CN106011150A - Rice grain number per ear Gn1a gene artificial site-directed mutant and application thereof - Google Patents
Rice grain number per ear Gn1a gene artificial site-directed mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a rice grain number per ear Gn1a gene artificial site-directed mutant and an application thereof. The 4th exon of the Gn1a gene is modified by the CRISPR/Cas9 gene targeting modification technology so that the base of the 4th exon is deleted or inserted to obtain the rice grain number per ear Gn1a gene artificial site-directed mutant; the length of the modification target is 20bp during the CRISPR/Cas9 modification; and the target sequence of the bases from the 164th site to the 183rd site in the 4th exon is AAGCAGTACCTGCCTTACTA. In the invention, the CRISPR/Cas9 is adopted for artificial mutation of the 4th exon of the Gn1a gene, and the mutant capable of remarkably increasing the rice yield is screened out. The Gn1a gene artificial site-directed mutant disclosed by the invention can increase the rice yield by 20% or over.
Description
Technical field
The present invention relates to rice genome editor's breeding field, particularly relate to a kind of Oryza sativa L. grain number per spike Gn1a gene
Artificial directed mutants and application thereof.
Background technology
Number of grain per ear is one of most important economical character of Oryza sativa L., is the Components of rice yield, directly determines
Determine rice yield, therefore increase the important channel that number of grain per ear is increasing production of rice.But, number of grain per ear is typical case
Quantitative trait, by polygenic quantitative character gene locus therefor (Quantitative Trait Loci, QTL) control.
2005, the Matsuoka seminar of Japan Nagoya university utilized Xian round-grained rice to hand over Habataki/Koshihikari,
1st the short arm of a chromosome navigates to a QTL-Gn1 controlling grain number.Further with NIL-Gn1
96 F producing of heterozygous plant (Gn1/gn1)2Individual plant, is decomposed into two sites by Gn1:
Gn1a and Gn1b, the effect in the two site is suitable, and Gn1a is positioned between R3192 and C12072S not
To the interval of 2cM, Gn1b is positioned at the upstream of Gn1a.
The Gn1a gene being positioned at the 1st chromosome is the major gene resistance controlling Oryza sativa L. number of grain per ear, can explain Xian
The phenotypic variation of 44% between round-grained rice kind Habataki, Koshihikari, from rice variety Habataki etc.
Position gene can increase the number of grain per ear of Oryza sativa L., such that it is able to increase the single plant yield of Oryza sativa L..
The research of Matsuoka discloses Gn1a gene and occurs that at First Exon the deletion of 6bp (lacks
Lose), cause the raising of rice yield, therefore found to be used for breeding by breeding man.This mutant (First Exon
The disappearance of 6bp) it is natural mutant, it is random generation.In recent years, genome editing technique is vigorously sent out
Exhibition, uses genome editing technique CRISPR/Cas9 system that the Gn1a of wild type is carried out artificial mutation, energy
Enough acquisitions are different from the Novel high yield mutant of nature mutant.
Summary of the invention
Utilize genome pointed decoration technology CRISPR/Cas9 system, the invention discloses a kind of Rice Panicle grain
The number artificial directed mutants of Gn1a gene and application thereof.The present invention enters by showing son to the 4th of Gn1a gene the
Row artificial mutation, affects the normal expression of the 4th aobvious son, causes gene function to be lost or weakens thus further
Improve the yield of Oryza sativa L..
Technical scheme is as follows: a kind of Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene, described
The Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene uses CRISPR/Cas9 gene target modification technique pair
4th exon of Gn1a gene is modified so that the base of the 4th exon is replaced, lacks and/or inserted
(three kinds mutational formats can random combine) and obtain；Target spot is modified a length of when CRISPR/Cas9 modifies
20bp, is positioned at the 164th bit base～the 183rd bit base in the 4th exon, modifies target sequence and is
AAGCAGTACCTGCCTTACTA。
Described CRISPR/Cas9 gene target modification technique modify target spot the 180th and 181 bit bases it
Between be inserted into single base A, obtain Gn1a gene mutation body Gn1a-G1, insert the 4th after base outer aobvious
The base sequence such as SEQ ID NO.1 of son, output increased about 3%.
