CN114395564B - Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening - Google Patents
Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening Download PDFInfo
- Publication number
- CN114395564B CN114395564B CN202210210632.5A CN202210210632A CN114395564B CN 114395564 B CN114395564 B CN 114395564B CN 202210210632 A CN202210210632 A CN 202210210632A CN 114395564 B CN114395564 B CN 114395564B
- Authority
- CN
- China
- Prior art keywords
- lrein3
- lycium ruthenicum
- gene
- ruthenicum murr
- seq
- 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
Links
Images
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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
The invention discloses application of a Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening. The Lycium ruthenicum LrEIN3 gene has a nucleotide sequence shown as SEQ ID No.1 or an amino acid sequence shown as SEQ ID No. 2. In the invention, the Lycium ruthenicum Murr EIN3 gene is edited by a CRIPSR-Cas9 technology, so that the Lycium ruthenicum Murr EIN3 gene is transferred into the Lycium ruthenicum Murr after mutation, and compared with the 32-day fruit ripening and color change of wild Lycium ruthenicum Murr, 34-day fruit color change of the LrEIN3 gene mutant plant begins, and the fruit ripening is delayed. The LrEIN3 gene can delay the natural ripening of fruits, thereby improving the shelf life of the lycium ruthenicum murr.
Description
Technical Field
The invention belongs to the technical field of biotechnology and genetic engineering, and particularly relates to an Lycium ruthenicum Murr EIN3 gene and application of a protein coded by the gene in delaying maturation of Lycium ruthenicum Murr.
Background
Lycium ruthenicum (Lycium ruthenicum) is a perennial deciduous shrub of Lycium of Solanaceae, and fruits of Lycium ruthenicum are rapidly softened after ripening, so that dry and ripe fruits are mainly used in the Lycium ruthenicum market, and the development of the industry of Lycium ruthenicum is greatly restricted.
The gene editing technology can realize the fixed-point knockout of specific genes so as to create artificial mutants. For non-model plants, the gene editing technology can accelerate the species domestication process, obtain mutants by a short-time efficient biotechnology means, and break the seed barriers of the traditional breeding mode. For example, the banana MaACO1 gene can be edited at a fixed point by a gene editing technology to delay the natural ripening process of bananas and improve the shelf life of bananas (Hu et al, 2021).
Therefore, the gene capable of delaying the fruit ripening of the lycium ruthenicum is researched and developed from the lycium ruthenicum, and the gene is edited at fixed points by a gene editing technology, so that the fruit ripening of the lycium ruthenicum is delayed, the ripening and softening process is slowed down, and the method has important significance for prolonging the shelf life of the lycium ruthenicum.
Disclosure of Invention
Based on the situation, one of the purposes of the invention is to provide an application of the Lycium ruthenicum Murr EIN3 gene in delaying the fruit ripening of the Lycium ruthenicum Murr.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
the application of the Lycium ruthenicum Murr EIN3 gene in delaying the maturation of Lycium ruthenicum Murr fruit is disclosed, wherein the Lycium ruthenicum Murr EIN3 gene codes a protein with an amino acid sequence shown as SEQ ID No. 2.
In some embodiments, the lycium ruthenicum LrEIN3 gene has a nucleotide sequence shown as SEQ ID No. 1.
The invention also provides application of the Lycium ruthenicum Murr EIN3 protein in delaying the fruit ripening of Lycium ruthenicum Murr, wherein the Lycium ruthenicum Murr EIN3 protein has an amino acid sequence shown in SEQ ID No. 2.
The invention also provides application of the Lycium ruthenicum Murr EIN3 gene or the Lycium ruthenicum Murr EIN3 protein in Lycium ruthenicum Murr genetic breeding.
The invention also provides a Lycium ruthenicum LrEIN3 gene editing vector, wherein the Lycium ruthenicum LrEIN3 gene codes a protein with an amino acid sequence shown in SEQ ID No. 2.
In some embodiments, the lycium ruthenicum LrEIN3 gene has a nucleotide sequence as shown in SEQ ID No. 1.
In some embodiments, the Lycium ruthenicum LrEIN3 gene editing vector is pAGM4723:: CR-LrEIN3, and the Lycium ruthenicum LrEIN3 gene editing vector has a nucleotide sequence shown as SEQ ID No. 8.
The invention also provides application of the Lycium ruthenicum Murr EIN3 gene editing vector in delaying maturation of Lycium ruthenicum Murr fruits.
The invention also provides application of the Lycium ruthenicum Murr EIN3 gene editing vector in Lycium ruthenicum Murr genetic breeding.
The invention also provides an engineering bacterium transformed with the Lycium ruthenicum LrEIN3 gene editing vector.
In some of these embodiments, the engineered bacterium is Agrobacterium GV3101 transformed with pAGM4723:: CR-LrEIN 3.
The invention also provides application of the engineering bacteria in delaying the fruit ripening of lycium ruthenicum.
The invention also provides application of the engineering bacteria in Lycium ruthenicum Murr genetic breeding.
The invention also provides a method for delaying the fruit ripening of lycium ruthenicum.
