CN108085319A - Plant tillering angle GAP-associated protein GAP and its encoding gene and application - Google Patents
Plant tillering angle GAP-associated protein GAP and its encoding gene and application Download PDFInfo
- Publication number
- CN108085319A CN108085319A CN201711424584.5A CN201711424584A CN108085319A CN 108085319 A CN108085319 A CN 108085319A CN 201711424584 A CN201711424584 A CN 201711424584A CN 108085319 A CN108085319 A CN 108085319A
- Authority
- CN
- China
- Prior art keywords
- plant
- sequence
- wox6
- gravity
- tillering angle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of plant tillering angle GAP-associated protein GAP and its encoding gene and applications.A kind of method for cultivating genetically modified plants of present invention protection, includes the following steps：The expression quantity and/or activity of WOX6 albumen and WOX11 albumen in purpose plant are reduced, obtains the genetically modified plants that tillering angle increase and/or gravity reaction reduce.The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps：It is the expression for inhibiting WOX6 genes and WOX11 genes in purpose plant, obtains the genetically modified plants that tillering angle increase and/or gravity reaction reduce.The present invention excavates rice tillering angle key gene WOX6 and WOX11, and carries out functional verification.The present invention provides important theoretical foundation for the improvement of plant type of rice, the cultivation of super rice kind and has the genetic resources of application value.
Description
Technical field
The present invention relates to a kind of plant tillering angle GAP-associated protein GAP and its encoding gene and applications.
Background technology
Rice is one of most important cereal crops in the whole world, and the population of more than half is using rice as staple food grain in the world.With
The increase year by year of population in the world, the continuous reduction effectively ploughed, the rice varieties for cultivating good quality and high output have become and urgently solve
Certainly the problem of.Plant-type Breeding plays an important role as a kind of safely and effectively solution route in agricultural production.
Tillering angle is one of Main Agronomic Characters for determining plant type of rice.The size of tillering angle for luminous energy utilization, it is resistant to lodging,
Resisting pest and disease damage has direct relation, and final yield is very significant considering that.Therefore, point of Study On Rice tillering angle
Sub- regulatory mechanism has great importance for improving rice yield.Rice tillering angle is influenced be subject to several factors, such as is lost
The environmental factors such as biography factor, light, planting density, in environmental factor, the gravity reaction of rice stem plays a crucial role.It is sent out in rice
During educating, aerial part basal part of stem and the leaf sheath pillow of rice constantly adjust the direction of growth of plant according to the direction of gravity,
The tiller of last rice and gravity direction reach a fixed angle (The gravitropic set-point angle,
GSA).The change of aboveground vegetation part gravity reaction frequently can lead to the variation of GSA.Different rice material gravity reactions are strong and weak not
Together, the change on the plant forms such as tillering angle is caused, so as to influence effective planting density of single plant growth conditions and group etc., most
Crop yield is influenced eventually.At present, the regulated and control network on the reaction of rice gravity is not still apparent.Therefore, Study On Rice gravity reacts
And deep functional study is carried out to relevant difference expressing gene, it will help the molecular regulation net that structure tillering angle is formed
Network excavates the key gene for having application value.
The content of the invention
The object of the present invention is to provide a kind of plant tillering angle GAP-associated protein GAP and its encoding gene and applications.
The present invention provides a kind of methods for cultivating genetically modified plants, include the following steps：Reduce WOX6 in purpose plant
The expression quantity and/or activity of albumen and WOX11 albumen obtain the transgenosis that tillering angle increase and/or gravity reaction reduce and plant
Object.
The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps：Inhibit WOX6 in purpose plant
The expression of gene and WOX11 genes obtains the genetically modified plants that tillering angle increase and/or gravity reaction reduce.
" expression for inhibiting WOX6 genes and WOX11 genes in purpose plant " is prominent by being imported into purpose plant
Become what carrier was realized.
The mutational vector can pass through Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, electricity
It the conventional biology methods such as leads, is agriculture bacillus mediated to be transformed into purpose plant.
" expression for inhibiting WOX6 genes and WOX11 genes in purpose plant " is prominent by being imported into purpose plant
What change carrier first and mutational vector second were realized.
The mutational vector first is in carrier pYLCRISPR/Cas9PubiSequence table is inserted in the BsaI restriction enzyme sites of-H
Sequence 9 shown in the obtained recombinant expression carrier of double chain DNA molecule.
The mutational vector second is in carrier pYLCRISPR/Cas9PubiSequence table is inserted in the BsaI restriction enzyme sites of-H
Sequence 10 shown in the obtained recombinant expression carrier of double chain DNA molecule.
Purpose plant described in any of the above is dicotyledon or monocotyledon.The monocotyledon can be Poales
Plant.The Poales plant can be grass.The grass can be oryza plant.The oryza plant is specific
Can be rice, such as in spend 11 rice.
The present invention also protects the application of WOX6 albumen, for following (e1) or (e2)：
(e1) plant tillering angle is regulated and controled；
(e2) reaction of plant gravity is regulated and controled.
The present invention also protects the application of WOX11 albumen, for following (f1) or (f2)：
(f1) plant tillering angle is regulated and controled；
(f2) reaction of plant gravity is regulated and controled.
The present invention also protects the application of WOX6 genes, for following (g1) or (g2)：
(g1) the big plant of tillering angle is cultivated；
(g2) plant that plant gravity reacts small is cultivated.
The present invention also protects the application of WOX11 genes, for following (h1) or (h2)：
(h1) the big plant of tillering angle is cultivated；
(h2) plant that plant gravity reacts small is cultivated.
The present invention also protects application of any of the above the method in plant breeding.
The purpose of the breeding is to cultivate the plant that tillering angle is big and/or gravity reaction is small.
The present invention also protects to inhibit the substance of WOX6 genes and WOX11 gene expressions or, for reducing in plant
Tillering angle is big and/or gravity reaction plantlet cultivating for the expression quantity of WOX6 albumen and WOX11 albumen and/or the substance of activity
In application.
WOX6 albumen described in any of the above is following (a1) or (a2) or the protein of (a3) or (a4)：
(a1) protein being made of the amino acid sequence shown in sequence in sequence table 2；
(a2) protein being made of the amino acid sequence shown in sequence in sequence table 4；
(a3) amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and react the relevant protein as derived from sequence 2 with plant tillering angle and/or gravity；
(a4) amino acid sequence of sequence 4 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and react the relevant protein as derived from sequence 4 with plant tillering angle and/or gravity.
WOX11 albumen described in any of the above is the protein of following (b1) or (b2)：
(b1) protein being made of the amino acid sequence shown in sequence in sequence table 6；
(b2) amino acid sequence of sequence 6 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and react the relevant protein as derived from sequence 6 with plant tillering angle and/or gravity.
WOX6 genes described in any of the above are following (c1) or (c2) or (c3) or (c4) or (c5)：
(c1) DNA molecular shown in the sequence 11 of sequence table；
(c2) DNA molecular of the code area as shown in the sequence 1 of sequence table；
(c3) DNA molecular of the code area as shown in the sequence 3 of sequence table；
(c4) the DNA sequence dna hybridization limited under strict conditions with (c1) or (c2) or (c3) and coded plant tillering angle
And/or gravity reacts the DNA molecular of relevant protein；
(c5) DNA sequence dna limited with (c1) or (c2) or (c3) or (c4) has more than 90% homology and plant tillering
Angle and/or gravity react the DNA molecular of relevant protein.
WOX11 genes described in any of the above are following (d1) or (d2) or (d3)：
(d1) DNA molecular of the code area as shown in the sequence 5 of sequence table；
(d2) DNA sequence dna limited under strict conditions with (d1) hybridizes and coded plant tillering angle and/or gravity are anti-
Answer the DNA molecular of relevant protein；
(d3) and the DNA sequence dna of (d1) or (d2) restriction is with more than 90% homology and plant tillering angle and/or again
Power reacts the DNA molecular of relevant protein.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is miscellaneous in DNA or RNA
It hands over and hybridizes in experiment at 65 DEG C and wash film.
Plant described in any of the above is dicotyledon or monocotyledon.The monocotyledon can be that Poales is planted
Object.The Poales plant can be grass.The grass can be oryza plant.The oryza plant specifically may be used
For rice, for example, in spend 11 rice.
