CN102234330B - Plant type related protein and coding gene thereof - Google Patents
Plant type related protein and coding gene thereof Download PDFInfo
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- CN102234330B CN102234330B CN 201010168639 CN201010168639A CN102234330B CN 102234330 B CN102234330 B CN 102234330B CN 201010168639 CN201010168639 CN 201010168639 CN 201010168639 A CN201010168639 A CN 201010168639A CN 102234330 B CN102234330 B CN 102234330B
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Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses plant type related protein and coding gene thereof. The protein is protein represented by the following a) or b): a) the protein comprises amino acid sequence represented by SEQ ID NO:2; b) the protein comprises amino acid sequence represented by SEQ ID NO:2, and is related to the plant type and/or related to output, and is derived from a), wherein the amino acid sequence are processed from substituting and/or deleting and/or adding one or a plurality of amino acid residues. Experiments results shows that: after inserting the gene provided by the present invention to a rice mutant nall, plant type of the resulting transgenic plant is changed significantly, in particular, height of the plant main stem, length of the plant main panicle, number of the plant main panicle, leaf width of the plant and leaf length of the plant are less than those of the mutant nall. The gene provided by the present invention has wide application prospect in the field of plant genetic breeding.
Description
Technical field
The present invention relates to and plant plant type associated protein and encoding gene thereof.
Background technology
Plant type (plant type) is meant stack features relevant with crop varieties output ability or plant materials at the spatial arrangement mode, i.e. the growing way appearance.Ideotype (Ideal plant type) also is called ideal type (Ideotype), finger by favourable plant photosynthesis, grow and idealized plant type that the proterties of grain yield is formed, it can improve colony's efficiency of light energy utilization to greatest extent, increases biological yield and improves economic coefficient etc.The research of relevant plant type of rice, phase late 1950s, Japan scientist angle Tian Chongsan youth sums up the kind that is suitable for how fertile intensive culture and should have thick, little, upright and bottle-green blade from his research practice to paddy rice, soybean, sugarcane etc., short and the tough and tensile stem stalk and the ideotype theory of medium ability for tillering (angle Tian Chongsan youth. agricultural and gardening .1987,62 (1): 25～29.).Constantly the improvement plant type also is the basic experience of China's super high-yielding rice breeding.Drape over one's shoulders blade profile from the high stalk of farm variety and progressively improve and be straight blade profile of short stem, the form improvement develops into form gradually and the function improvement is laid equal stress on from paying attention to, and has not only improved optical energy utilization efficiency, has increased lodging tolerance, has also strengthened the physiological function of paddy rice.
The rice leaf proterties is the important factor that plant type constitutes, be directly connected to the photosynthetic area and the efficiency of light energy utilization of blade, and then to output generation material impact, therefore the research to blade profile is one of breeding man, geneticist and molecular biologist Focal Point of Common Attention, and the width of blade and curling situation are important component parts wherein.Studies show that paddy rice narrow leaf and leaf roll proterties are controlled by qualitative trait gene mainly.The narrow leaf of report and leaf roll mutant body are many as classical morphological markers at present.
Summary of the invention
An object of the present invention is to provide a kind of albumen and encoding gene thereof.
Albumen provided by the present invention, be following a) or b) protein:
A) protein of forming by the aminoacid sequence shown in the SEQ ID NO:2;
B) with the aminoacid sequence shown in the SEQ ID NO:2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant plant type and/or be correlated with output by a) deutero-protein.
Described proteic encoding gene is following 1), 2), 3) or 4) gene:
1) its nucleotide sequence is from dna molecular shown in the Nucleotide of 5 ' terminal 140-1066 position among the SEQ ID NO:1;
2) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:1;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;
4) with 1) or 2) dna sequence dna that limits has the homology more than 90% and the dna molecular of encoding said proteins.
The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
In order to make the albumen in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the SEQ ID NO:2 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLI SEEDL |
Above-mentioned (a) but in the albumen synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Proteic encoding gene in above-mentioned (a) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the SEQ ID NO:1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Increase above-mentioned arbitrary described encoding gene total length or its any segmental primer to also belonging to protection scope of the present invention.
A primer sequence of described primer centering is shown in SEQ ID NO:3, and another primer sequence of described primer centering is shown in SEQ ID NO:4.
The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.
Described recombinant vectors obtains for the multiple clone site of described encoding gene being inserted carrier pCAMBIA2300-Actin.
