CN108949753A

CN108949753A - Expression vector WxbThe building of -10T, the preparation of transgenic paddy rice and primer

Info

Publication number: CN108949753A
Application number: CN201810836925.8A
Authority: CN
Inventors: 刘巧泉; 张昌泉; 范晓磊; 李钱峰; 陆彦; 陈盛杰
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2018-07-26
Filing date: 2018-07-26
Publication date: 2018-12-07
Anticipated expiration: 2038-07-26
Also published as: CN108949753B

Abstract

The invention belongs to field of biotechnology, are related to expression vectorWx ^bThe building of -10T, the preparation of transgenic paddy rice and primer.Primer is shown in sequence table SEQ ID NO.3-8.In conventional japonica riceWx ^bAllele is template,Construct the rice expression vector that single nucleotide acid is replaced at the tenth exon of alleleWx ^b-10T.The overall length of carrier carrying single base mutationWx ^bAllelic sequences obtain glutinous rice transfer using the method for mediated by agriculture bacillusWx ^bThe transgenic paddy rice of -10T gene.Breeding obtains the transgenic paddy rice of homozygous line, and generally 12% or so, gel consistence and Starch pasting viscosity index approach such transgenic paddy rice rice grain amylose content with the instantly popular soft rice of excellent flavour.The method in glutinous rice by importing to granule bound starch synzyme GBSSI encoding geneWxThe rice expression vector that specific nucleotide pointed decoration is crossed is achieved.

Description

Expression vector WxbThe building of -10T, the preparation of transgenic paddy rice and primer

Technical field

The invention belongs to plant genetic engineering fields, specifically, are repaired the present invention relates to a kind of using genetic engineering means ChangeWxGene particular bases site, the function of making it encode albumen (enzyme) suitably decline, and adjust amylose in rice using it and contain Amount is applied in the excellent soft rice quality breeding of rice.

Background technique

Rice (Oryza sativaIt L.) is the important cereal crops in China.The evaluation index of rice quality generally comprises: Milled quality, exterior quality, nutritional quality and Cooking and eating quality, wherein the most key is Cooking and eating quality (Rao etc., Plant Cell Reports, 2014,33 (4): 551-564；Zhang Changquan etc., Scientia Agricultura Sinica, 2016, 49 (22): 4267-4283.).Cooking and eating quality routine evaluations index have amylose content (amylose content, ), AC gel consistence (gel consistency, GC), and gelatinization point (gelatinization temperature, GT) Deng (Tian etc., PNAS, 2009,106 (51): 21760.).Starch is most important component part in rice, typically constitutes from endosperm 90 % of dry weight or so, therefore, the composition of starch, structure are closely related in the Cooking and eating quality and endosperm of rice.Starch It is to exist in endosperm in granular form, shape majority is in irregular polygonal, and corner angle are significant, and in the form of composite starch class In the presence of spherical in shape or oval.Starch can be divided into two classes according to architectural difference, i.e. amylose (Amylose) and branch forms sediment Powder (Amylopectin).In rice quality breeding, amylose content is an extremely important index, it is that rice steams The deciding factor for flavor quality quality of cooking.Dry and hard characteristic is shown after rice boiling with high amylose content, And being shown as compared with the rice of low amylose content soft and flexible, palatability is preferable.

Amylose in paddy endosperm by rice waxy gene (Waxy, Wx) coding granule bound starch synzyme (granule bound starch synthase I, GBSS I) catalyze and synthesize (Wang etc., The Plant Journal, 1995,7 (4): 613-622).It is poor that the different allelic variations of the gene cause content and activity of the GBSS I of coding etc. to exist It is different, to cause difference (Pandey etc., Biotechnology of the amylose content of rice between different cultivars Advances, 2012, 30(6):1697；Teng Bin etc., nuclear agricultural science report, 2014,28,10:1760-1764), thereforeWxGene It plays a crucial role to the regulation of rice cooking and eating quality.RiceWxGene is located on the galianconism of No. 6 chromosome, including 13 Introne and 14 exons encode albumen (Wang etc., the Nucleic Acids being made of 609 amino acid Research, 1990,18 (19): 5898).WxThere are multiple important allelic variation sites for gene, at presentWx ^mpWithWx ^op Allele is the favorable genes resource being widely used in conventional softpanel rice variety, but these genes are main when in use Breeding Application is carried out by hybridization and molecular marker assisted selection mode, this process is relatively more single than relatively time-consuming and genetic resources One.Therefore, this field creates new soft rice material up for developing new technology and methods, and then improves rice quality.

Summary of the invention

The object of the present invention is to provide a kind of method for cultivating the transgenic paddy rice of low amylose content in tool, this method The rice of acquisition is showed with preferable Cooking Quality.The present invention constructs riceWx ^bThe single base mutation expression vector of gene, It is conducted into the waxy near isogenic lines of rice varieties OryzasativaLcv.Nipponbare, is detected by technological means such as PCR amplifications, as a result table Bright target gene has been introduced into rice material and is expressed.

A kind of method for cultivating the soft rice of excellent flavour quality transgenosis of the present invention is prominent by expressing single base in glutinous rice The expression vector of changeWx ^b- 10T is achieved.Specifically constructWx ^bThe single base mutation expression vector of geneWx ^b- 10T is utilized The method of mediated by agriculture bacillus obtains expression vector Introduced into RiceWx ^bThe transgenic paddy rice of -10T expression.

