CN113430221B - Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield - Google Patents

Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield Download PDF

Info

Publication number
CN113430221B
CN113430221B CN202110815459.7A CN202110815459A CN113430221B CN 113430221 B CN113430221 B CN 113430221B CN 202110815459 A CN202110815459 A CN 202110815459A CN 113430221 B CN113430221 B CN 113430221B
Authority
CN
China
Prior art keywords
tomato
wrky37
protein
gene
yield
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.)
Expired - Fee Related
Application number
CN202110815459.7A
Other languages
Chinese (zh)
Other versions
CN113430221A (en
Inventor
张娜
王志荣
高明
赵冰
郭仰东
王馨曼
曹萌
董晓南
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.)
China Agricultural University
Original Assignee
China Agricultural 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 China Agricultural University filed Critical China Agricultural University
Priority to CN202110815459.7A priority Critical patent/CN113430221B/en
Publication of CN113430221A publication Critical patent/CN113430221A/en
Application granted granted Critical
Publication of CN113430221B publication Critical patent/CN113430221B/en
Expired - Fee Related 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
    • 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)
    • C12N15/8241—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
    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8266—Abscission; Dehiscence; Senescence
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of tomato WRKY37 protein in regulation and control of leaf senescence resistance of tomatoes and improvement of tomato yield. The invention discovers that the WRKY37 protein of the tomato can regulate the leaf senescence resistance of the tomato and regulate the fruit yield of the tomato. In practical application, the WRKY37 gene in the tomato can be knocked out by a gene editing means, and the expression of WRKY37 protein is inhibited, so that a tomato strain with stronger leaf blade aging resistance is obtained; or the slwrky37-KO plant is crossed with other tomato varieties to culture the tomato varieties with anti-aging and high yield. The method utilizes the CRISPR-Cas9 genome fixed-point editing system to mutate the tomato WRKY37 gene, creates a high-yield tomato strain, remarkably improves the leaf senescence resistance of the tomato strain, remarkably improves the yield, and has important application value.