The present invention excavates rice tillering angle key gene WOX6 and WOX11, and carries out functional verification.The present invention is rice
The improvement of plant type, the cultivation of super rice kind provide important theoretical foundation and have the genetic resources of application value.
Description of the drawings
Fig. 1 is WOX6 and WOX11 expression analysis results.
Fig. 2 is transfer-gen plant phenotypic analysis result.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Average.
11 are spent in rice varieties：Bibliography：Li Meifang, Zhou Kaida rice biologicals technology Breeding [M] Chinese agricultures section
Skill publishing house, 2001.；The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.
CRISPR carriers pYLCRISPR/Cas9Pubi-H：Bibliography：Ma,X.et al..A robust CRISPR/
Cas9system for convenient,high-efficiency multiplex genome editing in monocot
and dicot plants..Mol Plant.2015Aug；8(8):1274-84., can be in Addgene websites (http://
Www.addgene.org) check and buy.
The ingredient of NB culture mediums is shown in Table 1.
Table 1NB medium components
| KNO3 | 2830mg/L |
| (NH4)2SO4 | 463mg/L |
| KH2PO4 | 400mg/L |
| MgSO4·7H2O | 185mg/L |
| CaCl2·2H2O | 166mg/L |
| FeSO4·7H2O | 27.8mg/L |
| Na2-EDTA | 37.5mg/L |
| MnSO4·4H2O | 10mg/L |
| H3BO3 | 3mg/L |
| ZnSO4·7H2O | 2mg/L |
| Na2MoO4·2H2O | 0.25mg/L |
| CuSO4·5H2O | 0.025mg/L |
| CoCl2·6H2O | 0.025mg/L |
| KI | 0.75mg/L |
| Thiamine hydrochloride (VB1) | 10mg/L |
| Puridoxine hydrochloride (VB6) | 1mg/L |
| Niacin | 1mg/L |
| Inositol | 100mg/L |
| Caseinhydrolysate | 300mg/L |
| Glutamine | 500mg/L |
| Glycine | 2mg/L |
| Proline | 1000mg/L |
| 2,4-D | 2mg/L |
| Phytagel | 2.5g/L |
YEP solid mediums：Yeast extract 10g, soy peptone 10g, NaCl 5g, agar 15g, moisturizing to 1L,
120 DEG C sterilize 20 minutes.
YEP fluid nutrient mediums：Yeast extract 10g, soy peptone 10g, NaCl 5g, moisturizing to 1L, 120 DEG C of sterilizings
20 minutes.
EHA105 Agrobacteriums：Takara companies.
The discovery of embodiment 1, rice tillering angle key protein and its encoding gene
Extraction carries out RNA-Seq surveys by the rice seedling of stimulus of gravity and the total serum IgE of untreated rice seedling
Sequence.By a large amount of sequence analyses, cluster analysis, KEGG pathway and Gene Ontology (GO) analyses, two rice are obtained
Tillering angle key gene WOX6 genes and WOX11 genes).
WOX6 genes tool translates into two different albumen there are two different transcripts.
The genome sequence of WOX6 genes is as shown in the sequence 11 of sequence table.
1 corresponding unnamed gene of transcript is WOX6-1, and open reading frame is as shown in the sequence 1 of sequence table.WOX6-1
Protein (being named as WOX6-1 albumen) shown in the sequence 2 of polynucleotide, is made of 356 amino acid residues.
2 corresponding unnamed gene of transcript is WOX6-2, and open reading frame is as shown in the sequence 3 of sequence table.WOX6-2
Protein (being named as WOX6-2 albumen) shown in the sequence 4 of polynucleotide, is made of 328 amino acid residues.
The open reading frame of WOX11 genes is as shown in the sequence 5 of sequence table, 6 institute of sequence of WOX11 gene coded sequence tables
The protein (being named as WOX11 albumen) shown, is made of 262 amino acid residues.
Embodiment 2, WOX6 genes and the research of WOX11 gene expression patterns
1st, in growing 10 days 11 Rooted Cuttings being spent to handle 0,0.25,0.5,1,2,4,8 and 12h respectively with 1 μM of NAA,
It compares to spend 11 in the corresponding time point handled using DMSO.Basal part of stem 1.5cm is taken to extract RNA, and reverse transcription is cDNA, with
CDNA is template, and using qRT-PCR, the expression variation of analysis IAA20 (responsive genes of auxin), WOX6 and WOX11 use
The expression of the primer pair detection IAA2 of primer I AA20_F and primer I AA20_R compositions, using primer WOX6_F and primer
The expression (total expression of WOX6-1 and WOX6-2) of the primer pair detection WOX6 of WOX6_R compositions, using primer
The expression of the primer pair detection WOX11 of WOX11_F and primer WOX11_R compositions, using primer Ubi_F and primer Ubi_R
The expression of the primer pair detection reference gene Ubi of composition.
IAA20_F：5’-TGGCGGATATGTGAAGGTGAA-3’；
IAA20_R：5’-TATGAGCCGAGGATGGACAAG-3’；
WOX6_F：5’-TCCAATAGACTTGCGAGCCAT-3’；
WOX6_R：5’-GCATTAGGATTCCATAGTCGTT-3’；
WOX11_F：5’-CGGTGTTCATCAACGGAGTG-3’；
WOX11_R：5’-TCTGGAGAGAATGGAGGAGGAT-3’；
Ubi_F：5’-AACCAGCTGAGGCCCAAGA-3’；
Ubi_R：5’-ACGATTGATTTAACCAGTCCATGA-3’.
2nd, 11 seedling is spent in 12 days to turn over to turn 90 degrees and keep flat carry out stimulus of gravity to vertical growth in Nutrition Soil, are pierced in gravity
Sharp 0h and 6h, the near end for the rice basal part of stem 1.5cm that fetches water respectively and remote ground terminal material, simultaneously reverse transcription is extraction total serum IgE
CDNA, the expression for being analyzed IAA20 (responsive genes of auxin), WOX6 and WOX11 using qRT-PCR using cDNA as template are become
Change, using the expression of the primer I AA20_F and primer I AA20_R primer pair detection IAA20 formed, using primer WOX6_F
With the expression (total expression of WOX6-1 and WOX6-2) of the primer pair detection WOX6 of primer WOX6_R compositions, using drawing
The expression of the primer pair detection WOX11 of object WOX11_F and primer WOX11_R compositions, using primer Ubi_F and primer Ubi_
The expression of the primer pair detection reference gene Ubi of R compositions.
The results are shown in Figure 1.Figure 1A is WOX6 and WOX11 with the expression situation of change of NAA processing times, Figure 1B WOX6
With WOX11 stimulus of gravity the rice basal part of stem expression situation of change in half material up and down.The result shows that WOX6 and WOX11
Apparent and similar expression pattern is shown for stimulus of gravity.WOX6 and WOX11 can by NAA induced expressions, WOX6 with
The asymmetric expression pattern similar to IAA20 is presented in basal part of stem near ends and remote ground terminal of the WOX11 when stimulus of gravity 6 is small.
Embodiment 3, transgenic experiments
First, the structure of mutational vector
1st, mutational vector pYLCRISPR/Cas9-WOX6-gRNA1＆gRNA2：In CRISPR carriers pYLCRISPR/
Cas9PubiDouble chain DNA molecule in the BsaI restriction enzyme sites of-H shown in the sequence 7 of insetion sequence table, obtains mutational vector
pYLCRISPR/Cas9-WOX6-gRNA1&gRNA2。
2nd, mutational vector pYLCRISPR/Cas9-WOX11-gRNA1＆gRNA2：In CRISPR carriers pYLCRISPR/
Cas9PubiDouble chain DNA molecule in the BsaI restriction enzyme sites of-H shown in the sequence 8 of insetion sequence table, obtains mutational vector
pYLCRISPR/Cas9-WOX11-gRNA1&gRNA2。
3rd, mutational vector pYLCRISPR/Cas9-WOX6＆11-gRNA1：In CRISPR carriers pYLCRISPR/Cas9Pubi-
Double chain DNA molecule in the BsaI restriction enzyme sites of H shown in the sequence 9 of insetion sequence table, obtains mutational vector pYLCRISPR/
Cas9-WOX6&11-gRNA1。
4th, mutational vector pYLCRISPR/Cas9-WOX6＆11-gRNA2：In CRISPR carriers pYLCRISPR/Cas9Pubi-
Double chain DNA molecule in the BsaI restriction enzyme sites of H shown in the sequence 10 of insetion sequence table, obtains mutational vector pYLCRISPR/
Cas9-WOX6&11-gRNA2。
2nd, the structure of CR-wox6 transfer-gen plants
1st, middle spend is sterilized 45 minutes after 11 seed decladding with 2.5% (mass percent) liquor natrii hypochloritis, then
It is rinsed 6 times with aqua sterilisa, point is sowed at 30 DEG C of evoked callus in dark on NB culture mediums.After about 20 days, choose from maturation
The callus that embryonic shield piece director goes out subculture on NB culture mediums, once, when each subculture, picks out cause to subculture every two weeks later
Close, surface is smooth and the embryo callus of pale yellow.