The technical scheme for realizing the aim of the invention comprises the following steps:
a method of delaying the ripening of lycium ruthenicum fruit, the method comprising the steps of: editing the Lycium ruthenicum LrEIN3 gene by using a CRISPR-Cas9 system to lose the function of the Lycium ruthenicum LrEIN3 gene; the Lycium ruthenicum LrEIN3 gene codes a protein with an amino acid sequence shown in SEQ ID No. 2.
In some embodiments, the lycium ruthenicum LrEIN3 gene has a nucleotide sequence shown as SEQ ID No. 1.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, the LrEIN3 gene homozygous mutant plant is obtained by transferring the vector which is obtained by accurately editing the LrEIN3 gene by adopting the CRIPSR-Cas9 gene editing technology into Lycium ruthenicum, and compared with a wild type plant with 32 days, the fruit becomes mature in a color-changing way, and the fruit of the LrEIN3 gene mutant plant begins to change the color in 34 days. The LrEIN3 gene can delay the natural maturation of the lycium ruthenicum murr fruits and is beneficial to prolonging the shelf life of the lycium ruthenicum murr.
Drawings
FIG. 1 is a diagram showing the domain analysis of the LrEIN3 transcription factor of Lycium ruthenicum in example 1 of the present invention.
FIG. 2 is a schematic structural view of the recombinant vector constructed in example 1 of the present invention.
FIG. 3 shows the PCR result of the T0 LrEIN3 gene editing Lycium ruthenicum Murr plant in example 2 of the present invention; wherein, WT is an untransformed wild type plant, lrEIN3-4, lrEIN3-9, lrEIN3-10, lrEIN3-11 and LrEIN3-16 are T0 generation LrEIN3 gene editing Lycium ruthenicum Murr plant.
FIG. 4 shows the genotype analysis results of Lycium ruthenicum Murr plant edited by LrEIN3 gene of the T0 generation in example 2 of the present invention; wherein, WT is an untransformed wild type plant, lrEIN3-4, lrEIN3-9 and LrEIN3-11 are T0 generation LrEIN3 gene editing lycium ruthenicum plant.
FIG. 5 shows the fruit phenotype observed 37 days after the T0 generation LrEIN3 gene editing of Lycium ruthenicum Murr flowers in example 3 of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The experimental procedures used in the following examples are generally carried out under conventional conditions, for example, as described in the molecular cloning protocols (second edition, written by J. SammBruk et al, huang Petang et al, science publishers, 2002), or as recommended by the manufacturers, unless otherwise specified.
In the invention, the variety of the lycium ruthenicum can be a conventional variety of the lycium ruthenicum and can also be a germplasm resource of the wild lycium ruthenicum. In the following examples, lycium ruthenicum murr, a wild species, was used in zhongning county, zhongwei city, zhongxia hui nationality.
The nucleotide sequence of the LrEIN3 gene is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.
SEQ ID No.1
ATGATGATGTTTGAAGAGATCGGGTTTTGTGGTGATCTAGATTTCTTCCCCACTCCGCTAAAGGAGGTGGAAGCCGCTGCTCCACAGGGTGATCCGGAGCCGTTGATGGACGACGATTATAGTGATGAAGAGATTGAAGTTGATGAGTTGGAGAGGAGGATGTGGAGGGATAAAATGAAGCTTAAAAGGCTTAAAGAAATGAGTAGTAAGGGCAAGGAAGGTGTTGACTCGGTCAAACAACGCCAGTCTCAGGAGCAAGCGAGGAGGAAGAAGATGTCGAGGGCACAAGATGGGATCTTGAAGTACATGTTGAAGATGATGGAAGTATGTAAAGCTCAGGGTTTCGTTTACGGAATTATCCCTGAGAAAGGCAAACCGGTGACCGGGGCTTCTGATAATCTAAGGGAGTGGTGGAAGGATAAGGTCAGGTTCGATCGCAACGGGCCTGCCGCCATAGCGAAGTACCAAGCTGATAACGCCATCCCTGGCAAGAACGAGGGATCTAATCCGATTGGTCCGACCCCTCACACCTTGCAGGAGCTTCAAGATACCACTCTTGGTTCTTTATTGTCAGCTTTAATGCAACATTGTGATCCTCCTCAGAGGCGATTCCCATTGGAGAAAGGCGTTGCACCTCCATGGTGGCCTAATGGACTGGAGGATTGGTGGCCTCAATTGGGACTTCCGAAGGATCAAGGTCCTCCACCTTATAAGAAGCCTCATGATCTGAAGAAGGCCTGGAAGGTTGGTGTTCTCACAGCAGTGATCAAGCATATGTCCCCTGATATTGCTAAGATTCGCAACCTGGTAAGGCAATCAAAGTGCTTGCAGGACAAGATGACGGCGAAGGAAAGTGCAACTTGGCTTGCCATCATCAATCAGGAGGAAGTCTTGGCTCGAGAACTTTATCCTGATCGCTGTCCGCCTTTGTCCTCCGCTGGCGGTAGTGGAACTTTCACTATGAATGACAGCAGCGAGTATGATGTTGAAGGTGCCGTTGATGACCCTATTAACTTTGATGCTCAAGAGCAAAAACCAAACCATCTAGGTTTGCTGAATGCTAATGTCGATATGTTTAAGGAGAGGCTACCTCTGCAACAGCAATCTCATCCAATCAAGGATGAAATTATTGCCAGCTTAGATTTCACTCGGAAGAGAAAGCCGGCTGATGACCTGACTTTTTTGATGGATCAGAAGATATATGTTTGTGAGTGTCTTCAATGTCCGCATAGTGAGCTCCGCCATGGATTTCCGGACAGATCCATTAGAGACAATCATCAGTTAAGTTGCCCTTACAGAAATCCTTCGCAATTTGGAGTTTCAAACTTTCACGTGGATGAAGTCAAGCCGGTTTTCCCGCAACAATATGTTCAACCAAAGACGGCTTCTCTGGCGGTTAACCCAGCTCCACCGTCCTTCGATCTATCAGGACTTGGGGTTCCTGAAGACGGGCACAGGATGATCAATGACCTTATGTCATTCTATGATAGTAACGTACAAGGAAATAAAAGCTCAATGGTTGGGAATGTTGAGCAGCCTCGTAAACAACCTAGTGTTCAACAGAACAATTACCTACAAAGCCAAGGAATTGTGTTGGAGGGAAATGTCTTTGGGGACTCCAACATTTCTGCTAATCATAATTCCGTGTTCGCGCAAGGAGATCGGTTTGATCAGAGCAAGGCTTTAACTTCACCGTTCAATGCAGGCTCTAGTGACAATTTCCATTTCATGTTCGGGTCTTCATTCAATTTACAATCCACCGATTACTCTGAAGGTCTTTCTGGGATCTCACATGATAGCTTGCCGAAGCAAGATGTTCCGGTTTGGTACT
SEQ ID No.2
MMMFEEIGFCGDLDFFPTPLKEVEAAAPQGDPEPLMDDDYSDEEIEVDELERRMWRDKMKLKRLKEMSSKGKEGVDSVKQRQSQEQARRKKMSRAQDGILKYMLKMMEVCKAQGFVYGIIPEKGKPVTGASDNLREWWKDKVRFDRNGPAAIAKYQADNAIPGKNEGSNPIGPTPHTLQELQDTTLGSLLSALMQHCDPPQRRFPLEKGVAPPWWPNGLEDWWPQLGLPKDQGPPPYKKPHDLKKAWKVGVLTAVIKHMSPDIAKIRNLVRQSKCLQDKMTAKESATWLAIINQEEVLARELYPDRCPPLSSAGGSGTFTMNDSSEYDVEGAVDDPINFDAQEQKPNHLGLLNANVDMFKERLPLQQQSHPIKDEIIASLDFTRKRKPADDLTFLMDQKIYVCECLQCPHSELRHGFPDRSIRDNHQLSCPYRNPSQFGVSNFHVDEVKPVFPQQYVQPKTASLAVNPAPPSFDLSGLGVPEDGHRMINDLMSFYDSNVQGNKSSMVGNVEQPRKQPSVQQNNYLQSQGIVLEGNVFGDSNISANHNSVFAQGDRFDQSKALTSPFNAGSSDNFHFMFGSSFNLQSTDYSEGLSGISHDSLPKQDVPVWY
In the invention, firstly, a Lycium ruthenicum LrEIN3 gene editing vector pAGM4723: CR-LrEIN3 is provided, and the vector has a nucleotide sequence shown as SEQ ID No. 8. The lycium ruthenicum LrEIN3 gene editing vector is obtained by editing a lycium ruthenicum LrEIN3 gene by using a CRISPR/Cas9 system, wherein the CRISPR/Cas9 system comprises a Cas9 protein coding gene and two sgRNA coding genes, namely sgRNA1 (SEQ ID No. 3) and sgRNA2 (SEQ ID No. 4).
The invention also provides an engineering bacterium transformed with the Lycium ruthenicum LrEIN3 gene editing vector, wherein the engineering bacterium is agrobacterium GV3101 transformed with a recombinant vector pAGM4723:: CR-LrEIN 3.
pGreen II 62-SK, pGreen II0800, pGADT7, pAbAi, pICH 86966:. AtU6 p:. SgRNA _ PDS, pICSL 01009:. AtU6p, pICH47751, pICH47761, pAGM4723, pICH41780, pICH 47742:. 2x35S-5 dge UTR-hCas9 (STOP) -NOST and pICH 47732:. NOSp-NPTII-OCST vectors, pGreen II0800-LUC, all available from the Adne vector library (http:///w.adwdgen. Org.).
The PCR Buffer KOD-Plus and KOD-Plus DNA polymerases used in the following examples are products of Toyo textile Biotech Ltd; the restriction enzyme, T4 ligase and DNA ligation kit are products of NEB company; the PCR product purification kit and the plant genome DNA extraction kit are both products of Guangzhou Meiji biological technology limited company; MS culture medium is purchased from Beijing ZhengYongwei science and technology development Limited, and has a product number of M519; GV3101 is competence purchased from Shanghai Toyu Biotechnology, inc. (cat # AC 1001S); the primer is synthesized by Beijing Optimae Biotechnology Limited; sequencing was performed by Biotechnology engineering (Shanghai) Inc.; the rest reagents are analytically pure reagents. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.
The present invention is described in detail below with reference to specific embodiments and the attached drawings.