Expression vectorWx ^b- 10T primer special:

SEQ ID NO.3 5' TAAGCTTTAGATCCGCTGCCGCCCCGAAT3'；

SEQ ID NO.4 5'CGCCTGCAAAGAACACAAGAACACAACATT3'；

SEQ ID NO.5 5'AACAATTCAATTCAGTGCAGAGATCTTCCACA3'；

SEQ ID NO.6 5'CCATGACGTCAGAGCCCTTCTGTTCC3'；

SEQ ID NO.7 5'GGAACAGAAGGGCTCTGACGTCATGG3'；

SEQ ID NO.8 5’TGGTACCTGAACTTGACGTAGACAGACGTACGATA3’。

Expression vector of the present inventionWx ^bThe construction method of -10T, includes the following steps:

(1) to carryWx ^bThe paddy DNA of allele is template, and with the primer pair of sequence SEQ ID NO.3 and 4, amplification is obtained The segment of sequence such as SEQ ID NO.1；To carryWx ^bThe paddy DNA of allele is template, with bridging sequence SEQ ID The primer pair of NO.5 and 6 and sequence SEQ ID NO.7 and 8 sequences, amplification obtain the segment of sequence such as SEQ ID NO.2；

(2) building carries the expression vector that sequence SEQ ID NO.1 and SEQ ID NO.2 links togetherWx ^b-10T。

Above-mentioned sequence SEQ ID NO.1 is riceWx ^bOne section of sequence of gene, including its own promoter；SEQ ID NO.2 is riceWx ^bOne section of sequence of gene includesWx ^bTenth exons 1,15 base C sports the DNA sequence dna of T.

The invention also discloses a kind of preparation methods of transgenic paddy rice, obtain especially by following methods:

(2) building carries the expression vector that sequence SEQ ID NO.1 and SEQ ID NO.2 links togetherWx ^b-10T；

(3) expression vector of step (2) is imported in glutinous rice rice varieties, obtains transgenic paddy rice.

Amplified production SEQ ID NO.1 and SEQ ID NO.2 is to carryWx ^bThe paddy DNA of allele be template into The DNA fragmentation that row PCR amplification obtains；Amplified production SEQ ID NO.2 be obtained by bridging primer amplificationWx ^bEquipotential base 15 base C of the tenth exons 1 sports the DNA sequence dna of T on gene basis；Amplified production SEQ ID NO.1 and SEQ ID NO.2 points It does not connect through T/A clone into pMD18-T carrier, usesHinD III andNcoI double digestion recycles segment I；With NcoI andKpnI is bis- Segment II is recycled in digestion；2 target fragments are passed through into double enzyme siteHinD III andKpnI is inserted into carrier pCAMBIA1301 It obtainsWx ^b -10T expression vector.

Heretofore describedWx ^b -10T expression vector is by transgenic methods such as agrobacterium tumefaciens mediations, by it It imports in glutinous rice rice varieties, obtains transgenic paddy rice；It is normal relative to carryingWx ^bThe unconverted rice varieties of allele, This transgenic paddy rice rice has the apparent amylose content suitably reduced, has the flexible glue significantly improved thick, food flavor product Matter obviously improves.

The present invention provides a kind of soft rice method of transgenosis cultivated and there is appropriate apparent amylose content, to being transferred toWx ^bThe transgenic rice of -10T expression vector carries out the detection of every index of quality, and it is a series of to show that its quality characteristic has occurred Variation.The strain and carrying wild typeWx ^bThe OryzasativaLcv.Nipponbare of allele is compared, and rice apparent amylose content moderately declines, Gel consistence dramatically increases, and rice starch viscosity number moderately declines, and food flavor significantly improves.

The present invention in glutinous rice by importing to granule bound starch synzyme GBSSI encoding geneWxSpecific nucleotide is fixed The rice expression vector of point modified is achieved.In conventional japonica riceWx ^bAllele is template,Construct the allele The rice expression vector that single nucleotide acid is replaced at tenth exonWx ^b-10T.The overall length of carrier carrying single base mutationWx ^b Allelic sequences obtain glutinous rice transfer using the method for mediated by agriculture bacillusWx ^bThe transgenic paddy rice of -10T gene.Pass through PCR experiment shows that target gene has been integrated into rice genome.Breeding obtains the transgenic paddy rice of homozygous line, such turn Trans-genetic hybrid rice rice grain amylose content generally 12% or so, gel consistence and Starch pasting viscosity index with it is instantly popular excellent The soft rice of food flavor is close, illustrates that such rice belongs to the excellent soft rice type of Cooking Quality.

Detailed description of the invention

Fig. 1 is to containWx ^bThe structure in the area Agrobacterium binary vector T-DNA of -10T expression vector structure.Wherein RB and LB table Show the right boundary sequence in the area T-DNA；HinD III andKpnI is the slotting such as position of target fragment during vector construction；It opens Mover is the promoter region of purpose gene itself；ATG is the translation initiation codon of purpose gene；TGA is the end of purpose gene Only codon；CaMV 35S promoter and 35S polyA be respectively the 35S of cauliflower mosaic virus (CaMV) promoter and Terminator district；Hygromycin (R) is hygromycin gene.

Fig. 2 is to identify transgenic paddy rice using round pcr.Wherein WT is parent control, and other 5 swimming lanes are to carryWx ^b -The transgenosis system of 10T building.

Fig. 3 be containingWx ^b -The homozygous transgenic rice of 10T carrier and its it is unconverted control (Nip (wx)) and routinely take BandWx ^bAllele rice (Nip (Wx ^b)) amylose content.Nip is the abbreviation of japonica rice OryzasativaLcv.Nipponbare, and 1# -4# is to turnWx ^b - 4 transgenosis systems of 10T building.Wherein ' ☆ ☆ ' indicates that difference is extremely significant.