2nd, the mutational vector pYLCRISPR/Cas9-WOX6-gRNA1＆gRNA2 that step 1 is built is transferred to EHA105 agriculture bars
In bacterium, recombinational agrobacterium is obtained.
3rd, the recombinational agrobacterium for obtaining step 2 is inoculated in the YEP containing 50mg/L kanamycins and 25mg/L rifampins
On solid medium tablet, when 28 DEG C of cultures 48 are small, picking monoclonal is inoculated in kanamycins containing 50mg/L and 25mg/L profit good fortune
In flat YEP fluid nutrient mediums, 28 DEG C, 150rpm is cultivated to OD600nmThalline were collected by centrifugation by=0.8-1.0,3000rpm, with dip dyeing
The abundant suspension thalline of liquid, obtains bacteria suspension.
4th, the callus that is obtained in step 1 is carried out using the bacteria suspension that step 3 obtains Agrobacterium-mediated Transformation (method referring to
Document：Hiei,Y.,Ohta,S.,Komari,T.,and Kumashiro,T.(1994).Efficient
transformation of rice(Oryza SativaL)mediated by Agrobacterium and sequence-
analysis of the boundaries of the T-DNA.Plant J.6,271-282).Callus after conversion exists
After 25 DEG C of dark place culture 3 days, the screening kanamycin-resistant callus tissue and transfer-gen plant on the Selective agar medium containing 50mg/L hygromycin.
By hygromycin resistance plant hardening in the cool, after be transplanted in paddy field, the transfer-gen plant of acquisition is T0Generation.
5th, T is harvested0It for the seed of transgenic seedling, then plants in field, obtains T1For transfer-gen plant.Using primer GTR4_
Cas9JD_F and primer GTR4_Cas9JD_R are to T1It is expanded for the genomic DNA of transfer-gen plant, screening positive T1In generation, turns
Gene plant (positive T1The amplified production of 400bp is obtained for transfer-gen plant).
GTR4_Cas9JD_F:5’-GCAAGCATGGTTTAATTGTTGTTGCC-3’；
GTR4_Cas9JD_R:5’-CTTGCGGATGCGGACGGTCTC-3’。
Positive T1It is selfed to obtain T for transfer-gen plant2For transfer-gen plant.
3rd, the structure of CR-wox11 transfer-gen plants
1st, middle spend is sterilized 45 minutes after 11 seed decladding with 2.5% (mass percent) liquor natrii hypochloritis, then
It is rinsed 6 times with aqua sterilisa, point is sowed at 30 DEG C of evoked callus in dark on NB culture mediums.After about 20 days, choose from maturation
The callus that embryonic shield piece director goes out subculture on NB culture mediums, once, when each subculture, picks out cause to subculture every two weeks later
Close, surface is smooth and the embryo callus of pale yellow.
2nd, the mutational vector pYLCRISPR/Cas9-WOX11-gRNA1＆gRNA2 that step 1 is built is transferred to EHA105 agricultures
In bacillus, recombinational agrobacterium is obtained.
3rd, the recombinational agrobacterium for obtaining step 2 is inoculated in the YEP containing 50mg/L kanamycins and 25mg/L rifampins
On solid medium tablet, when 28 DEG C of cultures 48 are small, picking monoclonal is inoculated in kanamycins containing 50mg/L and 25mg/L profit good fortune
In flat YEP fluid nutrient mediums, 28 DEG C, 150rpm is cultivated to OD600nmThalline were collected by centrifugation by=0.8-1.0,3000rpm, with dip dyeing
The abundant suspension thalline of liquid, obtains bacteria suspension.
4th, the callus that is obtained in step 1 is carried out using the bacteria suspension that step 3 obtains Agrobacterium-mediated Transformation (method referring to
Document：Hiei,Y.,Ohta,S.,Komari,T.,and Kumashiro,T.(1994).Efficient
transformation of rice(Oryza SativaL)mediated by Agrobacterium and sequence-
analysis of the boundaries of the T-DNA.Plant J.6,271-282).Callus after conversion exists
After 25 DEG C of dark place culture 3 days, the screening kanamycin-resistant callus tissue and transfer-gen plant on the Selective agar medium containing 50mg/L hygromycin.
By hygromycin resistance plant hardening in the cool, after be transplanted in paddy field, the transfer-gen plant of acquisition is T0Generation.
5th, T is harvested0It for the seed of transgenic seedling, then plants in field, obtains T1For transfer-gen plant.Using primer GTR5_
Cas9JD_F and primer GTR5_Cas9JD_R are to T1It is expanded for the genomic DNA of transfer-gen plant, screening positive T1In generation, turns
Gene plant (positive T1The amplified production of 400bp is obtained for transfer-gen plant).
GTR5_Cas9JD_F:5’-AAGCCAATTAAGCGAATTAAGCAC-3’；
GTR5_Cas9JD_R:5’-GAAGCAGTCGGAGGAGATCCC-3’。
Positive T1It is selfed to obtain T for transfer-gen plant2For transfer-gen plant.
4th, the structure of CR-wox6wox11 transfer-gen plants
1st, middle spend is sterilized 45 minutes after 11 seed decladding with 2.5% (mass percent) liquor natrii hypochloritis, then
It is rinsed 6 times with aqua sterilisa, point is sowed at 30 DEG C of evoked callus in dark on NB culture mediums.After about 20 days, choose from maturation
The callus that embryonic shield piece director goes out subculture on NB culture mediums, once, when each subculture, picks out cause to subculture every two weeks later
Close, surface is smooth and the embryo callus of pale yellow.
2nd, the mutational vector pYLCRISPR/Cas9-WOX6＆11-gRNA1 that step 1 is built is transferred to EHA105 Agrobacteriums
In, obtain recombinational agrobacterium.
3rd, the mutational vector pYLCRISPR/Cas9-WOX6＆11-gRNA2 that step 1 is built is transferred to EHA105 Agrobacteriums
In, obtain recombinational agrobacterium.
4th, the recombinational agrobacterium for obtaining step 2 is inoculated in the YEP containing 50mg/L kanamycins and 25mg/L rifampins
On solid medium tablet, when 28 DEG C of cultures 48 are small, picking monoclonal is inoculated in kanamycins containing 50mg/L and 25mg/L profit good fortune
In flat YEP fluid nutrient mediums, 28 DEG C, 150rpm is cultivated to OD600nmThalline were collected by centrifugation by=0.8-1.0,3000rpm, with dip dyeing
The abundant suspension thalline of liquid, obtains bacteria suspension.
5th, the recombinational agrobacterium for obtaining step 3 is inoculated in the YEP containing 50mg/L kanamycins and 25mg/L rifampins
On solid medium tablet, when 28 DEG C of cultures 48 are small, picking monoclonal is inoculated in kanamycins containing 50mg/L and 25mg/L profit good fortune
In flat YEP fluid nutrient mediums, 28 DEG C, 150rpm is cultivated to OD600nmThalline were collected by centrifugation by=0.8-1.0,3000rpm, with dip dyeing
The abundant suspension thalline of liquid, obtains bacteria suspension.
6th, the bacteria suspension that the bacteria suspension and step 5 obtained using step 4 is obtained carries out the callus obtained in step 1
(method is referring to document for Agrobacterium-mediated Transformation：Hiei,Y.,Ohta,S.,Komari,T.,and Kumashiro,T.(1994)
.Efficient transformation of rice(Oryza SativaL)mediated by Agrobacterium and
sequence-analysis of the boundaries of the T-DNA.Plant J.6,271-282).After conversion
Callus at the dark after 25 DEG C of cultures 3 days, screened on the Selective agar medium containing 50mg/L hygromycin kanamycin-resistant callus tissue and
Transfer-gen plant.By hygromycin resistance plant hardening in the cool, after be transplanted in paddy field, the transfer-gen plant of acquisition is T0
Generation.