Example 1 CRIPSR-Cas9 Gene editing vector pAGM4723 construction of CR-LrEIN3 and transformation of Agrobacterium
In the embodiment, CRIPSR-Cas9 gene editing vector pAGM4723:: CR-LrEIN3 is firstly constructed, then the gene editing vector pAGM4723:: CR-LrEIN3 is transformed into agrobacterium GV3101 to obtain recombinant agrobacterium pAGM4723:: CR-LrEIN3/GV3101, and the method specifically comprises the following steps:
1. obtaining an LrEIN3 gene CRIPSR-Cas9 gene editing target sequence
Genomic information of LrEIN3 is obtained from the genomic information of Lycium ruthenicum Murr, the structural domain of transcription factor of LrEIN3 gene (SEQ ID No. 1) is analyzed, and the analysis result is shown in FIG. 1.
And selecting a sequence with high score, exon region and 20bp length at the front end of the NGG sequence on the LrEIN3 gene sequence by using online software http// skl.scau.edu.cn/targettdisign/. Two sgRNA targets capable of improving the editing efficiency are finally selected through design, and the specific sequences of the sgRNAs are as follows:
sgRNAl(SEQ ID NO.3)：
5'-GCGGCTTCCACCTCCTTTAG-3'
sgRNA2(SEQ ID NO.4)：
5'-GAGAAAGGCAAACCGGTGAC-3'
2. CRIPSR-Cas9 gene editing vector for constructing LrEIN3 gene
1. Design of sgRNA amplification primers
Designing and constructing an sgRNA amplification primer according to a selected target sequence, which specifically comprises the following steps:
LrEIN3-g1(SEQ ID No.5)：
5'-TGTGGTCTCAATTGGCGGCTTCCACCTCCTTTAGGTTTTAGAGCTAGAAATAGCAAG-3'
LrEIN3-g2(SEQ ID No.6)：
5'-TGTGGTCTCAATTGGAGAAAGGCAAACCGGTGACGTTTTAGAGCTAGAAATAGCAAG-3'
sgRNA-R(SEQ ID No.7)：
5’-TGTGGTCTCAAGCGTAATGCCAACTTTGTAC-3’
2. CRIPSR-Cas9 gene editing vector pAGM4723 construction of CR-LrEIN3
a. Construction of recombinant plasmids pICH47751, lrEIN3-sgRNA1 and pICH47761, lrEIN3-sgRNA2
PCR amplification is carried out by taking pICH86966:: atU6p:: sgRNA _ PDS as a template and LrEIN3-g1 (SEQ ID No. 5) and sgRNA-R (SEQ ID No. 7), lrEIN3-g2 (SEQ ID No. 6) and sgRNA-R (SEQ ID No. 7) as primers respectively.
The PCR reaction system is as follows: 10 XPCR Buffer KOD-Plus 5. Mu.L, 25mM MgSO 4 2 mu L, forward primer LrEIN3-g1/LrEIN3-g 2.5 mu L, reverse primer sgRNA-R1.5 mu L, 2mM dNTPs5 mu L, template 10-200 ng,KOD-Plus-DNA polymerase 1. Mu.L, double distilled water was added to 50. Mu.L.
The PCR reaction program is: denaturation at 94 ℃ for 120 seconds; denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 32 cycles; keeping the temperature at 4 ℃.
Amplifying to obtain target sgRNA-1 and sgRNA-2 nucleotide fragments with Bsa I enzyme cutting sites on two side bands; purifying a PCR amplification product by using a PCR product purification kit, carrying out enzyme digestion on the purified PCR amplification product by using Bsa I, and connecting sgRNA-1 with a pICH47751 vector and a pICHSL 01009 vector subjected to the same enzyme digestion under the action of a DNA connection kit to obtain a recombinant plasmid pICH47751 and LrEIN3-sgRNA1; the sgRNA-2 is connected with a pICH47761 vector and a pICHSL 01009 vector which are digested by the same enzyme under the action of a DNA connecting kit to obtain a recombinant plasmid pICH47761 vector and LrEIN3-sgRNA2.
b. CRIPSR-Cas9 gene editing vector pAGM4723 construction of CR-LrEIN3
According to the literature "Soyk S, muller NA, park SJ, schmalenbach I, jiang K, hayama R, et al.variation in the marketing gene SELF PRUNING 5G proteins day-neutral and early yield in the science genetics.2017;49 162-168 'using Golden Gate cloning technology to recombine pICH47751: lrEIN3-sgRNA1, pICH47761: lrEIN3-sgRNA2, pICH47732: NOSp-NPTII-OCST, pICH47742: 2x35S-5' UTR-hCas9 (STOP) -NOST, pICH41780 and pAGM 23 to obtain CRIPSR-Cas9 gene editing recombination vector pAGM4723: CR-LrEIN3 (wherein CR is an abbreviation for CRISPR and represents gene editing), the structure diagram of the recombination vector is shown in FIG. 2, and the nucleotide sequence is shown in SEQ ID No. 8.
3. The gene editing vector pAGM 4723:CR-LrEIN 3 is used for transforming agrobacterium
Placing 1 μ g of the gene editing vector pAGM4723 prepared in step two in 100 μ L of GV3101 competence, quickly freezing in liquid nitrogen for 5 minutes, water bathing at 37 ℃ for 5 minutes, adding 1mL of LB culture medium, and culturing at 28 ℃ for 4 hours; spread on LB plate containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL rifampicin, and cultured in dark at 28 ℃ for 2 days; a single colony was picked, inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL rifampicin, and cultured overnight at 28 ℃ with shaking.
