CN1800395A - Wheat seed hardness related gene and its encoding protein and uses - Google Patents

Abstract

The invention discloses a gene which is relative to wheat inner core hardness and its coding protein and application. The gene relative to wheat inner core hardness has one of the following nucleic acid sequences: 1) the polynucleotide of SEQ ID NO: 2; 2) the SEQ ID NO: 1 polypeptide sequence DNA; 3) the nucleic acid sequence which is cross with the limited DNA sequence SEQ ID NO: 2 on high austerity condition.

Description

Gene relevant and proteins encoded and application with wheat seed hardness

Technical field

The present invention relates to gene and proteins encoded thereof and application, particularly relate to a gene relevant and proteins encoded thereof and its application in Wheat Quality Improvement with wheat seed hardness.

Background technology

Grain hardness is one of important character of wheat quality, and it influences the flour extraction rate, flour particle size, wheat wetting amount of water, damage starch grain quantity of wheat and final food-processing quality etc., is the important evidence of on the market it being carried out classification, price.Can be divided into hard wheat, wheat mixture and soft wheat to common wheat by the endosperm quality.Grain hardness is mainly by the major gene Pina and Pinb (the being referred to as puroindoline) control that are positioned on the karyomit(e) 5D galianconism (5DS).1986, Greenwell and Schofield extracted the protein polypeptide that one group of molecular weight is about 15kD from wheat grain, and called after friabilin albumen has advanced the flow of research of grain hardness on the molecular level greatly.Studies show that, Puroindoline albumen is the proteic main component of friabilin, be the basis of decision wheat seed hardness, mainly form that its encoding gene is called after Pina and Pinb respectively by two kinds of protein ingredient puroindoline a (PINA) and puroindoline b (PINB).PINA protein delation or the coding proteic gene of PINB (Pinb) are undergone mutation and all can be caused the hardening of wheat endosperm quality.Giroux etc. study two hard wheat varieties, and finding has a site that sudden change has taken place in the Pinb gene, cause in the corresponding aminoacid sequence the 46th Gly to become Ser, with this mutation type called after Pinb-D1b.After this, there is multiple hardness mutation type to be found in succession.Lillemo and Morris have found two kinds of new mutation types of Pinb gene, and one is that the 60th Leu becomes Pro, and with this mutation type called after Pinb-D1c, another is that the 44th amino acids becomes Arg by Trp, with this mutation type called after Pinb-D1d.Discoveries such as Morris, the 39th amino acids Trp sports terminator codon in a kind of Pinb of hard red spring wheat gene, with this mutation type called after Pinb-D1e; The 44th amino acids Trp sports terminator codon in the Pinb of a kind of hard red winter wheat gene, with this mutation type called after Pinb-Dif; The 56th amino acids Cys sports terminator codon in the Pinb of the hard red winter wheat of another kind gene, with this mutation type called after Pinb-D1g.Recently, Massa etc. in goatweed new discovery the allelic variation of six kinds of Pina genes and four kinds of Pinb genes, but all show as soft.Xia etc. find that after deliberation Pinb-D1b is a hardness variation type the most common in China's wheat, and the new variation type that in 10 kinds (being) such as agricultural university 3213 and agricultural university 3395, has a base A to lack corresponding to the 42nd amino acids in the discovery Pinb gene, with its called after Pinb-D1p.The summary of relevant Pinb gene and mutant thereof is as shown in table 1:

The phenotype of the known Pinb gene of table 1, mutation type, molecule change and reference