5th, T is harvested0It for the seed of transgenic seedling, then plants in field, obtains T1For transfer-gen plant.Using primer GTR4_
The primer pair of Cas9JD_F and primer GTR4_Cas9JD_R compositions is to T1It is expanded for the genomic DNA of transfer-gen plant, together
Shi Caiyong primers GTR5_Cas9JD_F and primer GTR5_Cas9JD_R are to T1Expanded for the genomic DNA of transfer-gen plant
Increase, screening positive T1For transfer-gen plant (positive T1Primer GTR4_Cas9JD_F and primer GTR4_ is used for transfer-gen plant
The amplified production size that Cas9JD_R is expanded is 400bp, using primer GTR5_Cas9JD_F and primer GTR5_
The amplified production size that Cas9JD_R is expanded is 400bp).
Positive T1It is selfed to obtain T for transfer-gen plant2For transfer-gen plant.
5th, Phenotypic examination
Plant (each 10 plants of strain to be measured) to be measured：11 (WT), T are spent in rice varieties2For CR-wox6 transfer-gen plants, T2
For CR-wox11 transfer-gen plants and T2For CR-wox6wox11 transfer-gen plants.
1st, tillering angle, maturity period observation phenotype are measured in the plant tillering phase to be measured.
2nd, the plant seed to be measured of decladding with 2.5% (w/v) liquor natrii hypochloritis is sterilized 45 minutes, is rinsed with aqua sterilisa
6 times, vernalization in 37 DEG C of incubators is put into, consistent seed point of germinateing then is selected and is multicast to 0.4% (mass percentage) agar
On culture medium, plate is vertically placed in 28 DEG C of incubators and cultivates.Growth of seedling is rotated by 90 ° plate after 4 days and carries out gravity thorn
Swash, the angle that plants stems are bent upwards is measured when 12 is small.
The results are shown in Figure 2.In Fig. 2, Fig. 2A is in the mutational site of WOX6, CR-wox11 plant in CR-wox6 plant
The mutational site of WOX11 has mutational site and the transfer-gen plant of transfer-gen plant CR-wox6 in CR-wox6wox11 simultaneously
The mutational site of CR-wox11, Fig. 2 B are phenotype of the ripe plant in field, and Fig. 2 C are the tillering angle of the plant in Fig. 2 B,
Fig. 2 D be CR-wox6, CR-wox11 and CR-wox6wox11 rice seedling gravity response situations.The result shows that wox6 and wox11
Each single prominent tillering angle no notable difference with wild type compared with, wox6wox11 is double prominent to there is tillering angle to increase and again
The phenotype of power habituation.
<110>Inst. of Genetics and Development Biology, CAS
<120>Plant tillering angle GAP-associated protein GAP and its encoding gene and application
<160> 11
<210> 1
<211> 1071
<212> DNA
<213>Rice（Oryza sativa）
<400> 1
atggagggga gcagtaatag cccggatagg cagtcgtcgg gcggcagccc gccggaggag 60
cgcggcggcg ggggaagcgg tggaggagga gggcggagcg ccgccggcga gccggtgcgg 120
tcgcggtgga cgcccaagcc ggagcagata ctgatcctgg agtccatctt caacagcggc 180
atggtcaacc cgcccaagga cgagaccgtc cgcatccgca agctgctcga gaggttcggc 240
gccgtcggcg acgccaacgt cttctactgg ttccagaacc gccgctcgcg ctcgcgccgc 300
cgccagcgcc agatgcaggc ggccgccgcc gccgccgcag cggcggcctc ttcctcttcc 360
ccatccgcca acacctctcc cgcagccgcg agcgccgcca ccgtgcaggt gggcctcccg 420
cccggcgccg tcgtccacac catggccatg ggtgggagcg cgtgccagta cgagcagcag 480
gcgagctcgt cgtcgtcgtc cggcagcacg ggaggttcgt cgctggggct gttcgcgcac 540
ggcgcggggg cctccggcgc cggcgggtac ctgcaggcgt cgtgcggcgc gtccgcgtcg 600
gcgtcgtcgg cgctggcgcc cgggctgatg ggggatgtgg tggacagcgg gggaagcgac 660
gatctcttcg ccatctcgag gcagatgggg tttgtgggga gccctcgctg ctcgccggcc 720
agctcgccgg cgacgccgag ctccgcggcc accgccgcgc agcagcagtt ctactcatgc 780
caattacctg cagcgacgat cacggtgttc atcaacggag tcccaatgga gatgccgagg 840
ggtccaatag acttgcgagc catgttcggc caggatgtga tgctcgtcca ctctactggg 900
gccctcctcc cagtcaacga ctatggaatc ctaatgcaga gcctccaaat cggagagagc 960
tactttctgg taaccatcca tctccaagct cttactagct ggtctcatcg ctctctctct 1020
actccgatca gtcaatgcag tttcgtcttc atgaaagcac cacattttta g 1071
<210> 2
<211> 356
<212> PRT
<213>Rice（Oryza sativa）
<400> 2
Met Glu Gly Ser Ser Asn Ser Pro Asp Arg Gln Ser Ser Gly Gly Ser
1 5 10 15
Pro Pro Glu Glu Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Arg
20 25 30
Ser Ala Ala Gly Glu Pro Val Arg Ser Arg Trp Thr Pro Lys Pro Glu
35 40 45
Gln Ile Leu Ile Leu Glu Ser Ile Phe Asn Ser Gly Met Val Asn Pro
50 55 60
Pro Lys Asp Glu Thr Val Arg Ile Arg Lys Leu Leu Glu Arg Phe Gly
65 70 75 80
Ala Val Gly Asp Ala Asn Val Phe Tyr Trp Phe Gln Asn Arg Arg Ser
85 90 95
Arg Ser Arg Arg Arg Gln Arg Gln Met Gln Ala Ala Ala Ala Ala Ala
100 105 110
Ala Ala Ala Ala Ser Ser Ser Ser Pro Ser Ala Asn Thr Ser Pro Ala
115 120 125
Ala Ala Ser Ala Ala Thr Val Gln Val Gly Leu Pro Pro Gly Ala Val
130 135 140
Val His Thr Met Ala Met Gly Gly Ser Ala Cys Gln Tyr Glu Gln Gln
145 150 155 160
Ala Ser Ser Ser Ser Ser Ser Gly Ser Thr Gly Gly Ser Ser Leu Gly
165 170 175
Leu Phe Ala His Gly Ala Gly Ala Ser Gly Ala Gly Gly Tyr Leu Gln
180 185 190
Ala Ser Cys Gly Ala Ser Ala Ser Ala Ser Ser Ala Leu Ala Pro Gly
195 200 205
Leu Met Gly Asp Val Val Asp Ser Gly Gly Ser Asp Asp Leu Phe Ala
210 215 220
Ile Ser Arg Gln Met Gly Phe Val Gly Ser Pro Arg Cys Ser Pro Ala
225 230 235 240
Ser Ser Pro Ala Thr Pro Ser Ser Ala Ala Thr Ala Ala Gln Gln Gln
245 250 255
Phe Tyr Ser Cys Gln Leu Pro Ala Ala Thr Ile Thr Val Phe Ile Asn
260 265 270
Gly Val Pro Met Glu Met Pro Arg Gly Pro Ile Asp Leu Arg Ala Met
275 280 285
Phe Gly Gln Asp Val Met Leu Val His Ser Thr Gly Ala Leu Leu Pro
290 295 300
Val Asn Asp Tyr Gly Ile Leu Met Gln Ser Leu Gln Ile Gly Glu Ser
305 310 315 320
Tyr Phe Leu Val Thr Ile His Leu Gln Ala Leu Thr Ser Trp Ser His
325 330 335
Arg Ser Leu Ser Thr Pro Ile Ser Gln Cys Ser Phe Val Phe Met Lys
340 345 350
Ala Pro His Phe
355
<210> 3
<211> 987
<212> DNA
<213>Rice（Oryza sativa）
<400> 3
atggagggga gcagtaatag cccggatagg cagtcgtcgg gcggcagccc gccggaggag 60
cgcggcggcg ggggaagcgg tggaggagga gggcggagcg ccgccggcga gccggtgcgg 120
tcgcggtgga cgcccaagcc ggagcagata ctgatcctgg agtccatctt caacagcggc 180
atggtcaacc cgcccaagga cgagaccgtc cgcatccgca agctgctcga gaggttcggc 240
