CN102181444A - Rice callus-specific promoter and application thereof - Google Patents

Abstract

The invention provides a callus-specific promoter cloned from rice, an expression vector containing the promoter and a selective marker gene optimized by a rice codon, and a transformant. A nucleotide sequence of the promoter is shown in SEQ ID NO.1; and the promoter has the expression specificity of calli, and has higher activity in the calli. The expression of the selective marker gene (a hygromycin B phosphotransferase gene with a sequence shown in SEQ ID No.2) optimized by the codon in a normal rice plant is controlled by using the promoter, so that the transgenic rice can be efficiently screened, and the problem of the safety of the selective marker gene to the biological environment can be completely solved; therefore, the biological safety of the transgenic rice is improved, and the selection efficiency is improved at the same time.

Description

A kind of rice callus tissue-specific promoter and application thereof

Technical field

The invention belongs to plant genetic engineering field, be specifically related to a kind of rice callus tissue-specific promoter and application thereof.

Background technology

The selection markers gene is used for the screening of the positive transformant after plant genetic transforms as a kind of effective means, but mediate the marker gene expression at present and all adopt the composition promotor, wherein transforming at plant genetic is the CaMV35S promotor (Xu Chunbo of tobacco mosaic virus (TMV) the most widely, Wang Yong, Li Xing tenth of the twelve Earthly Branches, Zhao Haixia, the structure of the CBF4 gene plant expression vector under two kinds of promoter regulations, biotechnology, 2010), next is Ubiquitin promotor (Garbarino, J.E., T.Oosumi, and W.R.Belknap, Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.Plant Physiol, 1995), though these promotors transform at plant genetic and have obtained widespread use, but owing to be the constructive expression, the worry that has the security of marker gene at genetically modified biological safety, in order to overcome the exogenous and constructive expression's of the existing promotor that extensively adopts shortcoming, we attempt, and the clone has the specific expressed promotor of callus in the rice genome, can not only solve effectively to form and express the safety concerns that promotor mediation marker gene exists, can also solve the problem of exogenous DNA array, can break simultaneously and use the intellecture property barrier of external composition promotor at China's transgenic research.

In plant transgene research, the expression amount size of marker gene is most important to screening effect, the selectable marker gene that is applied to plant transgene at present mainly contains two big classes: a class is the gene of coding antibiotics resistance, neomycin phosphotransferase gene II (nptH), hygromycin phosphotransferase gene (hpt) and dihydrofolate reductase gene (dhfr) etc.; Another kind of is the gene of coding Herbicid resistant, for example, and careless fourth phosphinothricin acetyl transferase gene (bar), 5-enol pyruvoyl oxalic acid-3-phosphate synthase gene (epsps) etc.From the genetically modified screening of paddy rice, best with hygromycin phosphotransferase gene (hpt) screening effect, grass fourth phosphinothricin acetyl transferase gene (bar) takes second place, other marker gene utilizing on paddy rice is relatively poor, and the efficiency of selection that the expression amount that improves marker gene can improve, can starting with from transcriptional level and post-transcriptional level and translation skill and improve genetic expression, mainly is to select stronger promotor for use from improving transcriptional level, mainly is to increase translation efficiency transcribing back or translation skill.Because the selected marker who uses comes from fungi at present, because there is degeneracy in genetic code, between each species to existing on the codon usage frequency than big-difference, the a tree name bibliographical information, the gene expression amount of foreign gene after codon optimized improves 100～1000 times of (Mason, H.S., et al., Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.Vaccine, 1998), therefore carry out codon optimized according to the species difference to the selected marker, the expression of selectable marker gene can be improved, thereby the purpose that improves selection effect can be reached.

