativi is by the Peponidium of ovary and holder symbiotic developmental.The commodity of fruit Property, such as melon, length, fruit tumor size, fruit thorniness density etc., is to affect product market sale Principal element.Eat raw compared with cucumber fruits are stingless tumor type with American-European countries, China market The cucumber fruits of supply are then most to pierce tumor type and a small amount of dilute thorn tumor type kind for close.Cause This, while the Comprehensive Traits such as disease resistance, yielding ability are considered, selection-breeding fruit thorn warty The kind that the exterior qualities such as condition accord with the demands of the market becomes one of target of breed cucumber.

Epidermal hair is a kind of structure specific to most plants aerial partss epidermal tissue, epidermis The form of hair is varied, and has different functions in different biologies.Some epidermal hairs by Unicellular composition, have by multiple cellularities；Have with branch, what is had does not then have point ；What is had has body of gland, the then Non-gland body having.As the epidermal hair of plant tissue surface, it Major function is to increase skin layer thickness, to reduce scattering and disappearing for heat and moisture；Simultaneously it is also protected Infringement and reduction mechanical damage and ultraviolet light harms of the shield plant from insecticide and pathogen.One The epidermal hair of a little specializations also has critically important economic function.Such as, cotton fiber (is also called kind Sublist fur) it is the important raw material of textile industry as important natural fiber.Fructus Cucumidis sativi etc. The epidermal hair of vegetable not only has defencive function, also affects the commodity nature of fruit, studies table The epidermal hair of bright Fructus Cucumidis sativi is many cells, without branch's epidermal hair, on the fruit thorniness on fruit and blade Epidermal hair morphosiss are the same, are the eglandular epidermal hair of many cells, the epidermal hair on stem and leaf It is made up of 4-9 elongated cells, vertical shape is arranged in shaft-like, and the cell of base portion expands, top Cell is in shaft-like, and cell wall keratinization, quality is hard and crisp.Cucumber plant stem, leaf, tendril, Calyx, ovary surface are covered with epidermal hair, and most cucumber variety ovary surface spinosity tumors pierce tumor Grow up therewith as fruit development expands.

Model plant arabidopsiss epidermal hair is dispersed throughout blade, sepal, petiole and stem, Non-gland body, It is typical unicellular epidermal hair.It is general a length of 200-500 μm, 3-5 branch.Different On organ, the form and density of epidermal hair have very big difference, and lotus throne leaf epidermal hair has 3-4 points , and branch's number of stem leaf epidermal hair is less, the epidermal hair on stem does not have branch.Arabidopsiss Although epidermal hair does not secrete chemical substance, the infringement of plant-eating animal can be hindered.Arabidopsiss table The growth course of fur can study as a kind of good modular system cell fate determine, it is thin Problem in terms of the cell differentiations such as born of the same parents' cycle regulating, cell polarity and cell increase.In recent years, The Study on Molecular Mechanism that it is developed is had made great progress.

The epidermal hair research of arabidopsiss trichome development being concentrated mainly at present on lotus throne leaf, The procuticle cell development that they are quickly divided by new leaf base.Set up in morphological criteria On the basis of, arabidopsiss trichome development can be divided into 6 stages.Stage 1：Initial period, one Relative to the epidermis cell rapid expanding of surrounding, nucleus carry out multiple in core the epidermis cell of orientation System.Stage 2：When epidermal hair progenitor cells expand to bigger than peripheral cell diameter 2-3 times, open Begin the projection for occurring perpendicular to epidermis cell aspect, generates the rod-like structure of epidermis hair cell. Stage 3：Subsequently, the cellular apical for growing develops the conjugate focus of cell increase, table Fur branch starts generation.Arabidopsiss leaf epidermal hair can typically form 2-4 according to Geographic strain Branch.The direction of branch is axially aligned with each other to top with blade base, and the angle between branch It is fairly regular.Stage 4：After all branches occur, cell continues to increase by dispersivity growth Greatly, so the cell will increase on diameter and height.Originally, the table for growing Fur branch is blunt.Stage 5：Subsequently, top gradually comes to a point.Stage 6：In cell increase After stopping, the mastoid process that epidermal hair cell surface quantity of formation is not waited around is supported by a circle epidermis Cell is surrounded.Epidermal hair after the growth course that experienced this 6 stages, its shape, size, Spatial arrangement there occurs essence change, imply this process complete need the ginseng of lots of genes With.A large amount of Arabidopsis Mutants defective in terms of trichome development have been separated. Classified according to the period of gene mutation impact trichome development, mutant can be divided into 5 classes.The starting of 1st class and trichome development is related, shows as increasing or reducing for epidermal hair quantity； 2nd class is extremely related to the regulation and control of endoreduplication, and these include endoreduplication number of cycles extremely Change and the turn model from mitosiss to endoreduplication change；3rd class mutant Show as increasing or reducing for epidermal hair branch number；4th class mutant and epidermal hair stretching, extension side Position is related, shows as epidermal hair random growth；5th class mutant shows as the maturation of epidermal hair Pattern exception.

