CN110791525B - Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield - Google Patents

Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield Download PDF

Info

Publication number
CN110791525B
CN110791525B CN201911264946.8A CN201911264946A CN110791525B CN 110791525 B CN110791525 B CN 110791525B CN 201911264946 A CN201911264946 A CN 201911264946A CN 110791525 B CN110791525 B CN 110791525B
Authority
CN
China
Prior art keywords
rice
target
tillering number
gene
cas9
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.)
Active
Application number
CN201911264946.8A
Other languages
Chinese (zh)
Other versions
CN110791525A (en
Inventor
高清松
赵祥祥
王欢欢
李刚
纪剑辉
张雯霞
崔文星
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anyang Hongzhixing Agricultural Technology Co ltd
Dragon Totem Technology Hefei Co ltd
Original Assignee
Huaiyin Normal University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Huaiyin Normal University filed Critical Huaiyin Normal University
Priority to CN201911264946.8A priority Critical patent/CN110791525B/en
Publication of CN110791525A publication Critical patent/CN110791525A/en
Application granted granted Critical
Publication of CN110791525B publication Critical patent/CN110791525B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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

Abstract

The invention discloses a method for knocking out a rice tillering number regulating gene OsFW L to increase the tillering number and the yield of rice, belonging to the technical field of molecular biology and genetic engineering.