Performing PCR identification on the bacterial liquid, performing PCR amplification (a PCR reaction system and a reaction program are the same as the second step) by using Cas9-F (5.
Example 2 obtaining and identifying Lycium ruthenicum Murr plant edited by T0-generation LrEIN3 Gene
In the embodiment, a T0 generation LrEIN3 gene edited Lycium ruthenicum Murr plant is obtained and identified, and the specific method comprises the following steps:
1. the recombinant bacterium pAGM4723 of example 1, CR-LrEIN3/GV3101, was transformed into Lycium ruthenicum leaf disc explants (the transformation method is referred to university of Chinese academy of sciences, schering Master's paper), and then cultured in MS solid medium (pH 5.8) containing 0.5 mg/L6-benzylaminopurine and 0.4 mg/L1-naphthylacetic acid at 25 ℃ under dark conditions for 48 hours; transferring into MS solid culture medium (pH 5.8) containing 0.5 mg/L6-benzylaminopurine, 0.4 mg/L1-naphthalene acetic acid, 200mg/L cefuroxime and 50mg/L kanamycin, and culturing at 25 deg.C under photoperiod of 16h/d and illumination intensity of 3000lux until regeneration bud grows out; cutting the regeneration bud when the regeneration bud grows to 2-3 cm, transferring the regeneration bud into an MS solid culture medium (pH is 5.8) containing 200mg/L of cefuroxime and 50mg/L of kanamycin, and culturing the regeneration bud to root under the conditions of 25 ℃, 16h/d of photoperiod and 3000lux of illumination intensity, namely a T0 generation regeneration plant.
2. The T0 generation regeneration plant DNA is extracted by adopting a plant genome DNA extraction kit, and PCR amplification is carried out by taking the DNA as a template and CR-LrEIN3-F (SEQ ID No. 11) and CR-LrEIN3-R (SEQ ID No. 12) as primers.
The PCR reaction system is as follows: 10 XPCRBufferKOD-Plus 5. Mu.L, 25mM MgSO 4 2 mu L, 1.5 mu L of forward primer CR-LrEIN3-F, 1.5 mu L of reverse primer CR-LrEIN3-R, 5 mu L of 2 mdNTPs, 10-200 ng of template, 1 mu L of KOD-Plus-DNase and double distilled water to 50 mu L.
The primer sequences are as follows:
CR-LrEIN3-F(SEQ ID No.11)：
5'-GCCTTTGGTTGTTGCTATATGTAAT-3'
CR-LrEIN3-R(SEQ ID No.12)：
5'-GATGGCGTTATCAGCTTGGTACT-3'
the PCR reaction program is: denaturation at 94 ℃ for 120 seconds; denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 32 cycles; keeping the temperature at 4 ℃.
3. The results of nucleic acid electrophoresis analysis of the obtained PCR products are shown in FIG. 3, and the sizes of the PCR products of lrein3-4, lrein3-9, lrein3-10, lrein3-11 and lrein3-16 are consistent with those of the wild-type fragment compared with the wild-type plant WT, indicating that large fragment deletion does not occur at the genome level.
Further, the PCR products were analyzed by T-A clone sequencing, and lrein3-10 and lrein3-16 were found to exist in the wild-type genome and were not completely edited. The results of genotyping lrein3-4, lrein3-9 and lrein3-11 are shown in FIG. 4. As can be seen from fig. 4: compared with wild genome information, the lrein3-4 has a nucleic acid mutation type of-1 bp/+1bp, the lrein3-9 has a nucleic acid fragment deletion of-6 bp/-6bp, one site of the other allele has InDel of-36 bp/+27bp, and the other site has a mutant type of-6 bp; lrein3-11 shows the insertion of +1bp/-6bp bases. Sequencing results of the mutants indicate that LrEIN3-4, lrEIN3-9 and LrEIN3-11 are all LrEIN3 gene homozygous mutant plants.
Example 3 study of LrEIN3 Gene editing fruit ripening in Lycium ruthenicum plants
The phenotype of fruits of Lycium ruthenicum Murr plants LrEIN3-4 and LrEIN3-9 after flowering is continuously observed for the LrEIN3 gene obtained in example 2, and the result is shown in FIG. 5.
