CN101979563A - Soybean leaf specific promoter SRS4 and application thereof - Google Patents

Abstract

The invention discloses a soybean leaf specific promoter SRS4 and application in a transgenic plant. A soybean rbcS gene promoter SRS4 is cloned by using a uniquely designed primer and taking soybean 'Jinong13' genome DNA as a template; a plant expression vector is constructed and is named Pcambia-GrbcSP; and a specific light control promoter is obtained from soybeans by performing genetic transformation by adopting agrobacterium-mediated tobacco and performing functional verification in a mode of expressing a GUS gene. The soybean leaf specific promoter SRS4 can be used in the transgenic plant, in particular in genetically engineered soybeans, lays a theoretical basis for establishing a time, space and amount three-dimensional regulation and control system for plant transgene expression, and has great significance for researching the regulation and the control of gene expression, constructing a vector for genetic engineering, expressing a target protein, and improving yield.

Description

A kind of soybean leaves specificity promoter SRS4 and application thereof

Technical field

The invention belongs to biological technical field, exactly is soybean leaves light-operated gene SRS4 promotor and the application in transgenic plant thereof.

Background technology

Soybean is a Papilionaceae Glycine annual herb plant, and seed contains rich in protein, originates in China, so far the plantation history in existing 5000.Soybean is not only important food oils and egg-white food raw material, and is animal husbandry important protein feed resource, is one of important grain and oil crop of China.

China is soybean big producing country, but because the continuous growth of China's soybean acreage minimizing and edible oil consumption and aquaculture development are to the increase of dregs of beans demand, cause China's soybean demand to increase sharply, nowadays China also becomes soybean net importer the biggest in the world.

At present, utilize traditional breeding method, methods such as improvement cultivation improve soybean yields and have been absorbed in bottleneck; Therefore, utilize genetically engineered to obtain good quality and high output and the stable yields soybean is the task of top priority of China.

The expression of gene to the effect that and the regulation and control of plant genetic engineering research, but the exogenous gene expression quantity not sufficient is the major reason that can not obtain desirable transgenic plant.Promotor is playing keying action aspect the decision genetic expression, and therefore selecting suitable plant promoter is the matter of utmost importance that strengthens exogenous gene expression.

Constitutive promoter exposes some problems in transgenic plant are used, as: foreign gene is expressed in whole strain plant, produces a large amount of heterologous proteins or meta-bolites at plant interior accumulation, has broken the original metabolic balance of plant; Some product is also nonessential even poisonous to plant, thereby has hindered the normal growth of plant, even causes death.Therefore, the essential specificity promoter of seeking of people replaces constitutive promoter, with regulating expression of foreign genes better.

1,5-diphosphoribolose carboxylase/oxygenase (Rubisco) is present in all higher plants, is the rich in protein of content in the photosynthetic plant leaf, accounts for 50% of solubility total protein; It also is the key enzyme in the photosynthesis.Rubisco is made up of 8 big subunits (rbcL) and 8 small subunits (rbcS), and wherein the expression by the small ylidene gene of nuclear gene encoding is regulated and control by light, and has tissue specificity.

Summary of the invention

The purpose of this invention is to provide a kind of soybean leaves specificity promoter SRS4.

A kind of soybean leaves specificity promoter SRS4, its base sequence is shown in SEQ ID No.1;

It is to be template with soybean " Ji Nong 13 " genomic dna, with

SS:5`-GTGGATGACTCAAGTGCTGG-3`SA:5`-TCATTGAGGAAGCCATTTGC-3` is a primer, obtains by the PCR method amplification.

A kind ofly contain the plant expression vector that base sequence is a SEQ ID No.1 gene.

Another object of the present invention is a kind of soybean leaves specificity promoter SRS4, the application in the application in transgenic plant, particularly genetically engineered soybean.