Fig. 4 be containingWx ^b -The homozygous transgenic rice of 10T carrier and its it is unconverted control (Nip (wx)) and routinely take BandWx ^bAllele rice (Nip (Wx ^b)) gel consistence.Nip is the abbreviation of japonica rice OryzasativaLcv.Nipponbare, and 1# -4# is to turnWx ^b -10T structure 4 transgenosis systems built.Wherein ' ☆ ☆ ' indicates that difference is extremely significant.

Fig. 5 be containingWx ^b -The homozygous transgenic rice of 10T carrier and its it is unconverted control (Nip (wx)) and routinely take BandWx ^bAllele rice (Nip (Wx ^b)) viscosity of rice flour spectrum.Nip is the abbreviation of japonica rice OryzasativaLcv.Nipponbare, and 1# -4# is to turnWx ^b - 4 transgenosis systems of 10T building.Wherein ' ☆ ☆ ' indicates that difference is extremely significant.

Specific embodiment

It is not to limit that by following three examples, the invention is further described with definition.Pass through example, section The personnel of grinding can be more clearly understood the present invention, can make certain change and modification to the present invention on this basis, To obtain different research effects.Experimental method in following embodiments is conventional method unless otherwise specified.Experimentation In the reagent that is related to be conventional reagent, use and equal used referring to products instruction.

The acquisition of 1 DNA sequence dna of embodiment and the building of expression vector

According to rice full-length genome information, obtainWx ^bAfter the coded sequence of allele, it is divided to two sections of design specific primers pairWx ^bGenome is expanded (first segment primer pair 5 ' TAAGCTTTAGATCCGCTGCCGCCCCGAAT3 ', SEQ ID NO.3； With 5 ' CGCCTGCAAAGAACACAAGAACACAACATT3 ', SEQ ID NO.4;Second segment rite-directed mutagenesis primer 15 ' AACAATTCAATTCAGTGCAGAGATCTTCCACA3’, SEQ ID NO.5; CCATGACGTCAGAGCCCTTCTGTTCC3', SEQ ID NO.6;With rite-directed mutagenesis primer 25 ' GGAACAGAAGGGCTCTGACGTCATGG3’, SEQ ID NO.7; 5’ TGGTACCTGAACTTGACGTAGACAGACGTACGATA3 ', SEQ ID NO.8) it is used forWx ^bExon10 functionality alkali The fixed point of base replaces building.The sequence amplified such as SEQ ID NO.1 and SEQ ID NO.2.

It takes the tender rice leaf CTAB method of children of japonica rice OryzasativaLcv.Nipponbare to extract oryza sativa genomic dna, makees template with 1 μ L DNA, it is right In first segment sequence, standard PCR amplification directly is carried out with corresponding primer pair (sequence SEQ ID NO.3 and SEQ ID NO.4). For second segment sequence, first with rite-directed mutagenesis primer to 1(sequence SEQ ID NO.5 and SEQ ID NO.6) and pinpoint prominent Become primer pair 2(sequence SEQ ID NO.7 and SEQ ID NO.8) respectively carry out standard PCR amplification obtain PCR product, secondly with The mixture of two kinds of PCR products does template, with the upstream primer 1(SEQ ID NO.5 of rite-directed mutagenesis primer 1) and rite-directed mutagenesis draw The downstream primer 2(SEQ ID NO.8 of object 2) it is that primer carries out standard PCR amplification, obtain segment 2.

Two segments respectively through T/A clone connect into pMD18-T carrier (purchased from TaKaRa company, commodity serial number: 6011), screening positive clone serves the sequencing of Jin Sirui Biotechnology Co., Ltd.Filter out right-on positive gram of sequencing It is grand.For first segment (SEQ ID NO.1) with double digestion (HinD III andNcoI) after on 1% agarose gel electrophoresis Separation recycles target fragment (being named as I) using plastic recovery kit.Double enzymes are used for second segment (SEQ ID NO.2) Cut ( NcoI HeKpnI it is separated on 1% agarose gel electrophoresis after), using plastic recovery kit recycling target fragment (name Simultaneously for II), using double digestion (HinD III andKpnI) digested vector pCAMBIA1301 recycles carrier-pellet after electrophoretic separation Section.Target gene fragment I, II with the pCAMBIA1301 carrier segments that digestion is recycled are connected, it is thin to be transformed into DH5 α competence In born of the same parents；Correct positive colony is connected using digestion and/or the identification of PCR technology screening to be known asWx ^b- 10T(Fig. 1).

Embodiment 2 containsWx ^b -10TThe cultivation and identification of expression vector transgenic paddy rice

The established method in laboratory where rice tissue culture and Agrobacterium-medialed transformation program press inventor carries out (Liu Skilful spring etc., the foundation of Agrobacterium tumefaciens mediated Efficient Transformation System In Rice, plant physiology, 1998,24 (3): 259~ 271).Using OryzasativaLcv.Nipponbare mature embryo as explant, callus is induced on N6D2 culture medium as transformation receptor, passes through agriculture bar Bacterium mediates will be in the RNA interference structure Introduced into Rice callus of building；It, will more after being co-cultured 3 days on co-culturing base N6D2C Injured tissue is transferred to N6D2S1 Selective agar medium and screens 14 days or so, is then screened on N6D2S2 Selective agar medium again, root According to the number and number of days of the practical growing state adjustment screening of callus.More resistant calli is obtained, these resistances are cured Injured tissue moves into differentiation in differential medium and re-forms seedling, finally obtained a large amount of transgenic paddy rices after breaking up in advance.For Whether testing goal gene is integrated into transgenic rice plant, extracts transgenic paddy rice single plant total DNA.With tight with target gene Close chain hygromycin gene sequence is template, designs pair of primers Hyg-F (5 ' GCTTCTGCGGGCGATTTGTGT3 ', SEQ ID No.9) and Hyg-R (5 ' GGTCGCGGAGGCTATGGATGC 3 ', SEQ ID No.10) PCR amplification analysis is carried out, positive plant (Fig. 2) is selected, it is allowed to be selfed, it is pure for breeding acquisition by 2-3 Close transgenosis system.