Because the quantity (64) of codon is more than amino acid whose quantity (20),, most amino acid surpasses a codon coding so all having.Identical or the close amino acid whose codon of encoding is often similar on sequence, and the codon of four same seed amino acids of coding is only different on the 3rd, and the difference on this site can be that difference between purine and the pyrimidine causes sometimes.The phenomenon that last bit base specificity of codon reduces is called the degeneracy of the 3rd bit base.The translation of codon need be matched with the anticodon on corresponding aminoacyl-tRNA.Though a kind of tRNA often can discern and surpass a kind of codon, different tRNA frequencies of utilization are different.Different plant species is to the preferences difference of the son that accesses to your password, and each species changes not quite in the rate of utilization of codon first and second bit bases, but expresses very big difference on the 3rd bit base, has preferences.If when Plant Transformation, use plant-preference codon, can improve the recognition efficiency of tRNA, thereby improve translation efficiency.Therefore, under the prerequisite that does not change aminoacid sequence, be optimized according to the preferences of paddy rice codon codon the hpt gene, make the codon of the use paddy rice preference of its nucleotide sequence 100%, be cloned into the downstream of OsCSP promotor then, made up the carrier that is used for the expression vector cotransformation.And whole Expression element (comprising promotor, screening-gene, terminator) all has been cloned between the T-border of JH2600 carrier, has made up the carrier that can be used for Agrobacterium-mediated Transformation.

The cysteine proteinase gene of this research paddy rice has been cloned a callus specificity (Rice Callus-Specific Promoter, be called for short OsCSP) promotor, and be used for mediation screening property marker gene and express, codon optimized by to screening property marker gene hpt gene, improved the transformation efficiency that OsCSP selects in the paddy rice transgenosis significantly, at transformation efficiency, false positive rate, the cotransformation rate has all reached the effect of the Ubiquitin promotor mediation marker gene of pCambia, set up selection markers system, for the genetically modified organism security of setting up genetically modified crops provides a new approaches and methods with the genetic transformation of the paddy rice that utilizes OsCSP promotor mediation hygromycin phosphotransferase gene (hpt) of paddy rice monocot crops or monocot crops.

Summary of the invention

An object of the present invention is to provide a kind of promotor of rice callus tissue specific expression, promptly the promotor of cysteine proteinase gene-OsCSP promotor has improved the biological safety at the selection markers gene of genetically modified crops.

Another object of the present invention provides the plant expression vector that contains described OsCSP promotor.

A further object of the present invention provides the transformant that contains described OsCSP promoter sequence.

Another object of the present invention provide described OsCSP promotor and codon optimized after hygromycin phosphotransferase gene (hpt) merge application in the transgenic paddy rice screening.

According to an aspect of the present invention, from rice paddy seed, cloned L-Cysteine HCL Anhydrous (OsCSP) promotor, its nucleotide sequence is shown in SEQ ID No.1, show that in one embodiment OsCSP of the present invention starts and has the callus specificity, for the security of the selection markers genes of genetically modified crops provides new method.

According to a second aspect of the invention, described plant expression vector is to adopt gene engineering method, the OsCSP promotor is inserted in the suitable expression vector and obtains.Preferably in described expression vector, comprise hygromycin phosphotransferase gene (hpt gene) as screening-gene, more preferably, be optimized according to the preferences of paddy rice codon codon the hpt gene, make the codon of the use paddy rice preference of its nucleotide sequence 100%, its nucleotide sequence is shown in SEQ ID No.2, be cloned into the downstream of OsCSP promotor then, made up the carrier that is used for the expression vector cotransformation, made it reach or be better than the existing effect of carrier in the screening of paddy rice transgenosis that be equal in the world at the genetically modified screening effect of the positive.In one embodiment, made up the pOsPMP522 plant expression vector, it has structure as shown in Figure 2.

According to a third aspect of the invention we, above-mentioned plant expression vector is transformed suitable host and can obtain transformant of the present invention efficiently.In one embodiment, adopt the plant expression vector pOsPMP522 that the cotransformation method will contain the OsCSP promotor to be transformed into the rice conversion body that has obtained among the agrobacterium strains EHA105 with the promotor mediation selection markers gene that adopts external source in the world.