Further, described CRISPR/Cas9 gene target modification technique causes at modification target spot the 179th
Base T lacks, and obtains Gn1a gene mutation body Gn1a-G2, lacks the 4th exon alkali after single base
Basic sequence such as SEQ ID NO.2, rice yield is improved more than 10% by this sudden change physical ability.
Described CRISPR/Cas9 gene target modification technique is positioned at the 180th and 181 alkali at modification target spot
It is inserted into single base C between base, obtains Gn1a gene mutation body Gn1a-G3, insert the 4th after base
The base sequence of exon such as SEQ ID NO.3, rice yield is improved more than 20% by this sudden change physical ability.
Described CRISPR/Cas9 gene target modification technique causes base at modification target spot the 179th～182
TACT lacks, and obtains Gn1a gene mutation body Gn1a-G4, lacks the 4th exon after 4 bases
Base sequence such as SEQ ID NO.4, rice yield is improved more than 20% by this sudden change physical ability.
The present invention also provide for the described Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene at breeding of hybridized rice and
Application in genome editor's rice breeding.
Mechanism of the present invention is as follows: the present invention is by for Oryza sativa L. Gn1a gene, and the present invention uses CRISPR/Cas9
Technology carries out artificial mutation at the 4th exon design target spot of Gn1a gene, thus artificially generated different prominent
Become type, then look at these different types of sudden change impacts on rice yield, then filter out performance excellent
Different mutation type, designs target spot on the 4th exon and is difficult to miss the target, it is possible to obtain more mutation type.
Compared with prior art, the method have the advantages that the present invention uses CRISPR/Cas9 pair
The 4th of Gn1a gene shows son and carries out artificial mutation, and filters out the mutant that can significantly improve rice yield.
Rice yield can be brought up to more than 20% by the artificial directed mutants of Gn1a gene that the present invention provides.
Accompanying drawing explanation
Fig. 1 is Gn1a gene mutation body Gn1a-G1 the 4th exon base sequence figure；
Fig. 2 is Gn1a gene mutation body Gn1a-G2 the 4th exon base sequence figure；
Fig. 3 is Gn1a gene mutation body Gn1a-G3 the 4th exon base sequence figure；
Fig. 4 is Gn1a gene mutation body Gn1a-G4 the 4th exon base sequence figure；
Fig. 5 is Gn1a gene mutation body Gn1a-G5 the 4th exon base sequence figure；
Fig. 6 is Gn1a gene mutation body Gn1a-G6 the 4th exon base sequence figure；
Fig. 7 is wild rice grain number per spike Gn1a gene the 4th exon base sequence figure；
Note: in figure, black overstriking region is target sequence, the base of insertion uses italic to represent, the alkali of disappearance
Base uses "-" to represent.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme is described in further details.
Embodiment 1
The base sequence of Oryza sativa L. grain number per spike Gn1a gene is as shown in SEQ ID NO.7.
4th exon base sequence (wild type) of Gn1a gene, as shown in SEQ ID NO.8, is shown in Fig. 7
Modify target spot a length of 20bp when the present embodiment CRISPR/Cas9 modifies, be positioned in the 4th exon 164
Bit base～183 bit bases, modification target sequence is AAGCAGTACCTGCCTTACTA.The present embodiment
Target sequence is synthesized:
Oligo15’-GGCAAAGCAGTACCTGCCTTACTA-3 ',
Ologo2:5 '-AAACTAGTAAGGCAGGTACTGCTT-3 '.
The present embodiment uses containing cas9 expression cassette (35S promoter startup) and guid rna expression box (water
Rice OsU3 promoter) binary vector Pcambia1300 be that skeleton carrier carries out vector construction, by target spot sequence
Row insert in skeleton carrier, and specific practice is: Oligo1 Yu Oligo2 carries out phosphorylation of annealing, and use limit
Property restriction endonuclease Aar I processed carries out enzyme action to skeleton carrier, then uses T4 ligase by after annealing phosphorylation
Target sequence is connected between skeleton carrier OsU3 promoter and sgRNA-scaffold sequence, completes vector construction.