Description

Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and relates to a method for knocking out a rice tillering number regulation gene OsFW L4 to increase the tillering number and yield of rice.
Background
The rice is an important food crop, more than half of the population of China takes rice as main food, the rice yield is improved, the basic self-sufficiency of grains is ensured, the national food safety is guaranteed, and the rice yield is extremely important strategic significance, the tillering number is one of the most important agronomic traits of the rice, the tillering number directly determines the number of each ear, and further influences the rice yield, the generation of rice tillering can be divided into two steps, namely, the formation of axillary buds and the elongation of axillary buds, at present, cloned genes for controlling the formation of the axillary buds comprise MOC1, MOC3/TAB1, TAD1/TE, L AX1, L AX2 and the like, MOC1 is the first cloned gene for controlling the rice tillering, encodes a GRAS family transcription factor, the tillering is influenced by controlling the formation of the axillary buds, the mutant only has a main stem without producing tillering, the transgenic plant canolide (Strigolanectins, S L) is a novel plant hormone, the transgenic plant gene for controlling the transgenic plant tillering, the transgenic plant tillering gene for controlling the transgenic plant, the transgenic plant tillering of the transgenic plant, the transgenic plant with the transgenic plant gene encoding the transgenic plant gene for enhancing the transgenic plant with the transgenic plant gene for enhancing the transgenic plant gene for the transgenic plant with the transgenic plant with the transgenic plant transgenic.
FW2.2 is a major QT L controlling fruit size in tomato, determining 30% of fruit size variation, its corresponding gene FW2.2 affects fruit size by negatively regulating cell division FW2.2 homologous genes are widely present in plants, among which corn ZmCNR1, physiology floridana PfCNR1, rice OsFW L3, avocado pafw2.2-like, are all negative regulators of cell division and organ size, therefore, regulation of cell number and organ size by FW2.2-like (FW L) genes may be a common mechanism in plants, rice genome contains 8 FW L genes, among which OsFW L3 affects grain length and grain weight by negatively regulating glume cell division, OsFW L5/PCR 1 participates in metal ion transport and grain weight regulation.
The CRISPR/Cas9(Clustered regulated short linked polypeptides/CRISPR-associated protein 9) system is a novel genome editing technology newly developed in recent years. The system consists of two parts, a single guide rna (sgrna) and a Cas9 nuclease. By utilizing the technology, accurate cutting of different target points can be realized only by replacing a section of 20 nucleotide 'guide sequence' in the sgRNA, so that the protein engineering step is avoided, and the construction of an expression unit is very simple and convenient. Currently, this technique has been widely applied to targeted editing of genomes of various organisms including plants.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for knocking out a rice tillering number regulation gene OsFW L4 so as to increase the tillering number and yield of rice.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for knocking out a rice tillering number regulation gene OsFW L4 to increase the tillering number and yield of rice is characterized in that a CRISPR/Cas9 technology is used for carrying out targeted mutation on the rice tillering number regulation gene OsFW L4, and the nucleotide sequence of the rice tillering number regulation gene OsFW L4 is shown as SEQ ID No. 1.
Preferably, the method for targeting mutation of the rice tillering number regulation gene OsFW L4 by using the CRISPR/Cas9 technology comprises the following specific steps:
1) selecting a target fragment in a coding region of a rice tillering number regulating gene OsFW L4;
2) synthesizing a joint primer with a cohesive end according to the sequence information of the target fragment, and constructing a CRISPR/Cas9 recombinant vector for OsFW L4 gene targeting, wherein the recombinant vector comprises a sgRNA expression cassette and a Cas9 nuclease expression cassette which can be expressed in rice cells and have the target sequence;
3) transferring the recombinant vector into an agrobacterium tumefaciens engineering strain, and transforming a conventional japonica rice variety by using an agrobacterium-mediated method to obtain a transgenic rice plant;
4) amplifying and sequencing the genome DNA of the transgenic plant by using a genome primer covering a target site, thereby carrying out genotype identification on the T0 generation transgenic plant and obtaining a homozygous or double allelic mutant;
5) predicting potential off-target sites of a target sequence by using a bioinformatics tool; then, respectively carrying out PCR amplification and sequence determination on two off-target sites with highest prediction probability in homozygous or biallelic T0 mutant by using genome primers to obtain homozygous or biallelic mutant without detectable off-target effect;
6) the homozygous or biallelic mutant without the detectable off-target effect is planted in the T1 generation, and PCR detection is carried out on HPT, sgRNA and Cas9 transgenes in the T1 generation plants by using primers, so that the rice without transgene insertion, detectable off-target effect and remarkable increase of tillering number and yield of a single plant can be obtained.