Pinb	Phenotype	Molecule changes	Reference
				Pinb-D1a	Soft	Wild-type	Giroux M J and C.F Morris.A glycine to serine change in puroindoline b is asssociated with wheatgrain hardness and low levels of starch-surface friabilin. Theoretical Applied Genetics，1997，95：857-864
Pinb-D1a	Hard	The Pina disappearance	Giroux M J and Morris C F.Wheat grain hardness results from highly conserved mutations in friabilin components puroindoline a and b. Proceedings of National Academic Science of USA，1998，95：6262-6266
				Pinb-D1b	Hard	Gly-46 sports Ser-46, GGC → AGC	Giroux M J and C.F Morris.A glycine to serine change in puroindoline b is asssociated with wheatgrain hardness and low levels of starch-surface friabilin. Theoretical Applied Genetics，1997，95：857-864
Pinb-D1c	Hard	Leu-60 sports Pro-60, CTG → CCG	Lillemo M，Morris C F.Aleucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe. Theoretical Applied Genetics，2000，100：1100-1107
				Pinb-D1d	Hard	Trp-44 sports Arg-44, TGG → AGG	Lillemo M，Morris C F.Aleucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe. Theoretical Applied Genetics，2000，100：1100-1107
Pinb-D1e	Hard	Trp-39 sports terminator TGG → TGA	Morris C F，Lillemo M，Simeone M C，Giroux M J，Babb S L，Kimberlee K K. Prevalence of puroindoline grain hardness genotypes among historically significant North American spring and winter wheats.Crop Science，2001， 41：218-228
				Pinb-D1f	Hard	Trp-44 sports terminator TGG → TGA	Morris C F，Lillemo M，Simeone M C，Giroux M J，Babb S L，Kimberlee K K. Prevalence of puroindoline grain hardness genotypes among historically significantNorth American spring and winter wheats.Crop Science，2001， 41：218-228
Pinb-D1g	Hard	Cys-56 sports terminator TGC → TGA	Morris C F，Lillemo M，Simeone M C，Giroux M J，Babb S L，Kimberlee K K. Prevalence of puroindoline grain hardness genotypes among historically significantNorth American spring and winter wheats.Crop Science，2001， 41：218-228
				Pinb-D1l	Hard	Lys-45 sports Glu-45AAG → GAG	Pan Z，Song W，Meng F，Xu L，Liu B，Zhu J.Characterization of Genes Encoding Wheat Grain Hardness from Chinese Cultivar GaoCheng 8901. Cereal Chemistry，2004，81(2)：287-289
Pinb-D1p	Hard	Base A disappearance	Xia L Q，Chen F，He Z H，Chen X M，Morris C F.Occurrence of puroindoline alleles in Chinese winter wheat Cereal Chemistry，2004 in press

Summary of the invention

The purpose of this invention is to provide a gene relevant and proteins encoded thereof with wheat seed hardness.

The gene relevant provided by the present invention with wheat seed hardness, name is called Pinb-D1q, derives from wheat, has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 polynucleotide;

2) SEQ ID № in the code sequence tabulation: the DNA of 1 protein sequence;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.

SEQ ID № in the sequence table: 2 by 447 based compositions, and its encoder block is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 444th bit base; Compare with the encoding gene of the Pinb that does not undergo mutation, its nucleotide sequence has become t from 5 ' end the 218th bit base by g, makes the codon TGG of Ser be mutated into the codon TTG of Leu.

The proteins encoded of provided by the present invention and wheat seed hardness genes involved, name is called PinB-D1q, is the polypeptide with one of following amino acid residue sequences:

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

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or interpolation and the protein relevant with wheat seed hardness of one to ten amino-acid residue.

SEQ ID № in the sequence table: 1 is made up of 148 amino-acid residues, Pinb-D1a compares with wild-type, and the 44th of its aminoacid sequence sports Leu by Ser, simultaneously, Pinb gene after the sudden change is no longer expressed friabilin albumen, causes wheat grain SKCS hardness to improve therefrom.

Contain that arbitrary segmental primer also belongs to protection scope of the present invention in above-mentioned and wheat seed hardness Expression of Related Genes carrier, transgenic cell line and host bacterium and the amplification wheat seed hardness genes involved.

The variation type of different hardness gene is different to grain hardness influence, and the grain hardness of Pinb-D1q mutant wheat of the present invention improves a lot with comparing with the product grow wheat of not undergoing mutation of Pinb.Therefore, improvement will play an important role Pinb-D1q to the genetically engineered of wheat seed hardness, and the cultivation of good wheat breed also is significant.Utilize the flour of the different private types that this kind and other product grow wheat obtain, can satisfy the needs of each grow wheat goods, also help the heredity and the molecular biological further research of wheat seed hardness simultaneously.

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer is synthetic by Shanghai bio-engineering corporation.

The acquisition of embodiment 1, wheat Pinb-D1q and Puroindoline gene type assay

Wheat lines: select by Beijing agricultural academy of sciences cultivate, high-quality wheat variety capital winter 11 of authorization in 2002, its parent is Changfeng No. 3/041//capital winter No. 6.This kind has good noodles characteristic.

One, grain hardness is measured

Capital winters 11 wheat samples is placed 3d under identical conditions, the water content of wheat grain is controlled between the 11-13%.Measure the hardness value of 300 wheat samples with simple grain grain hardness instrument (SKCS 4100, Sweden Perten company), determine the hardness rank of seed to write down thousand seed weight and seed diameter simultaneously according to measurement result.The grain hardness value mostly is soft wheat less than 40, mostly is the hard wheat greater than 60, and the person mostly is wheat mixture between 40 and 60.

The SKCS analytical results shows, the SKCS hardness number (± standard deviation) in capital winter 11 and distribution are respectively 51 ± 12 and 07-22-45-26, and grade classification is 3 grades, belongs to wheat mixture, and wherein soft partially hard partially seed all has bigger distribution.

Two, the acquisition of wheat Pinb-D1q

Puroindoline (comprising PinA and PinB) in capital winters 11 wheat samples that SKCS analyzes analyzes to step 1, and concrete grammar may further comprise the steps:

1, extracts the wheat grain genomic dna

Choose a representational seed of hard (its cutting back is judged whether to coincide with the SKCS analytical results according to its vitreousness), extract the wheat grain genomic dna, concrete grammar is: put into the 1.5mL centrifuge tube after 1) smashing with hammer, add the 1mL sample extracting solution and (contain 288mM NaCl, 200mM Tris-HCl, 25mM EDTA, 0.5%SDS), shook 30 minutes, and made its abundant mixing; 2) at 4 ℃, 12, under the 000rpm centrifugal 15 minutes, move supernatant to another 2mL centrifuge tube, add isopyknic phenol/chloroform (1: 1); 3) 12, centrifugal 15 minutes of 000rpm moves supernatant to another 2mL centrifuge tube, adds the chloroform/primary isoamyl alcohol (24: 1) of 0.5 times of supernatant volume, fully shakes up; 4) 12, centrifugal 10 minutes of 000rpm moves supernatant to another new 2mL centrifuge tube, adds 3M NaAC (pH=5.2) and the isopropanol precipitating DNA of 0.6 times of supernatant volume, the mixing gently of 1/10 volume; 5) 12, centrifugal 15 minutes of 000rpm abandons supernatant, adds 0.5mL 70% ethanol, leaves standstill 5min; 6) 12,000 centrifugal 5min abandon supernatant.Add 100 μ L TE dissolution precipitations after the vacuum-drying, precipitation is the wheat grain genomic dna.At last, with the concentration of UV spectrophotometer measuring wheat grain genomic dna, preserve standby down for-20 ℃.

2, identify with PCR method whether the flint wheat in capital winter 11 is the Pinb-D1b mutant

Whether the flint wheat of identifying the capital winter 11 is the Pinb-D1b variation type, and whether the 46th site of promptly detecting the Pinb gene is become the codon (AGC) of Serine by the codon (GGC) of glycine.Used primer sequence is as follows:

The special primer series of identifying the Pinb-D1b mutant is:

Primer 1 (upstream): 5 '-ATGAAGACCTTATTCCTCCTA-3 '

Primer 2 (downstream): 5 '-CTCATGCTCACAGCCGCT-3 '

Identify non-Pinb-D1b type, promptly detect the type that Pinb gene 46 site glycine do not change, used primer sequence is:

Primer 1 (upstream): 5 '-ATGAAGACCTTATTCCTCCTA-3 '

Primer 3 (downstream): 5 '-CTCATGCTCACAGCCGCC-3 '

The seed genomic dna of capital winters 11 wheat that obtains with step 1 is a template; respectively under the guiding of Pinb-D1b special primer (primer 1 and primer 2) and the non-special primer of Pinb-D1b (primer 1 and primer 3); carry out pcr amplification, the composition of PCR reaction system is: 1.5mmol/L MgCl ₂, 0.3mmol/L dNTP, each 10pmol of upstream and downstream primer, template DNA 200ng, 0.5U Taq enzyme adds 1 * PCR damping fluid (contain 10mmol/L Tris-HCl, pH 9.0,50mmol/L KCl, 1.0%Triton X-100) and is settled to 25 μ L.The PCR response procedures is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, 58 ℃ of annealing 45sec, 72 ℃ are extended 1min, totally 35 circulations; At last, 72 ℃ are extended 5min.After pcr amplification finishes, get each 10 μ L of above-mentioned two kinds of pcr amplification products and carry out 1.5% agarose gel electrophoresis (employing 1 * TAE damping fluid respectively; Ethidium bromide (EB) dyeing, 160V, 1.5 hour), electrophoresis finishes the back and analyzes with gel imaging system (MultiGenius Gel Documentation and AnalysisSystem) scanning imagery and with computer, analytical results shows the dna fragmentation that has amplified 250bp with the non-special primer of Pinb-D1b (primer 1 and primer 3), and do not amplify the dna fragmentation of 250bp with Pinb-D1b special primer (primer 1 and primer 2), the flint wheat in proof capital winter 11 is not the Pinb-D1b mutant, and its method with step 3 is done further to identify.

3, the method for cutting with PCR and enzyme identifies whether the flint wheat in capital winter 11 is the Pinb-D1c mutant

Whether the flint wheat of identifying the capital winter 11 is the Pinb-D1c variation type, and whether the 60th site of promptly detecting the Pinb gene is become the codon (CCG) of proline(Pro) by leucic codon (CTG).The Pinb full length sequence that at first increases, used primer sequence is as follows:

Primer 1 (upstream): 5 '-ATGAAGACCTTATTCCTCCTA-3 '

Primer 4 (downstream): 5-TCACCAGTAATAGCCACTAGGGAA-3 '

The seed genomic dna of capital winters 11 wheat that obtains with step 1 is a template, under the guiding of Pinb full length sequence primer (primer 1 and primer 4), carries out pcr amplification, and the PCR reaction system is identical with step 2 with amplification program.Then, the PCR product is carried out enzyme with the PvuII restriction enzyme cut, the enzyme system of cutting is, 3 μ L, 10 * NE damping fluid, 2 (NEB companies), and 0.2U PvuII restriction endonuclease (NEB company), 20 μ L PCR products add ddH ₂O is settled to 30 μ L; The enzyme tangent condition is: 37 ℃ of enzymes are cut 2h.After enzyme is cut, getting enzyme cuts product 10 μ L and carries out (ethidium bromide (EB) dyeing of 2.0% agarose gel electrophoresis, 80-100V, 1.5 hour), electrophoresis finishes the back and analyzes with gel imaging system (MultiGenius Gel Documentation and Analysis System) scanning imagery and with computer, analytical results shows, amplified production can be cut into 264bp and two fragments of 183bp by PvuII, the flint wheat in proof capital winter 11 is not the Pinb-D1c mutant, and its method with step 4 is done further to identify.

4, identify with SDS-PAGE whether the flint wheat in capital winter 11 is Pina-D1b (PINA disappearance) mutant

May further comprise the steps:

(1) extracts seed protein

Choose the flint wheat in a capital winter 11, extract seed protein by following step: put into a 2mL centrifuge tube after 1) grinding, TBS solution (the Tris-buffered saline that adds the 1mL precooling, the Tris buffer salt solution) and 0.15mL 12%Triton-X114 (Sigma company), mix back 4 ℃ and place 12-24h; 2) 12000rpm is centrifugal 3 minutes, moves supernatant to the 1.5mL centrifuge tube, 37 ℃ of incubation half an hour; 3) the centrifugal 5min of 12000rpm abandons supernatant, moves in the new centrifuge tube of lower floor to, adds the TBS solution of 1mL precooling, 37 ℃ of incubation 30min; 4) the centrifugal 3min of 12000rpm abandons supernatant, adds the acetone of 900 μ L precoolings, places 30min behind the vortex in-20 ℃ of refrigerators; 5) the centrifugal 2min of 12000rpm abandons supernatant, washes to be placed on for one time with acetone again and carries out drying in the air, obtains wheat grain albumen.

(2) the SDS-PAGE electrophoresis is identified

1) in the seed protein that step 1 is extracted, adds 150 μ L sample buffers (containing 7.57mg/mL Tris, 10% glycerine, 0.02mg/mL SDS, 0.0125mg/mL bromjophenol blue); 2) seed protein that will add sample buffer is got 25 μ L and is carried out the SDS-PAGE electrophoresis at 70 ℃ of following incubation 15min.Deposition condition is: (concentration T is 4% with concentrated glue, glue connection degree C is 2.67%) electrophoresis under 40mA, treat to convert voltage power to 12W after indicator arrives separation gel (concentration T is 13.5%, and glue connection degree C is 2.6%), treat indicator arrive bottom the separation gel after electrophoresis 30min again.For reducing proteic diffusion, replace water preparation separation gel with 10% glycerine.Replace methene so that background is more clear with PDA (Piperiazine diacrylamide) during the gel preparation, the more solid and high resilience of glue.Press (Morris C F, Massa A N.Puroindoline genotype of the U.S.nationalinstitute of standards ﹠amp such as Morris; Technology reference material 8441, wheat hardness.CerealChemistry, 2003, method 80:674-678) dye (with trichoroacetic acid(TCA) and methyl alcohol fix, silver dyes colour developing, stops colour developing with citric acid).The SDS-PAGE electrophoresis result shows expresses the PINA that 15kDa is arranged in the hard seed in capital winter 11, prove that the flint wheat in capital winter 11 neither the Pina-D1b mutant, and its method with order-checking is done further evaluation.

5, the mutant of the flint wheat in capital winter 11 is identified in order-checking

The total length primer sequence of amplification Pinb is:

Primer 1 (upstream): 5 '-ATGAAGACCTTATTCCTCCTA-3 '

Primer 4 (downstream): 5 '-TCACCAGTAATAGCCACTAGGGAA-3 '

The total length primer sequence of amplification Pina is:

Primer 5 (upstream): 5 '-ATGAAGGCCCTCTTCCTCA-3 '

Primer 6 (downstream): 5 '-TCACCAGTAATAGCCAATAGTG-3 '

Identify through above-mentioned steps 2-4, proof is not Pinb-D1b through capital winters 11 flint wheat that SKCS detects, Pinb-D1c and Pina-D1b (PINA disappearance) mutant, for proving conclusively the variation type of its hardness gene, from capital winters 11 wheat samples, choose 3 soft grain seeds and 10 solids seeds respectively, with its genomic dna is template, respectively under the guiding of Pinb total length primer (primer 1 and primer 4) and Pina total length primer (primer 5 and primer 6), carry out pcr amplification, the PCR reaction system is identical with step 2 with reaction conditions, then the PCR product is delivered to the order-checking of Bo Ya and AudioCodes company.Sequencing result shows: the capital winters 11, the Pinb of flint wheat had SEQ ID № in the sequence table: 2 nucleotide sequence, SEQ ID № in the sequence table: 2 by 447 based compositions, its encoder block is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 444th bit base.The sequence of sequencing result and known wild-type Pina and Pinb is compared, find that the Pina in capital winters 11 flint wheat is a wild-type, and hold the 218th bit base from 5 ' in the nucleotide sequence of Pinb, sport T by bases G, (SEQ ID №: the 44th amino acids sports leucine by tryptophane 1) thereby cause Pinb amino acids coding residue sequence.This mutation type is all inequality with the known mutations type of previous all reports.The hardness gene rank of announcing on Genebank according to McIntosh etc. is with this mutator gene called after Pinb-D1q.And 10 solids seed sequencing result unanimities from the capital winter 11, selecting, be the Pinb-D1q mutation type, thereby further confirmed the reliability of The above results.And the sequencing result of 3 soft wheats in capital winter 11 shows, any site mutation does not take place Pinb, and is identical with the sequence of wild-type Pinb.Above-mentioned sequencing result shows that also the capital winter 11 is mixtures of two kinds of puroindoline genes.

Sequence table

<160>2

<210>1

<211>148

<212>PRT

＜213〉Triticum (Triticum aestivum L.)

<400>1

Met Lys Thr Leu Phe Leu Leu Ala Leu Leu Ala Leu Val Ala Ser Thr

1 5 10 15

Thr Phe Ala Gln Tyr Ser Glu Val Gly Gly Trp Tyr Asn Glu Val Gly

20 25 30

Gly Gly Gly Gly Ser Gln Gln Cys Pro Gln Glu Arg Pro Lys Leu Ser

35 40 45

Ser Cys Lys Asp Tyr Val Met Glu Arg Cys Phe Thr Met Lys Asp Phe

50 55 60

Pro Val Thr Trp Pro Thr Lys Trp Trp Lys Gly Gly Cys Glu His Glu

65 70 75 80

Val Arg Glu Lys Cys Cys Lys Gln Leu Ser Gln Ile Ala Pro Gln Cys

85 90 95

Arg Cys Asp Ser Ile Arg Arg Val Ile Gln Gly Arg Leu Gly Gly Phe

100 105 110

Leu Gly Ile Trp Arg Gly Glu Val Phe Lys Gln Leu Gln Arg Ala Gln

115 120 125

Ser Leu Pro Ser Lys Cys Asn Met Gly Ala Asp Cys Lys Phe Pro Ser

130 135 140

Gly Tyr Tyr Trp

145

<210>2

<211>447

<212>DNA

＜213〉Triticum (Triticum aestivum L.)

<400>2

atgaagacct tattcctcct agctctcctt gctcttgtag cgagcacaac cttcgcgcaa 60

tactcagaag ttggcggctg gtacaatgaa gttggcggag gaggtggttc tcaacaatgt 120

ccgcaggagc ggccgaagct aagctcttgc aaggattacg tgatggagcg atgtttcaca 180

atgaaggatt ttccagtcac ctggcccaca aaatggttga agggcggctg tgagcatgag 240

gttcgggaga agtgctgcaa gcagctgagc cagatagcac cacaatgtcg ctgtgattct 300

atccggcgag tgatccaagg caggctcggt ggcttcttgg gcatttggcg aggtgaggta 360

ttcaaacaac ttcagagggc ccagagcctc ccctcaaagt gcaacatggg cgccgactgc 420

aagttcccta gtggctatta ctggtga 447

Claims (8)

1, wheat seed hardness genes involved has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 polynucleotide;

2) SEQ ID № in the code sequence tabulation: the DNA of 1 peptide sequence;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

2, the proteins encoded of the described wheat seed hardness genes involved of claim 1.

3, proteins encoded according to claim 2 is characterized in that: described proteins encoded is the polypeptide with one of following amino acid residue sequences:

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

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or interpolation and the polypeptide relevant with wheat seed hardness of one to ten amino-acid residue.

4, contain the described wheat seed hardness Expression of Related Genes of claim 1 carrier.

5, the transgenic cell line that contains the described wheat seed hardness genes involved of claim 1.

6, the host bacterium that contains the described wheat seed hardness genes involved of claim 1.

7, arbitrary segmental primer in the described wheat seed hardness genes involved of amplification claim 1.

8, the application of the described wheat seed hardness genes involved of claim 1 in Wheat Quality Improvement.