gccgtcggcg acgccaacgt cttctactgg ttccagaacc gccgctcgcg ctcgcgccgc 300
cgccagcgcc agatgcaggc ggccgccgcc gccgccgcag cggcggcctc ttcctcttcc 360
ccatccgcca acacctctcc cgcagccgcg agcgccgcca ccgtgcaggt gggcctcccg 420
cccggcgccg tcgtccacac catggccatg ggtgggagcg cgtgccagta cgagcagcag 480
gcgagctcgt cgtcgtcgtc cggcagcacg ggaggttcgt cgctggggct gttcgcgcac 540
ggcgcggggg cctccggcgc cggcgggtac ctgcaggcgt cgtgcggcgc gtccgcgtcg 600
gcgtcgtcgg cgctggcgcc cgggctgatg ggggatgtgg tggacagcgg gggaagcgac 660
gatctcttcg ccatctcgag gcagatgggg tttgtgggga gccctcgctg ctcgccggcc 720
agctcgccgg cgacgccgag ctccgcggcc accgccgcgc agcagcagtt ctactcatgc 780
caattacctg cagcgacgat cacggtgttc atcaacggag tcccaatgga gatgccgagg 840
ggtccaatag acttgcgagc catgttcggc caggatgtga tgctcgtcca ctctactggg 900
gccctcctcc cagtcaacga ctatggaatc ctaatgcaga gcctccaaat cggagagagc 960
tactttctgg tcgctaggcc accttaa 987
<210> 4
<211> 328
<212> PRT
<213>Rice（Oryza sativa）
<400> 4
Met Glu Gly Ser Ser Asn Ser Pro Asp Arg Gln Ser Ser Gly Gly Ser
1 5 10 15
Pro Pro Glu Glu Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Arg
20 25 30
Ser Ala Ala Gly Glu Pro Val Arg Ser Arg Trp Thr Pro Lys Pro Glu
35 40 45
Gln Ile Leu Ile Leu Glu Ser Ile Phe Asn Ser Gly Met Val Asn Pro
50 55 60
Pro Lys Asp Glu Thr Val Arg Ile Arg Lys Leu Leu Glu Arg Phe Gly
65 70 75 80
Ala Val Gly Asp Ala Asn Val Phe Tyr Trp Phe Gln Asn Arg Arg Ser
85 90 95
Arg Ser Arg Arg Arg Gln Arg Gln Met Gln Ala Ala Ala Ala Ala Ala
100 105 110
Ala Ala Ala Ala Ser Ser Ser Ser Pro Ser Ala Asn Thr Ser Pro Ala
115 120 125
Ala Ala Ser Ala Ala Thr Val Gln Val Gly Leu Pro Pro Gly Ala Val
130 135 140
Val His Thr Met Ala Met Gly Gly Ser Ala Cys Gln Tyr Glu Gln Gln
145 150 155 160
Ala Ser Ser Ser Ser Ser Ser Gly Ser Thr Gly Gly Ser Ser Leu Gly
165 170 175
Leu Phe Ala His Gly Ala Gly Ala Ser Gly Ala Gly Gly Tyr Leu Gln
180 185 190
Ala Ser Cys Gly Ala Ser Ala Ser Ala Ser Ser Ala Leu Ala Pro Gly
195 200 205
Leu Met Gly Asp Val Val Asp Ser Gly Gly Ser Asp Asp Leu Phe Ala
210 215 220
Ile Ser Arg Gln Met Gly Phe Val Gly Ser Pro Arg Cys Ser Pro Ala
225 230 235 240
Ser Ser Pro Ala Thr Pro Ser Ser Ala Ala Thr Ala Ala Gln Gln Gln
245 250 255
Phe Tyr Ser Cys Gln Leu Pro Ala Ala Thr Ile Thr Val Phe Ile Asn
260 265 270
Gly Val Pro Met Glu Met Pro Arg Gly Pro Ile Asp Leu Arg Ala Met
275 280 285
Phe Gly Gln Asp Val Met Leu Val His Ser Thr Gly Ala Leu Leu Pro
290 295 300
Val Asn Asp Tyr Gly Ile Leu Met Gln Ser Leu Gln Ile Gly Glu Ser
305 310 315 320
Tyr Phe Leu Val Ala Arg Pro Pro
325
<210> 5
<211> 789
<212> DNA
<213>Rice（Oryza sativa）
<400> 5
atggacggcg gccacagccc ggacaggcat gcggcggcgg cggcggggga gccggtgagg 60
tcgcggtgga cgccgaagcc ggagcagata ctcatcctgg agtccatctt caacagcggc 120
atggtgaacc cgcccaagga cgagaccgtc cgcatccgca agctgctcga gcgcttcggc 180
gccgtcggcg acgccaacgt cttctactgg ttccagaacc gccgctcgcg ctcccgccgc 240
cgccagcgcc agctgcaggc gcaggcgcag gcggccgcgg ccgccgcctc gtcgggatct 300
cctccgactg cttcgtccgg tggcctcgcg cctggccacg ccggctcgcc ggcttcgtcg 360
ctcgggatgt tcgcgcacgg cgccgccggg tacagctcct cgtcgtcctc atcgtggccg 420
tcctcgccgc cgtcggtggg gatgatgatg ggggacgtgg actacggggg cggcggcgac 480
gacctgttcg ccatctcgag gcagatgggg tacatggacg gcggcggcgg ctcgtcgtcg 540
tcggcggccg ccggtcagca tcagcagcag cagctctact actcgtgtca acctgcgacg 600
atgacggtgt tcatcaacgg agtggcgacg gaggtgccaa ggggaccgat cgatctgaga 660
tcaatgtttg ggcaggacgt gatgctggtg cattcaacgg gtgctcttct tccagccaac 720
gagtacggca tcctcctcca ttctctccag atgggcgaga gctacttcct ggtcacgagg 780
tcgtcttga 789
<210> 6
<211> 262
<212> PRT
<213>Rice（Oryza sativa）
<400> 6
Met Asp Gly Gly His Ser Pro Asp Arg His Ala Ala Ala Ala Ala Gly
1 5 10 15
Glu Pro Val Arg Ser Arg Trp Thr Pro Lys Pro Glu Gln Ile Leu Ile
20 25 30
Leu Glu Ser Ile Phe Asn Ser Gly Met Val Asn Pro Pro Lys Asp Glu
35 40 45
Thr Val Arg Ile Arg Lys Leu Leu Glu Arg Phe Gly Ala Val Gly Asp
50 55 60
Ala Asn Val Phe Tyr Trp Phe Gln Asn Arg Arg Ser Arg Ser Arg Arg
65 70 75 80
Arg Gln Arg Gln Leu Gln Ala Gln Ala Gln Ala Ala Ala Ala Ala Ala
85 90 95
Ser Ser Gly Ser Pro Pro Thr Ala Ser Ser Gly Gly Leu Ala Pro Gly
100 105 110
His Ala Gly Ser Pro Ala Ser Ser Leu Gly Met Phe Ala His Gly Ala
115 120 125
Ala Gly Tyr Ser Ser Ser Ser Ser Ser Ser Trp Pro Ser Ser Pro Pro
130 135 140
Ser Val Gly Met Met Met Gly Asp Val Asp Tyr Gly Gly Gly Gly Asp
145 150 155 160
Asp Leu Phe Ala Ile Ser Arg Gln Met Gly Tyr Met Asp Gly Gly Gly
165 170 175
Gly Ser Ser Ser Ser Ala Ala Ala Gly Gln His Gln Gln Gln Gln Leu
180 185 190
Tyr Tyr Ser Cys Gln Pro Ala Thr Met Thr Val Phe Ile Asn Gly Val
195 200 205
Ala Thr Glu Val Pro Arg Gly Pro Ile Asp Leu Arg Ser Met Phe Gly
210 215 220
Gln Asp Val Met Leu Val His Ser Thr Gly Ala Leu Leu Pro Ala Asn
225 230 235 240
Glu Tyr Gly Ile Leu Leu His Ser Leu Gln Met Gly Glu Ser Tyr Phe
245 250 255
Leu Val Thr Arg Ser Ser
260
<210> 7
<211> 1162
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
ttcagaggtc tctctcgcac tggaatcggc agcaaaggaa ggaatcttta aacatacgaa 60
cagatcactt aaagttcttc tgaagcaact taaagttatc aggcatgcat ggatcttgga 120
ggaatcagat gtgcagtcag ggaccatagc acaagacagg cgtcttctac tggtgctacc 180
agcaaatgct ggaagccggg aacactgggt acgttggaaa ccacgtgtga tgtgaaggag 240
taagataaac tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt 300
attgcagtat gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc 360
acctcggcta tccacataga tcaaagctgg tttaaaagag ttgtgcagat gatccgtggc 420
atagcccgga taggcagtcg tgttttagag ctagaaatag caagttaaaa taaggctagt 480
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaagag cttggagtgg 540
atggaccctg acactggaat cggcagcaaa ggattttttc ctgtagtttt cccacaacca 600
ttttttacca tccgaatgat aggataggaa aaatatccaa gtgaacagta ttcctataaa 660
attcccgtaa aaagcctgca atccgaatga gccctgaagt ctgaactagc cggtcacctg 720
tacaggctat cgagatgcca tacaagagac ggtagtagga actaggaaga cgatggttga 780
ttcgtcaggc gaaatcgtcg tcctgcagtc gcatctatgg gcctggacgg aataggggaa 840
aaagttggcc ggataggagg gaaaggccca ggtgcttacg tgcgaggtag gcctgggctc 900
tcagcacttc gattcgttgg caccggggta ggatgcaata gagagcaacg tttagtacca 960
cctcgcttag ctagagcaaa ctggactgcc ttatatgcgc gggtgctggc ttggctgccg 1020