The carrier that sets out that is used to make up described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprise the polyadenylic acid signal and any other participation mRNA processing or the dna fragmentation of genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, induces non-translational region that (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end transcribes etc. all to have similar functions as the Agrobacterium crown-gall nodule.
Carry gene of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.
Above-mentioned arbitrary described encoding gene also belongs to protection scope of the present invention in the application that changes the plant plant type and/or change in the output phenotype.
Above-mentioned arbitrary described albumen also belongs to protection scope of the present invention in the application that changes the plant plant type and/or change in the output phenotype.
Last purpose of the present invention provides a kind of method that changes plant plant type and/or output phenotype.
The method of change plant plant type provided by the present invention and/or output phenotype is following 1) or 2):
1) is in the plant that sets out, to import above-mentioned arbitrary described encoding gene, obtains comparing the purpose plant of plant type change with the plant that sets out;
2) be in the plant that sets out, to import above-mentioned arbitrary described encoding gene, obtain comparing the purpose plant of output phenotypic alternation with the plant that sets out.
In the aforesaid method, described encoding gene imports by above-mentioned arbitrary described recombinant vectors.
In the method for above-mentioned arbitrary described application and arbitrary described change plant plant type, described plant type is a plant stem height, plant leaf is wide or plant leaf is long; Described output phenotype is plant master spike length or plant master grain number per spike;
The plant stem higher primary school that described plant type is changed into the purpose plant in the wide plant leaf length less than described set out plant and/or purpose plant of the plant leaf of the described plant that sets out, purpose plant less than the described plant that sets out;
The plant master spike length that described output phenotype is changed into the purpose plant is less than the described plant that sets out less than the plant master grain number per spike of described set out plant and/or purpose plant.
In the method for above-mentioned arbitrary described application and arbitrary described change plant plant type, the described plant that sets out is a monocotyledons; Described monocotyledons is a paddy rice; Described paddy rice is rice mutant nall.
Experimental results show that, after in rice mutant nall, importing gene of the present invention, the plant type of the transfer-gen plant that obtains obviously changes, and is in particular in: plant leaf is wide to be less than mutant nall in mutant nall, plant master spike length less than mutant nall, plant master grain number per spike less than mutant nall, plant stem higher primary school less than mutant nall, plant leaf length.If with this gene knockout then cause the increase of output.Gene of the present invention will have broad application prospects in the genetic breeding field of plant.
Description of drawings
Fig. 1 is the phenotype of transgenic paddy rice and adjoining tree.
Fig. 2 is the main fringe phenotype of transgenic paddy rice and adjoining tree.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, gene and functional verification
Wild-type paddy rice Zhejiang spoke 802 (zf802) are at document (Zheng Leiying, Zhu Xudong, before the money, Zhao Zhong, Zhang Jianjun, Hu Xiaohe, Lin Hongxuan, form and the positioning analysis of the Luo Da .2003. paddy rice fringe mutant Cl of portion. Science Bulletin the 48th the 3rd phase of volume) in (being provided by Inst. of Genetics and Development Biology, CAS) disclosed.
The pMD19-T carrier is available from takara, and catalog number is D102A.
Carrier pCAMBIA2300-Actin is at document (Kejian Wang, Ding Tang, Lilan Hong, WenyingXu, Jian Huang, Ming Li, Minghong Gu, Yongbiao Xue, Zhukuan Cheng.2010.DEPandAFORegulate Reproductive Habit in Rice.January 2010.Volume 6.) disclosed in.(providing) by Inst. of Genetics and Development Biology, CAS.
Agrobacterium tumefaciens EH105 document (Zhao Zhiqiang, Fu Yaping, poplar Kun, Zhang Yuman, the face Yongsheng, Fang Rongxiang, Sun Zongxiu, the clone and the functional analysis .Chin J Biotech2008 of Chen Xiao English .2008. paddy rice small G-protein OsPra2 gene, December 25; 24 (12): (providing by Inst. of Genetics and Development Biology, CAS) was provided among the 2027-2033.
Rice mutant nall (Dong Fenggao, Xiong Zhenmin, Qian Qian, Zu Xudong, Chen Shihua (1994) .Breeding Near-isogenic lines ofmorphological markers in indica rice.Chinese J.Rice Sci.8,135-139.(providing) by Inst. of Genetics and Development Biology, CAS.