Preferably, in step 1), one strand of the double-stranded structure of the target fragment has a structure of 5 '- (N) X-NGG-3', wherein N is A, T, C, G, and X is 19 or 20; the target fragment has two or more mismatches with similar sequences in the rice genome.
Preferably, in step 1), there are two target fragments, and the sequences are shown as SEQ ID No.5 and SEQ ID No. 6.
Preferably, in step 2), the nucleotide sequence of the sgRNA expression cassette is shown as Seq ID No. 3; the nucleotide sequence of the Cas9 nuclease expression cassette is shown in Seq ID No. 4.
Preferably, in step 6), the primer sequences for transgenic amplification of hpt (hygromycin phosphotransferase), sgRNA and Cas9 are:
HPTF：5’-GGGTGTCACGTTGCAAGACC-3’，
HPTR：5’-ATGCCTCCGCTCGAAGTAGC-3’，
sgRNAF：5’-TCCCAGTCACGACGTTGTAA-3’，
sgRNAR：5’-GGCCATTTGTCTGCAGAAT-3’，
Cas9F：5’-CACCATCTACCACCTGAGAA-3’，
Cas9R：5’-CGAAGTTGCTCTTGAAGTTG-3’。
preferably, in step 5), the bioinformatic tool is CRISPR-P2.0 (L iu H, Ding Y, Zhou Y, Jin W, Xie K, Chen L. CRISPR-P2.0: an improved CRISPR/Cas9 tool for genetic engineering in plants, molecular Plant, 2017, 10 (3): 530) or CRISPR-GE (Xie X, MaX, Zhu Q, Zeng D, L i G, L iu Y. CRISPR-GE: A genetic software tool for CRISPR-based genetic engineering, molecular Plant, 2017, 10 (9): 1246 1249).
Has the advantages that: compared with the prior art, the invention has the advantages that:
the invention provides a method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield, which is a method for obtaining a transgene-free inserted mutant by targeted mutation of OsFW L4 gene by using CRISPR/Cas9 technology, and can ensure that the transgene-free inserted mutant is obtained by using three pairs of primers for detection in the T1 generation, and is equal to a naturally occurring mutant, so that the safety risk possibly brought by transgene is avoided.
Drawings
FIG. 1 shows the results of sequencing and identification of different target mutants in example 1;
FIG. 2 shows the results of the detection of the T1 generation transgene-free insertion mutant in example 1; wherein 1-10 respectively represent different T1 generation plants, VC represents a vector control, WT represents a wild type, and M represents a DNA molecular weight marker;
FIG. 3 shows mutant and wild-type plants of example 1;
FIG. 4 shows the tillering numbers of mutant and wild type individuals in example 1, where n is 20;
FIG. 5 shows the yield of mutant and wild-type individuals in example 1, n 15;
FIG. 6 shows the mutant and wild-type flag leaf phenotype in example 2; wherein, fig. 6A is mutant and wild type flag leaf, scale bar 2 em; fig. 6B and C are the mutant and wild type leaf length and leaf width, respectively, with n-20.
Note: error bars in all figures of the specification indicate standard deviation: p is less than 0.001.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, unless otherwise specified, all experimental procedures were carried out according to conventional methods.
Example 1 method for increasing tillering number and yield of rice by knocking out gene OsFW L4 based on CRISPR/Cas9 technology
The rice tillering number regulating gene OsFW L4 has the sequence as shown in SEQ ID No. 1. sequence analysis shows that the gene includes 2 exons, including the 1 st-67 th exon and the 320 th-663 th exon of SEQ ID No. 1.
(1) Construction of OsFW L4 gene knockout recombinant expression vector
The method comprises the following steps of designing two CRISPR/Cas9 gene knockout target sites in an OsFW L4 gene coding region, and respectively targeting sense chains of exons 1 and 2, wherein an Osfwl4a target sequence is shown as SEQ ID No.5, and an Osfwl4b target sequence is shown as SEQ ID No.6, respectively synthesizing target sequence primers based on a CRISPR/Cas9 system, and the sequences are as follows:
Osfwl4a-P1：5’-TGTGATTGAAGCAGGCGAAGAGTC-3’，
Osfwl4a-P2：5’-AAACGACTCTTCGCCTGCTTCAAT-3’，
Osfwl4b-P1：5’-TGTGCGCAGCATGGGTCCTCGGGG-3’，
Osfwl4b-P2：5’-AAACCCCCGAGGACCCATGCTGCG-3’，
wherein the underlined section is the sticky end used for vector ligation and the non-underlined section is the target sequence or its complement.
The maize ZmUBI promoter was ligated to hSpCas9 gene (Cong L, Ran FA, Cox D, L in S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini L A, Zhang F.multiple genetic engineering using CRISPR/Cas system science, 2013, 339 (6121): 819 823), and pCAMBIA1300 binary vector was inserted to obtain intermediate vector, the original BsaI cleavage site of pCAMBIA1300 vector was removed using a point mutation kit (TransGen Biotech), and then a fragment containing OsU6 promoter, ccdB negative selection marker gene flanked by BsaI cleavage sites, and sgRNA was inserted into the intermediate vector to construct CRISPR/Cas9 binary vector, which was preserved in E.coli strain DB 3.1.
Osfwl4a-P1 and Osfwl4a-P2, Osfwl4b-P1 and Osfwl4b-P2 are respectively annealed to form double-stranded DNA with cohesive ends, and the double-stranded DNA is used as an insert for constructing a CRISPR/Cas9 gene knockout vector. The above CRISPR/Cas9 binary vector was cleaved with BsaI restriction endonuclease (NEB) at 37 ℃ to obtain a vector backbone fragment. And (3) connecting the insert and the vector framework by using T4 ligase (NEB) to obtain recombinant expression vectors targeting Osfwl4a and Osfwl4b target sites respectively. The recombinant expression vector is transferred into escherichia coli for amplification, and the extracted vector plasmid is transferred into agrobacterium tumefaciens strain EHA105 for agrobacterium-mediated rice genetic transformation after being verified to be correct by sequencing (Shanghai Bioengineering Co., Ltd.).