The quality of 3 transgenic paddy rice rice of embodiment shows

Rice grain amylose content is measured according to the document method that the number that the Ministry of Agriculture promulgates is NY147-88, in a ten thousandth day 50 mg rice flour are accurately weighed on flat to be fitted into 50 ml test tubes, after 0.5 ml dehydrated alcohol of addition makes sample dispersion, add 4.5 The NaOH solution of 1.0 mol/l of ml mixes, and is cooled to room temperature after 20 min in boiling water bath, distilled water constant volume.5 ml are drawn to disappear Change liquid, is added in the 100mL volumetric flask for having filled half bottle of distilled water, the acetic acid solution for adding 1.0 ml, 1.0 mol/l makes sample Product acidification；The iodine solution of 1.5 ml 0.02% is added, with distilled water constant volume, 15 min are stood after shaking up；With the NaOH of 4.5ml 1N 95% ethyl alcohol that 0.5ml is added replaces sample, prepares blank solution.It is adjusted at spectrophotometer wavelength 620nm with blank solution Zero point and the absorbance value for measuring colored samples liquid, the OD value and its relevant amylose finally measured with standard sample Content makes standard curve, and the amylose content of sample to be tested is calculated according to this.Each sample does 3 repetitions.Detection knot Fruit is as shown in figure 3, the amylose content of transgenosis system rice (carries normal compared with conventional japonica rice OryzasativaLcv.NipponbareWx ^bGene) rice is aobvious Writing reduces.

Rice grain amylose content is measured according to the document method that the number that the Ministry of Agriculture promulgates is NY147-88, ten thousand/ 100 mg of milled rice flour sample is accurately weighed on one balance, is placed in 10ml test tube, 0.2ml thymol blue indicator is added, with vibration After swinging device oscillation sufficiently wetting sample, the accurate potassium hydroxide solution that 2.0ml 0.2mol/l is added is vibrated with oscillator again； It is immediately placed in after mixing in the water-bath acutely to boil, covers test tube mouth with glass marble, maintain the rice glue height of boiling always 2/3rds or so of test tube length, it is gelatinized 8min..After gelatinization, test tube is taken out, glass marble is taken down, it is cold at room temperature But cooling 20min in ice-water bath is placed into after 5min.Under the conditions of 25 ± 2 DEG C of room temperature, test tube is lain on platform, after 1h, The length for measuring test tube bottom to cold glue forward position, is indicated with millimeter, as the gel consistence of the sample.Testing result is as shown in figure 4, turn The gel consistence of gene line rice (carries normal compared with conventional japonica rice OryzasativaLcv.NipponbareWx ^bGene) extremely significant increase.

ACC(US corn chemistry association is pressed in the measurement of rice starch viscosity) operating instruction (1995) [93] progress (RVA instrument Purchased from Australian Newport Scientific instrument company).3.00 g of sample that water content is 14% is weighed, distillation is added 25.00 mL of water.Temperature change in continuous mode in tank is as follows: 50 DEG C of 1 min of holding, is risen with the speed of 12 DEG C/min To 95 DEG C (3.75 min), 95 DEG C of 2.5 min of holding are dropped to 50 DEG C (3.75 min), 50 with the speed of 12 DEG C/min DEG C keep 1.4 min.Blender is 960 r/min originating velocity of rotation in 10 s, is maintained at 160 r/min later.It is viscous Property (Viscosity) value with " cP " office indicate.Data analysis is mating with TCW (Thermal Cycle for Windows) Software carries out.Analysis the result shows that, (carried normal with conventional japonica rice OryzasativaLcv.NipponbareWx ^bGene) it compares, the peak value of transgenic rice Viscosity and cold glue viscosity are remarkably decreased (Fig. 5), and the disintegration value of indication food flavor index increases, and recovery value decline illustrates transgenosis The Cooking Quality of rice (carries normal compared with conventional japonica rice OryzasativaLcv.NipponbareWx ^bGene) be improved significantly.

<110>Yangzhou University

<120>building of expression vector Wxb-10T, the preparation of transgenic paddy rice and primer