Description

Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield
Technical Field
The invention relates to the technical field of gene editing, in particular to application of tomato WRKY37 protein in regulation and control of tomato leaf senescence resistance and improvement of tomato yield.
Background
Aging is a ubiquitous, important physiological phenomenon, generally referred to as the process by which the organs of an organism or the physiological functions of the whole individual gradually decline and eventually die. Leaves are important places where plants synthesize organic compounds using light energy, and are also one of the most sensitive organs of plants to senescence (Woo et al, 2019). With the increasing knowledge of the genetic control of senescence in the field, transcription factors of the WRKY, MYB and NAM/ATAF1/CUC2(NAC) families become regulating factors of the process (Kim et al, 2018), and researches on regulating leaf senescence by the transcription factors of the WRKY family are frequently reported in recent years. WRKY transcription factors were first found in sweetpotato (Ishiguro and Nakamura, 1994), and then a large number of WRKY transcription factors, which are a large family of plant transcription factors, were sequentially found in various plants. The WRKY transcription factor is a plant-specific zinc finger-type transcription regulatory factor, and is named because it contains a highly conserved 7 amino acid sequence consisting of WRKYGQK at its N-terminus (Huang et al, 2012).
The WRKY transcription factor has the functions of participating in regulation and control of disease resistance, aging, developmental metabolism, abiotic stress and the like. The WRKY transcription factor is involved in regulating plant senescence, and the transcription factor AtWRKY6 in Arabidopsis is an important transcription factor related to senescence, and is hardly expressed in young leaves and mature leaves, but has a high expression level in senescent leaves (Robatzek., 2002). Arabidopsis AtWRKY53 is used as a positive regulator of leaf senescence, and regulates leaf senescence through a complex regulatory network (Miao et al, 2004). Arabidopsis AtWRKY57 served as a key node of jasmonic and auxin-mediated signaling pathway in jasmonic-induced leaf senescence, and wrky57 mutant produced typical leaf senescence symptoms such as leaf yellowing, low chlorophyll content, and high cell death rate (Jiang et al, 2014).
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the application of tomato WRKY37 protein in regulating and controlling the leaf-aging resistance of tomatoes and improving the yield of the tomatoes.
In a first aspect, the invention provides application of tomato WRKY37 protein or coding gene thereof or inhibitor of coding gene of tomato WRKY37 protein in regulation and control of tomato leaf senescence resistance.
The invention further provides application of the tomato WRKY37 protein or the coding gene thereof or the inhibiting factor of the coding gene of the tomato WRKY37 protein in improving the tomato yield.
The invention further provides application of the tomato WRKY37 protein or the coding gene thereof or the inhibiting factor of the coding gene of the tomato WRKY37 protein in tomato molecular breeding or preparation of gene-edited tomatoes;
the tomato molecular breeding is to cultivate high-yield tomatoes and/or leaf senescence-resistant tomatoes;
the gene-editing tomato is a high-yield gene-editing tomato and/or a leaf senescence-resistant gene-editing tomato.
Further, by reducing the expression of the tomato WRKY37 protein, the anti-aging capacity of the tomato is improved and/or the yield of the tomato is improved;
preferably, the method for reducing the expression level of tomato WRKY37 protein comprises the following steps:
the encoding gene of tomato WRKY37 protein is inactivated.
Further, the amino acid sequence of the tomato WRKY37 protein comprises a sequence shown as SEQ ID No. 1.
Further, the inhibitor of the encoding gene of the tomato WRKY37 protein comprises a biological material capable of inhibiting the expression of the tomato WRKY37 protein by editing the tomato WRKY37 gene;
preferably, the biological material is a gRNA, or an expression vector comprising the gRNA, or an engineered bacterium comprising the gRNA;
more preferably, the gRNA comprises a nucleotide sequence as set forth in SEQ ID No.3 or SEQ ID No. 4.