(2) Obtaining and identifying OsFW L4 gene knockout rice
With reference to the method of Nishimura et al (Nishimura A, Aichi I, Matsuoka M.A protocol for Agrobacterium-mediated transformation in rice. Nature protocols.2006, 1 (6): 2796-2802), the recombinant expression vector is transformed into conventional japonica rice variety Zhonghua 11, and 15T 0 generation transgenic rice plants are obtained from both recombinant vectors.
Extracting T0 generation transgenic rice plant DNA, amplifying DNA fragment containing target sites by using KOD DNA polymerase (TOYOBO) and genome primers, and performing sequence determination on the amplified product by Shanghai biological engineering corporation. The primer sequence is as follows:
Osfwl4aF：5’-CACACCGTCGTCAAGCAAC-3’，
Osfwl4aR：5’-TCATGCTCATCGCTCCTAGC-3’，
Osfwl4bF：5’-GGCCGGCAGCTAATTTGAAG-3’，
Osfwl4bR：5’-GTTGCTTGACGACGGTGTG-3’，
sequencing results show that 11 transgenic plants at the Osfwl4a target site have target mutation, wherein 3 homozygous mutants and 8 biallelic mutant strains are obtained; 10 transgenic plants of the Osfwl4b target site carry target mutations, wherein 1 homozygous mutant, 8 biallelic mutant and 1 chimera are obtained.
(3) Obtaining of homozygous mutant without transgene and detectable off-target effect
Since the CRISPR/Cas9 system can generate off-target effect, the analysis of phenotype is influenced, and potential off-target sites of target sites are predicted by using the CRISPR-P2.0 tool. Then, designing genome primers to carry out PCR amplification and sequence determination on two off-target sites with the highest probability of each target site of the T0 generation mutant so as to identify the off-target effect. The primer sequence is as follows:
Osfwl4aOFF-1F：5’-GTTGCAGGAAACTGAAACATG-3’，
Osfwl4aOFF-1R：5’-CTGTAGAACAATGCCAATCAC-3’，
Osfwl4aOFF-2F：5’-GGAGCAAACACCGAACAACT-3’，
Osfwl4aOFF-2R：5’-TTCTTCCTGGAGATCGCCTA-3’，
Osfwl4bOFF-1F：5’-ACAAATCGACAGAAAATCCAAA-3’，
Osfwl4bOFF-1R：5’-CTCCGTCTTTCTCGATATTCCT-3’，
Osfwl4bOFF-2F：5’-TCCTGGTTATTAAGGGTTT-3’，
Osfwl4bOFF-2R：5’-TCCTGGCTGTTGAGGTAGA-3’，
for each target site, 1T 0 generation mutant with no detectable off-target effect was selected for subsequent analysis. Wherein, the Osfwl4a target site selects a homozygous mutant Osfwl4a #7, the Osfwl4b target site selects a biallelic mutant Osfwl4b #6, and the mutant genotype is shown in figure 1.
The genomic DNA of the T1 generation plant of the mutant is amplified by HPT, sgRNA and Cas9 primers, 5 mutants without transgene insertion are obtained from 10T 1 generation individuals of Osfwl4a #7 and Osfwl4b #6 respectively (figure 2), and the number of T-DNA insertion sites of the transgenic plant obtained by the method is few, so that the mutant without transgene insertion is easily obtained from T1 generation. In the detection of the transgenes of Osfwl4b #6T1 generation plants, only HPT sequences are detected, and sgRNA and Cas9 sequences are not detected, which indicates that the mutant without transgene insertion can be obtained only by simultaneously detecting 3 transgenes by using the method provided by the invention.
Planting T1 generation mutant without transgene insertion and detectable off-target effect, and separating the offspring to obtain homozygous mutant without transgene insertion and detectable off-target effect. Tillering numbers of the two allelic mutant individuals are respectively increased by 45.9% and 41.1% compared with a control (figure 3 and figure 4). The grain number of each ear of the mutant has no obvious difference with the wild type, the thousand grain weight is slightly reduced (reduced by 2.4 percent and 2.7 percent respectively), but the single plant yield is respectively increased by 40.9 percent and 30.4 percent (figure 5), which shows that the method provided by the invention can obviously increase the single plant tillering number and yield of the conventional japonica rice. The rice material with increased tillering number and yield obtained by the invention does not contain transgenic components, avoids the safety risk possibly brought by transgenosis, only changes one gene, and does not affect other agronomic characters of rice varieties.
Example 2 leaf phenotype analysis of OsFW L4 Gene mutant
Analysis of the phenotype of homozygous mutant sword-leaf with no transgenic insertion and no detectable off-target effect of OsFW L4 gene shows that the length of the mutant sword-leaf is not significantly different from that of the wild type, but the width of the sword-leaf is significantly higher than that of the control (figure 6), which shows that the method provided by the invention can increase the width of rice leaf, enhance photosynthesis and further improve the yield of rice.
Observations of the sisal leaf epidermal cells using the method of Yoshikawa et al (Yoshikawa T, Eiguchi M, Hibara K, Ito J, Nagao Y. Ricesterder leaf 1 gene codes cell synthesis-like D4 and is specific layer expressed in M-phase cells to regulated cell proliferation. journal of Experimental Botany, 2013, 64 (7): 2049-2061) showed no significant difference in size between the mutant and wild type sisal leaf epidermal cells, indicating that the increase in the width of the mutant leaf is due to an increase in the number of cells rather than an increase in the size of cells.