Study at present more epidermal hair branch controlling gene have AN, NOK, STI, FRC, TFCA, TFCC and ZWI.In recent years, NOK genes are confirmed as MYB6 genes, a kind of MIXTA-like class transcription factor, serves weight in the generation of control arabidopsiss epidermal hair branch Act on.There are some researches show that micro-pipe and microfilament are served during the formation of epidermal hair branch important Effect, micro-pipe participates in starting and the positioning that epidermal hair projection is formed, and microfilament participates in ensuing prominent Play prolongation process.

Sharon Regan etc. have found that the MYB6 genes that epidermal hair branch is controlled in arabidopsiss exist Homologous geness PtaMYB186 in willow take part in the initial development process of willow epidermal hair. 35S::PtaMYB186 plant leaf epidermal hair quantity increases in a large number, while the life of transfer-gen plant Growing way and the repellence to eating grass insecticide also improve a lot.This illustrates to participate in arabidopsiss The gene that epidermal hair branch is formed plays no less important in the crop without epidermal hair branch Effect.Recent study shows that the morphogenesis of Cotton Gossypii epidermal hair may be by GhMYB25 genes Regulation and control, GhMYB25 is also a R2R3MYB genoid, a kind of MIXTA-like transcription because Son, enlivens in the early expression of cotton fiber cell development.Under the regulation and control of GL1 gene promoters, The over-express vector of GhMYB25 can produce complete epidermis in gl1 Arabidopsis Mutants Hair；While 35S::Epidermal hair has been differentiated on the seed of GhMYB25 Arabidopsis plants, GhMYB25 is included and can be implied that it may pass through with the conserved amino acid sequence of bHLH interactions Regulate and control fibrocellular differentiation with bHLH albuminoids interaction.In Gossypium hirsutum L., GhMYB109 Also express in the fiber of development, also comprising the conserved amino acid sequence with bHLH interactions； The gene of two coding WD-repeat albumen is cloned, the expression of the two genes can be with complementation Arabidopsiss ttg1 mutants.These results of study show to there may be phase in arabidopsiss and Cotton Gossypii As epidermal hair Forming Mechanism.Compared with unicellular epidermal hair, the formation machine of many cells epidermal hair System is likely more complexity, in addition it is also necessary to research and analyse epidermal hair shape in more other floristics Into mechanism.The relevant report with Fructus Cucumidis sativi MYB6 gene studiess is not yet found at present.

The content of the invention

It is an object of the invention to provide Fructus Cucumidis sativi CsMYB6 genes are in regulation and control plant epidermal hair and fruit thorniness Developmental application.

In order to realize the object of the invention, the present invention provides Fructus Cucumidis sativi CsMYB6 genes in regulation and control plant Epidermal hair and the developmental application of fruit thorniness, wherein the nucleotides sequence of the Fructus Cucumidis sativi CsMYB6 genes It is classified as：

I) nucleotide sequence shown in Seq ID No.1；

Ii) under strict conditions with sequence hybridization shown in Seq ID No.1 and express identical function egg The nucleotide sequence of white matter；Or

Iii) with i) or ii) nucleotide sequence there is more than 90% homology and the identical work(of expression The nucleotide sequence of energy protein；

The stringent condition is 0.1 in 0.1 × SSPE containing 0.1%SDS or containing 0.1%SDS In × SSC solution, hybridize at 65 DEG C, and film is washed with the solution.