After vector construction, carrier is proceeded to Agrobacterium EHA105, using in Oryza sativa L. japonica rice variety Japan fine as
Receptor carries out Agrobacterium-mediated Transformation.Through screening, differentiation, obtain T0 after rooting process for transfer-gen plant.
Gathering T0 and extract DNA for plant leaf, the target spot periphery design at Gn1a gene the 4th exon detects draws
Thing carries out PCR detection, and detection primer is:
Gn1a-test-f:ACATGAAAAACAATGTCCGTT,
Gn1a-test-r:CGTATGCACAGTACACCCAT。
PCR primer is carried out SANGER order-checking, determines that the T0 undergone mutation is for plant.
By detecting that the plant of jumping phenomenon carries out sowing in T0 generation, carry out the plantation in T1 generation, gather T1
Extract DNA for plant leaf, use detection primer as above to detect, determine Gn1a gene mutation class
Type is homozygous mutation or heterozygous mutant；Designing hygromycin (entirely writing) detection primer, primer sequence is simultaneously:
HYG-f:CTATTTCTTTGCCCTCGGAC, HYG-r:CCTGACCTATTGCATCTCCC,
With this, the existence of detection hygromycin fragment, determines whether foreign vector T-DNA fragment exists.Filter out 6 kinds
Gn1a mutation type is that homozygous mutation simultaneously HYG is detected as the plant of feminine gender and carries out sowing.
The plant that T1 generation filters out is carried out sowing, carries out the plantation in T2 generation, every kind of mutation type plantation 49
Strain.T2 is on behalf of the Mutants homozygous of 6 kinds of mutation types in theory.In order to verify this point, random screening portion
Plant is divided to carry out genotype detection, it was demonstrated that T2 is the Mutants homozygous of 6 kinds of mutation types really for plant.
In order to determine the impact on yield of 6 kinds of mutation types, then in the period of maturation in T2 generation, to every kind of genotype
Setting percentage, grain number per spike, effective fringe, mass of 1000 kernel and single plant yield Isoquant index are added up.
6 kinds of Gn1a gene mutation bodies are as follows:
Gn1a gene mutation body Gn1a-G1: be inserted between target spot the 180th and 181 bit bases modifying
Single base A, target sequence becomes AAGCAGTACCTGCCTTAACTA, inserts the after base
The base sequence of 4 exons such as SEQ ID NO.1, is shown in Fig. 1.
Gn1a gene mutation body Gn1a-G2: modifying target spot the 179th bit base T disappearance, target sequence becomes
For AAGCAGTACCTGCCT-ACTA, lack the 4th exon base sequence after single base such as
SEQ ID NO.2, is shown in Fig. 2.
Gn1a gene mutation body Gn1a-G3: in modification target spot is between the 180th and 181 bit bases
Inserting single base C, target sequence becomes AAGCAGTACCTGCCTTACCTA, after inserting base
The base sequence such as SEQ ID NO.3 of the 4th exon, see Fig. 3.
Gn1a gene mutation body Gn1a-G4: cause disappearance modifying target spot the 179th～182 bit base TACT,
Target sequence becomes AAGCAGTACCTGCCT----A, lacks the alkali of the 4th exon after 4 bases
Basic sequence such as SEQ ID NO.4, is shown in Fig. 4.
Gn1a gene mutation body Gn1a-G5: modifying target spot the 180th bit base A disappearance, target sequence becomes
For AAGCAGTACCTGCCTT-CTA, lack the base sequence of the 4th exon after 4 bases such as
SEQ ID NO.5, is shown in Fig. 5.
Gn1a gene mutation body Gn1a-G6: modifying target spot the 164th～184 bit bases disappearance, disappearance
Base is AAGCAGTACCTGCCTTACTAC, lacks the base of the 4th exon after 4 bases
Sequence such as SEQ ID NO.6, is shown in Fig. 6.
The impact on single plant yield of 6 kinds of Gn1a gene mutation bodies, as shown in table 1.