One, gene preparation
From the DNA shown in 5 ' the end 140-1066 bit base, also can be prepared as follows among the SEQ ID NO:1 among the synthetic SEQ ID NO:1 from the DNA shown in 5 ' the end 140-1066 bit base.Hold the proteic aminoacid sequence of the dna encoding shown in the 140-1066 bit base shown in SEQ ID NO:2 from 5 ' among the SEQ ID NO:1.
Extracting total RNA of wild-type paddy rice Zhejiang spoke 802 (zf802), is template with its reverse transcription product, carries out pcr amplification with following primer P1/P2, obtains pcr amplification product.
P1:5 '-ATGGACTCCCCGTCGCCTAT (forward primer) (SEQ ID NO:3);
P2:5 '-CTAGAGGCTCAAGTTGAGGT (reverse primer) (SEQ ID NO:4).
The PCR product is carried out 1% agarose gel electrophoresis detect, reclaim target DNA fragment, be connected, will connect the product transformed into escherichia coli, resistance screening, picking mono-clonal with the pMD-19T carrier; Mono-clonal is carried out liquid culture respectively, extract plasmid, plasmid is checked order.As a result, the gene order of inserting in the pMD-19T carrier as among the SEQ ID NO:1 from shown in 5 ' the end 140-1066 bit base, show that the recombinant vectors of structure is correct, with its called after pMD-19T-SUN1.
Two, gene transformation mutant nall
1, recombinant expression vector makes up
Cut pMD-19T-SUN1 with restriction enzyme Xba I and Sal I enzyme, reclaim target gene fragment; Cut carrier pCAMBIA2300-Actin with restriction enzyme Xba I and Sal I enzyme, reclaim the big fragment of carrier; Target gene fragment is connected with the big fragment of carrier, obtains recombinant vectors.With the recombinant vectors transformed into escherichia coli, resistance screening, picking mono-clonal; Mono-clonal is carried out liquid culture respectively, extract plasmid, plasmid is checked order.As a result, the gene order of inserting between the Xba I of pCAMBIA2300-Actin carrier and Sal I restriction enzyme site as among the SEQ ID NO:1 from shown in 5 ' the end 140-1066 bit base, show that the recombinant vectors of structure is correct, with its called after pActin::SUN1.
2, the reorganization Agrobacterium makes up
With the thermal shock method recombinant expression vector pActin::SUN1 is transformed agrobacterium tumefaciens EH105, screen positive recombinant, obtain containing the agrobacterium tumefaciens EH105 of recombinant expression vector pActin::SUN1, note is made EH105-pActin::SUN1.
3, transfer-gen plant makes up
, transformed plant is carried out PCR identify that PCR identifies that the primer is above-mentioned P1/P2 pActin::SUN1 rice transformation mutant nall with agriculture bacillus mediated rice callus metaplasia method, the result obtains 10 strain positive plants, is T0 for plant; The plant note of transgene SUN1 is made transfer-gen plant.
With the rice mutant nall that changes empty carrier pCAMBIA2300-Actin in contrast, compare simultaneously with the wild-type rice mutant nall that does not carry out any processing.
4, the phenotype analytical of transfer-gen plant
Observe the phenotype of statistics transfer-gen plant, adjoining tree.Again to the stem height of plant, main spike length degree, main grain number per spike, wide, the blade progress row statistical study of blade.
The phenotype of transfer-gen plant and adjoining tree as depicted in figs. 1 and 2.A is wild-type rice mutant nall, and B is a transfer-gen plant.The result shows, compares with adjoining tree, and the plant type of transfer-gen plant changes, and is specific as follows: the stem height becomes short, blade obviously narrows down, blade length obviously diminishes; The output phenotypic alternation of transfer-gen plant, specific as follows: main spike length degree diminishes, main grain number per spike tails off.Wild-type rice mutant nall is identical with the plant type of the rice mutant nall that changes empty carrier pCAMBIA2300-Actin over to.