Aforesaid application, the plant is dicotyledon.Preferably, the dicotyledon For arabidopsiss, Fructus Cucumidis sativi.

Aforesaid application includes Fructus Cucumidis sativi CsMYB6 genes being proceeded in purpose plant, obtains and institute State purpose plant to compare, epidermal hair and fruit thorniness develop downtrod transgenic plant.The epidermis Hair educate it is suppressed be presented as compared with purpose plant, the blade epidermis of the transgenic arabidopsis Approximate number and branch's number are reduced, and transgenosis cucumber fruit thorniness number is reduced.

Aforementioned applications are specially：The Fructus Cucumidis sativi CsMYB6 genes described in overexpression in purpose plant, The epidermal hair of render transgenic plant and fruit thorniness number are reduced.

Preferably, the expression vector that overexpression is used is pBI121.

The present invention further provides a kind of method of cucumber quality improvement, builds Fructus Cucumidis sativi CsMYB6 The restructuring over-express vector of gene, and using agriculture bacillus mediated method, the restructuring that will be built Expression vector is proceeded in Fructus Cucumidis sativi, obtains (stingless type) transgenosis cucumber of quality-improving.

Aforesaid method, by the multiple clone site of Fructus Cucumidis sativi CsMYB6 gene insertion vector pBI121 Between, that is, build and obtain the over-express vector CsMYB6-pBI121 that recombinates.

The present invention is carried by building the overexpression of gene C sMYB6 related to trichome development Body, and proceeded in arabidopsiss wild type and arabidopsiss MYB6 gene mutation bodies, it was demonstrated that it is different The overexpression of source CsMYB6 can cause Arabidopsis leaf epidermal hair quantity and epidermal hair branch number Purpose is reduced in a large number.This shows that CsMYB6 genes may have part with arabidopsiss MYB6 genes Identical function, it is suppressed that the formation of epidermal hair branch, and different from arabidopsiss MYB6, CsMYB6 genes suppress the formation of arabidopsiss epidermal hair and Fructus Cucumidis sativi fruit thorniness simultaneously, and the present invention is first The function of Fructus Cucumidis sativi CsMYB6 genes is demonstrated, for the improvement of cucumber quality has important function.

Description of the drawings

Fig. 1 is the phenotype of overexpression transgenic CsMYB6 Fructus Cucumidis sativi strains in the embodiment of the present invention 2.

Fig. 2 be the embodiment of the present invention 2 in overexpression transgenic CsMYB6 Fructus Cucumidis sativi strains Line1,2 Fructus Cucumidis sativi with 3 blooms fruit thorniness number statistical result on same day fruit cross section.

Fig. 3 is the table of overexpression transgenic CsMYB6 arabidopsiss strains in the embodiment of the present invention 2 Type；Wherein, Col represents wildtype Arabidopsis thaliana, and myb6 represents arabidopsiss myb6 mutants, 35S:CsMYB6::What myb6 represented CsMYB6 heterologous transformant arabidopsiss myb6 mutants turns base Because of plant, 35S:CsMYB6::Col represents turning for CsMYB6 heterologous transformant wildtype Arabidopsis thalianas Gene plant.

Specific embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not Specialize, embodiment is according to conventional laboratory conditions, the such as cloning experimentation of Sambrook equimoleculars Handbook (Sambrook J＆Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer's description suggestion.

The clone of the Fructus Cucumidis sativi CsMYB6 genes of embodiment 1

1.1 material

With Fructus Cucumidis sativi 3407 (being provided by China Agricultural University's agronomy and Biotechnology Institute) as examination material, The kind epidermal hair is obvious, thorn tumor is intensive.Cucumber seeds are sowed in heliogreenhouse, are carried out general Logical cultivation management, when plant to be planted starts to tie melon, takes buds liquid nitrogen flash freezer, is stored in -80 DEG C Refrigerator store, it is standby.

The extraction of 1.2 total serum IgEs synthesizes with cDNA

1.2.1 the extraction of total serum IgE

Bounties Gene science limited public affairs in Beijing day (are purchased from using pillar plant RNA out test kits Department, catalog number is 71203) to extract the total serum IgE of cucumber fruits, and is removed with DNase The DNA remained in RNA sample.Step is as follows：

(1) about 200mg cucumber fruits are added in mortar, liquid nitrogen grinding is added to powder. Abrasive material is transferred to into the clean 1.5ml plastic centrifuge tubes without RNase, adds 1ml cells to split Solution liquid (test kit is carried), fully vibration are mixed.