As can be seen from fig. 5: the fruit development of the Lycium ruthenicum Murr plant edited by the LrEIN3 gene is obviously influenced. The wild lycium ruthenicum plant has the advantages that fruits suddenly turn colors and mature 32 days after blooming, and then the fruits suddenly expand; the LrEIN3 gene edits the Lycium ruthenicum Murr plant LrEIN3-4, the fruit is slightly changed in color in 32 days after flowering, the color is slightly darker in 34 days, and the fruit is completely mature after 37 days; the LrEIN3 gene edited Lycium ruthenicum Murr plant LrEIN3-9 has smaller fruits after flowering, the fruits begin to turn color until 34 days, the sizes of the fruits do not change obviously, the colors of the fruits deepen after 37 days, and the sizes of the fruits begin to expand; the results show that the fruit development time of the Lycium ruthenicum Murr plant edited by the LrEIN3 gene is prolonged, and the fruit ripening is delayed.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> south China botanical garden of Chinese academy of sciences
Application of <120> Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening
<130> 1
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1833
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgatgatgt ttgaagagat cgggttttgt ggtgatctag atttcttccc cactccgcta 60
aaggaggtgg aagccgctgc tccacagggt gatccggagc cgttgatgga cgacgattat 120
agtgatgaag agattgaagt tgatgagttg gagaggagga tgtggaggga taaaatgaag 180
cttaaaaggc ttaaagaaat gagtagtaag ggcaaggaag gtgttgactc ggtcaaacaa 240
cgccagtctc aggagcaagc gaggaggaag aagatgtcga gggcacaaga tgggatcttg 300
aagtacatgt tgaagatgat ggaagtatgt aaagctcagg gtttcgttta cggaattatc 360
cctgagaaag gcaaaccggt gaccggggct tctgataatc taagggagtg gtggaaggat 420
aaggtcaggt tcgatcgcaa cgggcctgcc gccatagcga agtaccaagc tgataacgcc 480
atccctggca agaacgaggg atctaatccg attggtccga cccctcacac cttgcaggag 540
cttcaagata ccactcttgg ttctttattg tcagctttaa tgcaacattg tgatcctcct 600
cagaggcgat tcccattgga gaaaggcgtt gcacctccat ggtggcctaa tggactggag 660
gattggtggc ctcaattggg acttccgaag gatcaaggtc ctccacctta taagaagcct 720
catgatctga agaaggcctg gaaggttggt gttctcacag cagtgatcaa gcatatgtcc 780
cctgatattg ctaagattcg caacctggta aggcaatcaa agtgcttgca ggacaagatg 840
acggcgaagg aaagtgcaac ttggcttgcc atcatcaatc aggaggaagt cttggctcga 900
gaactttatc ctgatcgctg tccgcctttg tcctccgctg gcggtagtgg aactttcact 960
atgaatgaca gcagcgagta tgatgttgaa ggtgccgttg atgaccctat taactttgat 1020
gctcaagagc aaaaaccaaa ccatctaggt ttgctgaatg ctaatgtcga tatgtttaag 1080
gagaggctac ctctgcaaca gcaatctcat ccaatcaagg atgaaattat tgccagctta 1140
gatttcactc ggaagagaaa gccggctgat gacctgactt ttttgatgga tcagaagata 1200
tatgtttgtg agtgtcttca atgtccgcat agtgagctcc gccatggatt tccggacaga 1260
tccattagag acaatcatca gttaagttgc ccttacagaa atccttcgca atttggagtt 1320
tcaaactttc acgtggatga agtcaagccg gttttcccgc aacaatatgt tcaaccaaag 1380
acggcttctc tggcggttaa cccagctcca ccgtccttcg atctatcagg acttggggtt 1440
cctgaagacg ggcacaggat gatcaatgac cttatgtcat tctatgatag taacgtacaa 1500
ggaaataaaa gctcaatggt tgggaatgtt gagcagcctc gtaaacaacc tagtgttcaa 1560
cagaacaatt acctacaaag ccaaggaatt gtgttggagg gaaatgtctt tggggactcc 1620
aacatttctg ctaatcataa ttccgtgttc gcgcaaggag atcggtttga tcagagcaag 1680
gctttaactt caccgttcaa tgcaggctct agtgacaatt tccatttcat gttcgggtct 1740
tcattcaatt tacaatccac cgattactct gaaggtcttt ctgggatctc acatgatagc 1800
ttgccgaagc aagatgttcc ggtttggtac taa 1833
<210> 2
<211> 610
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Met Met Phe Glu Glu Ile Gly Phe Cys Gly Asp Leu Asp Phe Phe
1 5 10 15
Pro Thr Pro Leu Lys Glu Val Glu Ala Ala Ala Pro Gln Gly Asp Pro
20 25 30
Glu Pro Leu Met Asp Asp Asp Tyr Ser Asp Glu Glu Ile Glu Val Asp
35 40 45
Glu Leu Glu Arg Arg Met Trp Arg Asp Lys Met Lys Leu Lys Arg Leu
50 55 60
Lys Glu Met Ser Ser Lys Gly Lys Glu Gly Val Asp Ser Val Lys Gln
65 70 75 80
