CN1301266C

CN1301266C - High molecular wheat glutelin subunit, genes encoding same and use thereof

Info

Publication number: CN1301266C
Application number: CNB2003101038934A
Authority: CN
Inventors: 张学勇; 王汉潜
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-11-24
Filing date: 2003-11-24
Publication date: 2007-02-21
Anticipated expiration: 2023-11-24
Also published as: CN1621412A

Abstract

The present invention discloses a wheat high molecular weight glutenin subunit, a coding gene and the application thereof. The wheat high molecular glutelin subunit provided by the present invention is protein having the SEQ ID No. 2 amino acid residue sequence in the sequence table or protein which has the same activity as that of the SEQ ID No. 2 amino acid residue sequence and is derived from SEQ ID No. 2 by that one or a plurality of amino acid residues of the SEQ ID No. 2 amino acid residue sequence are replaced, deleted and added. The coding gene of the high molecular weight glutenin subunit has one of the following nucleotide sequences: 1), the SEQ ID No. 1 DNA sequence in the sequence table, 2), the SEQ ID No. 3 DNA sequence in the sequence table, 3), a polynucleotide of the SEQ ID No. 2 protein sequence in the coding sequence table and 4) a DNA sequence limited by SEQ ID No. 1 or SEQ ID No. 3 in the sequence table, having more than 90% homology with SEQ ID No. 1 or SEQ ID No. 3 and coding protein with the same function. The coding gene 1By15 of the wheat high molecular weight glutenin subunit can be widely used for grain crops, particularly for the transgenic breeding of wheat quality.

Description

The high-molecular-weight glutelin subunit of wheat and encoding gene thereof and application

Technical field

The present invention relates to high-molecular-weight glutelin subunit and the encoding gene and the application of wheat in the plant genetic engineering field.

Background technology

The wheat processing quality mainly is subjected to the influence of gluten (Glutenin) and prolamine (Gliadin) composition, amylose starch and amylopectin ratios in heredity, wherein high-molecular-weight glutelin subunit (HMW-GS) is formed the influence of quality the most outstanding (Payne et al, 1983; Shewry et al 1992).

Processing quality is the core of wheat competition in the international market.In influencing numerous genes of quality, the effect of high-molecular-weight glutelin subunit (HMW-GS) is very important, and for example " 5+10 " combination is normal closely related with good baking properties on the 1D karyomit(e), and other array mode often causes the decline of baking properties.The present inventor finds after to a wheat commercial variety electrophoresis surplus domestic 1000, the good source kind of northern China wheat main producing region has two, " in make 8131-1 " is (by the Ceng Daoxiao of Crop Breeding Cultivation Inst., Chinese Agriculture Academy, the refined favour of state etc. is bred, carry high quality subunit encoding gene 1Dx5+1Dy10, see Zhang Xueyong etc. for details: Scientia Agricultura Sinica, 34:355-362,2001) and " laying down for a short time No. 6 " (breed by people such as northwest plant research institute of former Chinese Academy of Sciences Li Zhenshengs, carry high quality subunit encoding gene 1Bx14+1By15, see Zhang Xueyong etc. for details: Scientia Agricultura Sinica, 34:355-362,2001).The 1Bx14+1By15 subunit is to having brought into play bigger effect in China's good quality wheat breeding and production, some famous fine quality are excellent 225 as lay down for a short time No. 54, high excellent 503, Shan, Shan excellent 229, Ph82-2-2 all carries this two genes, but this subunit only is that 5.01% (Zhang Xueyong etc. 2002 to the frequency of occurrences in China's improved variety, Scientia Agricultura Sinica, 35 (11): 1302-1310), its potential value is not fully exerted.

1Bx14 and promotor thereof are obtained by the clone, and its N end lacks two cysteine residues (number of patent applications: 01143428.7) than other subunit.But never be cloned into the closely linked 1By15 subunit of 1Bx14.

The innovation and creation content

The purpose of this invention is to provide high-quality high-molecular weight gluten subunit of common wheat and encoding gene thereof.

Wheat high-molecular-weight glutelin subunit 1By15 provided by the present invention, deriving from wheat breed lays down No. 6 for a short time, it is to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

SEQ ID № in the sequence table: the 2nd, the protein of forming by 723 amino-acid residues.

The encoding gene 1By15 of wheat high-molecular-weight glutelin subunit 1By15 is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the sequence table: 3 dna sequence dna;

3) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

4) with sequence table in SEQ ID №: 1 or SEQ ID №: 3 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.

The dna sequence dna of sequence 1 is by 2175 based compositions in the sequence table, and the reading frame of this gene is to hold the 1st to the 2175th bit base from 5 '; The dna sequence dna of sequence 3 is by 2363 based compositions in the sequence table, and the reading frame of this gene is to hold the 1st to the 2175th bit base from 5 '.Wherein, SEQ ID №: the 3rd, full length cDNA sequence (the SEQ ID №: 3) of the wheat high-molecular-weight glutelin subunit 1By15 that obtains by the nested deletion method.