(2) protein liquid removal (test kit is carried) and 200 μ l of 300 μ l are added in centrifuge tube Provide chloroform for oneself, vibrate on the oscillator 30 seconds and mix, now solution is in uniform milkiness shape.Shake Swinging shake must ttom of pipe solution when device vibrates.Room temperature 12000rpm is centrifuged 3-5min, The two alternate cell breakage things for having about 5-10mm thick.

(3) supernatant (about 600 μ l) is transferred to into another clean 1.5ml plastic centrifuge tubes In, DNA, protein and other impurities are contained in lower floor's organic faciess and intermediate layer, it is to avoid touch or Draw.Preferably leave 100 μ L of supernatant liquid not take.Isopyknic rinsing liquid is added, it is fully reverse Mix.The solution of half is transferred in centrifugal adsorbing column, 12000rpm room temperatures centrifugation 1min, Abandon and penetrate liquid.

(4) remaining half solution is transferred in same centrifugal adsorbing column, 12000rpm Room temperature is centrifuged 1min, abandons and penetrates liquid.

(5) plus 700 μ l are general washes post liquid, 12000rpm room temperatures centrifugation 1min is abandoned and is penetrated liquid. Plus 300 μ l it is general wash post liquid, 12000rpm room temperatures centrifugation 1min is abandoned and is penetrated liquid.12000rpm Room temperature is centrifuged 1min.This step is particularly significant, and the ethanol for otherwise remaining can affect the use of RNA. Central authorities toward adsorption column add 50 μ l DNAase reactant liquors, are stored at room temperature 10-15min, add 500 μ l are general to wash post liquid, and 12000rpm room temperatures centrifugation 1min is abandoned and penetrated liquid.12000rpm Room temperature is centrifuged 1min.

(6) centrifugal adsorbing column is transferred in RNase-free collecting pipes, adds 50 μ l RNA Eluent, room temperature places 1-2min.12000rpm room temperatures are centrifuged 1min, solution in centrifuge tube As RNA sample, deposit in -80 DEG C it is stand-by.

1.2.2cDNA the synthesis of the first chain

With 1 μ g cucumber fruits RNA as template, utilizeRTase synthesizes cDNA First chain.Reverse transcription operation carries out [test kit title with reference to test kit explanation：1st Strand cDNA Synthesis Kit (50 secondary amounts)；Beijing six directions is stimulated the menstrual flow Trade company limited；Catalog number：D6110A].

The synthesis of the chains of cDNA first is saved backup after -20 DEG C.

The clone of 1.3 Fructus Cucumidis sativi CsMYB6 genes

Transcript profile sequencing is carried out to a pair of wild types and mutant material of Fructus Cucumidis sativi, according to sequencing knot The analysis of fruit and the annotation of gene, select one of candidate gene to be studied, temporarily name For CsMYB6 genes, according to the gene I/D (Csa3M824850) of CsMYB6 in cucumber gene Group data base (http://cucumber.genomics.org.cn/page/cucumber/index.jsp) In find the CDS sequences of the gene, according to its CDS full length sequences design primer：

Forward primer：5’-ATGGGAAGGTCTCCTTACTGCG-3’(Seq ID No.3)

Reverse primer：5’-TCAGAATCTCAGGAATTCACCAAGA-3’(Seq ID No.4)

With cDNA as template, using above-mentioned forward and reverse primer, obtain yellow by PCR amplifications Melon CsMYB6 genes.The nucleotide sequence of Fructus Cucumidis sativi CsMYB6 genes as shown in Seq ID No.1, The aminoacid sequence of its encoding proteins is as shown in Seq ID No.2.

Pcr amplification reaction system：5U/ μ l Taq archaeal dna polymerases (TaKaRa) 0.25 μ l, 10 × PCR reaction buffer (Mg²⁺Plus the μ l of) 5 μ l, dNTP Mixture (each 2.5mM) 4, Template DNA 2.5ng, each 1 μ l of 20 μM of forward and reverse primers, sterile purified water complements to totality 50 μ l of product.