gagcagtaat agcccggatg ttttagagct agaaatagca agttaaaata aggctagtcc 1080
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 1140
ggacccggtc gagacccacg ct 1162
<210> 8
<211> 1163
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
ttcagaggtc tctctcgcac tggaatcggc agcaaaggaa ggaatcttta aacatacgaa 60
cagatcactt aaagttcttc tgaagcaact taaagttatc aggcatgcat ggatcttgga 120
ggaatcagat gtgcagtcag ggaccatagc acaagacagg cgtcttctac tggtgctacc 180
agcaaatgct ggaagccggg aacactgggt acgttggaaa ccacgtgtga tgtgaaggag 240
taagataaac tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt 300
attgcagtat gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc 360
acctcggcta tccacataga tcaaagctgg tttaaaagag ttgtgcagat gatccgtggc 420
acagcccgga caggcatgcg ggttttagag ctagaaatag caagttaaaa taaggctagt 480
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaagag cttggagtgg 540
atggaccctg acactggaat cggcagcaaa ggattttttc ctgtagtttt cccacaacca 600
ttttttacca tccgaatgat aggataggaa aaatatccaa gtgaacagta ttcctataaa 660
attcccgtaa aaagcctgca atccgaatga gccctgaagt ctgaactagc cggtcacctg 720
tacaggctat cgagatgcca tacaagagac ggtagtagga actaggaaga cgatggttga 780
ttcgtcaggc gaaatcgtcg tcctgcagtc gcatctatgg gcctggacgg aataggggaa 840
aaagttggcc ggataggagg gaaaggccca ggtgcttacg tgcgaggtag gcctgggctc 900
tcagcacttc gattcgttgg caccggggta ggatgcaata gagagcaacg tttagtacca 960
cctcgcttag ctagagcaaa ctggactgcc ttatatgcgc gggtgctggc ttggctgccg 1020
ccacagcccg gacaggcatg gttttagagc tagaaatagc aagttaaaat aaggctagtc 1080
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcaagagc ttggagtgga 1140
tggacccggt cgagacccac gct 1163
<210> 9
<211> 1162
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
ttcagaggtc tctctcgcac tggaatcggc agcaaaggaa ggaatcttta aacatacgaa 60
cagatcactt aaagttcttc tgaagcaact taaagttatc aggcatgcat ggatcttgga 120
ggaatcagat gtgcagtcag ggaccatagc acaagacagg cgtcttctac tggtgctacc 180
agcaaatgct ggaagccggg aacactgggt acgttggaaa ccacgtgtga tgtgaaggag 240
taagataaac tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt 300
attgcagtat gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc 360
acctcggcta tccacataga tcaaagctgg tttaaaagag ttgtgcagat gatccgtggc 420
acagcccgga caggcatgcg ggttttagag ctagaaatag caagttaaaa taaggctagt 480
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaagag cttggagtgg 540
atggaccctg acactggaat cggcagcaaa ggattttttc ctgtagtttt cccacaacca 600
ttttttacca tccgaatgat aggataggaa aaatatccaa gtgaacagta ttcctataaa 660
attcccgtaa aaagcctgca atccgaatga gccctgaagt ctgaactagc cggtcacctg 720
tacaggctat cgagatgcca tacaagagac ggtagtagga actaggaaga cgatggttga 780
ttcgtcaggc gaaatcgtcg tcctgcagtc gcatctatgg gcctggacgg aataggggaa 840
aaagttggcc ggataggagg gaaaggccca ggtgcttacg tgcgaggtag gcctgggctc 900
tcagcacttc gattcgttgg caccggggta ggatgcaata gagagcaacg tttagtacca 960
cctcgcttag ctagagcaaa ctggactgcc ttatatgcgc gggtgctggc ttggctgccg 1020
gagcagtaat agcccggatg ttttagagct agaaatagca agttaaaata aggctagtcc 1080
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat 1140
ggacccggtc gagacccacg ct 1162
<210> 10
<211> 1163
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
ttcagaggtc tctctcgcac tggaatcggc agcaaaggaa ggaatcttta aacatacgaa 60
cagatcactt aaagttcttc tgaagcaact taaagttatc aggcatgcat ggatcttgga 120
ggaatcagat gtgcagtcag ggaccatagc acaagacagg cgtcttctac tggtgctacc 180
agcaaatgct ggaagccggg aacactgggt acgttggaaa ccacgtgtga tgtgaaggag 240
taagataaac tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt 300
attgcagtat gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc 360
acctcggcta tccacataga tcaaagctgg tttaaaagag ttgtgcagat gatccgtggc 420
atagcccgga taggcagtcg tgttttagag ctagaaatag caagttaaaa taaggctagt 480
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaagag cttggagtgg 540
atggaccctg acactggaat cggcagcaaa ggattttttc ctgtagtttt cccacaacca 600
ttttttacca tccgaatgat aggataggaa aaatatccaa gtgaacagta ttcctataaa 660
attcccgtaa aaagcctgca atccgaatga gccctgaagt ctgaactagc cggtcacctg 720
tacaggctat cgagatgcca tacaagagac ggtagtagga actaggaaga cgatggttga 780
ttcgtcaggc gaaatcgtcg tcctgcagtc gcatctatgg gcctggacgg aataggggaa 840
aaagttggcc ggataggagg gaaaggccca ggtgcttacg tgcgaggtag gcctgggctc 900
tcagcacttc gattcgttgg caccggggta ggatgcaata gagagcaacg tttagtacca 960
cctcgcttag ctagagcaaa ctggactgcc ttatatgcgc gggtgctggc ttggctgccg 1020
ccacagcccg gacaggcatg gttttagagc tagaaatagc aagttaaaat aaggctagtc 1080
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcaagagc ttggagtgga 1140
tggacccggt cgagacccac gct 1163
<210> 11
<211> 3946
<212> DNA
<213>Rice（Oryza sativa）
<400> 11
gagtgtgcaa gcatggttta attgttgttg cctcctctca agctagctag ctatactact 60
ccgatcctct ctcgcttatt gaaacctagc tactgcttca catgtagcgc gtgctgcaac 120
tcgtgttgtg tcactcacac acacatccaa gaaccaagat tccgaggtaa ccaacgctcg 180
atcttttact taccctatct cccgacgatc gatcgatcga tcgatggagg ggagcagtaa 240
tagcccggat aggcagtcgt cgggcggcag cccgccggag gagcgcggcg gcgggggaag 300
cggtggagga ggagggcgga gcgccgccgg cgagccggtg cggtcgcggt ggacgcccaa 360
gccggagcag atactgatcc tggagtccat cttcaacagc ggcatggtca acccgcccaa 420
ggacgagacc gtccgcatcc gcaagctgct cgagaggttc ggcgccgtcg gcgacgccaa 480
cgtcttctac tggttccaga accgccgctc gcgctcgcgc cgccgccagc gccagatgca 540
ggcggccgcc gccgccgccg cagcggcggc ctcttcctct tccccatccg ccaacacctc 600
tcccgcagcc gcgagcgccg ccaccgtgca ggtgggcctc ccgcccggcg ccgtcgtcca 660
caccatggcc atgggtggga gcgcgtgcca gtacgagcag caggcgagct cgtcgtcgtc 720
gtccggcagc acgggaggtt cgtcgctggg gctgttcgcg cacggcgcgg gggcctccgg 780
cgccggcggg tacctgcagg cgtcgtgcgg cgcgtccgcg tcggcgtcgt cggcgctggc 840
gcccgggctg atgggggatg tggtggacag cgggggaagc gacgatctct tcgccatctc 900
gaggcagatg gggtttgtgg ggagccctcg ctgctcgccg gccagctcgc cggcgacgcc 960
gagctccgcg gccaccgccg cgcagcagca gttctactca tgccaattac ctgcaggtga 1020
gttgatcagt taccacatca atcttggttt tggccattgc taatttgcta tttcttccct 1080
tcaatatttg gttggttagc tactcagtac tcaccgatca atctcgtatt ctcatgcatg 1140
tgtttcccat gtctcgatcg cttaagggat taggttattt attaatttgt tcatcttttc 1200
atcgaaatgg ctctaatctt aacacactta ctcttgtgat tttacaagtt attgtgattt 1260