In a second aspect, the present invention provides a gRNA for editing tomato WRKY37 protein, the gRNA comprising a nucleotide sequence as set forth in SEQ ID No.3 or SEQ ID No. 4.
The invention further provides a biological material comprising the gRNA, and an expression cassette, a vector, a transgenic cell or an engineering bacterium of the biological material.
In a third aspect, the present invention provides a method for increasing tomato yield, comprising: reducing the expression level of the encoding gene of tomato WRKY37 protein in tomato; the encoding gene of the tomato WRKY37 protein comprises a nucleotide sequence shown as SEQ ID No. 2.
Further, knocking out a coding gene of tomato WRKY37 protein in tomato through a CRISPR/Cas9 system; the gRNA used in the CRISPR/Cas9 system includes a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 4.
The invention has the following beneficial effects:
the invention discovers that the tomato WRKY37 protein participates in regulation and control of leaf senescence resistance and fruit yield of tomatoes, and can obviously improve the leaf senescence resistance of the tomatoes and improve the fruit yield of the tomatoes by reducing the expression of the tomato WRKY37 protein. When the method is applied to actual production, on one hand, the SlWRKY37 gene of the tomato can be mutated to obtain a tomato line with stronger anti-aging capability; on the other hand, the mutant slwrky37-KO plant can be crossed with other tomato varieties to culture anti-aging and high-yield tomato varieties. The tomato WRKY37 protein, the coding gene thereof and the corresponding inhibitor have great application values in tomato leaf senescence resistance and high-yield breeding.
Drawings
FIG. 1 is a schematic diagram of a target site and gene editing of the SIWRKY37 gene provided in example 1 of the present invention.
FIG. 2 is a cloning gel diagram of a SIWRKY37 gene fragment provided in example 1 of the present invention; wherein lane 1 is slwrky-KO-1 and lane 2 is slwrky-KO-3.
FIG. 3 is a picture of the senescence status of the first true leaves of the SIWRKY37 gene editing strain provided in example 2 of the present invention.
FIG. 4 is a schematic diagram of fruit yield of the SIWRKY37 gene editing line provided in example 2 of the present invention.
FIG. 5 is a statistical chart of fruit yield of the SIWRKY37 gene editing line provided in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified.
Example 1 tomato SlWRKY37 Gene knockout based on CRISPR-Cas9 System
1. Background Material preparation
The variety used by the method is tomato Micro Tom, tomato seeds are subjected to surface disinfection for 15min by 3% sodium hypochlorite, are washed for 7-8 times by sterile water after disinfection, and are sown in an MS culture medium after being washed clean. Taking the cotyledon part of the tomato seedling as a tissue culture material, cutting off the leaf apex, cutting the rest part into a square of 5mm multiplied by 5mm, placing the processed cotyledon with the back face upward on a pre-culture medium, and carrying out dark culture.
2. Selection of 2gRNA targets
The NCBI website (https:// www.ncbi.nlm.nih.gov /) is logged in, and the sequence of tomato S1WRKY37 gene is queried. According to the sequence of SlWRKY37 gene, the selected target point is located on the antisense chain of the second exon of SlWRKY37 gene, and contains sequences WRKY37-gRNA1 and WRKY37-gRNA2 which are characterized by NGG sequence, based on the 2gRNA sequence characteristics of CRISPR-Cas9, and the nucleotide sequence is shown as SEQ ID NO.3 or SEQ ID NO. 4.
3. Construction of expression plasmid pHSE401-2gR containing 2gRNA target
According to two selected 2gRNA target sequences, primers WRKY37-DT1-Bs F, WRKY37-DT1-F0, WRKY37-DT2-R0 and WRKY37-DT2-BSR (S EQ ID NO.5-8), WRKY37-DT1-BsF/WRKY37-DT2-BSR each 2 mul, WRKY37-DT1-F0/WRKY37-DT2-R0 each 0.5 mul are synthesized, intermediate vector pCBC-DT1T2 plasmid is used as a template for carrying out four-primer PCR amplification, and pCBC-DT1T2-PCR products are recovered after electrophoresis. The ligation system was digested by BsaI, the pHSE401 plasmid was ligated to pCBC-DT1T2-PCR by digestion with BsaI, the pCBC-DT1T2-PCR sequence was ligated to the plasmid pHSE401 vector, and the fragment of pCBC-DT1T2-PCR containing AtU6 promoter and 2g RNA sequence was cloned into pHSE401 vector by T4 ligation, to obtain recombinant vector pHSE401-2gR (pHSE401+ pCBC-DT1T 2-PCR).