PCR amplification programs：95℃3min；95 DEG C of 10sec, 54 DEG C of 30sec, 72 DEG C of 60sec, Totally 30 circulations；72℃5min.

1.4 vector construction

1.4.1 recombinate over-express vector structure

The expression vector for using is pBI121, is XbaI and SmaI from restriction enzyme site.In purpose The ORF areas two ends design primer of gene, and add phase at primer (Seq ID No.3 and 4) 5' ends The restriction enzyme site answered and protection base, by template of Cucumber cDNA performing PCR amplification is entered.

Pcr amplification reaction system：5U/ μ l Taq archaeal dna polymerases (TaKaRa) 0.25 μ l, 10 × PCR reaction buffer (Mg²⁺Plus the μ l of) 5 μ l, dNTP Mixture (each 2.5mM) 4, Template DNA 2.5ng, each 1 μ l of 20 μM of forward and reverse primers, sterile purified water complements to totality 50 μ l of product.

PCR amplification programs：95℃3min；95 DEG C of 10sec, 54 DEG C of 30sec, 72 DEG C of 60sec, Totally 30 circulations；72℃5min.

Pcr amplification product is reclaimed, with carrier pBI121 respectively with special restricted enzyme enzyme Cut, endonuclease reaction system is：10 × NEB buffer 2：4.0μl；PCR primer or carrier：1μg； Enzyme XbaI：2.0μl；Enzyme SmaI：2.0μl；ddH₂O complements to 50 μ l.37 DEG C of enzyme action are overnight. Gel reclaims digestion products, is then connected with T4 ligases, converts bacillus coli DH 5 alpha, PCR Positive bacterium solution after identification send Shenzhen Huada Genetic Technology Co., Ltd to be sequenced, sequencing it is correct then into Work(builds restructuring over-express vector, is named as CsMYB6-pBI121 (i.e. 35S:CsMYB6). Extract plasmid standby.

1.5 Agrobacterium-mediated Transformation

Restructuring over-express vector CsMYB6-pBI121 conversion Agrobacteriums, electroporation are opened and are preheated The electricity revolving cup of pre-cooling simultaneously is some.The Agrobacterium competent cell of 80 μ l is placed in into thawed on ice, plus Enter the recombiant plasmid CsMYB6-PBI121 that 1-2 μ l build, flick mixing, be transferred to electric revolving cup Bottom.Electric revolving cup is placed on the seat of electroporation, is shocked by electricity.1ml is quickly added not contain antibiotic Liquid YEB culture medium into electric revolving cup, softly blow and beat suspension cell.By cell suction out to In Eppendorf pipes, 28 DEG C of slowly vibrating culture 1h.Draw 100 μ l bacterium solutions be added to containing card that It is with aseptic elbow glass rod with gentle that cell is uniform on the YEB solid mediums of rifampicin Spreadable, room temperature is placed after planar surface is dried, and with sealed membrane flat board is sealed.It is inverted flat board, 28 DEG C of culture 36-48hr.PCR evaluation and screening positive bacterias, PCR identifies that primer used is：

Forward primer：5’-ATGGGAAGGTCTCCTTACTGCG-3’(Seq ID No.5)

Reverse primer：5’-TCAGAATCTCAGGAATTCACCAAGA-3’(Seq ID No.6)

As a result the positive Agrobacterium of the purpose fragment containing 702bp is obtained.

Embodiment 2CsMYB6 gene overexpression carrier arabidopsis thaliana transformation and Fructus Cucumidis sativi

The acquisition of 2.1 transgenic arabidopsis

(1) arabidopsiss material used by is wild type Col and arabidopsiss myb6 mutant 49c (purchases From ABRC, catalog number is CS28169 and SALK_025449).Broadcast after seed disinfection On MS solid mediums, 4 DEG C process 2 days after be put into phjytotron, condition of culture is： 16hr illumination/8hr is dark, 22 DEG C of constant temperature.