<160>10

<210>1

<211>3541

<212>DNA

<213>artificial sequence

<400>1

TAGATCCGCT GCCGCCCCGA ATCGCCCGTG CCACCGTCGC AGGAGAAGAG ATGTGAGAGA 60

GAGTTGAAGA GATGGGAGAT GAGGGGAGAG GACAGGAAGA AGAGGGGTGG GAATGATACG 120

TGGGGCCCAC GTGGGCCCCA CCATTTTTTA TTAATTTCTG GGTGAAACTT ACAAGTGGGT 180

CCCATGAGGT TTATTATTTT TTCGGATAAA TTGCCACGTA AGCGTCACAT CAGCGCCACG 240

TCAGATCGAG ACCAAGTCAA ATTAGCCACG TAGGCGCCAC GTCAGCCAAA ACCGCCGTCA 300

AAACCGCCGA GGGACCTAAT CTGCACCGGT TTTAATAGTT GAGAGACCCG TTGTATCTGG 360

TTTTGCGGTT GAAGGACGGA AATTGGATTT ATTGACAAGT CAAGGGACCT TAGATGAACT 420

TATTCCTTTT TATATTTGCA CAGGCCTAAT TTCAAGTCCA GCCCAGCTTT CTTCAGCCTG 480

TTTGATAATT CTCTCTAGCT TATTACAGCC GTGGGAGAGG AGATATACAG CTACAAGATT 540

ACAAGTCGAT GTATACAGCA AACCCATGAG CTGATTGCCT GATTAGACGG TAAGAATGCA 600

TCCCTGAGAA GCAAATGCAT CACCAAATTT GTAGCTTAGA TAAATGCTGT GACCTGCAAG 660

AAAATAAAAT TAAAATCAAA ATAAAAGAAA AGCGCAGGTA ATTGACACCC CACGCATATA 720

AGTGTAGATA CATAACACGT TCATCTAATC ATCTTAATTA GACTTAGGTA AAACTACAAT 780

GAGGTTTATG TCCTACGGAA TGACGACAAG CTAGCAGCAC AGAGGCACAG ATCATATCGT 840

CTCCAGACTC AAGTGCACGT TGATCGTTCG CTCACTGCTT CATCGATCAT CCCTTTGTCG 900

AGGCGTTAGT TGGCAGGCAC TAATAGCTAC AGTAAAGTAA AGAGCAACGT GCCAACGTAC 960

GCACGCTAAC GTGAGTCATG TAGCGTAATT CCAAGTTCTT TTTTTTTTGT CAGCACGTAC 1020

AAGCAGCCGC TAGCCTCGCC CTGCATGAGA AGCTCGCGGC GCGCCACCAA ACTGGCAGGC 1080

ACTCAGCTCG CTGCTGGTCC CGCACGTCGC CACACGATCG ACGTACGCAC GCGAGCGAGA 1140

TCCACCGATG GTTTACGCGT ACGCCGACGG CTCACACATC CCCCGGTGCC CAACAGAAAC 1200

CACACACCAC CCGCACGAAA AAAACCGAAC CGCACGTGCG CGCGCGCTCC ACGCACACCC 1260

CAAACAGACG GCACGGCGGG AGCGCGCGCG CGCACGCGAG CCGAGGAGAA AACAAACGGG 1320

GGAAACAAGC TGGAAAAGCA AAAGGGGAAA AGAACGGAGC GGAGGCTTCA CCCACGGCCA1380

CCGCGACGCG CCACCAGCGT GCGGTGCAAT GCAACGTACG CCAAGCCGAA ACGGCAGGCA 1440

GCATCGCGCA CGCACGCACA CACAGGCCAC AGCACACGCG AGCGACGTAC GCGAGTGCAT 1500

GCAGATGCAT GCGCGGGGCT CGCGCGAGAC CGGCCGATGG GTTCGCTTCT CTTCTCTCTC 1560

CCGTCCCGTT GCGTCGTCAT AGACAAAAGT CGGTTTTGCT TTTGGTTTTT TGGCTCTGAG 1620

GCACTGACGT GCGGGCCAGC GTACGCCTGC GTGCCCCGCA TGTCATCGTC GACACCGGCC 1680

GGGGACCGGG TAAAATGTGT TGCGGGAGGG AGAGGGGGAG AGAGAGATCG CGCGGGCTTC 1740

ACGCAACGGC GCTACAAATA GCCACCCACA CCACCACCCC CTCTCTCACC ATTCCTTCAG 1800

TTCTTTGTCT ATCTCAAGAC ACAAATAACT GCAGTCTCTC TCTCTCTCTC TCTCTCTCTC 1860

TCTCTCTCTC TGCTTCACTT CTCTGCTTGT GTTGTTCTGT TGTTCATCAG GAAGAACATC 1920

TGCAAGTTAT ACATATATGT TTATAATTCT TTGTTTCCCC TCTTATTCAG ATCGATCACA 1980

TGCATCTTTC ATTGCTCGTT TTTCCTTACA AGTAGTCTCA TACATGCTAA TTTCTGTAAG 2040

GTGTTGGGCT GGAAATTAAT TAATTAATTA ATTGACTTGC CAAGATCCAT ATATATGTCC 2100

TGATATTAAA TCTTCGTTCG TTATGTTTGG TTAGGCTGAT CAATGTTATT CTAGAGTCTA 2160

GAGAAACACA CCCAGGGGTT TTCCAACTAG CTCCACAAGA TGGTGGGCTA GCTGACCTAG 2220

ATTTGAAGTC TCACTCCTTA TAATTATTTT ATATTAGATC ATTTTCTAAT ATTCGTGTCT 2280

TTTTTTATTC TAGAGTCTAG ATCTTGTGTT CAACTCTCGT TAAATCATGT CTCTCGCCAC 2340

TGGAGAAACA GATCAGGAGG GTTTATTTTG GGTATAGGTC AAAGCTAAGA TTGAAATTCA 2400

CAAATAGTAA AATCAGAATC CAACCAATTT TAGTAGCCGA GTTGGTCAAA GGAAAATGTA 2460

TATAGCTAGA TTTATTGTTT TGGCAAAAAA AAATCTGAAT ATGCAAAATA CTTGTATATC 2520

TTTGTATTAA GAAGATGAAA ATAAGTAGCA GAAAATTAAA AAATGGATTA TATTTCCTGG 2580