The vectors pCBC-DT1T2 and pHSE401 are from the biological college of Chinese university of agriculture.
4. Plasmid pHSE401-2gR was transformed into Agrobacterium tumefaciens GV3101
And (3) transforming the recombinant vector pHSE401-2gR in the step (3) into an agrobacterium GV3101 strain by a heat shock method to obtain a recombinant agrobacterium containing the recombinant expression vector pHSE401-2gR, which is named as GV3101-pHSE401-2 gR.
5. Agrobacterium-mediated transformation of tomato
Infecting young cotyledons of a tomato variety Micro Tom by recombinant agrobacterium tumefaciens GV3101-pHSE401-2gR, and taking plants which are successfully regenerated and rooted on a culture medium containing herbicide antibiotics as transgenic positive plants slwrky-KO-1 and slwrky-KO-3.
6. Transgenic tomato identification and mutation site detection
Extracting T0 generation tomato leaf slice genome DNA, and designing and identifying primers WRKY37-F and WRKY37-R (SEQ ID NO.9-10) according to target gene segments. A T0-generation transgenic plant genome is used as a template, WRKY37-F and WRKY37-R are used as primers to amplify an SlWRKY37 gene segment, a PCR product is used for sequencing and verifying a mutant tomato strain, and whether a target site is mutated or not is judged through a sequencing sequence.
The PCR reaction system is shown in Table 1.
TABLE 1 amplification system of target gene SlWRKY37 gene fragment sequence
Figure BDA0003169987360000051
Figure BDA0003169987360000061
The amplification result is shown in FIG. 2, lanes 2 and 3 in FIG. 2 show No.1 and No.3 of transgenic plants, which are named as slwrky-KO-1 and slwrky-KO-3, respectively, M represents a DNA molecule Marker, and the sizes of the bands are 2000bp, 1000bp and 750bp from top to bottom in sequence. The PCR fragment of the SlWRKY37 gene is sequenced, a sequencing primer is WRKY37-F, and the sequencing result is shown in figure 1. The results show that the WRKY37 genes of the two transgenic plants have deletion mutation near the PAM locus (namely, TGG and AGG sequences), and the two chromosomes have mutation at the same time. SlWRKY37 genes on two chromosomes of the SlWRKY-KO-1 plant are mutated, namely 34 bases are continuously deleted, a SlWRKY37 gene on the chromosome of the SlWRKY-KO-3 plant is mutated, and six bases are intermittently deleted.
The nucleotide sequence of the slwrky-KO-1 plant is shown in SEQ ID NO.11, and the deduced amino acid sequence after mutation is shown in SEQ ID NO. 12; the nucleotide sequence of the slwrky-KO-3 plant is shown in SEQ ID NO.13, and the deduced amino acid sequence after mutation is shown in SEQ ID NO. 13. Due to the deletion of the basic group, the translation dislocation of the post sequence of the PAM locus is caused, the protein translation is terminated in advance, and the function is changed.
Example 2 anti-aging test and use of transgenic lines tomato
1. Detection for delaying senescence and improving yield of SlWRKY37 gene editing strain
Selecting a knockout strain SlWRKY-KO-1SlWRKY-KO-3 which is homozygous through sequencing verification as a control group, selecting wild types with consistent growth vigor and the same quantity, knocking seedlings of the gene knockout strains SlWRKY-KO-1 and SlWRKY-KO-3, planting 30 strains of each strain in a sunlight greenhouse and a light culture box respectively, and observing the natural aging conditions of different strains of the slwrKY37 in a natural growth state until the whole life cycle of fruit maturity.
The setting of the illumination incubator: the light period is 16/8h (light/dark), the temperature is 25/18 deg.C (light/dark), and the light intensity is 125 μmol m-2s-1The relative humidity is about 60%.
In the later stage of plant growth, the leaves of the two knock-out lines slwrky-KO-1 and slwrky-KO-3 senesced more slowly and the fruiting rate was higher compared to the wild-type control group. As shown in figure 3, in the senescence condition of the first true leaves of the plants of different strains, the chlorophyll content and the photosynthetic property of the first true leaves of slwrky37-KO-1 and slwrky-KO-3 are obviously higher than those of the wild type, and the senescence is slower. Picking fruits of different strains in a mature period, as shown in figure 4, wherein the number of fruits of two gene knockout strains slwrky-KO-1 and slwrky-KO-3 is obviously more than that of wild WT, and as shown in figure 5, the yield of the two gene knockout strains is obviously higher than that of WT according to the statistics of yield measurement of different strains.