According to a forth aspect of the invention, by with the selection markers gene clone to the downstream that is positioned at rice callus of the present invention tissue-specific promoter, make up the plant expression vector that contains described rice callus tissue-specific promoter, selection markers gene and target gene; Utilize this plant expression vector to be used for paddy rice Agrobacterium-mediated Transformation callus; Utilize selection markers gene pairs callus to screen, and each tissue of the later etap except callus is not expressed, thereby improved the biological safety that in bread crop, utilizes the selected marker to carry out the transgenosis screening.

The invention provides a kind of rice callus tissue-specific promoter-OsCSP promotor, this promotor is specific expressed in the rice callus tissue, can come transgenic paddy rice is screened by this expression of promotor control selection markers marker gene in the normal water rice plants.Combination simultaneously is codon optimized to the selection markers gene, not only mainly carry out in the callus stage owing to the selection markers expression of gene, and improved of the expression of selection markers gene at callus, improved positive genetically modified screening efficiency, the constructive expression's promotor that has overcome because of using endogenous or external source reduces the risk that exogenous genetic material imports rice genome, make the selection markers expression of gene be limited to callus simultaneously, improve the biological safety of selection markers gene pairs transgenic paddy rice, reduce the safety concerns of the public simultaneously the selection markers gene in the transgenosis.

Description of drawings

Fig. 1: the agarose gel electrophoresis in the amplification of OsCSP promotor detects collection of illustrative plates;

Fig. 2: the structural representation of plant expression vector pOsPMP522;

Fig. 3: the tissue specific expression GUS coloration result of OsCSP promotor; Wherein 1 is the non-transgenic callus; 2,3,4,5 be respectively pOsPMP43 transgenosis kanamycin-resistant callus tissue, leaf, root, seed.

Embodiment

Below in conjunction with accompanying drawing the present invention is further described in detail, but following description do not limit the present invention, any to distortion of the present invention and change, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.

Material reagent

Vegetable material is paddy rice japonica rice (Oryza sativa.subsp.japonica) kind TP309.

Coli strain is DH10B, available from Invitrogen company;

Agrobacterium strains is EHA105, available from Invitrogen company;

Carrier pCAMBIA1300, pCAMBIA1301, available from Invitrogen company, pBI221, pUC57 is available from Nanjing Genscript Biotechnology Co., Ltd.; POsPMP05, pOsPMP122, pOsPMP209, pOsPMP522, the carrier that pOsPMP43 makes up for the present invention.

Used restriction enzyme in the vector construction process is available from NEB and Fermentas company; Taq enzyme, T4DNA Ligase are available from American I nvitrogen company; Gel reclaims test kit available from X-gene; Used chemical reagent is analytical pure except that specified otherwise, available from the Shanghai traditional Chinese medicines.

The clone of [embodiment 1] rice callus tissue-specific promoter (OsCSP)

1, extracts paddy rice TP309 genomic dna

A) get 0.1 gram spire and place mortar, add 0.6ml CTAB extracting solution, blade is ground evenly;

B) temperature is bathed 30min in 65 ℃ of water-baths, and gentleness is swayed 2～3 times therebetween, takes out after temperature is bathed and naturally cools to room temperature;

C) (chloroform: primary isoamyl alcohol=24: 1) the back jog is 5 minutes, centrifugal (10000rpm) 5 minutes to add the 0.6ml extract;

D) draw supernatant and place another 1.5ml centrifuge tube, add the 3M sodium-acetate of 75 μ L and the precooling dehydrated alcohol of 1ml;

E) at room temperature centrifugal (12000rpm) 10 minutes, supernatant discarded adds 1ml 70% ethanol, and the centrifuge washing precipitation is outwelled ethanol, dry DNA;

F) treat the DNA thorough drying after, add the aseptic ddH2O dissolving DNA of 200 μ l, 4 ℃ standby.

