CN102234650A - Nucleic acid sequence of barley grain B-hordein gene and application thereof - Google Patents

Nucleic acid sequence of barley grain B-hordein gene and application thereof Download PDF

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Publication number
CN102234650A
CN102234650A CN2010101539001A CN201010153900A CN102234650A CN 102234650 A CN102234650 A CN 102234650A CN 2010101539001 A CN2010101539001 A CN 2010101539001A CN 201010153900 A CN201010153900 A CN 201010153900A CN 102234650 A CN102234650 A CN 102234650A
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gln
pro
leu
hordein
val
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Inventor
余懋群
龙海
潘志芬
邓光兵
黄卓
韩召雪
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Chengdu Institute of Biology of CAS
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Chengdu Institute of Biology of CAS
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Abstract

The invention belongs to the field of plant genetic engineering, and in particular relates to a novel nucleic acid sequence of B-hordein grain protein coding gene from barley of Tibet Plateau, and application thereof. The nucleic acid sequence of the barley grain B-hordein gene contains a gene segment in seq.1, seq.2 or seq.3. The nucleic acid sequence of the barley grain B-hordein gene is used for crop quality improvement.

Description

Hullessbarley seed B-hordein gene nucleic acid sequence and uses thereof
Technical field
The present invention relates to the genetically engineered plant field, be specifically related to a kind of novel B-hordein seed protein encoding gene nucleotide sequence, and pass through the method for biotechnology in the application aspect the crop quality improvement based on this gene from Qinghai-Tibet highland barley.
Background technology
Barley ( Hordeum vulgareL.) be the important in the world raise crop and the model plant of genetic research, be distributed widely in agricultural planting district all over the world, have edible, feeding, brewage and multiple use such as health care.Highland barley claim again hull-less barley ( H. vulgareSsp. Vulgare), as important food and fodder crop, playing the part of very important role in Qinghai-Tibet area.Its seed is naked grain, for processing has brought very big facility.In addition, hullessbarley seed contains rich in protein, food fibre, vitamin-E and beta-glucan etc., makes it have better nutritivity and is worth, and more and more causes people's attention.The Qinghai-Tibet Platean is the area of the unique concentrated plantation highland barley in the world as one of origin center of cultivating barley, and its complicated and diversified geographical environment and ecological condition have bred abundant highland barley germ plasm resource.
The barley endosperm storage protein mainly is hordein (hordeins), and it is proteinic 50～60% to account for barley endosperm, is to influence food-processing, brewage and raise one of principal element of quality.According to the mobility size on the SDS-PAGE protein electrophoresis, hordein is divided into three types: rich sulfoprotein subclass (B, γ-hordeins), poor sulfoprotein subclass (C-hordeins) and high-molecular-weight protein subclass (D-hordeins).B group (B-hordeins) and C group (B-hordeins) prolamine are the main poly hordeins of two classes, and they account for the 70-80% and the 10-12% of barley endosperm prolamine composition respectively.B-hordein is by 1H karyomit(e) Hor2Coding, and structure and low molecular weight glutenin (low-molecular-weight glutenin subunit, LMW-GS) comparatively similar.The quantity of B-hordein and distribution are the material impact factors of barley brewing quality.High B-hordein content in the inferior aleurone layer and low D/B-hordein are than closely related with the raising of brewing quality.The B-hordein gene of having reported at present mainly from the common cultivation barley, ( H. vulgare), wild rough bran barley ( H. turkestanicum) and wild Chilean barley ( H. chilense), the report of B-hordein gene is very limited in the relevant highland barley.
Summary of the invention
The invention provides the coding proteic gene order of highland barley B-hordein and variant or fragment.The inventor utilizes round pcr amplification and 3 proteic coding gene sequences of B-hordein of order-checking acquisition from the highland barley with special B-hordein subunit composition according to known barley B-hordein homologous gene design primer.Through sequence alignment, highland barley B-hordein protein coding gene sequence provided by the present invention and known barley B-hordein gene order have the higher sequence similarity, and its consistence is 84%～95%.