Arg Gln Ser Gln Glu Gln Ala Arg Arg Lys Lys Met Ser Arg Ala Gln
85 90 95
Asp Gly Ile Leu Lys Tyr Met Leu Lys Met Met Glu Val Cys Lys Ala
100 105 110
Gln Gly Phe Val Tyr Gly Ile Ile Pro Glu Lys Gly Lys Pro Val Thr
115 120 125
Gly Ala Ser Asp Asn Leu Arg Glu Trp Trp Lys Asp Lys Val Arg Phe
130 135 140
Asp Arg Asn Gly Pro Ala Ala Ile Ala Lys Tyr Gln Ala Asp Asn Ala
145 150 155 160
Ile Pro Gly Lys Asn Glu Gly Ser Asn Pro Ile Gly Pro Thr Pro His
165 170 175
Thr Leu Gln Glu Leu Gln Asp Thr Thr Leu Gly Ser Leu Leu Ser Ala
180 185 190
Leu Met Gln His Cys Asp Pro Pro Gln Arg Arg Phe Pro Leu Glu Lys
195 200 205
Gly Val Ala Pro Pro Trp Trp Pro Asn Gly Leu Glu Asp Trp Trp Pro
210 215 220
Gln Leu Gly Leu Pro Lys Asp Gln Gly Pro Pro Pro Tyr Lys Lys Pro
225 230 235 240
His Asp Leu Lys Lys Ala Trp Lys Val Gly Val Leu Thr Ala Val Ile
245 250 255
Lys His Met Ser Pro Asp Ile Ala Lys Ile Arg Asn Leu Val Arg Gln
260 265 270
Ser Lys Cys Leu Gln Asp Lys Met Thr Ala Lys Glu Ser Ala Thr Trp
275 280 285
Leu Ala Ile Ile Asn Gln Glu Glu Val Leu Ala Arg Glu Leu Tyr Pro
290 295 300
Asp Arg Cys Pro Pro Leu Ser Ser Ala Gly Gly Ser Gly Thr Phe Thr
305 310 315 320
Met Asn Asp Ser Ser Glu Tyr Asp Val Glu Gly Ala Val Asp Asp Pro
325 330 335
Ile Asn Phe Asp Ala Gln Glu Gln Lys Pro Asn His Leu Gly Leu Leu
340 345 350
Asn Ala Asn Val Asp Met Phe Lys Glu Arg Leu Pro Leu Gln Gln Gln
355 360 365
Ser His Pro Ile Lys Asp Glu Ile Ile Ala Ser Leu Asp Phe Thr Arg
370 375 380
Lys Arg Lys Pro Ala Asp Asp Leu Thr Phe Leu Met Asp Gln Lys Ile
385 390 395 400
Tyr Val Cys Glu Cys Leu Gln Cys Pro His Ser Glu Leu Arg His Gly
405 410 415
Phe Pro Asp Arg Ser Ile Arg Asp Asn His Gln Leu Ser Cys Pro Tyr
420 425 430
Arg Asn Pro Ser Gln Phe Gly Val Ser Asn Phe His Val Asp Glu Val
435 440 445
Lys Pro Val Phe Pro Gln Gln Tyr Val Gln Pro Lys Thr Ala Ser Leu
450 455 460
Ala Val Asn Pro Ala Pro Pro Ser Phe Asp Leu Ser Gly Leu Gly Val
465 470 475 480
Pro Glu Asp Gly His Arg Met Ile Asn Asp Leu Met Ser Phe Tyr Asp
485 490 495
Ser Asn Val Gln Gly Asn Lys Ser Ser Met Val Gly Asn Val Glu Gln
500 505 510
Pro Arg Lys Gln Pro Ser Val Gln Gln Asn Asn Tyr Leu Gln Ser Gln
515 520 525
Gly Ile Val Leu Glu Gly Asn Val Phe Gly Asp Ser Asn Ile Ser Ala
530 535 540
Asn His Asn Ser Val Phe Ala Gln Gly Asp Arg Phe Asp Gln Ser Lys
545 550 555 560
Ala Leu Thr Ser Pro Phe Asn Ala Gly Ser Ser Asp Asn Phe His Phe
565 570 575
Met Phe Gly Ser Ser Phe Asn Leu Gln Ser Thr Asp Tyr Ser Glu Gly
580 585 590
Leu Ser Gly Ile Ser His Asp Ser Leu Pro Lys Gln Asp Val Pro Val
595 600 605
Trp Tyr
610
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgtggtctca attggcggct tccacctcct ttaggtttta gagctagaaa tagcaag 57
<210> 6
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgtggtctca attggagaaa ggcaaaccgg tgacgtttta gagctagaaa tagcaag 57
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgtggtctca agcgtaatgc caactttgta c 31
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcctttggtt gttgctatat gtaat 25
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gatggcgtta tcagcttggt act 23
Claims (5)
1. Lycium ruthenicum MurrLrEIN3Application of gene in delaying fruit ripening of lycium ruthenicum murr, wherein lycium ruthenicum murrLrEIN3The gene codes the protein with the amino acid sequence shown as SEQ ID No. 2.
2. The use of claim 1, wherein lycium ruthenicum isLrEIN3The nucleotide sequence of the gene is shown in SEQ ID No. 1.
3. Lycium ruthenicum MurrLrEIN3Application of protein in delaying fruit ripening of lycium ruthenicum murrLrEIN3The amino acid sequence of the protein is shown as SEQ ID No. 2.
4. A method for delaying the ripening of Lycium ruthenicum Murr fruits, which comprises the following steps: lycium ruthenicum Murr by using CRISPR-Cas9 systemLrEIN3Editing genes to obtain Lycium ruthenicum MurrLrEIN3Loss of function of the gene; the lycium ruthenicum murrLrEIN3The gene codes the protein with the amino acid sequence shown as SEQ ID No. 2.