A kind of soybean leaves specificity promoter of the present invention SRS4, be with the unique primer of sending out meter of the inventor, with soybean " Ji Nong 13 " genomic dna is template, adopt TAIL-PCR to clone soybean rbcS gene SRS4 promotor 5` flank, according to TAIL-PCR institute's calling sequence and known array design special primer, clone soybean rbcS gene SRS4 promotor, and make up its plant expression vector, with agriculture bacillus mediated tobacco genetic transformation, express the mode of gus gene and screen, sift out the light-operated promotor SRS4 of a kind of blade specific.

1,5-diphosphoribolose carboxylase/oxygenase (Rubisco) is present in all higher plants, has dual-use function, can catalysis RuBP and CO ₂Form the reaction of 3-phoshoglyceric acid, again can catalysis RuBP and O ₂Oxicracking forms the oxygenation reaction of 3-phoshoglyceric acid, phosphoric acid and phosphoglycollic acid; Therefore, the RuBP carboxylase is to be in photosynthetic carbon reduction with two reverse directions of photosynthetic carbon oxidation but mutually on the circulation point of crossing of interconnected lock, and its content and activity change play decisive role to plant net photosynthesis efficient; For set up that plant transgene expresses the time, sky, the three-dimensional regulator control system based theoretical of amount; Research gene expression regulation, structure engineering carrier, expression target protein, raising output are had great significance.

Description of drawings

Fig. 1 is the electrophoretic analysis of TAIL-PCR product, M:DL2000; The 1st, 2,3 of 1,2,3 swimming lane: AD2 and special primer combination gained taken turns the PCR product;

Fig. 2 is the electrophoretic analysis of soybean rbcS promotor SRS4, M:DL2000; 1: the PCR product of soybean rbcS promotor.

Fig. 3 cuts evaluation for the PCR and the enzyme of recombinant plasmid, M:DL2000, and 1: the PCR of recombinant plasmid identifies 2: the double digestion of recombinant plasmid is identified.

Embodiment

Embodiment 1 extracts soybean " Ji Nong 13 " genomic dna

Soybean " Ji Nong 13 " seed is broadcast in installing the nutrition pot of sand, be placed in the RXZ type intelligence artificial climate incubator and cultivate.Adopt dark the cultivation before seed sprouts, the back of sprouting adopts the Hoagland nutritive medium to cultivate 25 ℃ of daytimes, 16 ℃ of nights, illumination 16h/d, light intensity 3000lx, relative humidity 75%; Get the soybean seedling tender leaf in 2 weeks of growth, extract test kit in a small amount according to the AxyPrep genomic dna and carry out, extract soybean " Ji Nong 13 " genomic dna;

Measure DNA in 260nm and 280nm place absorbancy with ultraviolet spectrophotometer, calculate DNA concentration and purity, gained DNA concentration is OD ₂₆₀/ OD ₂₈₀=1.865, purity is 850ng/ μ l;

The clone of embodiment 2 soybean rbcS gene SRS4 promotor 5` flanking sequences

Adopt TAIL-PCR clone soybean rbcS promotor 5` flanking sequence, schedule of operation is with reference to the method for Liu Yaoguang etc.

Primer source: according to 3 special primer SP1 of soybean RuBP carboxylase small ylidene gene (SRS4) upstream of coding region dna sequence dna (GeneBank accession:M16889) the design annealing temperature higher (60 ℃-65 ℃) of NCBI login, SP2, SP3; The degenerate primer AD2 of the annealing temperature lower (40 ℃-45 ℃) of process unique design is stored in this laboratory (table 1).

Table 1: primer and based composition

With soybean " Ji Nong 13 " genomic dna is template, carries out 3 with degenerate primer AD2 respectively with specific nested primer SP1, SP2 and SP3 and takes turns pcr amplification; 50 times of first round PCR product dilutions are done second take turns template, take turns the dilution of PCR product with second and make the third round template for 100 times.