GCTAAAAGAA TTGTTGATTT GGCACAATTA AATTCAGTGT CAAGGTTTTG TGCAAGAATT 2640

CAGTGTGAAG GAATAGATTC TCTTCAAAAC AATTTAATCA TTCATCTGAT CTGCTCAAAG 2700

CTCTGTGCAT CTCCGGGTGC AACGGCCAGG ATATTTATTG TGCAGTAAAA AAATGTCATA 2760

TCCCCTAGCC ACCCAAGAAA CTGCTCCTTA AGTCCTTATA AGCACATATG GCATTGTAAT 2820

ATATATGTTT GAGTTTTAGC GACAATTTTT TTAAAAACTT TTGGTCCTTT TTATGAACGT 2880

TTTAAGTTTC ACTGTCTTTT TTTTTCGAAT TTTAAATGTA GCTTCAAATT CTAATCCCCA 2940

ATCCAAATTG TAATAAACTT CAATTCTCCT AATTAACATC TTAATTCATT TATTTGAAAA 3000

CCAGTTCAAA TTCTTTTAGG CTCACCAAAC CTTAAACAAT TCAATTCAGT GCAGAGATCT 3060

TCCACAGCAA CAGCTAGACA ACCACCATGT CGGCTCTCAC CACGTCCCAG CTCGCCACCT 3120

CGGCCACCGG CTTCGGCATC GCCGACAGGT CGGCGCCGTC GTCGCTGCTC CGCCACGGGT 3180

TCCAGGGCCT CAAGCCCCGC AGCCCCGCCG GCGGCGACGC GACGTCGCTC AGCGTGACGA 3240

CCAGCGCGCG CGCGACGCCC AAGCAGCAGC GGTCGGTGCA GCGTGGCAGC CGGAGGTTCC 3300

CCTCCGTCGT CGTGTACGCC ACCGGCGCCG GCATGAACGT CGTGTTCGTC GGCGCCGAGA 3360

TGGCCCCCTG GAGCAAGACC GGCGGCCTCG GTGACGTCCT CGGTGGCCTC CCCCCTGCCA 3420

TGGCTGTAAG CACACACAAA CTTCGATCGC TCGTCGTCGC TGACCGTCGT CGTCTTCAAC 3480

TGTTCTTGAT CATCGCATTG GATGGATGTG TAATGTTGTG TTCTTGTGTT CTTTGCAGGC 3540

G 3541

<210>2

<211>4439

<212>DNA

<213>artificial sequence

<400>2

AACAATTCAA TTCAGTGCAG AGATCTTCCA CAGCAACAGC TAGACAACCA CCATGTCGGC 60

TCTCACCACG TCCCAGCTCG CCACCTCGGC CACCGGCTTC GGCATCGCCG ACAGGTCGGC 120

GCCGTCGTCG CTGCTCCGCC ACGGGTTCCA GGGCCTCAAG CCCCGCAGCC CCGCCGGCGG 180

CGACGCGACG TCGCTCAGCG TGACGACCAG CGCGCGCGCG ACGCCCAAGC AGCAGCGGTC 240

GGTGCAGCGT GGCAGCCGGA GGTTCCCCTC CGTCGTCGTG TACGCCACCG GCGCCGGCAT 300

GAACGTCGTG TTCGTCGGCG CCGAGATGGC CCCCTGGAGC AAGACCGGCG GCCTCGGTGA 360

CGTCCTCGGT GGCCTCCCCC CTGCCATGGC TGTAAGCACA CACAAACTTC GATCGCTCGT 420

CGTCGCTGAC CGTCGTCGTC TTCAACTGTT CTTGATCATC GCATTGGATG GATGTGTAAT 480

GTTGTGTTCT TGTGTTCTTT GCAGGCGAAT GGCCACAGGG TCATGGTGAT CTCTCCTCGG 540

TACGACCAGT ACAAGGACGC TTGGGATACC AGCGTTGTGG CTGAGGTAGG AGCATATGCG 600

TGATCAGATC ATCACAAGAT CGATTAGCTT TAGATGATTT GTTACATTTC GCAAGATTTT 660

AACCCAAGTT TTTGTGGTGC AATTCATTGC AGATCAAGGT TGCAGACAGG TACGAGAGGG 720

TGAGGTTTTT CCATTGCTAC AAGCGTGGAG TCGACCGTGT GTTCATCGAC CATCCGTCAT 780

TCCTGGAGAA GGTGGAGTCA TCATTAGTTT ACCTTTTTTG TTTTTACTGA ATTATTAACA 840

GTGCATTTAG CAGTTGGACT GAGCTTAGCT TCCACTGGTG ATTTCAGGTT TGGGGAAAGA 900

CCGGTGAGAA GATCTACGGA CCTGACACTG GAGTTGATTA CAAAGACAAC CAGATGCGTT 960

TCAGCCTTCT TTGCCAGGTC AGTGATTACT TCTATCTGAT GATGGTTGGA AGCATCACGA 1020

GTTTACCATA GTATGTATGG ATTCATAACT AATTCGTGTA TTGATGCTAC TGCAGGCAGC 1080

ACTCGAGGCT CCTAGGATCC TAAACCTCAA CAACAACCCA TACTTCAAAG GAACTTATGG1140

TGAGTTATAA TTGATCTCAA GATCTTATAA CTTTCTTCGA AGGAATCCAT GATGATCAGA1200

CTAATTCCTT CCGGTTTGTT ACTGACAACA GGTGAGGATG TTGTGTTCGT CTGCAACGAC1260

TGGCACACTG GCCCACTGGC GAGCTACCTG AAGAACAACT ACCAGCCCAA TGGCATCTAC1320

AGGAATGCAA AGGTCTATGC TTGTTCTTGC CATACCAACT CAAATCTGCA TGCACACTGC1380

ATTCTGTTCA GAAACTGACT GTCTGAATCT TTTTCACTGC AGGTTGCTTT CTGCATCCAC1440

AACATCTCCT ACCAGGGCCG TTTCGCTTTC GAGGATTACC CTGAGCTGAA CCTCTCCGAG1500

AGGTTCAGGT CATCCTTCGA TTTCATCGAC GGGTATGAGT AAGATTCTAA GAGTAACTTA1560

CTGTCAATTC GCCATATATC GATTCAATCC AAGATCCTTT TGAGCTGACA ACCCTGCACT1620