The impact on yield of the table 1.Gn1a different mutants
Note: " ns " represents the most not notable at 0.01 and 0.05 level difference, and " *, * * " is illustrated respectively in 0.05,0.01 difference and reaches significant level；G1-G6 is mutant in 6, wide
For wild type control.
As shown in Table 1, Gn1a G5 and Gn1a G6 does not improve rice yield, even results in yield and reduces.
The effect of increasing production of Gn1a G3 and Gn1a-G4 is the most notable, has reached more than 20%.
SEQUENCE LISTING
<110>Yunnan Na Bo bio tech ltd
Yunnan University
<120>a kind of Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene and application thereof
<130> 2016
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 320
<212> DNA
<213>artificial sequence
<400> 1
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgcctta 180
actacggcag ccaggcagag tggcagaagc ggcacttcgg tgccaatctc
tggccaagat 240
tcgtgcagcg gaagagcaag tatgatccaa aggccatcct gtcccgtggc
caggggattt 300
tcacgtcacc
actcgcatga
320
<210> 2
<211> 318
<212> DNA
<213>artificial sequence
<400> 2
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgcctac 180
tacggcagcc aggcagagtg gcagaagcgg cacttcggtg ccaatctctg
gccaagattc 240
gtgcagcgga agagcaagta tgatccaaag gccatcctgt cccgtggcca
ggggattttc 300
acgtcaccac
tcgcatga
318
<210> 3
<211> 320
<212> DNA
<213>artificial sequence
<400> 3
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgcctta 180
cctacggcag ccaggcagag tggcagaagc ggcacttcgg tgccaatctc
tggccaagat 240
tcgtgcagcg gaagagcaag tatgatccaa aggccatcct gtcccgtggc
caggggattt 300
tcacgtcacc
actcgcatga
320
<210> 4
<211> 315
<212> DNA
<213>artificial sequence
<400> 4
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgcctac 180
ggcagccagg cagagtggca gaagcggcac ttcggtgcca atctctggcc
aagattcgtg 240
cagcggaaga gcaagtatga tccaaaggcc atcctgtccc gtggccaggg
gattttcacg 300
tcaccactcg
catga
315
<210> 5
<211> 318
<212> DNA
<213>artificial sequence
<400> 5
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgccttc 180
tacggcagcc aggcagagtg gcagaagcgg cacttcggtg ccaatctctg
gccaagattc 240
gtgcagcgga agagcaagta tgatccaaag gccatcctgt cccgtggcca
ggggattttc 300
acgtcaccac
tcgcatga
318
<210> 6
<211> 298
<212> DNA
<213>artificial sequence
<400> 6
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacggcagcc
aggcagagtg 180
gcagaagcgg cacttcggtg ccaatctctg gccaagattc gtgcagcgga
agagcaagta 240
tgatccaaag gccatcctgt cccgtggcca ggggattttc acgtcaccac
tcgcatga 298
<210> 7
<211> 5137
<212> DNA
<213>Oryza sativa L.