2, from cysteine proteinase gene sequence (the Genbank accession number is AL732346) amplification callus specificity promoter (OsCSP)

With paddy rice TP309 genomic dna is template, the required primer (two ends have the restriction enzyme site of HindIII and NaeI) of design clone's OsCSP promotor:

Forward primer: 5 '-cgccaagcttGCATGCCTGCAGCCA-3 ' (SEQ ID NO.4)

Reverse primer: 5 '-ccatgccggcGACGTGGAG ACGAGC-3 ' (SEQ ID NO.5)

Utilize above-mentioned primer to carry out the PCR reaction, amplification OsCSP promotor from rice genome.

Amplification system:

Amplification program: 94 ℃ of 5min, 94 ℃ of 30s, 58 ℃ of 30s, 68 ℃ of 1min, 35 circulations, 68 ℃ are extended 10min, 25 ℃ of preservations.After reaction finishes, detect amplified production, reclaim purpose band (Fig. 1) by agarose gel electrophoresis.

To reclaim fragment and deliver to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, sequencing result shows that the OsCSP promotor has the nucleotide sequence shown in the SEQ ID NO.1, and is by 1111 based compositions, consistent with the sequence of online announcement.

[embodiment 2] make up the carrier that contains the OsCSP promotor

1, the structure of OsCSP-stuff-nos carrier

Amplified fragments with HindIII/NaeI difference double digestion OsCSP promotor reclaims product and pBI221 plasmid.Precipitation reclaims the promotor after enzyme is cut, and is inserted into the pBI221 carrier that enzyme is cut the 4.8kb that reclaims the back, the correct transformant of screening behind the conversion DH10B.Gained carrier OsCSP-stuff-nos is numbered pOsPMP209.

2, the structure of OsCSP-gus-nos carrier

With pCAMBIA1301 is masterplate, the amplification gus gene.Forward primer is 5 '-cggtgccggcATGGTAGATCTGAGGG-3 ' (SEQ ID NO.6), and reverse primer is 5 '-ccgctcgagTCACACGTGGT GGTG-3 ' (SEQ ID NO.7), 60 ℃ of amplification temperature.Reclaim product with NaeI/XhoI digestion PCR gel, enzyme is cut the product precipitation and is reclaimed, and is inserted into the plasmid through the pOsPMP209 of NaeI/XhoI double digestion, the correct transformant of screening behind the conversion DH10B.Gained carrier OsCSP-gus-nos is numbered pOsPMP43.

3, the structure of JH-OsCSP-hpt-nos carrier

With pCAMBIA1301 is masterplate, amplification hpt gene.Forward primer: 5 '-aaaagtactATGAAAAAGCCTGAACTCACCGCGA-3 ' (SEQ ID NO.8), reverse primer: 5 '-ggagaaactcgagCTTGTCGATCGACAGATCCGGT-3 ' (SEQ ID NO.9), the amplification temperature is 60 ℃.Reclaim product with ScaI/XhoI digestion PCR gel, enzyme is cut the product precipitation and is reclaimed, and is inserted into the plasmid through the pOsPMP209 of NaeI/XhoI double digestion, the correct transformant of screening behind the conversion DH10B.Gained carrier OsCSP-hpt-nos is numbered pOsPMP05;

With HindIII/EcoR I difference double digestion pOsPMP05 plasmid and JH2600 carrier, reclaim the OsCSP-hpt fragment of 3.1kb, be inserted into the 9.6kb JH2600 carrier after enzyme is cut, the correct transformant of screening behind the conversion DH10B.Gained carrier JH-OsCSP-hpt-nos is numbered pOsPMP122.Vector construction well transforms afterwards agrobacterium strains EHA105.