5. The method of claim 4, wherein the Lycium ruthenicum fruit ripening is delayedLrEIN3The nucleotide sequence of the gene is shown in SEQ ID No. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210210632.5A CN114395564B (en) | 2022-03-04 | 2022-03-04 | Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210210632.5A CN114395564B (en) | 2022-03-04 | 2022-03-04 | Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114395564A CN114395564A (en) | 2022-04-26 |
| CN114395564B true CN114395564B (en) | 2023-02-17 |
Family
ID=81234578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210210632.5A Active CN114395564B (en) | 2022-03-04 | 2022-03-04 | Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114395564B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005953A2 (en) * | 1999-07-15 | 2001-01-25 | Pioneer Hi-Bred International, Inc. | Maize ethylene signalling pathway ein3 genes and uses thereof |
| CN101368183A (en) * | 2008-05-22 | 2009-02-18 | 重庆大学 | Preparation of tomato EIL1 recombinant protein and uses thereof |
| CN101932712A (en) * | 2007-11-20 | 2010-12-29 | 先锋国际良种公司 | Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants |
| CN103103200A (en) * | 2013-02-28 | 2013-05-15 | 上海交通大学 | Cucumber ethylene signal transduction pathway EIN3 (Ethylene Insensitive 3) gene and encoded protein thereof |
| CN113307853A (en) * | 2021-06-18 | 2021-08-27 | 宁夏农林科学院枸杞科学研究所 | MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3186380A1 (en) * | 2014-08-26 | 2017-07-05 | Københavns Universitet | Plants with reduced ethylene sensitivity |
-
2022
- 2022-03-04 CN CN202210210632.5A patent/CN114395564B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005953A2 (en) * | 1999-07-15 | 2001-01-25 | Pioneer Hi-Bred International, Inc. | Maize ethylene signalling pathway ein3 genes and uses thereof |
| CN101932712A (en) * | 2007-11-20 | 2010-12-29 | 先锋国际良种公司 | Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants |
| CN101368183A (en) * | 2008-05-22 | 2009-02-18 | 重庆大学 | Preparation of tomato EIL1 recombinant protein and uses thereof |
| CN103103200A (en) * | 2013-02-28 | 2013-05-15 | 上海交通大学 | Cucumber ethylene signal transduction pathway EIN3 (Ethylene Insensitive 3) gene and encoded protein thereof |
| CN113307853A (en) * | 2021-06-18 | 2021-08-27 | 宁夏农林科学院枸杞科学研究所 | MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Fruit ripening inLycium barbarumandLycium ruthenicumis associated with distinct gene expression patterns;Jianhua Zhao等;《FEBS Open Bio.》;20200629;第10卷(第8期);第1550-1567页 * |
| Transcriptomic and metabolomic analyses of Lycium ruthenicumand Lycium barbarum fruits during ripening;Jianhua Zhao等;《Scientific Reports》;20200509;第10卷;文献号4354，第1-17页 * |
| 乙烯信号转导及其在植物逆境响应中的作用;张弢等;《生物技术通报》;20161231;第32卷(第10期);第11-17页 * |
| 黑果枸杞和宁夏枸杞FT(Flowering Locus T)基因的克隆及表达分析;薛定磊等;《基因组学与应用生物学》;20150325;第34卷(第3期);第565-570页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114395564A (en) | 2022-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104846010B (en) | A kind of method for deleting transgenic paddy rice riddled basins | |
| CN111705365A (en) | CRISPR-supported multiplexed genome engineering | |
| AU2019297209B2 (en) | Method of obtaining multi-leaf alfalfa material by means of MsPALM1 artificial site-directed mutant | |
| CN109705198B (en) | Application of OsCKX7 protein and coding gene thereof in regulation and control of resistance to plant sheath blight | |
| CN110607320B (en) | Plant genome directional base editing framework vector and application thereof | |
| CN109371040B (en) | Application of rice OsARF6 gene in regulation and control of rice seed grain type | |
| WO2021169925A1 (en) | Fusion protein and application thereof | |
| CN111718954B (en) | Genome editing tool and application thereof | |
| CN107338265B (en) | Gene editing system and method for editing plant genome by applying same | |
| CN110551736B (en) | ThHAM1 gene related to development of taxus chinensis root and application thereof | |
| CN112126652B (en) | Application of rice OsAUX3 gene in regulation of rice seed grain length | |
| CN107142271A (en) | The PL LbCpf1 RR genes with high mutation efficiency and its application in gene targeting | |
| US11365423B2 (en) | Method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants | |
| CN114540406B (en) | Genome editing expression frame, vector and application thereof | |
| CN113265422A (en) | Method for targeted knockout of rice grain type regulatory gene SLG7, rice grain type regulatory gene SLG7 mutant and application thereof | |
| CN113265406A (en) | Soybean FDL12 gene editing site and application thereof | |
| CN114395564B (en) | Application of Lycium ruthenicum Murr EIN3 gene in delaying fruit ripening | |
| CN113106120B (en) | Needle-leaved tree plant gene editing vector, construction method and application thereof | |
| CN112430612A (en) | SpRY gene capable of being efficiently cut and application thereof | |
| CN112430613A (en) | SpG gene with wide editing range and application thereof | |
| CN111909956A (en) | Method for improving drought resistance of rice by blocking or weakening gene expression of OsNAC092 of rice | |
| KR20190122595A (en) | Gene Construct for Base Editing in Plant, Vector Comprising the Same and Method for Base Editing Using the Same | |
| KR102551064B1 (en) | Novel U6 promoter separated form grapevine and use of the same | |
| CN109593768A (en) | Application of the Platanus acerifolia PaGL1 gene in regulation plant epidermal hair | |
| CN112626049B (en) | SpCas9-NRRH mutant for recognizing specific sites in rice gene targeting and application thereof |
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 |