ctactactat actgcatata tcacctgatc gaagaaactt atgaaggtgt aaatatgctg 1320
atcatttcac cactaaaatt ttgtctgttt gtattgtagt gctgcagaat tcaaggagat 1380
ctagtttgga tcttttgcgt gtgcatactt tatttggcct tccatataat taattgtgtc 1440
agttcttgtt ctttagctct tcactatact atttcggtta caacttacac ttcctagcta 1500
gtgtaccggc acataagatc atactacata ataattcacc atgcatgaca ctttcctttt 1560
gaaagatcac aatgatatgt tcctacgttt tgcgaagcct tgctcttgtg tctagctatg 1620
ttcttaatta cttgtacggt gtattccaga ctgaattgta caattttcaa aattttaggg 1680
agccttgcct ggtgttaatg atatgcatac actagaaagt actccctcca tctcaaaata 1740
taagaatcta gtaatggatg gaacatatcc tacttcgaat ctggatagat agtctatcca 1800
aattcgtagt attagaatgt gtctcatcta gtattatgtt gctatattat aggacggagg 1860
aaatactcct ataattctag gatgtatcat acaattttaa aaattaacca atacccaaaa 1920
atgagaatta caaaataaac acattagtaa ttttccatag attctaagca tgcatgcacc 1980
cacccaactg tcttcagatt cttctcaccg tgtatatgtt ttttttgtca attatatcca 2040
tttgaagctc catgtagcac acaatagagt tagcacacat gtttatgcat gatccaatca 2100
tttgatgcct gtatgtcaaa tagtaaatta attttggcaa tgtaactttg gttacattat 2160
tatgatttac taaatgcatg catgcatgag caatccatcc tactttggta atttttcttg 2220
ctttcttact gacatgttat atgtacaatc tatcatcaat taatttacca actagctact 2280
taattctgag ctaaaaacaa gccacagtat aattcatgca tgtcgcatgc atgcaaatgt 2340
gtgtaatgcc catgcatgct attgcagatg gagcaacaat ataatttcag aatatcctct 2400
catgcatttg cagaactctt attggaatca aatagactag gtgcatatgc caatatgata 2460
aaggattaga ggtcccaaag tcccaactcc caactgggga gtaaacggta gtaccgagta 2520
ccgaccaact aattaacgat attgataatc tcatcattgg ataatttgtt tggctagctt 2580
ctgcatctgt ttgttgctaa ttttgtcgtc aatgagctgt tgatatttct tgtgagtgga 2640
ttatatatat gctggctaga tgctgattgg ttgctggttt gattgcctgc taattccagc 2700
gacgatcacg gtgttcatca acggagtccc aatggagatg ccgaggggtc caatagactt 2760
gcgagccatg ttcggccagg atgtgatgct cgtccactct actggggccc tcctcccagt 2820
caacgactat ggaatcctaa tgcagagcct ccaaatcgga gagagctact ttctggtaac 2880
catccatctc caagctctta ctagctggtc tcatcgctct ctctctactc cgatcagtca 2940
atgcagtttc gtcttcatga aagcaccaca tttttaggca tgcattcatg cttatatatg 3000
tgtatcatgt ttgaatgtgc aaaactaatt actattaatt taattaattg catgcatgtt 3060
tgtgtgaatt gtgaaatgca tagctagcta ttgttgtgat cttcttaatt tcctgcttaa 3120
tgaggagtac atttatatgt gtgtatgtgt gtttctgatc atcttcaatg tgttgtgttt 3180
cctgatcgac aggtcgctag gccaccttaa aaacgagttc tcgatctatc cttgcagtag 3240
atagtcagat atatatctct actcgatact cattggatca gatgcatgag ccggccgggg 3300
tgcagctagt tctcagttaa tcatcaatca agaatgctgt cttggattca agaccttgga 3360
gtatgtctaa atcgatcaat cgtctatcga gaacaagact atatatatta gctaggtacg 3420
agaagcaaat taatgcatgc attgcagcat atcgatcggc aaaagtacaa ctacgtactt 3480
atatatatgc cccttaatta aaaacctatg aaagcgtggg cggtcgagtt tgttgtcctt 3540
agaatacaga ttacaatatg gtcgcgtctc ttctcatcat tttcacaatt ccattacaat 3600
atgcatgtat gcatgcctgc ccttggcagt cgagctatat atattcttta atttgtgcta 3660
gctatagatg cacgtttgta ttgttttttt ttaccgtgtg atgtaatgct atatgtgtca 3720
gccaaacgcg cctggaatgt tggtgcttga ttcaaggccg gttggaattt aaactaccat 3780
ttttctttgt acttaatttg tctttagcgt gcagctagcc gtgcatatgt cggcaaaggc 3840
ttgactcctt ttcactctac agctaagcta tcaatgttca agtgtatact atatatgata 3900
tcaacaatat attgagtagt gaagattcta atgtttgtat gcatca 3946
Claims (9)
1. a kind of method for cultivating genetically modified plants, includes the following steps：Reduce WOX6 albumen and WOX11 albumen in purpose plant
Expression quantity and/or activity, obtain tillering angle increase and/or gravity reaction reduce genetically modified plants；
The WOX6 albumen is following (a1) or (a2) or the protein of (a3) or (a4)：
(a1) protein being made of the amino acid sequence shown in sequence in sequence table 2；
(a2) protein being made of the amino acid sequence shown in sequence in sequence table 4；
(a3) by the amino acid sequence of sequence 2 by the substitution and/or missing and/or addition of one or several amino acid residues and
The relevant protein as derived from sequence 2 is reacted with plant tillering angle and/or gravity；
(a4) by the amino acid sequence of sequence 4 by the substitution and/or missing and/or addition of one or several amino acid residues and
The relevant protein as derived from sequence 4 is reacted with plant tillering angle and/or gravity；
The WOX11 albumen is the protein of following (b1) or (b2)：
(b1) protein being made of the amino acid sequence shown in sequence in sequence table 6；
(b2) by the amino acid sequence of sequence 6 by the substitution and/or missing and/or addition of one or several amino acid residues and
The relevant protein as derived from sequence 6 is reacted with plant tillering angle and/or gravity.
2. a kind of method for cultivating genetically modified plants, includes the following steps：Inhibit WOX6 genes and WOX11 genes in purpose plant
Expression, obtain tillering angle increase and/or gravity reaction reduce genetically modified plants；
The WOX6 genes are following (c1) or (c2) or (c3) or (c4) or (c5)：
(c1) DNA molecular shown in the sequence 11 of sequence table；
(c2) DNA molecular of the code area as shown in the sequence 1 of sequence table；
(c3) DNA molecular of the code area as shown in the sequence 3 of sequence table；
(c4) under strict conditions with (c1) or (c2) or (c3) limit DNA sequence dna hybridization and coded plant tillering angle and/
Or gravity reacts the DNA molecular of relevant protein；
(c5) DNA sequence dna limited with (c1) or (c2) or (c3) or (c4) has more than 90% homology and plant tillering angle
And/or gravity reacts the DNA molecular of relevant protein；
The WOX11 genes are following (d1) or (d2) or (d3)：
(d1) DNA molecular of the code area as shown in the sequence 5 of sequence table；
(d2) the DNA sequence dna hybridization limited under strict conditions with (d1) and coded plant tillering angle and/or gravity reaction phase
The DNA molecular of the protein of pass；
(d3) DNA sequence dna limited with (d1) or (d2) has more than 90% homology and plant tillering angle and/or gravity are anti-
Answer the DNA molecular of relevant protein.