The expression vector and the clone that contain the wheat high-molecular-weight glutelin subunit encoding gene also belong to protection scope of the present invention; utilize existing molecular biological method can obtain different expression vectors and engineering bacteria, as recombinant expression vector PET-3a-1By15 (M) with contain the E.coliBL21 (DE3) of recombinant expression vector PET-3a-1By15 (M).

The single-minded bonded monoclonal antibody of employing of the present invention and wheat high-molecular-weight glutelin subunit (number of patent application: 01144781.8) make probe, No. 6 endosperm development expression libraries in period are implemented screening to laying down for a short time, separate and cloned high-molecular-weight glutelin subunit encoding gene 1By15, sequential analysis finds that structurally there are bigger difference in high-molecular-weight glutelin subunit 1By15 and this genoid of having cloned, and finds that its expression amount becomes positive correlation with processing quality.

The encoding gene 1By15 of wheat high-molecular-weight glutelin subunit of the present invention will be widely used in the transgenic breeding of cereal crop, particularly wheat quality, and the present invention has simultaneously also opened up a new thinking for clone gene from big genome plant.

Description of drawings

Fig. 1 1By15 gene clone schema of laying down for a short time No. 6

The bloom cDNA expression library result of 16 days seeds of Fig. 2 monoclonal antibody screening wheat

Fig. 3 adopts the PCR method screening to insert the electrophoretogram of fragment greater than the positive plaque of 2kb

Restriction analysis electrophoretogram behind Fig. 4 positive colony subclone

Fig. 5 adopts the fixedly electrophoretogram of the deletion mutant of the other end positive colony different in size of a series of ends that the nested deletion method makes up

The SDS-PAGE of the prokaryotic expression of the excellent high-molecular-weight glutelin subunit 1By15 of Fig. 6 wheat analyzes (A) and immunoblotting (B) is analyzed

Fig. 7 carries out the sequence alignment analysis with CLUSTAL W (1.82) to HMW-GS1By15,1By8,1By9 and 1Dy10

Fig. 8 is the physical map of PET-3a

It is that Y152 and Y224 and the baking properties that contrasts capital 411 compare that Fig. 9 selects for No. 6 for laying down for a short time

Embodiment

The clone of embodiment 1, wheat high-molecular-weight glutelin subunit encoding gene 1By15

Clone's flow process of wheat high-molecular-weight glutelin subunit encoding gene 1By15 specifically may further comprise the steps as shown in Figure 1:

One, lay down for a short time structure of the seed cDNA expression library that No. 6 wheats bloom back 16 days

Get No. 6 wheats of laying down for a short time back 16 days seed of blooming, adopt RNeasy Plant Mini Kit (QIAGEN) to extract total RNA.Adopting the SMART cDNA of CLONTECH company library construction test kit to finish the cDNA expression library then makes up.

Two, the immunoscreening of cDNA expression library

Method is with reference to Sambrook et al (1989), and concrete steps are as follows:

1, with the single-minded bonded monoclonal antibody of wheat high-molecular-weight glutelin subunit (number of patent application: pre-treatment 01144781.8)

Adopt intestinal bacteria lysate absorption process.Being prepared as follows of E.coli XL1-Blue lysate:

(1) cultivating E.coli XL1-Blue bacterium in 500ml LB substratum reaches capacity.37 ℃, the 200rpm shaken overnight.4 ℃, centrifugal 10 minutes of 4000rpm, results bacterial precipitation.

(2) bacterial precipitation is resuspended in 15ml 50M Tris-Cl (PH8.0), among the 10mM EDTA (PH8.0).-70 ℃ are freezing, room temperature or 37 ℃ of thawings, and multigelation is repeatedly.In 0 ℃, use ultrasonication 6 times, each 20 seconds again.

Under (3) 4 ℃, centrifugal 10 minutes of 15000rpm transfers to another in vitro with supernatant liquor, the gained lysate be stored in-20 ℃ standby.

To add 1 milliliter of intestinal bacteria lysate in every 10ml monoclonal antibody cell conditioned medium liquid, mix the back and spend the night more than 4 hours or in 4 ℃ when (4) using in the room temperature incubation.It is 0.05%NaN that attention can add final concentration ₃, be stored in 4 ℃ and use in order to immunology when screening.

2, the preparation of host bacterium bacteria suspension

The single colony inoculation of picking E.coli XL1-Blue is in 50ml MgSO ₄In/maltose LB the substratum, 37 ℃, after the 200rpm shaken overnight, be sub-packed in two aseptic round bottom centrifuge tubes of 50ml, centrifugal 10 minutes of 4000rpm room temperature, the supernatant that inclines is used 10ml 0.01M MgSO respectively ₄Resuspended precipitation, it is standby to put 4 ℃ of storages.Available 3 weeks.

3, immunoscreening cDNA expression library

Bed board

(1) at first in the eppendorf pipe, adds 0.15ml XL1-Blue bacteria suspension and 0.15ml and contain 3 * 10 ⁴Pfu contains the segmental λ TriplEx2 phage of insertion (referring to the SMART of CLONTECH company ^TMCDNAlibrary construction kit user mannual) SM (wanting the high-density bed board during primary dcreening operation) of expression library mixes being placed on 37 ℃ of incubations 20 minutes, so that phage absorption intestinal bacteria.