ACTGTCCATC GTTCAAATCC GGTTAAATTT CAGGTATGAC ACGCCGGTGG AGGGCAGGAA1680

GATCAACTGG ATGAAGGCCG GAATCCTGGA AGCCGACAGG GTGCTCACCG TGAGCCCGTA1740

CTACGCCGAG GAGCTCATCT CCGGCATCGC CAGGGGATGC GAGCTCGACA ACATCATGCG1800

GCTCACCGGC ATCACCGGCA TCGTCAACGG CATGGACGTC AGCGAGTGGG ATCCCAGCAA1860

GGACAAGTAC ATCACCGCCA AGTACGACGC AACCACGGTA AGAACGAATG CATTCTTCAC1920

AAGATATGCA ATCTGAATTT TCTTTGAAAA AGAAATTATC ATCTGTCACT TCTTGATTGA1980

TTCTGACAAG GCAAGAATGA GTGACAAATT TCAGGCAATC GAGGCGAAGG CGCTGAACAA2040

GGAGGCGTTG CAGGCGGAGG CGGGTCTTCC GGTCGACAGG AAAATCCCAC TGATCGCGTT2100

CATCGGCAGG CTGGAGGAAC AGAAGGGCCC TGACGTCATG GCCGCCGCCA TCCCGGAGCT2160

CATGCAGGAG GACGTCCAGA TCGTTCTTCT GGTATAATAT AATACACTAC AAGACACACT2220

TGCACGATAT GCCAAAAATT CAGAACAAAT TCAGTGGCAA AAAAAAAACT CAAATATTAG2280

GGAAGAACCT AATATCAAAT AATTAGAAGG GGTGAGGCTT TGAACCCAGG TCATCTAGCC2340

CACCACCTTG TAGAGCTAGC CGGAAGAGCT CTGAGCATTT CTCGATTCAG TGGCAAATGA2400

TGTGTATAAT TTTGATCCGT GTGTGTTTCA GGGTACTGGA AAGAAGAAGT TCGAGAAGCT2460

GCTCAAGAGC ATGGAGGAGA AGTATCCGGG CAAGGTGAGG GCCGTGGTGA AGTTCAACGC2520

GCCGCTTGCT CATCTCATCA TGGCCGGAGC CGACGTGCTC GCCGTCCCCA GCCGCTTCGA2580

GCCCTGTGGA CTCATCCAGC TGCAGGGGAT GAGATACGGA ACGGTATACA ATTTCCATCT2640

ATCAATTCGA TTGTTCGATT TCATCTTTGT GCAATGCAAT GCAATTGCAA ATGCAAATGC2700

ATGATGATTT TCCTTGTTGA TTTCTCCAGC CCTGTGCTTG CGCGTCCACC GGTGGGCTCG2760

TGGACACGGT CATCGAAGGC AAGACTGGTT TCCACATGGG CCGTCTCAGC GTCGACGTAA2820

GCCTATACAT TTACATAACA ATCAGATATG ACACATTCTA ATACCGATAA GTCAGTACAC2880

TACTACACAT TTACATGGTT GCTGGTTATA TGGTTTTTTT GGCAGTGCAA GGTGGTGGAG2940

CCAAGCGACG TGAAGAAGGT GGCGGCCACC CTGAAGCGCG CCATCAAGGT CGTCGGCACG3000

CCGGCGTACG AGGAGATGGT CAGGAACTGC ATGAACCAGG ACCTCTCCTG GAAGGTATAA3060

ATTACGAAAC AAATTTAACC CAAACATATA CTATATACTC CCTCCGCTTC TAAATATTCA 3120

ACGCCGTTGT CTTTTTAAAA TATGTTTGAC CGTTCGTCTT ATTAAAAAAA TTAAATAATT 3180

ATAAATTATT TTCCTATCAT TTGATTCATT GTTAAATATA CTTATATGTA TACATATAGT 3240

TTTACATATT TCATAAAAGT TTTTGAACAA GACGAACGGT CAAACATGTG CTAAAAAGTT 3300

AACGGTGTCG AATATTCAGA AACGGAGGGA GTATAAACGT CTTGTTCAGA AGTTCAGAGA 3360

TTCACCTGTC TGATGCTGAT GATGATTAAT TGTTTGCAAC ATGGATTTCA GGGGCCTGCG 3420

AAGAACTGGG AGAATGTGCT CCTGGGCCTG GGCGTCGCCG GCAGCGCGCC GGGGATCGAA 3480

GGCGACGAGA TCGCGCCGCT CGCCAAGGAG AACGTGGCTG CTCCTTGAAG AGCCTGAGAT 3540

CTACATATGG AGTGATTAAT TAATATAGCA GTATATGGAT GAGAGACGAA TGAACCAGTG 3600

GTTTGTTTGT TGTAGTGAAT TTGTAGCTAT AGCCAATTAT ATAGGCTAAT AAGTTTGATG 3660

TTGTACTCTT CTGGGTGTGC TTAAGTATCT TATCGGACCC TGAATTTATG TGTGTGGCTT 3720

ATTGCCAATA ATATTAAGTA ATAAAGGGTT TATTATATTA TTATATATGT TATATTATAC 3780

TCCCCCTGTT CCATATTATG CCATGCCATT TTTGTTTTAT GCCAAGTCAA ACTTTTTATA 3840

TTTAACCAAA TTTATAAAAA TAAATATAGC AACATTTGTA ATACTGAACT ATTTTTGTTA 3900

GACAGACTGT CAAAACTTAA ATTATAGGTA CTATATTTGT CTCAAAATAT AATAATTTTT 3960

AGTTATGTAT CTGGGTATGT GTCTGTCTAT ATGTCTAGTT AAAAGTTGTT TTGTGTAAAA 4020

AAATGTTATT ATATTTTTCT ATAAATTTAT TTAAGTTTGA AGGAGCAGTA GTTTGACTCA 4080

GGATAAAATG TAAAATAATT TATAATATAC TCTCTCGTCC CATTTTAAAT GCAACCACAA 4140

CTTTGATCGT TCATCTTATT TATTTTTTTA TAATTAATAC TTTTATTGTT ATGGGATAAT 4200

AAAACATGAA TAGTACTTAT GTTTTTAATT TTTTTTTAAT TTTTTTTAAA TAAAACGAAT 4260

GATTAAAATT GTGCACAAAA AATTATAGTT GCACTTAAAA TATGACGGAG GGGGTGGATA 4320