4, the structure of JH-OsCSP-opt hpt-nos carrier

According to the aminoacid sequence of hpt, adopt the paddy rice preference codon to design the dna sequence dna (SEQ ID NO.2) of goal gene opt hpt.The Hpt gene is 1026bp altogether, 341 amino acid of encoding.Under the prerequisite that does not change aminoacid sequence, according to the preferences of paddy rice codon the codon of this gene is optimized, change 207 of bases altogether, account for 21% of base sum, codon has changed 188, accounts for 55% of codon sum.Synthetic by Nanjing Genscript Biotechnology Co., Ltd. through codon optimized gene, the synthetic gene order is inserted in the pUC57 carrier.

Cut the pUC57 carrier that contains goal gene with SchI/XhoI substep enzyme,, reclaim the goal gene of 1kb, be inserted into the carrier segments of about 4kb, the correct transformant of screening behind the conversion DH10B with NaeI/XhoI double digestion pOsPMP43 carrier.Plasmid and JH2600 carrier after double digestion transforms respectively with HindIII/EcoR I, the OsCSP-opt hpt fragment of recovery 2kb is inserted into the JH2600 carrier after enzyme is cut, the correct transformant of screening behind the conversion DH10B.Gained carrier JH2600-OsCSP-opt hpt-nos is numbered pOsPMP522 (SEQ ID NO.3).Vector construction well transforms afterwards agrobacterium strains EHA105.

[embodiment 3] agriculture bacillus mediated conversion

1, the cultivation of callus

Get sophisticated rice paddy seed, shelling back clorox (adding 1～2 polysorbas20) with 20% on super clean bench soaks 30～40min, aseptic water washing 4～5 times.Use the aseptic filter paper suck dry moisture, place on the callus of induce substratum, after 26 ℃ of dark 4 weeks of cultivation, picking is light yellow, fine and close, the callus of relatively dry is transferred on the subculture medium, 26 ℃ of dark cultivations for 2 weeks.

2, Agrobacterium-mediated Transformation

With pCAMBIA1300 (35S-hpt-nos), pOsPMP122 (JH-OsCSP-hpt-nos), the Agrobacterium bacterial classification of pOsPMP522 (JH-OsCSP-opt hpt-nos) and pOsPMP43 (OsCSP-gus-nos) plasmid is rule respectively and is connect bacterium on the YEB solid medium that contains the 50mg/L kantlex, 28 ℃ of dark cultivations 2 days.Agrobacterium is transferred to the suspension culture base respectively, and 28 ℃ were shaken bacterium 1～2 hour, made the final concentration of bacterium liquid reach OD ₆₀₀0.8～1.0.

Collect callus to aseptic triangular flask, add Agrobacterium bacterium liquid respectively, callus is fully contacted with bacterium liquid, room temperature is placed 30min.Take out callus, inhale with aseptic filter paper and remove unnecessary bacterium liquid, callus is transferred to common substratum, in 19～20 ℃ of dark cultivations 3 days.

With the callus after cultivating altogether several times, be soaked in 30min in the sterilized water that contains the 300mg/L cephamycin then with sterile water wash.Take out callus, place on the sterilization filter paper moisture is blotted, be transferred to screening culture medium, 26 ℃ of dark cultivations for 4～5 weeks.

3, the statistics of positive callus

With pCAMBIA1300 (35S-hpt-nos), three plasmids of pOsPMP122 (JH-OsCSP-hpt-nos) and pOsPMP522 (JH-OsCSP-Opt hpt-nos) are done three repetitions altogether with aforesaid method difference transformed calli.Choose the callus that is used to transform (300) of same quantity, the each number that transforms the kanamycin-resistant callus tissue that grows of statistics, and these kanamycin-resistant callus tissues of picking carry DNA, with primer amplification hpt gene, statistics can expand the callus number that the purpose band, the results are shown in Table 1.