Sequence listing
<110> Huaiyin college of learning professions
<120> method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield
<130>100
<160>22
<170>SIPOSequenceListing 1.0
<210>1
<211>663
<212>DNA
<213>Oryza sativa
<400>1
atggcgaggc cgcaacacaa tgactggtca tccggactct tcgcctgctt caatgactgc 60
gaagtttgtg cgtatatatt acttccatat tgtcgatcag tactatacta cttgcgatga 120
ttctatcgat cgtccttcac cagattatat atattgcatt tattgtatag accatctatt 180
aattctaagc taatttaagc ttatttcccg gaatatatat attgattgag taattttacc 240
atataacatt tggtaccata ttagatcatt ccttaatatt cacgtcttta tgtgacacaa 300
actaattata tatatgcagg ttgcttgacg acggtgtgcc cgtgcatcac gttcgggcgg 360
agcgcggaga tcgtgtcgag gggggagagg acgtgctgcg cggcgggcgt gatgtgcgtg 420
ctgctcggct tcttcgccca ctgccactgc ctctactcct gctgctaccg cggcaagatg 480
cgcgacagct tccacctccc cgaggaccca tgctgcgact gctgcgtcca cgccctctgc 540
ctgcagtgcg cgctgtgcca ggagtacagg cacctcaaga gcctcggcta caaaccctcg 600
cttggctggc tcggaaacaa ccagcacgtc ccgcccaagc acaacccgcc gatgcgacgc 660
taa 663
<210>2
<211>136
<212>PRT
<213>Oryza sativa
<400>2
Met Ala Arg Pro Gln His Asn Asp Trp Ser Ser Gly Leu Phe Ala Cys
1 510 15
Phe Asn Asp Cys Glu Val Cys Cys Leu Thr Thr Val Cys Pro Cys Ile
20 25 30
Thr Phe Gly Arg Ser Ala Glu Ile Val Ser Arg Gly Glu Arg Thr Cys
35 40 45
Cys Ala Ala Gly Val Met Cys Val Leu Leu Gly Phe Phe Ala His Cys
50 55 60
His Cys Leu Tyr Ser Cys Cys Tyr Arg Gly Lys Met Arg Asp Ser Phe
65 70 75 80
His Leu Pro Glu Asp Pro Cys Cys Asp Cys Cys Val His Ala Leu Cys
85 90 95
Leu Gln Cys Ala Leu Cys Gln Glu Tyr Arg His Leu Lys Ser Leu Gly
100 105 110
Tyr Lys Pro Ser Leu Gly Trp Leu Gly Asn Asn Gln His Val Pro Pro
115 120 125
Lys His Asn Pro Pro Met Arg Arg
130 135
<210>3
<211>348
<212>DNA
<213> sgRNA expression cassette (Artificial)
<400>3
ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60
aagcccgatt ctcttcgctg tgatgggctg gatgcatgcg ggggagcggg aggcccaagt 120
acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180
agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240
tgtgtgattg aagcaggcga agagtcgttt tagagctaga aatagcaagt taaaataagg 300
ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttt 348
<210>4
<211>6550
<212>DNA
<213> Cas9 nuclease expression cassette (Artificial)
<400>4
tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat 60
aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta 120
tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag 180
tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt 240
tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc 300
aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag 360
ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta ttttagcctc 420
taaattaaga aaactaaaac tctattttag tttttttatt taataattta gatataaaat 480
agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa 540
ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg tcgacgagtc 600
taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac 660
ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc 720
tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg 780
cggcctcctc ctcctctcac ggcacggcag ctacggggga ttcctttccc accgctcctt 840
cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc tctttcccca 900
acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat ccacccgtcg 960
gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct accttctcta 1020
gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca tgtttgtgtt 1080
agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc gacctgtacg 1140
tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc tgggatggct 1200
ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg catagggttt 1260
ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg gtcatctttt 1320
catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg ttctagatcg 1380
gagtagaatt ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt 1440
gccatacata ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag 1500
gtatacatgt tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg 1560
ttgtgatgat gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact 1620
gtttcaaact acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt 1680
catagttacg agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg 1740
tgggttttac tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc 1800
ttgagtacct atctattata ataaacaagt atgttttata attattttga tcttgatata 1860
cttggatgat ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc 1920
tatttatttg cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt 1980
ctgcagccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 2040
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 2100
cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg 2160
ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg 2220
accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag 2280
ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga 2340
tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc 2400
acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct 2460
tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga 2520
gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg 2580
cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca 2640
gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa 2700
accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga 2760
gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg 2820
gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg 2880
ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc 2940
tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg 3000
ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg 3060
cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg 3120
tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct 3180
acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca 3240
tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc 3300
tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc 3360
tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa 3420
agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa 3480
acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg 3540
aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg 3600
ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca 3660
ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct 3720
tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag 3780
tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg 3840
aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga 3900
aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata 3960
tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct 4020
atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct 4080
ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa 4140
tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg 4200
acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata 4260
gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc 4320
agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca 4380
tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg 4440
agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac 4500
accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg 4560
ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg 4620
accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca 4680
gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga 4740
tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca 4800
atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga 4860
gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga 4920
tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga 4980
agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca 5040
acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca 5100
aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc 5160
ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct 5220
acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga 5280
agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg 5340
attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg 5400
aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc 5460
tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg 5520
tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga 5580
gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca 5640
tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc 5700
ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg 5760
aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg 5820
ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg 5880
tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga 5940
gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg 6000
ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg 6060
gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca 6120
ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac 6180
ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc 6240
aggcaaaaaa gaaaaagtaa ggatcctgat tgatcgatag agctcgaatt tccccgatcg 6300
ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 6360
tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 6420
gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 6480
agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 6540
actagatcgg 6550
<210>5
<211>20
<212>DNA
<213>Oryza sativa
<400>5
attgaagcag gcgaagagtc 20
<210>6
<211>20
<212>DNA
<213>Oryza sativa
<400>6
cgcagcatgg gtcctcgggg 20
<210>7
<211>24
<212>DNA
<213>Osfwl4a-P1(Artificial)
<400>7
tgtgattgaa gcaggcgaag agtc 24
<210>8
<211>24
<212>DNA
<213>Osfwl4a-P2(Artificial)
<400>8
aaacgactct tcgcctgctt caat 24
<210>9
<211>24
<212>DNA
<213>Osfwl4b-P1(Artificial)
<400>9
tgtgcgcagc atgggtcctc gggg 24
<210>10
<211>24
<212>DNA
<213>Osfwl4b-P2(Artificial)
<400>10
aaacccccga ggacccatgc tgcg 24
<210>11
<211>19
<212>DNA
<213>Osfwl4aF(Artificial)
<400>11
cacaccgtcg tcaagcaac 19
<210>12
<211>20
<212>DNA
<213>Osfwl4aR(Artificial)
<400>12
tcatgctcat cgctcctagc 20
<210>13
<211>20
<212>DNA
<213>Osfwl4bF(Artificial)
<400>13
ggccggcagc taatttgaag 20
<210>14
<211>19
<212>DNA
<213>Osfwl4bR(Artificial)
<400>14
gttgcttgac gacggtgtg 19
<210>15
<211>21
<212>DNA
<213>Osfwl4aOFF-1F(Artificial)
<400>15
gttgcaggaa actgaaacat g 21
<210>16
<211>21
<212>DNA
<213>Osfwl4aOFF-1R(Artificial)
<400>16
ctgtagaaca atgccaatca c 21
<210>17
<211>20
<212>DNA
<213>Osfwl4aOFF-2F(Artificial)
<400>17
ggagcaaaca ccgaacaact 20
<210>18
<211>20
<212>DNA
<213>Osfwl4aOFF-2R(Artificial)
<400>18
ttcttcctgg agatcgccta 20
<210>19
<211>22
<212>DNA
<213>Osfwl4bOFF-1F(Artificial)
<400>19
acaaatcgac agaaaatcca aa 22
<210>20
<211>22
<212>DNA
<213>Osfwl4bOFF-1R(Artificial)
<400>20
ctccgtcttt ctcgatattc ct 22
<210>21
<211>19
<212>DNA
<213>Osfwl4bOFF-2F(Artificial)
<400>21
tcctggttat taagggttt 19
<210>22
<211>19
<212>DNA
<213>Osfwl4bOFF-2R(Artificial)
<400>22
tcctggctgt tgaggtaga 19

Claims (6)

1. Rice tillering number knockout regulation and control geneOsFWL4The method for increasing the tillering number and the yield of the rice is characterized in that the method utilizes the CRISPR/Cas9 technology to target and mutate the rice tillering number regulation and control geneOsFWL4The rice tillering number regulating geneOsFWL4The nucleotide sequence is shown in SEQ ID NO. 1.
2. The method as claimed in claim 1, wherein the CRISPR/Cas9 technology is used for targeting the rice tillering number controlling geneOsFWL4The method comprises the following specific steps:
1) gene for regulating and controlling tillering number of riceOsFWL4Selecting a target fragment from the coding region;
2) synthesis of adapter primers with sticky Ends based on sequence information of the target fragmentObject constructed forOsFWL4A gene-targeted CRISPR/Cas9 recombinant vector; the recombinant vector comprises an sgRNA expression cassette having the target sequence and a Cas9 nuclease expression cassette capable of being expressed in rice cells;
3) transferring the recombinant vector into an agrobacterium tumefaciens engineering strain, and transforming a conventional japonica rice variety by using an agrobacterium-mediated method to obtain a transgenic rice plant;
4) amplifying and sequencing the genome DNA of the transgenic plant by using a genome primer covering a target site, thereby carrying out genotype identification on the T0 generation transgenic plant and obtaining a homozygous or double allelic mutant;
5) predicting potential off-target sites of a target sequence by using a bioinformatics tool; then, respectively carrying out PCR amplification and sequence determination on two off-target sites with highest prediction probability in homozygous or biallelic T0 mutant by using genome primers to obtain homozygous or biallelic mutant without detectable off-target effect;
6) planting homozygous or biallelic mutant without detectable off-target effect in T1 generation, and using primer pair to obtain T1 generation plantHPTsgRNA andCas9and carrying out PCR detection on the transgenes to obtain the rice which has no transgene insertion, no detectable off-target effect and obviously increased single-plant tillering number and yield.
3. The method of claim 2, wherein in step 1), the sequence of the target fragment is SEQ ID No.5 or SEQ ID No. 6.
4. The method according to claim 2, wherein in step 2), the nucleotide sequence of the sgRNA expression cassette having the target sequence is shown in Seq ID No. 3; the nucleotide sequence of the Cas9 nuclease expression cassette is shown in Seq id No. 4.
5. The method of claim 2, wherein step 6) is used forHPTsgRNA andCas9the primer sequences of the transgenic amplification are respectively as follows:
HPTF：5’-GGGTGTCACGTTGCAAGACC-3’，
HPTR：5’-ATGCCTCCGCTCGAAGTAGC-3’，
sgRNAF：5’-TCCCAGTCACGACGTTGTAA-3’，
sgRNAR：5’-GGCCATTTGTCTGCAGAAT-3’，
Cas9F：5’-CACCATCTACCACCTGAGAA-3’，
Cas9R：5’-CGAAGTTGCTCTTGAAGTTG-3’。
6. the method according to claim 2, wherein in step 5) the bioinformatic tool is CRISPR-P2.0 or CRISPR-GE.
CN201911264946.8A 2019-12-10 2019-12-10 Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield Active CN110791525B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911264946.8A CN110791525B (en) 2019-12-10 2019-12-10 Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911264946.8A CN110791525B (en) 2019-12-10 2019-12-10 Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield

Publications (2)

Publication Number Publication Date
CN110791525A CN110791525A (en) 2020-02-14
CN110791525B true CN110791525B (en) 2020-07-14

Family

ID=69447941

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911264946.8A Active CN110791525B (en) 2019-12-10 2019-12-10 Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield

Country Status (1)

Country Link
CN (1) CN110791525B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112997966A (en) * 2021-03-08 2021-06-22 国家卫生健康委科学技术研究所 Mouse model knocking-in miRNA-125a based on CRISPR/Cas9 technology and construction method
CN113897372B (en) * 2021-10-08 2022-03-08 淮阴师范学院 Application of OsFWL7 gene in increasing content of metal trace elements in rice grains

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106939316A (en) * 2016-01-05 2017-07-11 复旦大学 The method for knocking out rice Os PDCD5 gene Second Exons is pinpointed using CRISPR/Cas9 systems
CN109306358A (en) * 2018-12-17 2019-02-05 湖南杂交水稻研究中心 The method for formulating not packet neck two-line sterile line of rice using CRISPR/Cas9 technology
CN109371041A (en) * 2018-12-19 2019-02-22 山东农业大学 It is a kind of increase grain number per spike paddy gene OsHGN and its application
CN109721649A (en) * 2019-03-14 2019-05-07 中国水稻研究所 A kind of plant type of rice regulation related gene, protein and application
CN110527687A (en) * 2019-07-25 2019-12-03 南京大学 A kind of rice transcription factor gene Osspl10 and its application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106939316A (en) * 2016-01-05 2017-07-11 复旦大学 The method for knocking out rice Os PDCD5 gene Second Exons is pinpointed using CRISPR/Cas9 systems
CN109306358A (en) * 2018-12-17 2019-02-05 湖南杂交水稻研究中心 The method for formulating not packet neck two-line sterile line of rice using CRISPR/Cas9 technology
CN109371041A (en) * 2018-12-19 2019-02-22 山东农业大学 It is a kind of increase grain number per spike paddy gene OsHGN and its application
CN109721649A (en) * 2019-03-14 2019-05-07 中国水稻研究所 A kind of plant type of rice regulation related gene, protein and application
CN110527687A (en) * 2019-07-25 2019-12-03 南京大学 A kind of rice transcription factor gene Osspl10 and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Molecular characterization and functional analysis of "fruit‑weight2.2‑like" gene family in rice;Jun Xu et al;《Planta》;20130621;全文 *
The rice "fruit‑weight 2.2‑like" gene family member OsFWL4 is involved in the translocation of cadmium from roots to shoots;Wentao Xiong et al;《Planta》;20180207;全文 *

Also Published As

Publication number Publication date
CN110791525A (en) 2020-02-14

Similar Documents

Publication Publication Date Title
CN107109427B (en) Methods and compositions for identifying and enriching cells comprising site-specific genomic modifications
CN100381465C (en) Bacterial leaf spot resistance related protein and its coding gene and uses
CN107164401A (en) A kind of method and application that rice Os PIL15 mutant is prepared based on CRISPR/Cas9 technologies
WO2003087313A2 (en) Enhanced silk exsertion under stress
CN110791525B (en) Method for knocking out rice tillering number regulation gene OsFW L4 to increase rice tillering number and yield
CN114540369B (en) Application of OsBEE1 gene in improving rice yield
CN113265422B (en) Method for targeted knocking out rice grain type regulatory gene SLG7, rice grain type regulatory gene SLG7 mutant and application thereof
CN111088263A (en) Tomato mSLBZR1L gene and application thereof
CN110592134A (en) Application of SDG40 gene or coding protein thereof
JP3051874B2 (en) How to make plants dwarf
CN114438114A (en) Method for improving rice blast resistance of rice
CN108341857B (en) Rice yield related protein and coding gene and application thereof
CN108409844B (en) Application of protein TaNRT2.5 in regulation and control of plant yield
CN115894646B (en) OsJDG1 gene and application thereof in regulation of rice grain type and thousand grain weight
CN114350687B (en) Rice bacterial leaf blight resistance gene, protein and application thereof
CN114231539B (en) Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof
CN113817033B (en) Application of ZmELF3.1 protein and its functional deletion mutant in regulating and controlling crop aerial root number or layer number
CN112195187B (en) Rice tillering angle regulation gene and protein coded by same and application of gene
CN109734786B (en) Plant pollen fertility restorer related protein TaDMT25, and coding gene and application thereof
CN110950944B (en) OsHCRF1 functional protein and application of coding gene thereof in rice breeding
CN110452914B (en) Gene BnC04BIN2-like1 for regulating brassinolide signal transduction and application thereof
CN108841840B (en) Application of protein TaNADH-GoGAT in regulation and control of plant yield
EP3913059A1 (en) Nematode resistance in plants
CN113897372B (en) Application of OsFWL7 gene in increasing content of metal trace elements in rice grains
CN108892712B (en) Application of protein TabZIP60 in regulation and control of plant yield

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
TR01 Transfer of patent right

Effective date of registration: 20231108

Address after: No. 85 North Street, Jingdian Town, Neihuang County, Anyang City, Henan Province, 455000

Patentee after: Anyang Hongzhixing Agricultural Technology Co.,Ltd.

Address before: 230000 floor 1, building 2, phase I, e-commerce Park, Jinggang Road, Shushan Economic Development Zone, Hefei City, Anhui Province

Patentee before: Dragon totem Technology (Hefei) Co.,Ltd.

Effective date of registration: 20231108

Address after: 230000 floor 1, building 2, phase I, e-commerce Park, Jinggang Road, Shushan Economic Development Zone, Hefei City, Anhui Province

Patentee after: Dragon totem Technology (Hefei) Co.,Ltd.

Address before: No. 111, Changjiang West Road, Huaian, Jiangsu, Jiangsu

Patentee before: HUAIYIN NORMAL University

TR01 Transfer of patent right