Highland barley B-hordein protein coding gene seq. 1 sequence provided by the invention is as follows:
seq.1：
ATGAAGACCTTCCTCATCTTTGCACTCCTCGCCATTGCGGCAACAAGTACGATTGCACAGCAACAACCATTTCCACAACAACCCTTCCCACAACAGCCACAACCATACCCACAACAACCACAACCATATCCACAACAACCATTCCAACCGCAACAACCATTTCCACAACAAACCATCCCACAACAACCACAACCATACCCACAACAACCACAACCATATCCACAACAACCCTTCCCACCGCAACAAGAATTTCCACAACAACCGCCATTTTGGCCACAACAACCATTTCCACAACAACCACCATTTGGGCTACAACAACCAATTCTATGGCAGCAACAACCATGTACACCACAACAAACACCACTCCCACAAGGACAACTGTATCAAACGCTTCTGCAACTACAAATACCCTATGTTCATCCTTCTATTTTGCAACAACTAAACCCATGCAAGGTATTCCTCCAGCAGCAGTGCAACCCCGTGCGAATGCCACAACTTATTGCTAGGTTGCAAATGTTGCAGCAGAGCAGTTGTCATGTGTTGCAGCGACAATGTTGCCAGCAACTGCCGCAAATCTCCGAACAATTCCGCCATGAGGCAATCCGTGCAATCGTCTACTCTATCTTTCTGCAAGAACAACCCCAACAGTCGGTCCAAGGTGTATCCCAACCCCAAAAACAGTTGCAGCAGGAGCAAGTTGGACAATGTTCTTTCCAACAACCTCAACCACAACAACTTGGTCAACCACAGCAGGTACCACACAGTGTTTTCTTGCAGCCACACCAGATAGCTCAGCTTGAGGCGACGACTTCCATTGCGCTGCGTACCCTACCAAGGATGTGCAATGTTAATGTGCCATTGTACGACATCATGCCAGTCGACTTTTGGCACTAG?。
According to highland barley B-hordein protein coding gene sequence (seq.1), obtain following aminoacid sequence:
MKTFLIFALLAIAATSTIAQQQPFPQQPFPQQPQPYPQQPQPYPQQPFQPQQPFPQQTIPQQPQPYPQQPQPYPQQPFPPQQEFPQQPPFWPQQPFPQQPPFGLQQPILWQQQPCTPQQTPLPQGQLYQTLLQLQIPYVHPSILQQLNPCKVFLQQQCNPVRMPQLIARLQMLQQSSCHVLQRQCCQQLPQISEQFRHEAIRAIVYSIFLQEQPQQSVQGVSQPQKQLQQEQVGQCSFQQPQPQQLGQPQQVPHSVFLQPHQIAQLEATTSIALRTLPRMCNVNVPLYDIMPVDFWH?。
Highland barley B-hordein protein coding gene seq. 2 sequences provided by the invention are as follows:
seq.?2：
ATGAAGACCTTCCTCATCTTTGCACTCCTCGCCATTGCGGCAACAAGCACGATTGCGCAGCAACAACCATTTCCACAACAACCCATCCCACAACAACCACAACCATACCCACAACAACCACAACCATATCCACAACAACCCTTTCCACCGCAACAACCATTTCCACAACAACCCGTCCCACAACAACCACAACCATACCCACAACAACCCTTCCCACCGCAACAACCATTTCCACAACAACCACCATTTTGGCAACAAAAACCATTTCCACAACAACCACCATTTGGGCTACAACAACCAATTCTATCGCAGCAACAACCATGTACACCACAACAAACACCACTCCCACAAGGACAACTGTACCAAACGCTTCTGCAACTACAAATACAATATGTTCATCCATCTATTTTGCAACAGCTAAACCCATGCAAGGTATTCCTCCAGCAGCAGTGCAGCCCTGTGCCAGTGCCACAACGTATTGCTAGGTCGCAAATGTTGCAGCAGAGCAGTTGCCATGTGTTGCAGCAACAATGTTGCCAGCAACTACCCCAAATCCCCGAACAACTCCGCCATGAGGCAGTCCGTGCAATCGTCTACTCTATCGTCCTGCAAGAACAATCCCTACAATTGGTCCAAGGTGTCTCCCAACCCCAACAACAGTCACAACAGCAACAAGTCGGACAATGTTCTTTCCAACAACCTCAACCACAACAGGGTCAACAACAGCAAGTGCCACAGAGTGTTCTCTTGCAGCCACACCAAATAGCTCAACTTGAGGCGACAACTTCCATTGCGCTACGTACCCTACCAACGATGTGCAGTGTTAATGTGCCGTTGTACCGCATAGTGCCATTAGCCATTGACACCAGAGTTGGTGTCTAATGATAA?。
According to highland barley B-hordein protein coding gene sequence (seq.2), obtain following aminoacid sequence:
MKTFLIFALLAIAATSTIAQQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPFPQQPVPQQPQPYPQQPFPPQQPFPQQPPFWQQKPFPQQPPFGLQQPILSQQQPCTPQQTPLPQGQLYQTLLQLQIQYVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQLRHEAVRAIVYSIVLQEQSLQLVQGVSQPQQQSQQQQVGQCSFQQPQPQQGQQQQVPQSVLLQPHQIAQLEATTSIALRTLPTMCSVNVPLYRIVPLAIDTRVGV?。
Highland barley B-hordein protein coding gene seq. 3 sequences provided by the invention are as follows:
seq.?3：
ATGAAGACCTTCCTCATCTTTGCACTCCTCGCCATTGTGGCAACAAGTACCATTGCACAACAACAACCATACCCACAACAACCACAACCATTTCCACAACAACCCATCCCACAACAACCACAACCATTTCCACAACAACCACAACCATACCCACAACAACCACAACCATTTCCACAACAACCCATCCCACAACAACCACAACCATACCCACAACAACCACAACCATTTCCACAACAACCCATCCCACAACAACCACAACCATACCCACAACAACCACAACCATTTCCCCTACAACCCTTCCCATCACAACAACCATTTCCACAACAACCACCATTTTGGCAACAACAACCAGTTCTATCGCAGCAACAACCATGTACACAAGAACAAACACCACTCCTACAAGAACAACAAGATCAAATGCTTCTGCAAGTACAAATACCATTTGTTCATCCATCTATTTTGCAGCAGCTAAACCCATGCAAGGTATTCCTCCAGCAGCAGTGCAGCCCTGTGGCAATGTCACAACGTATTGCAAGGTCGCAGATGTTGCAACAGAGCAGTTGCCATGTGTTGCAGCAACAGTGTTGCCAACAACTGCCGCAAATCCCCGAACAACTCCGCCATGAGGCAGTCCGTGCAATCGTCTACTCTATCGTCCTGCAAGAACAATCCCTACAATTGGTCCAAGGTGTCTCCCAACCCCAACAACAGTCACAACAGCAACAAGTCGGACAATGTTCTTTCCAACAACCTCAACCACAACAGGGTCAACAACAGCAAGTGCCACAGAGTGTTCTCTTGCAGCCACACCAAATAGCTCAACTTGAGGCGACAACTTCCATTGCGCTGCGTACCCTACCAACGATGTGCAGTGTTAATGTGCCGTTGTACCGCATAGTGCCATTAGCCATTGACACCAGAGTTGGTGTCTAATGATAA?。
According to highland barley B-hordein protein coding gene sequence (seq.3), obtain following aminoacid sequence:
MKTFLIFALLAIVATSTIAQQQPYPQQPQPFPQQPIPQQPQPFPQQPQPYPQQPQPFPQQPIPQQPQPYPQQPQPFPQQPIPQQPQPYPQQPQPFPLQPFPSQQPFPQQPPFWQQQPVLSQQQPCTQEQTPLLQEQQDQMLLQVQIPFVHPSILQQLNPCKVFLQQQCSPVAMSQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQLRHEAVRAIVYSIVLQEQSLQLVQGVSQPQQQSQQQQVGQCSFQQPQPQQGQQQQVPQSVLLQPHQIAQLEATTSIALRTLPTMCSVNVPLYRIVPLAIDTRVGV?。
Beneficial effect of the present invention is:
The special B-hordein protein protomer of highland barley B-hordein protein coding gene coding provided by the invention, they and known have higher homology.The processing quality of B-hordein albumen and highland barley is closely related, has the potential using value aspect crop improvement.Highland barley B-hordein protein coding gene of the present invention, can import in the crops such as vulgare, highland barley, wheat, paddy rice by the method that this area researchists such as particle gun, agriculture bacillus mediated, pollen tube channel know altogether, be used to change its original processing characteristics.
Beneficial effect of the present invention also shows:
Highland barley B-hordein protein coding gene provided by the present invention can be built up in protokaryon and the Yeast expression carrier, the research method of knowing altogether by this area imports it respectively carries out great expression in intestinal bacteria and the yeast, the albumen of this genetic expression of purifying and it is added in the flour then is to change the processing characteristics of flour or dough.
The following term that uses in the application's specification sheets and claims has following implication:
" variant " is meant a target DNA sequence carried out any replacement, variation, modification, replacement, the disappearance of one or more base or the sequence that interpolation produced, and this sequence still shows the activity that is similar to described target DNA sequence;
" fragment " is meant one or more zones of basic dna sequence dna, and it still has the activity that is similar to basic dna sequence dna.
Embodiment
(1) adopt the CTAB method to extract the highland barley genomic dna, extraction step:
1) get the fresh young leaflet tablet of 2g, liquid nitrogen grinding adds 2 * CTAB extracting solution (2% CTAB that is preheated to 65 ℃ after becoming fine powder; 1.4M NaCl, 0.1 MTris-HCl, PH8.0,0.1M EDTA, PH8.0) 15 ml, mixing;
2) 65 ℃ of water-bath 30-45 min, jog mixings therebetween.Be cooled to and add isopyknic chloroform after the room temperature: primary isoamyl alcohol (24:1), mixing to supernatant liquor is the milk shape gently, centrifugal 10 min of 4000 rpm;
3) get supernatant liquor, add the equal-volume Virahol, place ice bath deposit D NA;
4) tick DNA, wash 2 times with 70% alcohol, dehydrated alcohol is washed once gas and is done DNA, is dissolved in 1 * TE solution of an amount of pH8.0.Add the RNA enzyme to final concentration 100 μ g/ μ L;
5) under the 100V constant voltage, 1% agarose gel electrophoresis 30 minutes detects DNA concentration and quality.
(2) according to three B-hordein gene orders (GenBank No. X03103, X53690 and X53691) among the GenBank, utilize a pair of primer of Primer Premier5 software design:
HoB1：5’-AACCTCGGGATTTCTATACTTT-3’
HoB2：5’-ACACTTTATTTGTCACCGCTAC-3’。
(3) pcr amplification:
Reaction system is as follows: DNA 100-150ng
dNTPs 100μM
Primer HoB1 5 pmol
Primer HoB2 5 pmol
Taq plus polysaccharase 1U
10 * PCR damping fluid, 2.5 μ L
Mg2+ 1.5mM
DdH2O adds to final volume 25 μ L
The PCR response procedures:
1) 94 ℃ 4 minutes
2) 94 ℃ 40 seconds
3) 56 ℃ 40 seconds
4) 72 ℃ 1 minute
Repeat 35 circulations in (2) → (4)
5) 72 ℃ 10 minutes
PCR is reflected at PTC-200 type PCR instrument, and (MJ Research carries out in U.S.A.).Amplified production separates on 1% sepharose, 100V constant voltage electrophoresis 30 minutes.
(4) the PCR product reclaims and purifying
Use the recovery test kit of sky, Beijing root, all operations all carries out to specifications.
(5) connection and conversion
The reaction system that the recovery product is connected to the pMD-18T carrier is as follows:
The about 50ng of pMD-18T carrier 1 μ L()
10 * ligase enzyme damping fluid, 2 μ L
T4DNA ligase enzyme 350 U
Reclaim the PCR product and add to end reaction volume 20 μ L
16 ℃ connect 10 hours
Transformed into escherichia coli DH5 α
The preparation of competent cell
1) be taken at well-grown single DH5 α bacterium colony on the antibiotic-free flat board, be inoculated in the 2 μ L LB liquid nutrient mediums, 37 ℃ of shaken overnight are cultivated (220 rpm);
2) get bacterium liquid that 500 μ L activation spends the night in 50 ml LB liquid nutrient mediums, 37 ℃ of shaking culture are to OD600=0.3;
3) bacterium liquid is poured in the 50 ml centrifuge tubes, ice bath is placed 10 min;
4) in being chilled to 4 ℃ whizzer in advance, remove supernatant liquor behind centrifugal 10 min of 4000 rpm, collect thalline;
5) add the ice-cold 0.1M CaCl2 of 20 ml in centrifuge tube, behind the thalline that evenly suspends, 30 min in the ice bath;
6) under 4 ℃, remove supernatant liquor behind centrifugal 10 min of 4000 rpm, collect thalline, add the ice-cold 0.1M CaCl2 solution of 2 ml and evenly suspend behind the thalline, it is stand-by to put into ice;
Step of converting is as follows:
1) will connect product and add in the 200 μ L competent cells, fully mixing places 30 min on ice;
2) 42 ℃ of thermal shock 2 min 30 s, adding is preheated to 37 ℃ LB liquid nutrient medium 400 μ L behind ice bath 1 min;
3) 37 ℃ of low speed (100 rpm) shaking culture, 40 min;
4) add 200 μ L bacterium liquid on each contains the LB solid medium flat board of penbritin (50 μ g/ml), the X-gal 40 μ L of 0.1 M IPTG, 4 μ L and 20 mg/ml evenly are applied on the flat board;
5) cultivated 16 hours in 37 ℃ of incubators;
(6) screening of positive colony
1) the single bacterium colony of well-grown white on the picking culture plate is rule on the LB solid medium flat board that contains penbritin (50 μ g/ml), enlarged culturing (cultivating 12 hours for 37 ℃);
2) bacterium after the picking enlarged culturing carries out pcr amplification, and reaction system is as follows:
A little thalline of DNA
dNTPs 100μM
Primer P1 5 pmol
Primer P2 5 pmol
Taq plus polysaccharase 1U
10 * PCR damping fluid, 2.5 μ L
Mg2+ 1.5mM
DdH2O adds to final volume 25 μ L
3) the PCR response procedures is the same.Amplified production separates on 1% sepharose, 100V constant voltage electrophoresis 30 minutes, and detection has or not the purpose fragment.
(7) sequencing of gene is finished by Shanghai branch office of Invitrogen biotech firm.Sequential analysis adopts the related software on the NCBI network address to carry out.Comprise blast program (http://www.ncbi.nlm.nih.gov), DNAman5.2 software and multiple sequence comparison software ClustalW(http: //www.ebi.ac.uk/clustal w).Utilize ORF Finder software that the open reading frame of B-hordein gene is translated into aminoacid sequence.
SEQUENCE?LISTING
＜110〉Chengdu Inst. of Biology, Chinese Academy of Sciences
＜120〉hullessbarley seed B-hordein gene nucleic acid sequence and uses thereof
＜130〉specification sheets
<160> 6
<170> PatentIn?version?3.3
<210> 1
<211> 894
<212> DNA
<213> H.?vulgare?ssp.?vulgare
<400> 1
atgaagacct?tcctcatctt?tgcactcctc?gccattgcgg?caacaagtac?gattgcacag 60
caacaaccat?ttccacaaca?acccttccca?caacagccac?aaccataccc?acaacaacca 120
caaccatatc?cacaacaacc?attccaaccg?caacaaccat?ttccacaaca?aaccatccca 180
caacaaccac?aaccataccc?acaacaacca?caaccatatc?cacaacaacc?cttcccaccg 240
caacaagaat?ttccacaaca?accgccattt?tggccacaac?aaccatttcc?acaacaacca 300
ccatttgggc?tacaacaacc?aattctatgg?cagcaacaac?catgtacacc?acaacaaaca 360
ccactcccac?aaggacaact?gtatcaaacg?cttctgcaac?tacaaatacc?ctatgttcat 420
ccttctattt?tgcaacaact?aaacccatgc?aaggtattcc?tccagcagca?gtgcaacccc 480
gtgcgaatgc?cacaacttat?tgctaggttg?caaatgttgc?agcagagcag?ttgtcatgtg 540
ttgcagcgac?aatgttgcca?gcaactgccg?caaatctccg?aacaattccg?ccatgaggca 600
atccgtgcaa?tcgtctactc?tatctttctg?caagaacaac?cccaacagtc?ggtccaaggt 660
gtatcccaac?cccaaaaaca?gttgcagcag?gagcaagttg?gacaatgttc?tttccaacaa 720
cctcaaccac?aacaacttgg?tcaaccacag?caggtaccac?acagtgtttt?cttgcagcca 780
caccagatag?ctcagcttga?ggcgacgact?tccattgcgc?tgcgtaccct?accaaggatg 840
tgcaatgtta?atgtgccatt?gtacgacatc?atgccagtcg?acttttggca?ctag 894
<210> 2
<211> 297
<212> PRT
<213> H.?vulgare?ssp.?vulgare
<400> 2
Met?Lys?Thr?Phe?Leu?Ile?Phe?Ala?Leu?Leu?Ala?Ile?Ala?Ala?Thr?Ser
1 5 10 15
Thr?Ile?Ala?Gln?Gln?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Phe?Pro?Gln?Gln
20 25 30
Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Phe
35 40 45
Gln?Pro?Gln?Gln?Pro?Phe?Pro?Gln?Gln?Thr?Ile?Pro?Gln?Gln?Pro?Gln
50 55 60
Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Phe?Pro?Pro
65 70 75 80
Gln?Gln?Glu?Phe?Pro?Gln?Gln?Pro?Pro?Phe?Trp?Pro?Gln?Gln?Pro?Phe
85 90 95
Pro?Gln?Gln?Pro?Pro?Phe?Gly?Leu?Gln?Gln?Pro?Ile?Leu?Trp?Gln?Gln
100 105 110
Gln?Pro?Cys?Thr?Pro?Gln?Gln?Thr?Pro?Leu?Pro?Gln?Gly?Gln?Leu?Tyr
115 120 125
Gln?Thr?Leu?Leu?Gln?Leu?Gln?Ile?Pro?Tyr?Val?His?Pro?Ser?Ile?Leu
130 135 140
Gln?Gln?Leu?Asn?Pro?Cys?Lys?Val?Phe?Leu?Gln?Gln?Gln?Cys?Asn?Pro
145 150 155 160
Val?Arg?Met?Pro?Gln?Leu?Ile?Ala?Arg?Leu?Gln?Met?Leu?Gln?Gln?Ser
165 170 175
Ser?Cys?His?Val?Leu?Gln?Arg?Gln?Cys?Cys?Gln?Gln?Leu?Pro?Gln?Ile
180 185 190
Ser?Glu?Gln?Phe?Arg?His?Glu?Ala?Ile?Arg?Ala?Ile?Val?Tyr?Ser?Ile
195 200 205
Phe?Leu?Gln?Glu?Gln?Pro?Gln?Gln?Ser?Val?Gln?Gly?Val?Ser?Gln?Pro
210 215 220
Gln?Lys?Gln?Leu?Gln?Gln?Glu?Gln?Val?Gly?Gln?Cys?Ser?Phe?Gln?Gln
225 230 235 240
Pro?Gln?Pro?Gln?Gln?Leu?Gly?Gln?Pro?Gln?Gln?Val?Pro?His?Ser?Val
245 250 255
Phe?Leu?Gln?Pro?His?Gln?Ile?Ala?Gln?Leu?Glu?Ala?Thr?Thr?Ser?Ile
260 265 270
Ala?Leu?Arg?Thr?Leu?Pro?Arg?Met?Cys?Asn?Val?Asn?Val?Pro?Leu?Tyr
275 280 285
Asp?Ile?Met?Pro?Val?Asp?Phe?Trp?His
290 295
<210> 3
<211> 888
<212> DNA
<213> H.?vulgare?ssp.?vulgare
<400> 3
atgaagacct?tcctcatctt?tgcactcctc?gccattgcgg?caacaagcac?gattgcgcag 60
caacaaccat?ttccacaaca?acccatccca?caacaaccac?aaccataccc?acaacaacca 120
caaccatatc?cacaacaacc?ctttccaccg?caacaaccat?ttccacaaca?acccgtccca 180
caacaaccac?aaccataccc?acaacaaccc?ttcccaccgc?aacaaccatt?tccacaacaa 240
ccaccatttt?ggcaacaaaa?accatttcca?caacaaccac?catttgggct?acaacaacca 300
attctatcgc?agcaacaacc?atgtacacca?caacaaacac?cactcccaca?aggacaactg 360
taccaaacgc?ttctgcaact?acaaatacaa?tatgttcatc?catctatttt?gcaacagcta 420
aacccatgca?aggtattcct?ccagcagcag?tgcagccctg?tgccagtgcc?acaacgtatt 480
gctaggtcgc?aaatgttgca?gcagagcagt?tgccatgtgt?tgcagcaaca?atgttgccag 540
caactacccc?aaatccccga?acaactccgc?catgaggcag?tccgtgcaat?cgtctactct 600
atcgtcctgc?aagaacaatc?cctacaattg?gtccaaggtg?tctcccaacc?ccaacaacag 660
tcacaacagc?aacaagtcgg?acaatgttct?ttccaacaac?ctcaaccaca?acagggtcaa 720
caacagcaag?tgccacagag?tgttctcttg?cagccacacc?aaatagctca?acttgaggcg 780
acaacttcca?ttgcgctacg?taccctacca?acgatgtgca?gtgttaatgt?gccgttgtac 840
cgcatagtgc?cattagccat?tgacaccaga?gttggtgtct?aatgataa 888
<210> 4
<211> 293
<212> PRT
<213> H.?vulgare?ssp.?vulgare
<400> 4
Met?Lys?Thr?Phe?Leu?Ile?Phe?Ala?Leu?Leu?Ala?Ile?Ala?Ala?Thr?Ser
1 5 10 15
Thr?Ile?Ala?Gln?Gln?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Ile?Pro?Gln?Gln
20 25 30
Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Phe
35 40 45
Pro?Pro?Gln?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Val?Pro?Gln?Gln?Pro?Gln
50 55 60
Pro?Tyr?Pro?Gln?Gln?Pro?Phe?Pro?Pro?Gln?Gln?Pro?Phe?Pro?Gln?Gln
65 70 75 80
Pro?Pro?Phe?Trp?Gln?Gln?Lys?Pro?Phe?Pro?Gln?Gln?Pro?Pro?Phe?Gly
85 90 95
Leu?Gln?Gln?Pro?Ile?Leu?Ser?Gln?Gln?Gln?Pro?Cys?Thr?Pro?Gln?Gln
100 105 110
Thr?Pro?Leu?Pro?Gln?Gly?Gln?Leu?Tyr?Gln?Thr?Leu?Leu?Gln?Leu?Gln
115 120 125
Ile?Gln?Tyr?Val?His?Pro?Ser?Ile?Leu?Gln?Gln?Leu?Asn?Pro?Cys?Lys
130 135 140
Val?Phe?Leu?Gln?Gln?Gln?Cys?Ser?Pro?Val?Pro?Val?Pro?Gln?Arg?Ile
145 150 155 160
Ala?Arg?Ser?Gln?Met?Leu?Gln?Gln?Ser?Ser?Cys?His?Val?Leu?Gln?Gln
165 170 175