(2) transformation of Arabidopsis thaliana

By the positive Agrobacterium inoculation containing purposeful carrier in containing kanamycin and rifampicin YEB fluid medium (Kan：50μg/mL；Rif：50 μ g/mL) in, 28 DEG C of vibration trainings Support overnight to OD₆₀₀=1.0-2.0.4000rpm room temperatures are centrifuged 15min, collects thalline.With 200mL MS saline solution (1/2MS, 5% sucrose, 200 μ L/L Silwet L-77) suspended bacteria Body.Arabidopsis plant is inverted, 5s in inflorescence immersion Agrobacterium-mediated Transformation liquid is made, is repeated twice. The erect plants contaminated are positioned in pallet, are covered to protect with the plastic bag of black non transparent Hold humidity.Plastic bag is removed after 24hr, continues to cultivate to results T₀For seed.

Meanwhile, if unconverted Col is wild type control Arabidopsis plant.

(3) the PCR detections of transgenic Arabidopsis plants

Prepare the MS screening culture medium of the final concentration of 20 μ g/ml of Kan.Harvest the T of transformed plant₀ For seed, it is seeded in after sterilization in MS screening culture medium, 4 DEG C of vernalization treatments are transferred to two days later It is dark in 16hr illumination/8hr in illumination box, cultivated under the conditions of 22 DEG C of constant temperature.Two weeks The positive plant of energy normal growth in screening culture medium is chosen at afterwards, to be moved into continue in hole tray and is trained Support.After arabidopsiss bolting, transformant T is taken₀Generation (3 strains) lotus throne leaf, respectively intending south Mustard plant, the cDNA of the lotus throne leaf of wild type control Arabidopsis plant are template, are designed as follows Primer：

Forward primer：5′-CCAAAGCCGGTCTTGAGAGA-3′(Seq ID No.7)

Downstream primer：5′-GCTATGGCAGACCACCTGTT-3′(Seq ID No.8)

The total length CDS sequence of amplification CsMYB6.After obtaining transfer-gen plant, reference pair is according to plant Strain, observation turns CsMYB6 gene plants phenotype and finds transformed plant blade epidermal hair number and divide Prop up number to significantly reduce (table 1).

The 35S of table 1:CsMYB6 arabidopsis thaliana transformations T2 is for blade epidermal hair quantity statistics

The acquisition of 2.2 transgenosis cucumbers

(1) full complete cucumber seeds are selected, kind of a shell is peelled off, first sterilize 30s in 75% ethanol, The 15min of sterilizing is placed in 3.0% liquor natrii hypochloritises again, and aseptic water washing is broadcast in MS several times later In culture medium, after its cotyledon is sprouted lower step experiment (about 2 days) is carried out.

(2) Agrobacterium of activation is prepared.

(3) the young tender cotyledon of cucumber seeds is taken, every cotyledon after growing point is removed and is divided into uniform 2 Block, only retains the half near growing point.The cucumber cotyledons for cutting are immersed into bacterium solution 15 minutes； Then unnecessary bacterium solution is blotted with the filter paper of sterilizing, accesses MS division culture mediums；It is under the low light level or dark Place, 28 DEG C co-culture two days.

(4) explant after co-culturing is proceeded to containing kanamycin 50mg/L, Carbenicillin On the MS division culture mediums of 500mg/L, and constant temperature culture (25 DEG C, light application time 14h/d, light According to intensity 2000LX).

(5) about 15-20d, when resistant budses length to 1cm or so, is cut immigration MS cultures Root induction in base (100mg/L containing kanamycin, Carbenicillin 200mg/L), obtains Resistant plant.

(6) after transgenosis cucumber root system development is good, immigration is filled in the flowerpot of sterile soil, temperature Room Routine Management.

(7) the PCR detections of transgenosis cucumber plant and fluorescence quantitative PCR detection.To all turns The cucumber plant of gene takes buds and carries out the synthesis of first chain of RNA extractions and cDNA, together The description of embodiment 1.Quantitative fluorescent PCR analysis used kit is SYBR Premix Ex Taq (TaKara, DRR041S), quantitative real time PCR Instrument is ABI 7500, is designed as follows Primer：

CsMYB6-F:5′-CCAAAGCCGGTCTTGAGAGA-3′(Seq ID No.9)

CsMYB6-R:5′-GCTATGGCAGACCACCTGTT-3′(Seq ID No.10)

The reaction system of quantitative fluorescent PCR is：

Amplification program is：95℃,30sec；95℃,5sec；60 DEG C, 40sec, 40 circulations.With ACTIN genes are internal reference, utilize 2^-△△CtAlgorithm calculates each gene expression amount change.