Therefore, the anti-aging tomato can be enhanced by using the CRISPR-Cas9 system containing the gRNA sequence to edit the SlWRKY37 gene at a fixed point, and the SlWRKY37-KO plant is hybridized with other tomato varieties, so that anti-aging strains can be cultivated, anti-aging germplasm resources of the tomato are enriched, and a new idea is provided for high-yield breeding of the tomato.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of agriculture in China
Application of tomato WRKY37 protein in regulation of tomato leaf aging resistance and improvement of tomato yield
<130> KHP211117953.0
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 248
<212> PRT
<213> tomato (Solanum lycopersicum)
<400> 1
Met Gly Arg Ser Lys Pro Thr Lys Ser Gln Ser Lys Thr Ser Arg Leu
1 5 10 15
Leu Gln Pro Gln Phe Leu Ala Ser Lys Asn Ser Ser Glu Tyr Ser Ser
20 25 30
His Val Arg Lys Met Ala Gln Lys Val Val Met Thr Val Gln Met Glu
35 40 45
Ala Lys Ser Leu Lys Gln Lys Asn Glu Gly Pro Pro Ser Asp Cys Trp
50 55 60
Ser Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys Gly Ser Pro Tyr Pro
65 70 75 80
Arg Gly Tyr Tyr Arg Cys Ser Ser Ser Lys Gly Cys Ser Ala Lys Lys
85 90 95
Gln Val Glu Arg Cys Ser Lys Asp Ala Ser Leu Phe Ile Ile Thr Tyr
100 105 110
Thr Ser Ser His Asn His Pro Gly Pro Asn Leu Pro Lys Asp Ser Val
115 120 125
Lys Gln Glu Pro Val Val Asp Gln His Val Ser Leu Glu Asp Asn Asn
130 135 140
Asp Glu Asp Asp Asp Asp Asp Asp Asp Asn Asp Thr Gln Ser Cys Lys
145 150 155 160
Thr Pro Leu Asn Ser Thr Thr Thr Ser Thr Ser Ile Pro Gln Gly Ile
165 170 175
Leu Glu Glu Asn Leu Phe Thr Asp Asn Phe Leu Gly Thr Ile Ser Tyr
180 185 190
Asp Asp Phe Leu Pro Leu Ser Tyr Pro Gln Leu Met Lys Phe Pro Lys
195 200 205
Ser Glu Leu Ser Glu Glu Asn Asp Phe Tyr Asp Glu Leu Gly Glu Leu
210 215 220
Glu Leu Pro Pro Ser Ser Ser Thr Ser Phe Ala Gly Ile Phe Glu Glu
225 230 235 240
Ala Ile Leu Val Asp Pro Ser Ser
245
<210> 2
<211> 1177
<212> DNA
<213> tomato (Solanum lycopersicum)
<400> 2
atgggtagaa gtaagccaac aaaatcacaa tctaaaacat caagattgct acaaccacaa 60
tttcttgcct caaaaaacag gtaactttcc atctcaaagg tttcgtttat tttcaaatat 120
ctgtttggtc atgaaatttc aaaatatgat ctttgagttt ttgggacttt tactcacaaa 180
acacaaaact ttgtgtatag ttatggtttg gtcatggttt ttatataaag gggacctttt 240
tttttttttt ttctgacagt tctgagtatt cttctcatgt tagaaagatg gctcagaagg 300
ttgttatgac agtgcaaatg gaagcaaaat ctttaaagca gaaaaatgaa gggcctcctt 360
cagattgttg gtcttggaga aaatatggac aaaaacccat taaaggatcc ccttacccca 420
ggtagattta caattctctt ctccacacct cagatgtaga ggcgaattca agattcagag 480
tctgtgtgat cttgatgtat ttggaatgag ttattagttg aaaaaatgtt ttttgtgttt 540
gattgagtga gtaatatgaa acgaaaattt gtgataattt ctgaaaagga aaaagataag 600
ataatcctga tgtattttgc ttgaaaatgt acatttcacc taacatgttg taatgtatga 660
ttgtggatac aggggttact acagatgcag cagttcaaag ggttgttcag ctaaaaaaca 720
agtagaaagg tgtagtaaag atgcatcttt gttcatcatc acatatacat ccagccataa 780
tcatccaggt ccaaatttgc ctaaagactc tgttaaacaa gagccagtag tggatcaaca 840
tgtgtccctc gaagacaaca acgatgaaga tgatgatgat gatgatgata atgatacaca 900
aagctgcaaa accccattga atagtaccac tactagtacc agtattcccc aaggcatact 960
tgaagagaat ctgttcactg ataatttctt gggaacaatt tcatatgatg attttctgcc 1020
cctttcttac cctcaactaa tgaaatttcc aaaatccgaa ttgtcagaag agaatgactt 1080
ttatgatgaa ttaggagaat tggagctacc tccatcttct tctacatcct tcgcgggcat 1140
ttttgaggag gcaatccttg tagatccctc ctcttag 1177
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agggcctcct tcagattgt 19
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggacaaaa acccattaa 19
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atatatggtc tcgattgagg gcctccttca gattgtgtt 39
<210> 6
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgagggcctc cttcagattg tgttttagag ctagaaatag c 41
<210> 7
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aacttaatgg gtttttgtcc ataatctctt agtcgactct ac 42
<210> 8
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
attattggtc tcgaaactta atgggttttt gtccataa 38
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgggtagaa gtaagccaac aaaatc 26
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gcatctttac tacacctttc tacttg 26
<210> 11
<211> 1143