<400> 7
gcaagaacac acaaattcac acacacactg acacacacaa accgatcgat
tgattgattg 60
ataatgaagc aagagcaggt caggatggca gtgctcctca tgctcaactg
cttcgtcaag 120
gccacggcgc cgccgccatg gccgccgtcg gcttcgtccg cctccttcct
cgacgacctc 180
ggcgacctcg gcatcgcgcc gctcatccgc gccgacgagg cgggcaccgc
gcgcgcctcc 240
gccgactttg gcaacctctc cgtcgccggc gtcggggcgc ctcggctcgc
cgccgccgcc 300
gccgtgctct acccgtcgcg ccccgccgac atcgccgcgc tgctgcgcgc
gtcgtgcgca 360
cgcccggcgc cgttcgcggt gtccgcgcgg gggtgtggcc actcggtgca
cggccaggcc 420
tccgcgcccg acggcgtcgt cgtcgacatg gcgtcgctcg gccgcctgca
gggcggcggc 480
gcgcggcgcc tcgccgtgtc agtggagggg cggtacgtcg acgccggcgg
cgagcagctg 540
tgggtggacg tgctgcgcgc gtccatggcg cacgggctca cgccggtgtc
gtggacagac 600
tacctccacc tcaccgtcgg cggcacgctg tccaacgccg gcatcagcgg
ccaggccttc 660
cgccatggcc cccagatttc caacgtgcta gagctcgacg tcatcaccgg
tacgtagatc 720
catcacatct actaagacac gcgccgccat gatcgaggta attaaggtat
aggtgttttg 780
acgtatacat gtatctgcag gtgtcgggga gatggtgacg tgctcgaagg
agaaggcgcc 840
ggacctgttc gacgcggtgc tgggcgggct ggggcagttc ggcgtcatca
cgcgggcgcg 900
catcccgctc gcgccggcgc cggcgagggc gcggtgggtg cggttcgtgt
acacgacggc 960
ggcggcgatg acggccgacc aggagcgcct catcgccgtc gatcgcgccg gcggcgccgg
1020
cgcggtgggc gggctgatgg actacgtcga gggctcggtc cacctgaacc
agggcctggt 1080
cgagacctgg cgcacgcagc cgcagccgcc ttcgccgtcc tcctcctcct
cctcatcctt 1140
cttctccgac gccgacgagg cccgcgtcgc cgcgctcgcc aaggaggccg
gcggcgtgct 1200
gtatttcctc gagggcgcca tctacttcgg cggcgccgcc gggccgtccg
ccgccgacgt 1260
tgacaaggta tactagctag ctactagctt gctctgcgct gagccgacca
gagcgggtcc 1320
cacctcgtga tgatggcggg aacaactaag ctgcaaaaac ttttggcgcc
acctggggct 1380
tacgcttacg cacgcatgca attaaggggt gttctagatg gggctaaaac
tttttagccc 1440
atgtcacatc ggatgtttgg acgctaattt ggagtattaa atatagacta
ataaaaaaac 1500
taatttcata aatgagagct aatccgcgag acgaattttt taagcctaat
taatctataa 1560
ttataaaagt ttattgtagc atcacattgt caaaatcatg acataattag actcaaaaga
1620
ttcgtctcgt gaattagtcc aagatatgga atatgtttta taattagtgt
atgtttaata 1680
ctccaaatta gtattcaaac atctggtgtg acatggactt ggaataagtc
cgtggaaacc 1740
aaacagaccc taacggtgca tgaaattgaa gtctcttgcg ccgtcgacat
cgtcgtactt 1800
ggcctaccac ttttgtctgc cacgcgatgc acctctcgct atcacacacc
taactggaag 1860
taattaaata attattcgat tctgtgttaa ttttttttta tcttccttag
ttcccggaga 1920
gacaaagatt agatactata gtagcaactt agtaagctag tatatggagt
attaggttag 1980
tcgctctcac taagcttaaa caggtgtata aaatatatgc atcgtctgat
cgtgacatat 2040
tcttttagct acttatggtg aaaacttttt cgtccaaaac agtgaaaagc
atgcgtgcta 2100
gtgtaggtag tagctaccag gacgaattat atcattaaca gtatttgtag
cacatcaagg 2160
aaaaacttgt ctttttaaac actgttacag tcttcagaac gcacaacttt
aacaggtatt 2220
tttgtattat atttttttaa aaaaaaataa aggtaataaa attatggtat
tgtaaaagta 2280
tatttttaag gaaaatcata taaccaatca aaagtttatg aagatataca
tattgatgtt 2340
caaagttact aaaagttgac ttaaacatca cattttcatc ttgaccaaag
agggttcata 2400
tatatactcc ctcaatttta aaatataagc atttctaatt atatgcatct
agacaaatgc 2460
atataaaaat actttatttt ttaaagtgag ggagtatcaa ttttgagcat
gtagctagac 2520
tagattagtg tatgtctacg cacatatctg ttgttctgca caaaactact actcatcggt
2580