(2) then, eppendorf is managed content be transferred to diameter be that the corresponding sterile test tube of 150mm plate is (in 10 * 130mm).Each in vitro continues to add the top agarose that 7.5ml melts, and is poured into immediately on the LB agar plate behind the mixing.Wait to solidify and be placed on 42 ℃ of incubation 4-5 hours.

Change film

(3) identical with dull and stereotyped size approximately, after saturated nitrocellulose filter dries in the 10mM IPTG aqueous solution in advance, be covered on the flat board 37 ℃ of overnight incubation.

(4) raise the plate loam cake, continued incubation 20 minutes in 37 ℃.Flat board is moved on under the room temperature, film and flat board are made asymmetric labeling with syringe needle.Then, take film carefully off, film is put into a large amount of TNT.

Immuning hybridization

(5) wash film twice with the TNT damping fluid, each 20 minutes.Then, soaked film 30-60 minute with TNT+5% skim-milk confining liquid, and shake gently.

(6) the deblocking liquid that inclines soaked film 30-60 minute with TNT+5% skim-milk+monoclonal antibody (dilution in 1: 20), and shakes gently.

(7) wash film 3 times with TNT, each 10 minutes.

(8) with 10mM Tris-Cl (PH7.5), 150mM NaCl solution washing 10 minutes, film is transferred to 7.5ml contain the 10mMTris-Cl (PH7.5) of second antibody (alkaline phosphatase coupling sheep anti-mouse antibody) with dilution in 1: 20000, in the 150mM NaCl solution, under room temperature, shook gently 1.5-2 hour.

(9) with 10mM Tris-Cl (PH7.5), 150mM NaCl solution is washed film 3 times, each 10 minutes.

The immunity colour developing

(10) film is transferred in the freshly prepared colour developing liquid, developed the color 1-4 hour.

(11) slightly wash 2 times color development stopping then with distilled water.

Insert the screening of fragment greater than the positive plaque of 2kb

(12) find out corresponding to positive plaque clone on the film, use toothpick picking positive plaque then, with the distinguished sequence of λ TriplEx2 carrier: 5 '-CTCCGAGATCTGGACGAGC-3 ' is 5 ' end primer; 5 '-TAATACGACTCACTATAGGG-3 ' is 3 ' end primer.With the insertion fragment in the round pcr amplification positive plaque, insert segmental size to infer.Because the length of the high-molecular wheat glutelin subunit gene of wheat all greater than 2kb, is inserted the positive plaque of fragment greater than 2kb so only keep, the result screens and obtains an insertion fragment greater than 2kb positive plaque (arrow indication among the figure) as shown in Figure 3.

Follow-up screening

(13) agar block (containing phage) corresponding to positive colony on the film sucks out carefully with wide mouthful of pipette, is put among the 1ml SM that is added with 2 chloroforms.Spend the night or following 60 minutes of room temperature in 4 ℃ of placements, phage wash-out from agar block is come out.Determine the titre of elutriant pnagus medius then,, hybridize colour developing as described above, up to screening consistent immune positive plaque with the low density bed board.The result shows as shown in Figure 2 through twice screening, obtains consistent immune positive plaque, and among Fig. 2, stain represents to screen the positive plaque that obtains, and A is the The selection result first time, and B is the programmed screening result.

Three, will have the positive colony plasmidization of inserting segmental λ TriplEx2 phage vector

1, plasmidization

The segmental λ TriplEx2 phage vector of insertion that has that obtains is inserted segmental pTriplEx2 plasmid vector by deletion system transition in the Crex/Loxp for having, and experimentation is as follows:

(1) the single bacterium colony of picking E.coli BM25.8 is inoculated in 10ml MgSO ₄In/LB the substratum, 31 ℃, the 150rpm shaken overnight reaches 1.1-1.4 until the OD of culture.In culture, add 100 μ l 1M MgCl ₂, make its concentration reach 10mM.

(2) positive plaque of picking from several flat boards of taking turns after the screening immerses among the 350 μ l SM, and 4 ℃ are spent the night, and its wash-out is come out.

(3) 200 μ l BM25.8 bacterial culturess and 150 μ l plaque elutriants are mixed, 31 ℃ of following incubations 30 minutes add 400 μ l LB substratum then, 31 ℃, 225rpm acutely jolts 1 hour, takes out 5 μ l, evenly coat on the LB/Amp flat board 31 ℃ of overnight incubation.Several bacterium colonies of picking are to extract plasmid DNA.

2, plasmid extracts

(1) will transform good single colony inoculation and go in the 2ml LB/Amp liquid nutrient medium, 37 ℃ of shaking culture are spent the night.Get the 1.5ml culture and pour in the eppendorf pipe, centrifugal 2 minutes of 4000rpm.Abandon supernatant, make bacterial precipitation dry as far as possible.

(2) bacterial precipitation is suspended in the 100 μ l solution I, abundant mixing, room temperature was placed 10 minutes.Add the fresh obtain solution II of 200 μ l then, the tight pipe lid of lid, the mixing content is placed on 5 minutes on ice with centrifuge tube.The solution III that adds 200 μ l precoolings then, the tight pipe lid of lid is put upside down mixing for several times, and centrifuge tube is placed on 15 minutes on ice.Centrifugal 15 minutes of 12000rpm room temperature changes supernatant liquor in another eppendorf pipe over to.

(3) add equal-volume phenol to supernatant liquor: chloroform (1: 1), mixing repeatedly, centrifugal 5 minutes of 12000rpm room temperature changes supernatant liquor in another eppendorf pipe over to.

(4) add 2 times of volume ethanol mixings to supernatant liquor, room temperature was placed 5-10 minute, and centrifugal 5 minutes of 12000rpm room temperature is removed supernatant liquor, and the eppendorf pipe is tipped upside down on the thieving paper, blots liquid.

(5) with 1ml 70% washing with alcohol plasmid DNA precipitation, vibration is also centrifugal, abandons supernatant, and vacuum is drained.Add 50 μ l TE damping fluids DNA is dissolved fully ,-20 ℃ of preservations.

Four, will have the segmental pTriplEx2 plasmid of insertion and carry out subclone

Owing to have the restriction enzyme site of single EcoRI and SalI on the pTriplEx2 plasmid vector, carry out the EcoRI/SalI double digestion to having the segmental pTriplEx2 plasmid of insertion, agarose gel electrophoresis separates and the recovery endonuclease bamhi, be connected with pBSK (pBluescriptII SK the is abbreviated as pBSK) carrier of same double digestion purifying, obtain recombinant plasmid pBSK/1By15, Transformed E .coli DH5 α cell.Finish the subclone of positive colony from carrier pTriplEx2 to carrier pBSK.The pBSK/1By15 that obtains is carried out EcoRI single endonuclease digestion and the analysis of EcoRI/SalI double digestion, and the result shows and inserts segmental length (arrow indication among the figure) between 2-3kb among the plasmid pBSK/1By15 as shown in Figure 4.Among Fig. 4,1 is plasmid pB SK/1By15, and 2 cut through the EcoRI enzyme for plasmid pB SK/1By15, and 3 is that plasmid pBSK/1By15 analyzes through the EcoRI/SalI double digestion, and M is a standard molecular weight.

Five, the full length cDNA sequence of positive colony is measured

Make up the deletion mutant of the positive colony that a series of ends are fixed, the other end is different in size with the nested deletion method.Explain the principle of nested deletion method with restriction enzyme ApaI and SalI: the ApaI enzyme is cut the back and is produced 3 ' overhang, the effect that can resist excision enzyme ExoIII; And the SalI enzyme is cut the back and is produced 3 ' recessed end, is subjected to the effect of excision enzyme ExoIII and progressively removes Nucleotide.Because the SalI restriction enzyme site is nearer from inserting fragment, inserts fragment and progressively degrade, and remain unchanged away from an end of SalI restriction enzyme site near an end of SalI restriction enzyme site.ExolI difference action time just can produce fragment different in size.These fragments are scabbled overhang with nuclease S, and Transformed E .coli DH5 α after the connection cyclisation can the nested deletion mutant of a cover.Detailed process is as follows: with ApaI and SalI double digestion pBSK/1By15 recombinant plasmid.After phenol/chloroform extracting, digest linear plasmid with ExoIII, ExoIII difference action time just can produce fragment different in size.Then scabble overhang, connect Transformed E .coliDH5 α with nuclease S.Use the ABI3700 sequenator to finish the sequencing of deletion mutant, sequence is spliced, finish the mensuration of full length cDNA sequence with the Seqman program of DNAstar software.

Its electrophoresis result shows the pBSK/1By15 recombinant plasmid as shown in Figure 5 with behind ApaI and the SalI double digestion, with the electrophoretogram of ExoIII digestion different time gained linear plasmid.The time of ExoIII digestion every sampling in 1 minute, obtained 16 samples since 0 o'clock altogether.Point sample on 1% sepharose at last.ExoIII difference action time just can produce linear plasmid different in size (fragment) as shown in Figure 5, from the longest 5kb to 3kb.Use the ABI3700 sequenator to finish the sequencing of nested deletion mutant, sequence is spliced, finish full length cDNA sequence (SEQ ID №: mensuration 3) with the Seqman program of DNAstar software.SEQ ID №: in 3, being initiator codon ATG from the 1st to the 3rd at 5 ' end, is terminator codon TGATAG from the 2170th to the 2175th at 5 ' end, is poly A tailing signal AATAAA from the 2226th to the 2231st at 5 ' end.

Six, the prokaryotic expression of the excellent high-molecular-weight glutelin subunit 1By15 of wheat

Sequential analysis shows: one BamHI restriction enzyme site is arranged in the HMW-GS1By15cDNA in-line coding district.For 1By15 being cloned into the prokaryotic expression carrier PET-3a (its physical map as shown in Figure 8) that has the NdeI/BamHI restriction enzyme site, need to adopt big primer (Megaprimer) PCR method, 1By15cDNA is eliminated the synonym point mutation of BamHI restriction enzyme site.Specific as follows: as to design one and contain and come base (primer (15mer, the 5 '-GGGTAGAA of G → A) by point mutation in the original BamHI restriction enzyme site AGATCCAnd the proteinic aminoterminal of encoding mature and be another primer (27mer, 5 '-ACC in the sequence of its 5 ' end affix Nde I restriction enzyme site in addition G-3 '), CATATGGAAGGTGAGGCCTCTAGG-3 '), with pBSK/1By15 is template, adopts PCR method, and the one section sequence that amplifies thus is as big primer, hold the sequence of other affix BamHI restriction enzyme site to form upstream and downstream primer (34mer, 5 '-AAT with the proteinic carboxyl terminal of encoding mature and its 5 ' then GGATCCGCAAGTTGCAAAGAGTTCTATCACT-3 '), finally be eliminated BamHI restriction enzyme site and through the correct 1By15 of sequence verification (M).

With CLUSTAL W (1.82) HMW-GS1By15,1By8,1By9 and 1Dy10 are carried out sequential analysis, the result shows that structurally there are bigger difference in 1By15 gene and this genoid of having cloned as shown in Figure 7.

Then 1By15 (M) gene is linked to each other with expression vector PET-3a through the NdeI/BamHI double digestion, make up recombinant expression vector PET-3a/1By15 (M).Transformed E .coli BL21 (DE3), expressed protein is carried out SDS-PAGE (A) and immunoblotting [adopt with the single-minded bonded monoclonal antibody of wheat high-molecular-weight glutelin subunit (number of patent application: 01144781.8)] to be analyzed, the result as shown in Figure 6, show 1By15 (M) gene in the E.coli successful expression, 1By15 (M) subunit (arrow indication among the figure) that 1By15 (M) gene is expressed at E.Coli.1By15 (M) has one to have more methionine(Met) though its N of subunit holds, its electrophoretic mobility and " laying down 54 for a short time " seed storage protein 1By15 basically identical.The proof cloned genes is the gene of coding high-molecular-weight glutelin subunit 1By15.A is that SDS-PAGE analyzes collection of illustrative plates among Fig. 6, and B is for being the immunoblotting assay collection of illustrative plates, and 1 for laying down seed extract for a short time No. 54; 2 are recombinant expression vector PET-3a/1By15 (M) expression product after IPTG induces in E.coli BL21 (DE3); 3 is that recombinant expression vector PET-3a/1By15 (M) is at E.coliBL21 (DE3), without IPTG inductive expression product; 4 is that expression vector PET-3a is at E.coli BL21 (DE3), the expression product after IPTG induces.

Embodiment 2,1By15 are to the influence of bread baking properties

Taking by weighing that laying down for a short time of equivalent weight select for No. 6 is the flour in Y152 and Y224 (1Bx14 and 1By15 carrier) and contrast capital 411 (not carrying 1Bx14 and 1By15), under similarity condition, utilize the dough baking bread of identical weight, the result as shown in Figure 9, as can be seen from the figure, the strain bread coefficient of expansion that carries 1Bx14 and 1By15 is big, and honeycomb is even, color is turned white, the strain loaf volume that does not carry 1Bx14 and 1By15 is less, and honeycomb is inhomogeneous, the color jaundice.

Sequence table

<160>3

<210>1

<211>2175

<212>DNA

＜213〉Triticum wheat (Triticum aestivum L.)

<400>1

atggctaagc?ggttggtcct?ctttgcgaca?gtagtcatca?ccctcgtggc?tctcgctgct 60

gctgaaggtg?aggcctctag?gcaactacag?tgtgagcgcg?agctccagga?gagctcgctt 120

gaggcatgcc?gacaggtcgt?ggaccaacag?ttggccggtc?ggctgccatg?gagcacgggg 180

ctccagatgc?gatgctgcca?gcagctccga?gatgttagcg?ctaagtgccg?ccccgtcgcc 240

gtcagccaag?tcgtaagaca?atatgagcaa?accgtggtgc?cgcccaaggg?cggatccttc 300

taccctggcg?agaccacacc?actgcagcaa?ctccaacaag?taatattttg?gggaacatct 360

tcacaaacag?tacaagggta?ttacccaagc?gtaagttctc?ctcagcaggg?gccatattat 420

ccaggccaag?cttctccaca?acagccagga?caaaggcaac?agccaggcaa?atggcaagaa 480

ctgggacaag?ggcaacaagg?gtattaccca?acttctctgc?atcagtcagg?acaaggacaa 540

caagggtact?acccatcttc?tctgcagcaa?ccaggacaag?ggcaacagac?aggacaaggg 600

caacaaggat?actacccaac?ttctctgcag?cagccaggac?aagggcaaca?gataggacaa 660

gggcaacaag?ggtactaccc?aacttctccg?cagcacccag?gacaaaggca?acaaccagga 720

caagggcagc?aaataggaca?agggcaacaa?ccaggacaag?ggcggcaaat?aggacaaggg 780

caacaatcag?gacaagagca?acaagggtac?tatgcaactt?ctccacagca?gctaggacaa 840

gggcaacaac?caggacaatg?gcaacaatca?ggacaagggc?aacaaaggta?ctacccaact 900

tctcagcagc?agccaggaca?agggcaacag?gggcagtacc?cagcttctca?gcagcagcca 960

gcacaagggc?aacaagggca?gtacccagca?tctcagcagc?agccagcaca?agggcaacaa 1020

gggcagtacc?cagcttctca?gcagcagcca?ggacaagggc?aacaagggca?gtacccagct 1080

tctcagcagc?agccagcaca?agggcaacaa?gggcagtacc?cagcttctca?acagcagcca 1140

ggacaagggc?aacaagggca?ctacccagct?tctgagcagc?agccaggaca?agggcaacaa 1200

cggcactacc?cagcttctct?gcagcaacca?ggacaagggc?aacaaaggca?ttacgcagct 1260

tctctgcagc?aaccaggaca?agggcaacaa?gggcattacc?cagcttctct?gcagcaggta 1320

ggacaaggac?aacaaatagg?acagccagga?caaaggcaac?aaccaggaca?agggcaacaa 1380

acagaacaag?ggcaacaact?agaacaaggg?caacaaccag?gacaagggca?acaagggtac 1440

tatccaactt?ctccacaaca?gtcaggacaa?gggcaacaac?caggacaatc?gcaacaacca 1500

ggacaagggc?aacaagggta?ctactcaact?tctctacaac?agccaggaca?agggcaacaa 1560

gggcactacc?caacttctct?gcagcagcca?ggacaaggac?atccaggaca?aaggcaacaa 1620

ccaggacaag?ggcaacaacc?agaacaaggg?caacaaccag?gacaggggca?acaagggtat 1680

tatccaactt?ctccgcagca?gccaggacaa?gggaaacaac?taagacaagg?gcaacaaggg 1740

tactacccaa?cttctctgca?acagccagga?caagggcaac?aaccaggaca?agggcaacaa 1800

tcaggacaag?ggcaacaagg?gcactgccca?acttctccgc?aacagacagg?acaagcgcaa 1860

caaccaggac?aaggccaaca?aataggacaa?gtgcaacaac?caggacaagg?gcaacaaggg 1920

tactacccaa?tttctctgca?gcagtcagga?caagggcaac?agtcaggaca?agggcaacaa 1980

tcaggacaag?gacaccaact?aggacaaggg?cagcaatcag?gacaagagca?acaaggctac 2040

gacaacccat?accatgttaa?cacagagcag?caaacggcca?gcccaaaggt?ggcaaaggtg 2100

cagcaacccg?cgacacagct?gccgataatg?tgtcggatgg?aggggggcga?cgcattatcg 2160

gctagccagt?gatag 2175

<210>2

<211>723

<212>PRT

＜213〉Triticum wheat (Triticum aestivum L.)

<400>2

Met?Ala?Lys?Arg?Leu?Val?Leu?Phe?Ala?Thr?Val?Val?Ile?Thr?Leu?Val

1 5 10 15

Ala?Leu?Ala?Ala?Ala?Glu?Gly?Glu?Ala?Ser?Arg?Gln?Leu?Gln?Cys?Glu

20 25 30

Arg?Glu?Leu?Gln?Glu?Ser?Ser?Leu?Glu?Ala?Cys?Arg?Gln?Val?Val?Asp

35 40 45

Gln?Gln?Leu?Ala?Gly?Arg?Leu?Pro?Trp?Ser?Thr?Gly?Leu?Gln?Met?Arg

50 55 60

Cys?Cys?Gln?Gln?Leu?Arg?Asp?Val?Ser?Ala?Lys?Cys?Arg?Pro?Val?Ala

65 70 75 80

Val?Ser?Gln?Val?Val?Arg?Gln?Tyr?Glu?Gln?Thr?Val?Val?Pro?Pro?Lys

85 90 95

Gly?Gly?Ser?Phe?Tyr?Pro?Gly?Glu?Thr?Thr?Pro?Leu?Gln?Gln?Leu?Gln

100 105 110

Gln?Val?Ile?Phe?Trp?Gly?Thr?Ser?Ser?Gln?Thr?Val?Gln?Gly?Tyr?Tyr

115 120 125

Pro?Ser?Val?Ser?Ser?Pro?Gln?Gln?Gly?Pro?Tyr?Tyr?Pro?Gly?Gln?Ala

130 135 140

Ser?Pro?Gln?Gln?Pro?Gly?Gln?Arg?Gln?Gln?Pro?Gly?Lys?Trp?Gln?Glu

145 150 155 160

Leu?Gly?Gln?Gly?Gln?Gln?Gly?Tyr?Tyr?Pro?Thr?Ser?Leu?His?Gln?Ser

165 170 175

Gly?Gln?Gly?Gln?Gln?Gly?Tyr?Tyr?Pro?Ser?Ser?Leu?Gln?Gln?Pro?Gly

180 185 190

Gln?Gly?Gln?Gln?Thr?Gly?Gln?Gly?Gln?Gln?Gly?Tyr?Tyr?Pro?Thr?Ser

195 200 205

Leu?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Ile?Gly?Gln?Gly?Gln?Gln?Gly

210 215 220

Tyr?Tyr?Pro?Thr?Ser?Pro?Gln?His?Pro?Gly?Gln?Arg?Gln?Gln?Pro?Gly

225 230 235 240

Gln?Gly?Gln?Gln?Ile?Gly?Gln?Gly?Gln?Gln?Pro?Gly?Gln?Gly?Arg?Gln

245 250 255

Ile?Gly?Gln?Gly?Gln?Gln?Ser?Gly?Gln?Glu?Gln?Gln?Gly?Tyr?Tyr?Ala

260 265 270

Thr?Ser?Pro?Gln?Gln?Leu?Gly?Gln?Gly?Gln?Gln?Pro?Gly?Gln?Trp?Gln

275 280 285

Gln?Ser?Gly?Gln?Gly?Gln?Gln?Arg?Tyr?Tyr?Pro?Thr?Ser?Gln?Gln?Gln

290 295 300

Pro?Gly?Gln?Gly?Gln?Gln?Gly?Gln?Tyr?Pro?Ala?Ser?Gln?Gln?Gln?Pro

305 310 315 320

Ala?Gln?Gly?Gln?Gln?Gly?Gln?Tyr?Pro?Ala?Ser?Gln?Gln?Gln?Pro?Ala

325 330 335

Gln?Gly?Gln?Gln?Gly?Gln?Tyr?Pro?Ala?Ser?Gln?Gln?Gln?Pro?Gly?Gln

340 345 350

Gly?Gln?Gln?Gly?Gln?Tyr?Pro?Ala?Ser?Gln?Gln?Gln?Pro?Ala?Gln?Gly

355 360 365

Gln?Gln?Gly?Gln?Tyr?Pro?Ala?Ser?Gln?Gln?Gln?Pro?Gly?Gln?Gly?Gln

370 375 380

Gln?Gly?His?Tyr?Pro?Ala?Ser?Glu?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln

385 390 395 400

Arg?His?Tyr?Pro?Ala?Ser?Leu?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Arg

405 410 415

His?Tyr?Ala?Ala?Ser?Leu?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly?His

420 425 430

Tyr?Pro?Ala?Ser?Leu?Gln?Gln?Val?Gly?Gln?Gly?Gln?Gln?Ile?Gly?Gln

435 440 445

Pro?Gly?Gln?Arg?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Thr?Glu?Gln?Gly

450 455 460

Gln?Gln?Leu?Glu?Gln?Gly?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly?Tyr

465 470 475 480

Tyr?Pro?Thr?Ser?Pro?Gln?Gln?Ser?Gly?Gln?Gly?Gln?Gln?Pro?Gly?Gln

485 490 495

Ser?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly?Tyr?Tyr?Ser?Thr?Ser?Leu

500 505 510

Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly?His?Tyr?Pro?Thr?Ser?Leu?Gln

515 520 525

Gln?Pro?Gly?Gln?Gly?His?Pro?Gly?Gln?Arg?Gln?Gln?Pro?Gly?Gln?Gly

530 535 540

Gln?Gln?Pro?Glu?Gln?Gly?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly?Tyr

545 550 555 560

Tyr?Pro?Thr?Ser?Pro?Gln?Gln?Pro?Gly?Gln?Gly?Lys?Gln?Leu?Arg?Gln

565 570 575

Gly?Gln?Gln?Gly?Tyr?Tyr?Pro?Thr?Ser?Leu?Gln?Gln?Pro?Gly?Gln?Gly

580 585 590

Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Ser?Gly?Gln?Gly?Gln?Gln?Gly?His

595 600 605

Cys?Pro?Thr?Ser?Pro?Gln?Gln?Thr?Gly?Gln?Ala?Gln?Gln?Pro?Gly?Gln

610 615 620

Gly?Gln?Gln?Ile?Gly?Gln?Val?Gln?Gln?Pro?Gly?Gln?Gly?Gln?Gln?Gly

625 630 635 640

Tyr?Tyr?Pro?Ile?Ser?Leu?Gln?Gln?Ser?Gly?Gln?Gly?Gln?Gln?Ser?Gly

645 650 655

Gln?Gly?Gln?Gln?Ser?Gly?Gln?Gly?His?Gln?Leu?Gly?Gln?Gly?Gln?Gln

660 665 670

Ser?Gly?Gln?Glu?Gln?Gln?Gly?Tyr?Asp?Asn?Pro?Tyr?His?Val?Asn?Thr

675 680 685

Glu?Gln?Gln?Thr?Ala?Ser?Pro?Lys?Val?Ala?Lys?Val?Gln?Gln?Pro?Ala

690 695 700

Thr?Gln?Leu?Pro?Ile?Met?Cys?Arg?Met?Glu?Gly?Gly?Asp?Ala?Leu?Ser

705 710 715 720

Ala?Ser?Gln

<210>3

<211>2363

<212>DNA

＜213〉Triticum wheat (Triticum aestivum L.)

<400>3

atggctaagc?ggttggtcct?ctttgcgaca?gtagtcatca?ccctcgtggc?tctcgctgct 60

gctgaaggtg?aggcctctag?gcaactacag?tgtgagcgcg?agctccagga?gagctcgctt 120

gaggcatgcc?gacaggtcgt?ggaccaacag?ttggccggtc?ggctgccatg?gagcacgggg 180

ctccagatgc?gatgctgcca?gcagctccga?gatgttagcg?ctaagtgccg?ccccgtcgcc 240

gtcagccaag?tcgtaagaca?atatgagcaa?accgtggtgc?cgcccaaggg?cggatccttc 300

taccctggcg?agaccacacc?actgcagcaa?ctccaacaag?taatattttg?gggaacatct 360

tcacaaacag?tacaagggta?ttacccaagc?gtaagttctc?ctcagcaggg?gccatattat 420

ccaggccaag?cttctccaca?acagccagga?caaaggcaac?agccaggcaa?atggcaagaa 480

ctgggacaag?ggcaacaagg?gtattaccca?acttctctgc?atcagtcagg?acaaggacaa 540

caagggtact?acccatcttc?tctgcagcaa?ccaggacaag?ggcaacagac?aggacaaggg 600

caacaaggat?actacccaac?ttctctgcag?cagccaggac?aagggcaaca?gataggacaa 660

gggcaacaag?ggtactaccc?aacttctccg?cagcacccag?gacaaaggca?acaaccagga 720

caagggcagc?aaataggaca?agggcaacaa?ccaggacaag?ggcggcaaat?aggacaaggg 780

caacaatcag?gacaagagca?acaagggtac?tatgcaactt?ctccacagca?gctaggacaa 840

gggcaacaac?caggacaatg?gcaacaatca?ggacaagggc?aacaaaggta?ctacccaact 900

tctcagcagc?agccaggaca?agggcaacag?gggcagtacc?cagcttctca?gcagcagcca 960

gcacaagggc?aacaagggca?gtacccagca?tctcagcagc?agccagcaca?agggcaacaa 1020

gggcagtacc?cagcttctca?gcagcagcca?ggacaagggc?aacaagggca?gtacccagct 1080

tctcagcagc?agccagcaca?agggcaacaa?gggcagtacc?cagcttctca?acagcagcca 1140

ggacaagggc?aacaagggca?ctacccagct?tctgagcagc?agccaggaca?agggcaacaa 1200

cggcactacc?cagcttctct?gcagcaacca?ggacaagggc?aacaaaggca?ttacgcagct 1260

tctctgcagc?aaccaggaca?agggcaacaa?gggcattacc?cagcttctct?gcagcaggta 1320

ggacaaggac?aacaaatagg?acagccagga?caaaggcaac?aaccaggaca?agggcaacaa 1380

acagaacaag?ggcaacaact?agaacaaggg?caacaaccag?gacaagggca?acaagggtac 1440

tatccaactt?ctccacaaca?gtcaggacaa?gggcaacaac?caggacaatc?gcaacaacca 1500

ggacaagggc?aacaagggta?ctactcaact?tctctacaac?agccaggaca?agggcaacaa 1560

gggcactacc?caacttctct?gcagcagcca?ggacaaggac?atccaggaca?aaggcaacaa 1620

ccaggacaag?ggcaacaacc?agaacaaggg?caacaaccag?gacaggggca?acaagggtat 1680

tatccaactt?ctccgcagca?gccaggacaa?gggaaacaac?taagacaagg?gcaacaaggg 1740

tactacccaa?cttctctgca?acagccagga?caagggcaac?aaccaggaca?agggcaacaa 1800

tcaggacaag?ggcaacaagg?gcactgccca?acttctccgc?aacagacagg?acaagcgcaa 1860

caaccaggac?aaggccaaca?aataggacaa?gtgcaacaac?caggacaagg?gcaacaaggg 1920

tactacccaa?tttctctgca?gcagtcagga?caagggcaac?agtcaggaca?agggcaacaa 1980

tcaggacaag?gacaccaact?aggacaaggg?cagcaatcag?gacaagagca?acaaggctac 2040

gacaacccat?accatgttaa?cacagagcag?caaacggcca?gcccaaaggt?ggcaaaggtg 2100

cagcaacccg?cgacacagct?gccgataatg?tgtcggatgg?aggggggcga?cgcattatcg 2160

gctagccagt?gatagaactc?tttgcaactt?gcatggtgct?tggtatgcat?gccccttagc 2220

tatccaataa?acatgacgtg?tgttcacagg?ttttcatgta?actagagtaa?aacccaataa 2280

taatgcaaaa?tgaaaagctt?ctccaactaa?aaaacaacaa?aacgggcgtt?gtgcaaaaaa 2340

aaaaaaaaaa?aaaaaaaaaa?aaa 2363

Claims

1, high-quality high-molecular weight gluten subunit of common wheat, it is to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

2, wheat high-molecular-weight glutelin subunit according to claim 1 is characterized in that: described wheat high-molecular-weight glutelin subunit is the SEQ ID № in the sequence table: 2.

3, a kind of encoding gene of wheat high-molecular-weight glutelin subunit is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the sequence table: 3 dna sequence dna;

3) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.

4, contain the described expression carrier of claim 3.

5, expression vector according to claim 4 is characterized in that: described expression vector is recombinant expression vector PET-3a-1Byl5 (M).

6, the transgenic cell line that contains the described gene of claim 3.

7, clone according to claim 6 is characterized in that: described cell is the E.coli BL21 (DE3) that contains recombinant expression vector PET-3a-1Byl5 (M).