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atgggtagaa gtaagccaac aaaatcacaa tctaaaacat caagattgct acaaccacaa 60
tttcttgcct caaaaaacag gtaactttcc atctcaaagg tttcgtttat tttcaaatat 120
ctgtttggtc atgaaatttc aaaatatgat ctttgagttt ttgggacttt tactcacaaa 180
acacaaaact ttgtgtatag ttatggtttg gtcatggttt ttatataaag gggacctttt 240
tttttttttt ttctgacagt tctgagtatt cttctcatgt tagaaagatg gctcagaagg 300
ttgttatgac agtgcaaatg gaagcaaaat ctttaaagca gaaaaatgaa gggcctcctt 360
cagatttaaa ggatcccctt accccaggta gatttacaat tctcttctcc acacctcaga 420
tgtagaggcg aattcaagat tcagagtctg tgtgatcttg atgtatttgg aatgagttat 480
tagttgaaaa aatgtttttt gtgtttgatt gagtgagtaa tatgaaacga aaatttgtga 540
taatttctga aaaggaaaaa gataagataa tcctgatgta ttttgcttga aaatgtacat 600
ttcacctaac atgttgtaat gtatgattgt ggatacaggg gttactacag atgcagcagt 660
tcaaagggtt gttcagctaa aaaacaagta gaaaggtgta gtaaagatgc atctttgttc 720
atcatcacat atacatccag ccataatcat ccaggtccaa atttgcctaa agactctgtt 780
aaacaagagc cagtagtgga tcaacatgtg tccctcgaag acaacaacga tgaagatgat 840
gatgatgatg atgataatga tacacaaagc tgcaaaaccc cattgaatag taccactact 900
agtaccagta ttccccaagg catacttgaa gagaatctgt tcactgataa tttcttggga 960
acaatttcat atgatgattt tctgcccctt tcttaccctc aactaatgaa atttccaaaa 1020
tccgaattgt cagaagagaa tgacttttat gatgaattag gagaattgga gctacctcca 1080
tcttcttcta catccttcgc gggcattttt gaggaggcaa tccttgtaga tccctcctct 1140
tag 1143
<210> 12
<211> 78
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Gly Arg Ser Lys Pro Thr Lys Ser Gln Ser Lys Thr Ser Arg Leu
1 5 10 15
Leu Gln Pro Gln Phe Leu Ala Ser Lys Asn Arg Lys Met Ala Gln Lys
20 25 30
Val Val Met Thr Val Gln Met Glu Ala Lys Ser Leu Lys Gln Lys Asn
35 40 45
Glu Gly Pro Pro Ser Asp Leu Lys Asp Pro Leu Thr Pro Gly Val Thr
50 55 60
Thr Asp Ala Ala Val Gln Arg Val Val Gln Leu Lys Asn Lys
65 70 75
<210> 13
<211> 1171
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgggtagaa gtaagccaac aaaatcacaa tctaaaacat caagattgct acaaccacaa 60
tttcttgcct caaaaaacag gtaactttcc atctcaaagg tttcgtttat tttcaaatat 120
ctgtttggtc atgaaatttc aaaatatgat ctttgagttt ttgggacttt tactcacaaa 180
acacaaaact ttgtgtatag ttatggtttg gtcatggttt ttatataaag gggacctttt 240
tttttttttt ttctgacagt tctgagtatt cttctcatgt tagaaagatg gctcagaagg 300
ttgttatgac agtgcaaatg gaagcaaaat ctttaaagca gaaaaatgaa gggcctcctt 360
cagatgttgg tcttggagaa aatatggaca aaactaaagg atccccttac cccaggtaga 420
tttacaattc tcttctccac acctcagatg tagaggcgaa ttcaagattc agagtctgtg 480
tgatcttgat gtatttggaa tgagttatta gttgaaaaaa tgttttttgt gtttgattga 540
gtgagtaata tgaaacgaaa atttgtgata atttctgaaa aggaaaaaga taagataatc 600
ctgatgtatt ttgcttgaaa atgtacattt cacctaacat gttgtaatgt atgattgtgg 660
atacaggggt tactacagat gcagcagttc aaagggttgt tcagctaaaa aacaagtaga 720
aaggtgtagt aaagatgcat ctttgttcat catcacatat acatccagcc ataatcatcc 780
aggtccaaat ttgcctaaag actctgttaa acaagagcca gtagtggatc aacatgtgtc 840
cctcgaagac aacaacgatg aagatgatga tgatgatgat gataatgata cacaaagctg 900
caaaacccca ttgaatagta ccactactag taccagtatt ccccaaggca tacttgaaga 960
gaatctgttc actgataatt tcttgggaac aatttcatat gatgattttc tgcccctttc 1020
ttaccctcaa ctaatgaaat ttccaaaatc cgaattgtca gaagagaatg acttttatga 1080
tgaattagga gaattggagc tacctccatc ttcttctaca tccttcgcgg gcatttttga 1140
ggaggcaatc cttgtagatc cctcctctta g 1171
<210> 14
<211> 238
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Gly Arg Ser Lys Pro Thr Lys Ser Gln Ser Lys Thr Ser Arg Leu
1 5 10 15
Leu Gln Pro Gln Phe Leu Ala Ser Lys Asn Arg Lys Met Ala Gln Lys
20 25 30
Val Val Met Thr Val Gln Met Glu Ala Lys Ser Leu Lys Gln Lys Asn
35 40 45
Glu Gly Pro Pro Ser Asp Val Gly Leu Gly Glu Asn Met Asp Lys Thr
50 55 60
Lys Gly Ser Pro Tyr Pro Arg Gly Tyr Tyr Arg Cys Ser Ser Ser Lys
65 70 75 80
Gly Cys Ser Ala Lys Lys Gln Val Glu Arg Cys Ser Lys Asp Ala Ser
85 90 95
Leu Phe Ile Ile Thr Tyr Thr Ser Ser His Asn His Pro Gly Pro Asn
100 105 110
Leu Pro Lys Asp Ser Val Lys Gln Glu Pro Val Val Asp Gln His Val
115 120 125
Ser Leu Glu Asp Asn Asn Asp Glu Asp Asp Asp Asp Asp Asp Asp Asn
130 135 140
Asp Thr Gln Ser Cys Lys Thr Pro Leu Asn Ser Thr Thr Thr Ser Thr
145 150 155 160
Ser Ile Pro Gln Gly Ile Leu Glu Glu Asn Leu Phe Thr Asp Asn Phe
165 170 175
Leu Gly Thr Ile Ser Tyr Asp Asp Phe Leu Pro Leu Ser Tyr Pro Gln
180 185 190
Leu Met Lys Phe Pro Lys Ser Glu Leu Ser Glu Glu Asn Asp Phe Tyr
195 200 205
Asp Glu Leu Gly Glu Leu Glu Leu Pro Pro Ser Ser Ser Thr Ser Phe
210 215 220
Ala Gly Ile Phe Glu Glu Ala Ile Leu Val Asp Pro Ser Ser
225 230 235