cctaaaatat aagaatttaa aattggatgg gacataccct aatacaatga
atttagacat 2640
ggacatatac tagtaatacc atgtactacc tccatcccaa aataagttca
cttttcatcc 2700
atctcacaca taccaataga aagtactaca aatttcggtt attctctatt
ttcacaaact 2760
ccgatgcaat gattatttta aaaataaact tattttagaa taaatggaat
gagcaaaata 2820
taaactggtg tgtttgagga gaaggggatt gaggagattg ggaagatacg
caaaacgagg 2880
tgagccatta gctcatgatt aattgagtat taactatttt aaatttcaaa
aatggattaa 2940
tatgattttt taaagcaact ttcctatata aaatttttac aaaaaacaca
ccgtttaata 3000
gtttggaaag cgtacttgcg gaaaacgagg tgctttctcc ctcaatgtcg
tccaaacgaa 3060
cgctgcctta ttacgggact gaggaattag agctttgcca gaaagaaatc
agcatcgcca 3120
gcttggacct accatccatg catgcatcat gtggccattg acacatcaca
tagtatgtgc 3180
tagctagcta gcttttgatc atagttacat gtatctagct aggctagaag
ctggaaaccg 3240
atggatatga tggatctctc atggatgaca ggccagccaa agatctgtgc
gccactagat 3300
acagtgcatg catcagcttg tatggttata accctagcta gccagcttta gcacacacat
3360
gcatatgcat gcatgagccc ccatcttttg caacacgacc gaccaactat
gttggctcta 3420
tatagatagc tagctagtta ttccatgcat atacagtttg catttcctag
ctatagcttt 3480
tgctatgtga tccgagaaga tcctgcatgc ccacacgtga cacgtcacac
acacatgtgg 3540
acaaagtact gcctcacttt atccttgcat gacgtcacgt cgccacctgt
ccatccacgc 3600
tgctagtgct ggcaaaatta ataactcgat caaatttcgg tgatctctct
gcaaagaatt 3660
tgatgaattt taccaacata tatgctttaa tttctttgct tgattttatt
tgcagaggat 3720
ggatgtgctg cgtcgcgagc tgcggcacga gcgcgggttc gtgttcgcgc
aggacgtggc 3780
gtacgccggg ttcctggacc gcgtccacga cggcgagctc aagctccgcg
ccgcggggct 3840
ctgggacgtg ccgcacccat ggctgaacct gttcctcccc cgctccggcg
tcctcgcctt 3900
cgccgacggc gtcttccacg gcatcctcag ccgcaccccc gccatgggcc
ccgtcctcat 3960
ctaccccatg aaccgcaaca agtaataata ataataaaaa gctttactac
atatacacat 4020
gtatataatt tttacggggt ggattttttc gttcaaaatg acgacccctc
atattgtgcg 4080
tgtcgtctga aaacttatta aaatgtttaa ataaaaaatt aatatgatac ataaatatat
4140
tatatatcac tatataaaca ttgtaatctt aaactcaact tgcacaagta
gtaaaaaaac 4200
aaatttgact gcaaatagtg tgtactaagt tatttattta cttatgctag
tatgctactt 4260
gaatttaaac gtacatattt atgaagtggt atattatata tttccagagt
atttttatgg 4320
ttcttttacg acatgaaaaa caatgtccgt tctcttgaag gatgaataga
ctttccttaa 4380
ttttaacata tatggtggta actaaacata cacacacctg gatatgtttc
aggtgggaca 4440
gtaacatgtc ggcagtgatc accgacgacg acggtgacga ggtgttctac
acggtgggga 4500
tcctgcggtc ggcggcggcg gccggcgacg tggggaggct ggaggagcag
aacgacgaga 4560
tcttgggttt ctgcgaggtg gccgggatag cctacaagca gtacctgcct
tactacggca 4620
gccaggcaga gtggcagaag cggcacttcg gtgccaatct ctggccaaga
ttcgtgcagc 4680
ggaagagcaa gtatgatcca aaggccatcc tgtcccgtgg ccaggggatt
ttcacgtcac 4740
cactcgcatg aaatgacaca tgtatgcaaa tgcatatcta catgcgtata
tatacacgta 4800
tatatacgta tgtatgcata cacatatggg tgtactgtgc atacgttata
gcacactgca 4860
gctaattaag cttgacaggg agatcgatca atggacaatg ctctagtcaa gctaatataa
4920
ataatggagt agtagtatat atgtagtgcg agataattaa gtagtgtgtt
tgcctactaa 4980
aaggagaggc aaagtagtac tgtgatgcat gcatgccaac taataggtga
taagtacgtg 5040
tgtgtggccg catgtatgat tagaagaagt tggtttttaa ttaattaatt
aggtcatgta 5100
tgtaaatata tagtacagta ctacgtacta
ctagtgt
5137
<210> 8
<211> 319
<212> DNA
<213>Oryza sativa L.