As a result：6 plants of overexpression plant are obtained, CsMYB6 gene expression amounts are obviously higher than control.

The Phenotypic examination of 2.3 overexpression gene C sMYB6 plant

Treat above-mentioned turn CsMYB6 gene cucumber plants, turn empty vector control plant and wild type pair During according to plant length to fruiting period, taking the fruit of different developmental phases carries out observing its phenotype, as a result As shown in figure 1, the number of the fruit thorniness in the transgenic line cucumber fruits of CsMYB6 gene overexpressions Amount is substantially few than turning the adjoining tree of empty carrier.Statistics overexpression transgenosis cucumber strain Line1,2 Fructus Cucumidis sativi bloom fruit thorniness number on same day fruit cross section, as a result as shown in Figure 2.

Transgenic CsMYB6 arabidopsiss are as shown in Figure 3 with the phenotype for compareing wildtype Arabidopsis thaliana.

Although above having made in detail to the present invention with a general description of the specific embodiments Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this It is obvious for art personnel.Therefore, on the basis without departing from spirit of the present invention Upper these modifications or improvements, belong to the scope of protection of present invention.

List of references

Sharon Regan,Endogenous overexpression of Populus MYB186increases trichome density,improves insect pest resistance,and impacts plant growth.The Plant Journal,2010,64,419–432

Adriane Machado,The MYB transcription factor GhMYB25regulates early fibre and trichome development.The Plant Journal.2009,59,52–62

Claims (8)

1. Fructus Cucumidis sativi CsMYB6 genes are in the regulation and control plant epidermal hair and developmental application of fruit thorniness, its It is characterised by, the nucleotides sequence of the Fructus Cucumidis sativi CsMYB6 genes is classified as：

I) nucleotide sequence shown in Seq ID No.1；

Ii) under strict conditions with sequence hybridization shown in Seq ID No.1 and express identical function egg The nucleotide sequence of white matter；Or

Iii) with i) or ii) nucleotide sequence there is more than 90% homology and the identical work(of expression The nucleotide sequence of energy protein；

The stringent condition is 0.1 in 0.1 × SSPE containing 0.1%SDS or containing 0.1%SDS In × SSC solution, hybridize at 65 DEG C, and film is washed with the solution.

2. application according to claim 1, it is characterised in that the plant is dicotyledonous Plant.

3. application according to claim 2, it is characterised in that the dicotyledon is Arabidopsiss, Fructus Cucumidis sativi.

4. the application according to any one of claim 1-3, it is characterised in that by Fructus Cucumidis sativi CsMYB6 genes are proceeded in purpose plant, are obtained compared with the purpose plant, epidermal hair and Fruit thorniness develops downtrod transgenic plant.

5. application according to claim 4, it is characterised in that table is crossed in purpose plant Up to the Fructus Cucumidis sativi CsMYB6 genes, the epidermal hair of render transgenic plant and fruit thorniness number are reduced.

6. application according to claim 5, it is characterised in that the expression that overexpression is used Carrier is pBI121.

7. the method that cucumber quality is improved, it is characterised in that build Fructus Cucumidis sativi CsMYB6 genes Restructuring over-express vector, and using agriculture bacillus mediated method, the restructuring overexpression for building is carried Body is proceeded in Fructus Cucumidis sativi, obtains the transgenosis cucumber of quality-improving；

The nucleotides sequence of the Fructus Cucumidis sativi CsMYB6 genes is classified as：

I) nucleotide sequence shown in Seq ID No.1；

Ii) under strict conditions with sequence hybridization shown in Seq ID No.1 and express identical function egg The nucleotide sequence of white matter；Or

Iii) with i) or ii) nucleotide sequence there is more than 90% homology and the identical work(of expression The nucleotide sequence of energy protein；

The stringent condition is 0.1 in 0.1 × SSPE containing 0.1%SDS or containing 0.1%SDS In × SSC solution, hybridize at 65 DEG C, and film is washed with the solution.

8. method according to claim 7, it is characterised in that by Fructus Cucumidis sativi CsMYB6 bases Because insertion vector pBI121 multiple clone site between, that is, build obtain it is described restructuring overexpression carry Body.