Claims (4)

1. The application of the inhibitor of the encoding gene of tomato WRKY37 protein in improving the leaf aging resistance of tomato;
the inhibitory factor is gRNA with a sequence shown in SEQ ID NO.3 or SEQ ID NO. 4;
the application is as follows: the encoding gene of the tomato WRKY37 protein is mutated by the inhibitor of the encoding gene of the tomato WRKY37 protein so as to improve the leaf aging resistance of tomatoes;
the amino acid sequence of the tomato WRKY37 protein is shown in SEQ ID NO. 1.
2. The application of the inhibitor of the encoding gene of tomato WRKY37 protein in improving the tomato yield;
the inhibitory factor is gRNA with a sequence shown in SEQ ID NO.3 or SEQ ID NO. 4;
the application is as follows: the encoding gene of the tomato WRKY37 protein is mutated by the inhibitor of the encoding gene of the tomato WRKY37 protein, so that the tomato yield is improved;
the amino acid sequence of the tomato WRKY37 protein is shown in SEQ ID NO. 1.
3. The application of the inhibitor of the encoding gene of the tomato WRKY37 protein in tomato molecular breeding or preparing gene-edited tomatoes;
the tomato molecular breeding is to cultivate high-yield tomatoes and/or leaf senescence-resistant tomatoes;
the gene-editing tomato is a high-yield gene-editing tomato and/or a leaf senescence-resistant gene-editing tomato;
the inhibitory factor is gRNA with a sequence shown in SEQ ID NO.3 or SEQ ID NO. 4;
the application is as follows: mutating the encoding gene of the tomato WRKY37 protein by using an inhibitor of the encoding gene of the tomato WRKY37 protein so as to carry out tomato molecular breeding or prepare a gene-editing tomato;
the amino acid sequence of the tomato WRKY37 protein is shown in SEQ ID NO. 1.
4. A method for increasing tomato yield, comprising: adopting the gRNA of the nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO.4 to mutate the encoding gene of tomato WRKY37 protein in tomato;
the amino acid sequence of the tomato WRKY37 protein is shown in SEQ ID NO. 1.
CN202110815459.7A 2021-07-19 2021-07-19 Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield Expired - Fee Related CN113430221B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110815459.7A CN113430221B (en) 2021-07-19 2021-07-19 Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110815459.7A CN113430221B (en) 2021-07-19 2021-07-19 Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield

Publications (2)

Publication Number Publication Date
CN113430221A CN113430221A (en) 2021-09-24
CN113430221B true CN113430221B (en) 2022-07-05

Family

ID=77761161

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110815459.7A Expired - Fee Related CN113430221B (en) 2021-07-19 2021-07-19 Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield

Country Status (1)

Country Link
CN (1) CN113430221B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438104B (en) * 2022-03-21 2023-06-20 重庆大学 SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content
CN114807072B (en) * 2022-06-06 2024-06-21 中国农业科学院蔬菜花卉研究所 Tomato SlDAO gene and application thereof
CN116555301B (en) * 2023-07-03 2023-09-22 中国农业大学 SlMETS1 gene and application thereof in regulation and control of tomato growth and development

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102775483A (en) * 2012-07-13 2012-11-14 复旦大学 Transcription factor for adjusting and controlling catabiosis and multi-resistance of leaves and application thereof
CN102964439A (en) * 2012-12-24 2013-03-13 复旦大学 Transcription factor for regulating and controlling aging of leaf blades and improving multiple stress tolerance and application thereof
CN102964440A (en) * 2012-12-24 2013-03-13 复旦大学 WRKY transcription factor for regulating and controlling aging of leaf blades and multiple stress tolerance and application thereof
CN112430261B (en) * 2020-11-24 2022-06-28 上海交通大学 WRKY32 regulation and control YFT1 expression to influence tomato fruit color and application thereof in tomato quality improvement

Also Published As

Publication number Publication date
CN113430221A (en) 2021-09-24

Similar Documents

Publication Publication Date Title
CN113430221B (en) Application of tomato WRKY37 protein in regulation of leaf senescence resistance of tomatoes and improvement of tomato yield
CN110724183B (en) Application of GmXTH91 protein in regulation and control of plant stress resistance and plant height
CN113388016B (en) Protein GSW8 for regulating rice grain type and thousand grain weight, and coding gene and application thereof
CN108503700B (en) Rice grain type protein and coding gene and application thereof
CN110066774A (en) Corn receptoroid kinase gene ZmRLK7 and its application
CN109971763A (en) Florescence control gene C MP1 and relevant carrier and its application
CN113186200B (en) A pair of genes ClTFL/Cltfl1 for controlling existence and lateral branch of watermelon tendril and application thereof
CN113564176B (en) Wheat TaHAL3-7A gene and application thereof in regulating drought resistance of crops
CN112342236B (en) Application of rice histone methyltransferase in enhancing crop drought resistance and improving single plant yield
CN105504035A (en) Rice immune negative regulation and control protein and encoding genes and application thereof
CN109097367B (en) Rubber tree HbWRKY82 gene and application thereof
CN106318923B (en) The protein and its gene of a kind of High Temperature Stress down regulation Development of Chloroplasts and application
CN116064568A (en) Alfalfa MsASG166 gene and application thereof in improving drought tolerance of plants
CN114480417A (en) Gene ZmSAG39 for regulating and controlling leaf senescence, encoding protein and application thereof
CN114921583A (en) QTL for controlling wheat plant height, candidate gene TaDHL-7B thereof and application
CN114395580A (en) Gene for controlling plant height of corn
CN110468138B (en) Gene TSG2 for controlling cold resistance of rice and application thereof
CN113563439A (en) Fruit shape development related protein and coding gene and application thereof
CN108892714B (en) Plant salt tolerance associated protein GmLURP17 and application of coding gene thereof
CN112301034B (en) Rice low light response gene RLL1, mutant and application thereof
CN113308489B (en) Creation method of novel salt-tolerant oat germplasm
CN116445497B (en) Cabbage BoDMP9 gene and application thereof in maternal haploid induction
WO2024037338A1 (en) Plant grain size regulation gene and use thereof
CN118028307B (en) Application of TaNAC22-A1 gene in negative regulation of small flower number in wheat ears
CN109082436A (en) Utilize the method for BCS1L gene and guide RNA/CAS endonuclease enzyme system improvement plant agronomic character

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
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20220705