1% agarose gel electrophoresis detects 3 and takes turns PCR product (Fig. 1), with the single band in the dna gel recovery test kit recovery third round PCR product; To reclaim product and be connected with the pMD18-T carrier, Transformed E .coliJM109 competent cell carries out Amp (100mg/ml), IPTG, X-gal screening; Extract recombinant plasmid after choosing white single bacterium colony enlarged culturing, recombinant plasmid is served the Hai Shenggong order-checking with positive colony bacterium liquid after PCR identifies;

Sequencing result shows: the gained sequence length is 867bp, and the upstream and downstream primer is contained in both sides, and the 5` end parts sequence of 3` terminal sequence and known array is identical simultaneously, shows and successfully is cloned into soybean rbcS promotor 5` flanking sequence.

The acquisition of embodiment 3 soybean rbcS gene SRS4 promotor SRS4 (1538bp)

Design special primer according to TAIL-PCR institute's calling sequence and known array:

SS：5`-GTGGATGACTCAAGTGCTGG-3`

SA：5`-TCATTGAGGAAGCCATTTGC-3`

With soybean " Ji Nong, 13 " genomic dna is template, and with SS, SA is the primer amplification goal gene, detects through 1% agarose gel electrophoresis, and the result shows: at about 2000bp place single specific band (Fig. 2) is arranged, size conforms to the expectation theoretical value.

Return, receive test kit with dna gel and reclaim the PCR product; Purified product is connected with the pMD18-T carrier, and Transformed E .coliJM109 competent cell carries out the screening of Amp, blue hickie, chooses recombinant plasmid PCR and enzyme and cuts evaluations (Fig. 3) and be male clone bacterium liquid and serve Hai Shenggong and check order; The sequencing result analysis shows: fragment length is 1538bp, and the partial sequence of cloned sequence is almost completely overlapping with TAIL-PCR institute calling sequence, known array part respectively, shows and successfully is cloned into soybean rbcS promoter sequence; Its base sequence is shown in sequence table SEQ ID No.1;

Soybean rbcS gene SRS4 promoter sequence is analyzed

With the soybean rbcS promoter sequence (699bp) of DNAman comparison gained soybean rbcS gene SRS4 promoter sequence (1538bp) and design primer, both reach 97% at the respective regions homology, and similarity reaches 45%; Also it is compared with the rbcS promoter sequence of Kidney bean rbcS2 promoter sequence, pea in the ncbi database simultaneously, homology is respectively 73.43%, 33.25%; Analyze clone's soybean rbcS promoter sequence with PLACE, discovery has 10 GATA boxes (GATA), 5 I boxes (GATAAG), 1 GT-1 site (GGTTAA), 5 GT-1 conserved sequences (GRWAAW), 3 REalpha elements (AACCAA), these all are to express conservative element in the promotor in photoinduction, can regulatory gene be subjected to the transcriptional activity after the photoinduction.

Embodiment 4

One, soybean rbcS gene SRS4 promotor plant expression vector construction

According to the promoter sequence design primer that obtains, introduce the PstI restriction enzyme site at SSPAS 5` end, SSPAA 5` end is introduced the NcoI restriction enzyme site, is that template is carried out pcr amplification with the recombinant plasmid that contains soybean SRS4 promotor.PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60.℃30s，72。℃ 2min; 72 ℃ of 10min, 4 ℃ of 15min pcr amplifications, the promoter sequence of acquisition band restriction enzyme site.With PstI/NcoI double digestion amplification PCR products and plant expression vector Pcambia1301, purifying reclaims, connects respectively, makes up the plant expression vector of soybean rbcS gene SRS4 promotor, called after Pcambia-GrbcSP.

Two, the functional verification of the plant expression vector of soybean rbcS gene SRS4 promotor

1, the agriculture bacillus mediated tobacco genetic transformation and the positive-selecting of transfer-gen plant

Utilize triparental mating that the plant expression vector Pcambia-GrbcSP that builds is imported agrobacterium tumefaciens bacterial strain LBA4404, infect tobacco seed then and sprout the leaf explant of back 20d seedling age (size: 0.5cm * 0.5cm), cultivate the back altogether and screen, simultaneously in contrast with the Pcambia1301 carrier with the 50mg/L kantlex.The reference operation is pressed in experiment such as the screening of tobacco tissue culture, agrobacterium mediation converted, resistant calli and plant regeneration, slightly changes.To obtain resistant plant and carry out positive detection with the PCR method, primer is the inner primer of gus gene, and the product size is about 450bp.