The conversion results of three carriers of table 1

By The above results as can be known, in agriculture bacillus mediated rice callus metaplasia, pOsPMP522 has better screening effect than pOsPMP122, the quantity of the kanamycin-resistant callus tissue that the former can grow almost is the latter's twice, and when the antagonism callus is carried DNA expansion goal gene, it is also more to expand the callus number that the purpose band, basic identical with the result of pCAMBIA1300 on the whole.So the hpt after optimizing is higher than the transformation efficiency of not optimizing, false-positive ratio also decreases in the resistant calli that grows.In Agrobacterium-mediated Transformation, the effect of pOsPMP522 and pCAMBIA1300 is roughly the same.

4, the acquisition of pOsPMP43 (OsCSP-gus-nos) positive plant

To the kanamycin-resistant callus tissue that grows on the screening culture medium after pOsPMP43 (OsCSP-gus-nos) the plasmid Agrobacterium-mediated Transformation, under gnotobasis, get the tissue block of 1 * 1 * 1mm size, immerse (GUS staining fluid: 0.2M Na in the staining fluid ₃PO ₄Damping fluid, pH 7.0; 0.1mol/L K ₃[Fe (CN) ₆]; 0.1mol/LK ₄[Fe (CN) ₆] 3H ₂O; 1.0mol/L Na ₂EDTA; 0.1%X-Gluc), 37 ℃ are spent the night.The tissue block of stained positive is selected corresponding callus to division culture medium, cultivates for 2～3 weeks in 26 ℃ of light.

When seedling length to 2～3 centimetre of differentiation, it is transferred on the root media.Treat that regeneration plant on the root media about two weeks, takes out seedling, carefully rinse out agar on the seedling root, be transplanted in the little alms bowl with clear water.Seedling placed transparent watertight chest (18～26 ℃) lower refining seedling 3～5 days, progressively opened the watertight chest lid and slowly conformed by seedling.Hardening went to seedling in the big alms bowl after the phase.Undertaken by normal rice field management.

By being expressed, transgenic callus, root, leaf, seed gus gene carry out GUS dyeing detection, the result shows, the GUS activity can detect at callus, do not express at its hetero-organization such as root, leaf, seed, especially in dividing vigorous callus, be higher than ripe callus combination (Fig. 3), confirm that thus OsCSP is a promotor that callus is specific expressed.

In addition, than what do not optimize higher expression efficiency and better screening effect arranged through the hpt gene optimized, its efficient and pCAMBIA1300 are basic identical.And in selection markers carrier of the present invention, have only goal gene and expression vector segment in the middle of the T-DNA, be different from the pCAMBIA1300 carrier, the transfer-gen plant genetic background of Huo Deing does not contain unnecessary fragment like this, thereby more helps implant mass and commercially produce.

Because the callus specificity of OsCSP promotor, it can be used to start selection markers in the expression of screening stage just of callus stage, and do not express in the normal plant rhizome leaf, so just can be reduced to selection markers minimum to the pollution problem of environment.On a selection markers carrier and destination gene expression carrier cloning to two carrier, the method by cotransformation obtains transfer-gen plant then.The selection markers of the transfer-gen plant that obtains thus just can obtain separating by offspring's exchange reorganization, obtains only to contain the transfer-gen plant of destination gene expression carrier at last, so just can thoroughly avoid the pollution problem of selection markers to environment.

Claims (8)

1. rice callus tissue-specific promoter, it has the nucleotide sequence shown in SEQ ID NO.1.

2. the expression vector that contains the described promotor of claim 1.

3. the described expression vector of claim 2, it comprises the codon optimized hygromycin phosphotransferase gene of paddy rice.

4. the described expression vector of claim 3, the codon optimized hygromycin phosphotransferase gene of wherein said paddy rice has the sequence shown in SEQ ID NO.2.

5. as each described expression vector of claim 2-4, it has structure as shown in Figure 2.

6. expression vector as claimed in claim 5, it has the sequence shown in SEQ ID NO.3

7. transform the transformant that the host obtains by each described expression vector of claim 2-6.

8. described promotor of claim 1 or the described expression vector of claim 2 application in the preparation transgenic plant.

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