<400> 8
gtgggacagt aacatgtcgg cagtgatcac cgacgacgac ggtgacgagg
tgttctacac 60
ggtggggatc ctgcggtcgg cggcggcggc cggcgacgtg gggaggctgg
aggagcagaa 120
cgacgagatc ttgggtttct gcgaggtggc cgggatagcc tacaagcagt
acctgcctta 180
ctacggcagc caggcagagt ggcagaagcg gcacttcggt gccaatctct
ggccaagatt 240
cgtgcagcgg aagagcaagt atgatccaaa ggccatcctg tcccgtggcc
aggggatttt 300
cacgtcacca
ctcgcatga
319
Claims (6)
1. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene, it is characterised in that described Rice Panicle
The grain number artificial directed mutants of Gn1a gene uses CRISPR/Cas9 gene target modification technique to Gn1a base
4th exon of cause is modified so that the base of the 4th exon is replaced, lacked and/or insert and obtain；
Modify target spot a length of 20bp when CRISPR/Cas9 modifies, be positioned in the 4th exon the 164th～the 183rd
Bit base, modification target sequence is AAGCAGTACCTGCCTTACTA.
2. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene as claimed in claim 1, its feature exists
In, described CRISPR/Cas9 gene target modification technique modify target spot the 180th and 181 bit bases it
Between be inserted into single base A, obtain Gn1a gene mutation body Gn1a-G1, insert the 4th after base outer aobvious
The base sequence such as SEQ ID NO.1 of son.
3. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene as claimed in claim 1, its feature exists
In, described CRISPR/Cas9 genetic modification technology causes 179 bit base T disappearances at modification target spot, obtains
Gn1a gene mutation body Gn1a-G2, the 4th exon base sequence such as SEQ ID NO.2 after disappearance base.
4. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene as claimed in claim 1, its feature exists
In, described CRISPR/Cas9 gene target modification technique is positioned at the 180th and 181 alkali at modification target spot
It is inserted into single base C between base, obtains Gn1a gene mutation body Gn1a-G3, insert the 4th after base
The base sequence of exon such as SEQ ID NO.3.
5. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene as claimed in claim 1, its feature exists
In, described CRISPR/Cas9 gene target modification technique causes base at modification target spot the 179th～182
TACT lacks, and obtains Gn1a gene mutation body Gn1a-G4, lacks the 4th exon after 4 bases
Base sequence such as SEQ ID NO.4.
6. the Oryza sativa L. grain number per spike artificial directed mutants of Gn1a gene as described in Claims 1 to 5 is arbitrary
Application in breeding of hybridized rice and genome editor's rice breeding.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392024A (en) * | 2005-03-31 | 2009-03-25 | 中国科学院遗传与发育生物学研究所 | Rice tillering associated protein and encoding gene and use thereof |
| CN105063061A (en) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | Rice thousand kernel weight gene tgw6 mutant as well as preparation method and application thereof |
-
2016
- 2016-08-01 CN CN201610615319.4A patent/CN106011150A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392024A (en) * | 2005-03-31 | 2009-03-25 | 中国科学院遗传与发育生物学研究所 | Rice tillering associated protein and encoding gene and use thereof |
| CN105063061A (en) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | Rice thousand kernel weight gene tgw6 mutant as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| MEIRU LI.ET AL: "Reassessment of the Four Yield-related Genes Gn1a,DEP1,GS3, and IPA1 in Rice Using a CRISPR/Cas9 System", 《FRONT PLANT SCI.》 * |
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