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉with plant plant type associated protein and encoding gene thereof
<160>4
<210>1
<211>1374
<212>DNA
＜213〉Oryza paddy rice (Oryza sativa)
<400>1
tgaccccaac cccaaaccca ctctactcta ctgtgcctca cctcttgcca ctactatttc 60
tagtagtcgt gtatcatcat ttcagatatc atatcgccac ctctcgtttt tttaataata 120
tcagcggcga gcgagcgaga tggactcccc gtcgcctatg gcggcgcagg cggccgacct 180
gtcgctgacg ctggcgccgt cgggaggggg tggtggggga ggaggaggcg gcggcggtgg 240
tgggtcgtcg tcggcgtgca tcgacggcaa ggacgtgcgg ctgttcccgt gcttgttctg 300
caacaagaag ttcttgaagt cgcaggcgct aggcgggcac cagaacgcgc acaagaagga 360
gcggagcatc gggtggaatc cctacttcta catgccgccg acgccgcacc ccgccggcaa 420
tgccgccgcc gccgccgcgg cggcgacgcc cggtgggatg tcgtccgtca cgacgccgtc 480
cgggagctac ggcgtcgtcg gtggtgccgc cgccgcggcg gcggctgtcg tcggggctac 540
tgctggcgtt gggggcggag gtggagtggg aggggggctt ctcccggcgc acgcgtacgc 600
cgggcacggg tacgccgcgg tgccgacgtc gttccccatc gcgtcgcaca gctcgagcgt 660
ggttggctcc ggtgggctgc agtactacgc tggtaccgac tgcggcgcgg cggcggcggg 720
tgcggcgaag acgacgacga cgacggcggc ggcggcggcg acggccgtgg cggggagcga 780
gagcggcgtg caggtgcccc ggttcgcgac gcaccagcac catctcctgg cggtggtgag 840
cagcgggcgc gcgatgctgg cggcgcccga ccagccgggc gccgggcgcg acgacatgat 900
cgacatgctc aactggaggc gaggctccca cggccccacc gcctccgccg ccgccaccac 960
gccctccccg gcaagcacca ccaccacgct caccaccttc gccagcgccg acggcagcaa 1020
caacggcgag gagaacgagg agctcgacct caacttgagc ctctagctcc caccaccacc 1080
acctcctcct ccgccgccgc tgttgtcgcc gcgcaatcca agaaggcaag gtcaatcaat 1140
cgccatgttc ttcttctcca agctccacct actcctcttc caattcctcc tcgtgtgtga 1200
ttaatccccc tcttcttgct gcctgcgtac gtactcctta attaattagc tcttagggac 1260
gttaattaat ctcagttctt ggctctcttc tcctctcctc tcctctcctc tcatctcact 1320
tgtatgttaa tgttagtact ccttgtaatc gatcaatcag tcctcttttt ttgc 1374
<210>2
<211>308
<212>PRT
＜213〉Oryza paddy rice (Oryza sativa)
<400>2
Met Asp Ser Pro Ser Pro Met Ala Ala Gln Ala Ala Asp Leu Ser Leu
1 5 10 15
Thr Leu Ala Pro Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Ser Ser Ala Cys Ile Asp Gly Lys Asp Val Arg Leu
35 40 45
Phe Pro Cys Leu Phe Cys Asn Lys Lys Phe Leu Lys Ser Gln Ala Leu
50 55 60
Gly Gly His Gln Asn Ala His Lys Lys Glu Arg Ser Ile Gly Trp Asn
65 70 75 80
Pro Tyr Phe Tyr Met Pro Pro Thr Pro His Pro Ala Gly Asn Ala Ala
85 90 95
Ala Ala Ala Ala Ala Ala Thr Pro Gly Gly Met Ser Ser Val Thr Thr
100 105 110
Pro Ser Gly Ser Tyr Gly Val Val Gly Gly Ala Ala Ala Ala Ala Ala
115 120 125
Ala Val Val Gly Ala Thr Ala Gly Val Gly Gly Gly Gly Gly Val Gly
130 135 140
Gly Gly Leu Leu Pro Ala His Ala Tyr Ala Gly His Gly Tyr Ala Ala
145 150 155 160
Val Pro Thr Ser Phe Pro Ile Ala Ser His Ser Ser Ser Val Val Gly
165 170 175
Ser Gly Gly Leu Gln Tyr Tyr Ala Gly Thr Asp Cys Gly Ala Ala Ala
180 185 190
Ala Gly Ala Ala Lys Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Thr
195 200 205
Ala Val Ala Gly Ser Glu Ser Gly Val Gln Val Pro Arg Phe Ala Thr
210 215 220
His Gln His His Leu Leu Ala Val Val Ser Ser Gly Arg Ala Met Leu
225 230 235 240
Ala Ala Pro Asp Gln Pro Gly Ala Gly Arg Asp Asp Met Ile Asp Met
245 250 255
Leu Asn Trp Arg Arg Gly Ser His Gly Pro Thr Ala Ser Ala Ala Ala
260 265 270
Thr Thr Pro Ser Pro Ala Ser Thr Thr Thr Thr Leu Thr Thr Phe Ala
275 280 285
Ser Ala Asp Gly Ser Asn Ash Gly Glu Glu Asn Glu Glu Leu Asp Leu
290 295 300
Asn Leu Ser Leu
305
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atggactccc cgtcgcctat 20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
ctagaggctc aagttgaggt 20
Claims (9)
1. protein, the protein of forming by the aminoacid sequence shown in the SEQ ID NO:2.
2. the described proteinic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described proteinic encoding gene is following 1) or 2) gene:
1) its nucleotide sequence is from dna molecular shown in the Nucleotide of 5 ' terminal 140-1066 position among the SEQ ID NO:1;
2) its nucleotide sequence is a dna molecular shown in the SEQ ID NO:1.
4. in the amplification claim 3 1) shown in the primer of dna molecular right, a primer sequence is shown in SEQ ID NO:3, another primer sequence is shown in SEQ ID NO:4.
5. the recombinant vectors, reorganization bacterium or the expression cassette that contain claim 2 or 3 described encoding genes.
6. recombinant vectors according to claim 5 is characterized in that: described recombinant vectors obtains for the multiple clone site with claim 2 or 3 described encoding genes insertion carrier pCAMBIA2300-Actin.
7. claim 2 or the 3 described encoding genes application in changing plant plant type and/or change output phenotype, or the application of the described albumen of claim 1 in changing plant plant type and/or change output phenotype; Described plant is rice mutant nal1;
Described plant type is a plant stem height, plant leaf is wide or plant leaf is long; Described output phenotype is plant master spike length or plant master grain number per spike;
The plant stem higher primary school that described plant type is changed into the purpose plant in the wide plant leaf length less than described set out plant and/or purpose plant of the plant leaf of the described plant that sets out, purpose plant less than the described plant that sets out;
The plant master spike length that described output phenotype is changed into the purpose plant is less than the described plant that sets out less than the plant master grain number per spike of described set out plant and/or purpose plant.
8. a method that changes plant plant type and/or output phenotype is following 1) or 2):
1) is in the plant that sets out, to import claim 2 or 3 described encoding genes, obtains comparing the purpose plant of plant type change with the plant that sets out;
2) be in the plant that sets out, to import claim 2 or 3 described encoding genes, obtain comparing the purpose plant of output phenotypic alternation with the plant that sets out;
Described plant is rice mutant nal1;
Described plant type is a plant stem height, plant leaf is wide or plant leaf is long; Described output phenotype is plant master spike length or plant master grain number per spike;
The plant stem higher primary school that described plant type is changed into the purpose plant in the wide plant leaf length less than described set out plant and/or purpose plant of the plant leaf of the described plant that sets out, purpose plant less than the described plant that sets out;
The plant master spike length that described output phenotype is changed into the purpose plant is less than the described plant that sets out less than the plant master grain number per spike of described set out plant and/or purpose plant.
9. method according to claim 8 is characterized in that: described claim 2 or 3 described encoding genes import by recombinant vectors described in claim 5 or 6.
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| CN 201010168639 CN102234330B (en) | 2010-05-04 | 2010-05-04 | Plant type related protein and coding gene thereof |
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| CN 201010168639 CN102234330B (en) | 2010-05-04 | 2010-05-04 | Plant type related protein and coding gene thereof |
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| CN102234330A CN102234330A (en) | 2011-11-09 |
| CN102234330B true CN102234330B (en) | 2013-07-31 |
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| CN102584973B (en) * | 2012-03-12 | 2013-09-25 | 中国科学院遗传与发育生物学研究所 | Rice plant type related protein LPA1 and coding gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544691A (en) * | 2008-03-26 | 2009-09-30 | 中国科学院遗传与发育生物学研究所 | Gene TUD1 for controlling rice height and grain shape and application thereof |
| CN101607989A (en) * | 2008-06-20 | 2009-12-23 | 中国科学院遗传与发育生物学研究所 | A kind of rice dwarf-related protein and encoding gene thereof and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101544691A (en) * | 2008-03-26 | 2009-09-30 | 中国科学院遗传与发育生物学研究所 | Gene TUD1 for controlling rice height and grain shape and application thereof |
| CN101607989A (en) * | 2008-06-20 | 2009-12-23 | 中国科学院遗传与发育生物学研究所 | A kind of rice dwarf-related protein and encoding gene thereof and application |
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