CGAAGGAACT AGTCCTGTTA AATATCCTGT TCGTGTGTTT TTTAGGTTGT GGCTCTGGTT 4380

GATCAGATGC CACTGTCATT ACTAGTGCTC CATATATCGT ACGTCTGTCT ACGTCAAGT 4439

<210>3

<211>29

<212>DNA

<213>artificial sequence

<400>3

TAAGCTTTAG ATCCGCTGCC GCCCCGAAT 29

<210>4

<211>30

<212>DNA

<213>artificial sequence

<400>4

CGCCTGCAAA GAACACAAGA ACACAACATT 30

<210>5

<211>32

<212>DNA

<213>artificial sequence

<400>5

AACAATTCAA TTCAGTGCAG AGATCTTCCA CA 32

<210>6

<211>26

<212>DNA

<213>artificial sequence

<400>6

CCATGACGTC AGAGCCCTTC TGTTCC 26

<210>7

<211>26

<212>DNA

<213>artificial sequence

<400>7

GGAACAGAAG GGCTCTGACG TCATGG 26

<210>8

<211>35

<212>DNA

<213>artificial sequence

<221>

<400>8

TGGTACCTGA ACTTGACGTA GACAGACGTA CGATA 35

<210>9

<211>21

<212>DNA

<213>artificial sequence

<221>

<400>9

GCTTCTGCGG GCGATTTGTG T 21

<210>10

<211>21

<212>DNA

<213>artificial sequence

<221>

<400>10

GGTCGCGGAG GCTATGGATG C 21

Claims

1. expression vectorWx ^b- 10T primer special:

SEQ ID NO.3 5' TAAGCTTTAGATCCGCTGCCGCCCCGAAT3'；

SEQ ID NO.4 5'CGCCTGCAAAGAACACAAGAACACAACATT3'；

SEQ ID NO.5 5'AACAATTCAATTCAGTGCAGAGATCTTCCACA3'；

SEQ ID NO.6 5'CCATGACGTCAGAGCCCTTCTGTTCC3'；

SEQ ID NO.7 5'GGAACAGAAGGGCTCTGACGTCATGG3'；

SEQ ID NO.8 5’TGGTACCTGAACTTGACGTAGACAGACGTACGATA3’。

2. a kind of expression vectorWx ^bThe construction method of -10T, characterized in that it is characterized in that, the method specifically includes following Step:

(1) to carryWx ^bThe paddy DNA of allele is template, with the primer of sequence SEQ ID NO.3 and SEQ ID NO.4 Right, amplification obtains the segment of sequence such as SEQ ID NO.1；To carryWx ^bThe paddy DNA of allele is template, with sequence of putting up a bridge The primer pair for arranging SEQ ID NO.5 and SEQ ID NO.6 and sequence SEQ ID NO.7 and SEQ ID NO.8 sequence, expands To the segment of sequence such as SEQ ID NO.2；

3. expression vector according to claim 1Wx ^bThe construction method of -10T, characterized in that the sequence SEQ ID NO.1 is riceWx ^bOne section of sequence of gene, including its own promoter；SEQ ID NO.2 is riceWx ^bOne section of gene Sequence includesWx ^bTenth exons 1,15 base C sports the DNA sequence dna of T.

4. expression vector according to claim 1Wx ^bThe construction method of -10T, characterized in that step (2) structure Build expression vectorWx ^b- 10T's method particularly includes: amplified production SEQ ID NO.1 and SEQ ID NO.2 is respectively through T/A grams It is grand to connect into pMD18-T carrier, it usesHinD III andNcoI double digestion recycles segment I；With NcoI andKpnThe recycling of I double digestion Segment II；Segment I, segment II are passed through into double enzyme siteHinD III andKpnI insertion carrier pCAMBIA1301 is obtainedWx ^b- 10T expression vector.

5. a kind of method of prepare transgenosis rice, which is characterized in that the preparation method is that: it will be any in claim 2-4 Expression vector described inWx ^b- 10T is imported into glutinous rice using the method for mediated by agriculture bacillus, is obtainedWx ^b- 10T expression Homozygous transgenic rice.