Gln?Cys?Cys?Gln?Gln?Leu?Pro?Gln?Ile?Pro?Glu?Gln?Leu?Arg?His?Glu
180 185 190
Ala?Val?Arg?Ala?Ile?Val?Tyr?Ser?Ile?Val?Leu?Gln?Glu?Gln?Ser?Leu
195 200 205
Gln?Leu?Val?Gln?Gly?Val?Ser?Gln?Pro?Gln?Gln?Gln?Ser?Gln?Gln?Gln
210 215 220
Gln?Val?Gly?Gln?Cys?Ser?Phe?Gln?Gln?Pro?Gln?Pro?Gln?Gln?Gly?Gln
225 230 235 240
Gln?Gln?Gln?Val?Pro?Gln?Ser?Val?Leu?Leu?Gln?Pro?His?Gln?Ile?Ala
245 250 255
Gln?Leu?Glu?Ala?Thr?Thr?Ser?Ile?Ala?Leu?Arg?Thr?Leu?Pro?Thr?Met
260 265 270
Cys?Ser?Val?Asn?Val?Pro?Leu?Tyr?Arg?Ile?Val?Pro?Leu?Ala?Ile?Asp
275 280 285
Thr?Arg?Val?Gly?Val
290
<210> 5
<211> 939
<212> DNA
<213> H.?vulgare?ssp.?vulgare
<400> 5
atgaagacct?tcctcatctt?tgcactcctc?gccattgtgg?caacaagtac?cattgcacaa 60
caacaaccat?acccacaaca?accacaacca?tttccacaac?aacccatccc?acaacaacca 120
caaccatttc?cacaacaacc?acaaccatac?ccacaacaac?cacaaccatt?tccacaacaa 180
cccatcccac?aacaaccaca?accataccca?caacaaccac?aaccatttcc?acaacaaccc 240
atcccacaac?aaccacaacc?atacccacaa?caaccacaac?catttcccct?acaacccttc 300
ccatcacaac?aaccatttcc?acaacaacca?ccattttggc?aacaacaacc?agttctatcg 360
cagcaacaac?catgtacaca?agaacaaaca?ccactcctac?aagaacaaca?agatcaaatg 420
cttctgcaag?tacaaatacc?atttgttcat?ccatctattt?tgcagcagct?aaacccatgc 480
aaggtattcc?tccagcagca?gtgcagccct?gtggcaatgt?cacaacgtat?tgcaaggtcg 540
cagatgttgc?aacagagcag?ttgccatgtg?ttgcagcaac?agtgttgcca?acaactgccg 600
caaatccccg?aacaactccg?ccatgaggca?gtccgtgcaa?tcgtctactc?tatcgtcctg 660
caagaacaat?ccctacaatt?ggtccaaggt?gtctcccaac?cccaacaaca?gtcacaacag 720
caacaagtcg?gacaatgttc?tttccaacaa?cctcaaccac?aacagggtca?acaacagcaa 780
gtgccacaga?gtgttctctt?gcagccacac?caaatagctc?aacttgaggc?gacaacttcc 840
attgcgctgc?gtaccctacc?aacgatgtgc?agtgttaatg?tgccgttgta?ccgcatagtg 900
ccattagcca?ttgacaccag?agttggtgtc?taatgataa 939
<210> 6
<211> 310
<212> PRT
<213> H.?vulgare?ssp.?vulgare
<400> 6
Met?Lys?Thr?Phe?Leu?Ile?Phe?Ala?Leu?Leu?Ala?Ile?Val?Ala?Thr?Ser
1 5 10 15
Thr?Ile?Ala?Gln?Gln?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Phe?Pro
20 25 30
Gln?Gln?Pro?Ile?Pro?Gln?Gln?Pro?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Gln
35 40 45
Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Ile?Pro?Gln
50 55 60
Gln?Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Phe?Pro?Gln?Gln?Pro
65 70 75 80
Ile?Pro?Gln?Gln?Pro?Gln?Pro?Tyr?Pro?Gln?Gln?Pro?Gln?Pro?Phe?Pro
85 90 95
Leu?Gln?Pro?Phe?Pro?Ser?Gln?Gln?Pro?Phe?Pro?Gln?Gln?Pro?Pro?Phe
100 105 110
Trp?Gln?Gln?Gln?Pro?Val?Leu?Ser?Gln?Gln?Gln?Pro?Cys?Thr?Gln?Glu
115 120 125
Gln?Thr?Pro?Leu?Leu?Gln?Glu?Gln?Gln?Asp?Gln?Met?Leu?Leu?Gln?Val
130 135 140
Gln?Ile?Pro?Phe?Val?His?Pro?Ser?Ile?Leu?Gln?Gln?Leu?Asn?Pro?Cys
145 150 155 160
Lys?Val?Phe?Leu?Gln?Gln?Gln?Cys?Ser?Pro?Val?Ala?Met?Ser?Gln?Arg
165 170 175
Ile?Ala?Arg?Ser?Gln?Met?Leu?Gln?Gln?Ser?Ser?Cys?His?Val?Leu?Gln
180 185 190
Gln?Gln?Cys?Cys?Gln?Gln?Leu?Pro?Gln?Ile?Pro?Glu?Gln?Leu?Arg?His
195 200 205
Glu?Ala?Val?Arg?Ala?Ile?Val?Tyr?Ser?Ile?Val?Leu?Gln?Glu?Gln?Ser
210 215 220
Leu?Gln?Leu?Val?Gln?Gly?Val?Ser?Gln?Pro?Gln?Gln?Gln?Ser?Gln?Gln
225 230 235 240
Gln?Gln?Val?Gly?Gln?Cys?Ser?Phe?Gln?Gln?Pro?Gln?Pro?Gln?Gln?Gly
245 250 255
Gln?Gln?Gln?Gln?Val?Pro?Gln?Ser?Val?Leu?Leu?Gln?Pro?His?Gln?Ile
260 265 270
Ala?Gln?Leu?Glu?Ala?Thr?Thr?Ser?Ile?Ala?Leu?Arg?Thr?Leu?Pro?Thr
275 280 285
Met?Cys?Ser?Val?Asn?Val?Pro?Leu?Tyr?Arg?Ile?Val?Pro?Leu?Ala?Ile
290 295 300
Asp?Thr?Arg?Val?Gly?Val
305 310

Claims (6)

1. hullessbarley seed B-hordein gene nucleic acid sequence is characterized in that containing seq.1 or seq.2 or seq.3 gene fragment.
2. the described hullessbarley seed B-hordein of claim 1 gene nucleic acid sequence, it is characterized in that: the aminoacid sequence that obtains according to seq.1 is:
MKTFLIFALLAIAATSTIAQQQPFPQQPFPQQPQPYPQQPQPYPQQPFQPQQPFPQQTIPQQPQPYPQQPQPYPQQPFPPQQEFPQQPPFWPQQPFPQQP PFGLQQPILWQQQPCTPQQTPLPQGQLYQTLLQLQIPYVHPSILQQLNPCKVFLQQQCNPVRMPQLIARLQMLQQSSCHVLQRQCCQQLPQISEQFRHEA
3. the described hullessbarley seed B-hordein of claim 1 gene nucleic acid sequence, it is characterized in that: the aminoacid sequence that obtains according to seq.2 is:
MKTFLIFALLAIAATSTIAQQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPFPQQPVPQQPQPYPQQPFPPQQPFPQQPPFWQQKPFPQQPPFGLQQPILSQQQPCTPQQTPLPQGQLYQTLLQLQIQYVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQLRHEAVRAIVYSIVLQEQSLQLVQGVSQPQQQSQQQQVGQCSFQQPQPQQGQQQQVPQSVLLQPHQIAQLEATTSIALRTLPTMCSVNVPLYRIVPLAIDTRVGV。
4. the described hullessbarley seed B-hordein of claim 1 gene nucleic acid sequence, it is characterized in that: the aminoacid sequence that obtains according to seq.3 is:
MKTFLIFALLAIVATSTIAQQQPYPQQPQPFPQQPIPQQPQPFPQQPQPYPQQPQPFPQQPIPQQPQPYPQQPQPFPQQPIPQQPQPYPQQPQPFPLQPFPSQQPFPQQPPFWQQQPVLSQQQPCTQEQTPLLQEQQDQMLLQVQIPFVHPSILQQLNPCKVFLQQQCSPVAMSQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQLRHEAVRAIVYSIVLQEQSLQLVQGVSQPQQQSQQQQVGQCSFQQPQPQQGQQQQVPQSVLLQPHQIAQLEATTSIALRTLPTMCSVNVPLYRIVPLAIDTRVGV。
5. a hullessbarley seed B-hordein gene nucleic acid sequence is variant or the fragment of the described seq.1 of claim 1 or seq.2 or seq.3.
6. claim 1 or the 5 described hullessbarley seed B-hordein gene nucleic acid sequences purposes in crop quality improvement.
CN2010101539001A 2010-04-23 2010-04-23 Nucleic acid sequence of barley grain B-hordein gene and application thereof Pending CN102234650A (en)

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Application publication date: 20111109