2, GUS tissue chemical analysis

Blade, leaf sheath, stem stalk, root with positive transfer-gen plant are cut into suitable size, immerse in an amount of GUS dye liquor, and 37 ℃ are spent the night, and with the decolouring of 75% alcohol, observe.Coloration result is found: the rbcS promotor only drives the gus gene and expresses in the leaf of transfer-gen plant, leaf sheath, does not express in stem stalk, root; And 35S promoter driving gus gene is all expressed in whole transfer-gen plant.

3, the active detection of GUS is expressed in the transfer-gen plant photoinduction

The positive transfer-gen plant that PCR detects was cultivated 6 or 7 days at the dark place, got its rationing blade and analyze the GUS activity; Subsequently it is cultivated (16h light, 8h dark) and behind 24h, 48h and 96h, get its blade respectively GUS is carried out activation analysis under normal illumination.

The result shows that the plant that will cultivate 6 or 7 days at the dark place moves into when continuing to cultivate under the normal illumination, the GUS activity when its GUS activity is cultivated apparently higher than the dark place; And along with the prolongation of light application time, the GUS activity is also more and more higher.

Three, soybean rbcS gene SRS4 promoter deletion body plant expression vector construction

According to the promoter sequence design primer that obtains, introduce the PstI restriction enzyme site at SSPAS 5` end, SSPAA 5` end is introduced the NcoI restriction enzyme site.With the recombinant plasmid that contains soybean SRS4 promotor is that template is carried out the pcr amplification target fragment.

SSPAS1：5`-AACTGCAGTCACTGGAACTAACATCTTG-3`

SSPAS2：5`-AACTGCAGAGGCAGTGGCTTCTTATGAG-3`

SSPAS3：5`-AACTGCAGCATCACCTACCATCCCCTTC-3`

SSPAS4：5`-AACTGCAGAACTCCACCACCATCACACA-3`

SSPAA：5`-CTAGCCATGGTCATTGAGGAAGCCATTTGC-3`

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min; 72 ℃ of 10min, 4 ℃ of 15min.PCR amplifications obtain the promoter deletion body sequence of band restriction enzyme site, and length is respectively: 1089bp, 938bp, 712bp and 334bp.With PstI/NcoI double digestion amplification PCR products and plant expression vector Pcambia1301, purifying reclaims, connects respectively, make up the plant expression vector of soybean rbcS gene SRS4 promoter deletion body, called after Pcambia-GrbcSP1, Pcambia-GrbcSP2, Pcambia-GrbcSP3 and Pcambia-GrbcSP4.

The same method is carried out the functional verification of promoter deletion body plant expression vector.

The result shows:

1. in the transgenic plant that obtain, Pcambia-GrbcSP1, Pcambia-GrbcSP2, Pcambia-GrbcSP3 and Pcambia-GrbcSP4 and Pcambia-GrbcSP GUS tissue chemical analysis come to the same thing.

2. detection by quantitative shows: in the transgenic plant that obtain, Pcambia-GrbcSP driven GUS activity is the highest.

3. photoinduction is expressed in the active detection of GUS, and Pcambia-GrbcSP driven GUS gene soybean rbcS gene illumination susceptibility is the highest.

Claims (5)

1. soybean leaves specificity promoter SRS4, its base sequence is shown in SEQ ID No.1;

2. the described a kind of soybean leaves specificity promoter SRS4 of claim 1 is characterized in that: it is to be template with soybean " Ji Nong 13 " genomic dna, uses following primer:

SS：5`-GTGGATGACTCAAGTGCTGG-3`

SA：5`-TCATTGAGGAAGCCATTTGC-3`

Obtain by the PCR method amplification.

3. one kind contains the plant expression vector that base sequence is a SEQ ID No.1 gene.

4. soybean leaves specificity promoter SRS4, the application in transgenic plant.

5. soybean leaves specificity promoter SRS4, the application in genetically engineered soybean.

Images