3. the application of the WOX6 albumen described in claim 1, for following (e1) or (e2)：
(e1) plant tillering angle is regulated and controled；
(e2) reaction of plant gravity is regulated and controled.
4. the application of the WOX11 albumen described in claim 1, for following (f1) or (f2)：
(f1) plant tillering angle is regulated and controled；
(f2) reaction of plant gravity is regulated and controled.
5. the application of the WOX6 genes described in claim 2, for following (g1) or (g2)：
(g1) the big plant of tillering angle is cultivated；
(g2) plant that plant gravity reacts small is cultivated.
6. the application of the WOX11 genes described in claim 2, for following (h1) or (h2)：
(h1) the big plant of tillering angle is cultivated；
(h2) plant that plant gravity reacts small is cultivated.
7. application of the method described in claim 1 or 2 in plant breeding.
8. the use as claimed in claim 7, it is characterised in that：The purpose of the breeding is to cultivate tillering angle greatly and/or again
Power reacts small plant.
9. being used to inhibit the substance of the WOX6 genes and WOX11 gene expressions described in claim 2 or, for reducing plant
The substance of the expression quantity and/or activity of WOX6 albumen described in middle claim 1 and WOX11 albumen is big in cultivation tillering angle
And/or the application in gravity reaction plantlet.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711424584.5A CN108085319B (en) | 2017-12-25 | 2017-12-25 | Plant tillering angle related protein and its coding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711424584.5A CN108085319B (en) | 2017-12-25 | 2017-12-25 | Plant tillering angle related protein and its coding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108085319A true CN108085319A (en) | 2018-05-29 |
| CN108085319B CN108085319B (en) | 2020-05-22 |
Family
ID=62179084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711424584.5A Active CN108085319B (en) | 2017-12-25 | 2017-12-25 | Plant tillering angle related protein and its coding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108085319B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851755A (en) * | 2022-07-11 | 2023-03-28 | 内蒙古农业大学 | Meng nong hybrid wheatgrass tillering angle regulation factor and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477112A (en) * | 2002-08-20 | 2004-02-25 | 中国科学院遗传与发育生物学研究所 | Rice tiller control gene MOC1 and its application |
| CN104450777A (en) * | 2014-10-13 | 2015-03-25 | 南京农业大学 | Method for improving potassium absorption efficiency of plant and resisting against potassium deficiency stress and recombinant expression vector used therein |
| CN106046132A (en) * | 2016-06-30 | 2016-10-26 | 中国农业科学院作物科学研究所 | Rice RPS3a gene and application of encoded protein of rice RPS3a gene |
| WO2016173475A1 (en) * | 2015-04-29 | 2016-11-03 | 华中农业大学 | Gene for controlling erectness growth of rice leaf blades, and application thereof |
| US20170088846A1 (en) * | 2015-09-25 | 2017-03-30 | The United States Of America, As Represented By The Secretary Of Agriculture | Functional Analysis of LAZY1 in Arabidopsis thaliana and Prunus Trees |
| CN106676128A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院上海生命科学研究院 | Paddy rice OsWOX11 protein and application of paddy rice OsWOX11 protein for coding gene |
-
2017
- 2017-12-25 CN CN201711424584.5A patent/CN108085319B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477112A (en) * | 2002-08-20 | 2004-02-25 | 中国科学院遗传与发育生物学研究所 | Rice tiller control gene MOC1 and its application |
| CN104450777A (en) * | 2014-10-13 | 2015-03-25 | 南京农业大学 | Method for improving potassium absorption efficiency of plant and resisting against potassium deficiency stress and recombinant expression vector used therein |
| WO2016173475A1 (en) * | 2015-04-29 | 2016-11-03 | 华中农业大学 | Gene for controlling erectness growth of rice leaf blades, and application thereof |
| US20170088846A1 (en) * | 2015-09-25 | 2017-03-30 | The United States Of America, As Represented By The Secretary Of Agriculture | Functional Analysis of LAZY1 in Arabidopsis thaliana and Prunus Trees |
| CN106676128A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院上海生命科学研究院 | Paddy rice OsWOX11 protein and application of paddy rice OsWOX11 protein for coding gene |
| CN106046132A (en) * | 2016-06-30 | 2016-10-26 | 中国农业科学院作物科学研究所 | Rice RPS3a gene and application of encoded protein of rice RPS3a gene |
Non-Patent Citations (3)
| Title |
|---|
| HE JIWAI等: "Fine mapping and candidate gene analysis of qTAC8, a major quantitative trait locus controlling tiller angle in rice (Oryza sativa L.)", 《PLOS ONE》 * |
| ZHANG NING等: "A Core Regulatory Pathway Controlling Rice Tiller Angle Mediated by the LAZY1-Dependent Asymmetric Distribution of Auxin", 《THE PLANT CELL》 * |
| 武迪等: "植物重力反应的分子调控机制", 《遗传》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851755A (en) * | 2022-07-11 | 2023-03-28 | 内蒙古农业大学 | Meng nong hybrid wheatgrass tillering angle regulation factor and application thereof |
| CN115851755B (en) * | 2022-07-11 | 2024-05-24 | 内蒙古农业大学 | Mongolian hybrid agro-grass tillering angle regulating factor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108085319B (en) | 2020-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110105438B (en) | Alfalfa drought-resistant gene MsTHI1, protein coded by same and application thereof | |
| CN106222182B (en) | The IbERF5 genes of coding sweet potato ERF transcription and application | |
| CN110643618A (en) | Jatropha curcas MYB transcription factor JcMYB16 gene and application thereof in improving drought resistance of plants | |
| CN109762795A (en) | A kind of relevant Sesame SiGolS2 of drought resisting and its application | |
| CN110819635A (en) | Application of HAN homologous gene of leguminous plant in regulating and controlling number of root nodules of leguminous plant | |
| CN104862320B (en) | A kind of IbERF4 genes of coding sweet potato ERF transcription and application | |
| CN112725351A (en) | Application of gene OsWRKY43 in resisting bacterial blight of rice | |
| CN110592114B (en) | Application of oryza sativa auxin glycosyl transferase gene | |
| CN110643630B (en) | Application of KNAT1 gene in improving salt stress resistance of plants | |
| CN116769795A (en) | Melon CmADF1 gene and cloning method and application thereof | |
| CN113621625B (en) | Application of sesame SiERF103 gene in enhancing plant resistance | |
| CN105294847A (en) | Stress tolerance-related protein of plants and encoding gene and application of stress tolerance-related protein | |
| CN112626084B (en) | Strawberry MYB transcription factor FvMYB24 gene, expression protein and application | |
| CN102304532B (en) | Method for culturing anti-stress transgenic plants by using gene CYP710A11 | |
| CN102168106A (en) | Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance | |
| CN107304422A (en) | Control the application of the OsSBR1 genes and its RNA interference fragments of resisting rice sheath blight | |
| CN105671058B (en) | The gene of coding sweet potato ERF transcription and application | |
| CN103045639B (en) | Application of AtTGA 4gene in improving plant adverse resistance | |
| CN109068642A (en) | Improvement plant containing the apyrase assortment of genes and the method for being used to prepare the improvement plant with apyrase combination | |
| CN108085319A (en) | Plant tillering angle GAP-associated protein GAP and its encoding gene and application | |
| CN110184253A (en) | Application of the Caragana intermedia CiCPK32 gene in regulation stress resistance of plant | |
| CN102732553B (en) | Improve the gene engineering method and material of plant products | |
| CN107267552A (en) | A kind of method of regulation and control crop economical character and the method for obtaining genetically modified crops | |
| Haq et al. | Barley vacuolar pyrophosphatase (HVP1) gene confers salinity tolerance in locally adapted wheat (Triticum aestivum). | |
| CN109810984A (en) | A kind of relevant Sesame SiGolS6 of drought resisting and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |