CN116555321A - Application of PpERF98 gene in regulation and control of gummosis resistance of peach - Google Patents
Application of PpERF98 gene in regulation and control of gummosis resistance of peach Download PDFInfo
- Publication number
- CN116555321A CN116555321A CN202211154241.2A CN202211154241A CN116555321A CN 116555321 A CN116555321 A CN 116555321A CN 202211154241 A CN202211154241 A CN 202211154241A CN 116555321 A CN116555321 A CN 116555321A
- Authority
- CN
- China
- Prior art keywords
- peach
- gummosis
- gene
- pperf98
- resistance
- 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.)
- Pending
Links
- 235000006040 Prunus persica var persica Nutrition 0.000 title claims abstract description 81
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 230000033228 biological regulation Effects 0.000 title abstract description 4
- 240000006413 Prunus persica var. persica Species 0.000 title 1
- 244000144730 Amygdalus persica Species 0.000 claims abstract description 70
- 241000196324 Embryophyta Species 0.000 claims abstract description 27
- 239000012634 fragment Substances 0.000 claims abstract description 21
- 239000013598 vector Substances 0.000 claims abstract description 16
- 230000030279 gene silencing Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000001404 mediated effect Effects 0.000 claims abstract description 8
- 230000001105 regulatory effect Effects 0.000 claims abstract description 8
- 230000001276 controlling effect Effects 0.000 claims abstract description 5
- 238000010353 genetic engineering Methods 0.000 claims abstract description 4
- 241000700605 Viruses Species 0.000 claims abstract description 3
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 3
- 230000028604 virus induced gene silencing Effects 0.000 claims description 8
- 101150055707 98 gene Proteins 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 abstract description 16
- 230000009261 transgenic effect Effects 0.000 abstract description 14
- 240000005809 Prunus persica Species 0.000 abstract description 10
- 241000589158 Agrobacterium Species 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 5
- 230000003321 amplification Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 4
- 108700026244 Open Reading Frames Proteins 0.000 abstract description 3
- 238000012226 gene silencing method Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 24
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 24
- 238000011081 inoculation Methods 0.000 description 17
- 230000014509 gene expression Effects 0.000 description 16
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 14
- 240000003768 Solanum lycopersicum Species 0.000 description 13
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 10
- 230000002018 overexpression Effects 0.000 description 10
- 208000024891 symptom Diseases 0.000 description 8
- 238000011529 RT qPCR Methods 0.000 description 7
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 7
- 229960004889 salicylic acid Drugs 0.000 description 7
- 241000190144 Lasiodiplodia theobromae Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 108020005089 Plant RNA Proteins 0.000 description 3
- 241001631271 Prunus fasciculata Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 241000219194 Arabidopsis Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 108091061403 ERF family Proteins 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- 108020005120 Plant DNA Proteins 0.000 description 2
- 244000194806 Solanum sisymbriifolium Species 0.000 description 2
- 235000018724 Solanum sisymbriifolium Nutrition 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 241001344837 Ceratobasidium theobromae Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 241000498271 Necator Species 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 244000141353 Prunus domestica Species 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000407 epitaxy Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
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)
- C12N15/8241—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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
-
- 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]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates toPpERF98The invention relates to an application of a gene in regulating and controlling gummosis resistance of peach, which is characterized in that the gene is applied to the regulation and control of gummosis resistance of peach by a genetic engineering meansPpERF98A specific target fragment is obtained from the open reading frame sequence of the gene, and a target fragment primer is designed for amplification; fusing the target fragment with a virus-mediated gene silencing (VIGS) vector pCaRNA3 by an enzyme digestion method to successfully construct the VIGS vector; transformation of VIGS vectors into peach plants by agrobacterium-mediated methods, reductionPpERF98Obtaining plant material with gummosis resistance; the full-length sequence primer of the open reading frame is SEQ ID NO:1 and SEQ ID NO:2. the invention improves the resistance of peach trees to gummosis bacteria by a transgenic means and obviously reducesThe incidence rate of gummosis reduces the economic loss caused by gummosis of peaches.
Description
Technical Field
The invention relates to the field of prevention and treatment methods of gummosis of peaches, in particular to application of a PpERF98 gene in regulation and control of gummosis resistance of peaches.
Background
Peach gummosis is an accumulated branch disease, commonly occurs in Yangtze river basin and the south thereof, and is one of the most serious diseases in peach production in China. The disease causes tissue necrosis of trunk, main branch and other parts of peach tree, causes the weakening of tree vigor, reduces the yield and quality of fruits, shortens the service life of peach tree and seriously affects economic benefit. Most pathogenic bacteria causing gummosis of peach belong to the family Botrytis cinerea, and the bacteria belong to semi-living nutritive fungi, can infect xylem of peach tree, and cause difficulty in preventing and treating gummosis of peach. At present, no resistant material against gummosis of peach has been found. Therefore, analyzing the resistance gene of peach tree is a urgent problem to be solved in peach production, and the research result provides important theoretical basis for prevention and treatment of gummosis and creation of gummosis-resistant materials.
Disclosure of Invention
The invention aims to solve the problem that no effective control means and gummosis-resistant peach resources exist at present, and provides application of a PpERF98 gene in regulating and controlling gummosis resistance of peach.
The technical scheme for solving the technical problems is as follows:
an application of a pepre 98 gene in regulating resistance to gummosis of peach, which is characterized in that the sequence of the pepre 98 gene is SEQ ID NO:5.
the application of the PpERF98 gene in regulating and controlling gummosis resistance of peaches is to silence the PpERF98 gene by a genetic engineering method to improve gummosis resistance of peaches.
Selecting a target fragment from the full-length sequence of the PpERF98 gene, designing a primer pair VIGS-PpERF98 of the target fragment, connecting the target fragment with a virus-mediated silencing vector pCaRNA3 by an enzyme digestion method, constructing a VIGS vector, and introducing the VIGS vector into peach plants to improve the resistance of the peaches to gummosis; the sequence of the target fragment is SEQ ID NO:6.
further, the primer pair VIGS-PpERF98 sequence of the target fragment is SEQ ID NO:3 and SEQ ID NO:4.
the beneficial effects of the invention are as follows: the invention discovers that the PpERF98 gene is related to gummosis resistance of peaches, and can silence the PpERF98 gene by a genetic engineering means to improve the gummosis resistance, obviously reduce the incidence of gummosis and reduce the economic loss caused by gummosis of peaches.
Drawings
FIG. 1 is an electrophoretic picture of the full length of the PpERF98 gene;
FIG. 2 is a schematic representation of an ERF98 amino acid sequence alignment analysis in different species;
FIG. 3 is a comparative schematic diagram of the symptoms and lesions of different peach varieties inoculated with gummosis of peach;
FIG. 4 is a graph showing the relative expression level of the PpERF98 gene after inoculating gummosis bacteria in different peach varieties;
FIG. 5 is a schematic representation of silencing efficiency of the peach plant silencing the PpERF98 gene;
FIG. 6 is a schematic diagram of the disease symptoms and lesion areas of peach plants inoculated with gummosis bacteria that silence the PpERF98 gene;
FIG. 7 is a graph showing the relative expression levels of genes related to the course of disease after gummosis inoculation of peach plants silencing the PpERF98 gene;
FIG. 8 is a schematic diagram of the efficiency of over-expression of the PpERF98 gene by peach plants;
FIG. 9 is a schematic representation of the disease symptoms and lesion areas of peach plants inoculated with gummosis bacteria over-expressing the PpERF98 gene;
FIG. 10 is a graph showing the relative expression levels of genes related to the course of disease after gummosis inoculation of peach plants over-expressing the PpERF98 gene;
FIG. 11 is a graph showing the relative expression levels of PpERF98 from transgenic tomato plants;
FIG. 12 is a schematic diagram of the symptoms of the transgenic tomato plants and control plants inoculated with Blackstone mold;
FIG. 13 is a graph showing the relative expression levels of genes related to disease course after inoculation of transgenic tomato plants and control plants with gummosis bacteria;
FIG. 14 is a schematic representation of salicylic acid content in PpERF 98-silenced plants and over-expressed plants.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
The transcription factor PpERF98 responding to the peach gummosis Lasiodiplodia theobromae strain is screened out through transcriptome data, and the expression level of the transcription factor PpERF98 in different sensitive peach varieties is found to have positive correlation with the sensitivity of the peach gummosis. Based on the transgenic technology, the gene is heterologously and overexpressed in the common tomato A57, and the resistance of the transgenic plant to gummosis peach bacteria is found to be reduced. At present, a stable transgenic genetic system does not exist in peach, and the invention adopts a VIGS technology to silence a target gene in peach seedlings by an agrobacterium-mediated infection system, so that the resistance of the peach plants silencing PpERF98 to gummosis bacteria is enhanced.
The main experimental procedure is as follows:
1. full length cloning of the PpERF98 Gene
Based on transcriptome data obtained earlier, a transcription factor of ERF family responding to peach gummosis is selected, and the transcription factor is Prupe.8G224700. The amino acid sequence was analyzed by the NCBI database BlastP alignment and found to be most homologous to ERF98 in Arabidopsis (67.74%), hence the name PpERF98.
Taking phloem tissue of current annual branch of peach inoculated with gummosis bacteria as a sample, extracting total RNA by using an EASY spin Plus plant RNA rapid extraction kit (Aidlab, beijing), detecting the concentration and quality of RNA by using a Nanodrop one (Thermo, USA), and using a reverse transcription kitRT Reagent Kit with gDNA Eraser (TaKaRa, dalia, china) reverse transcribing the qualified RNA samples to obtain cDNA, and finally performing open reading frame fragment amplification using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, nanjin), the amplification primers using primers based on the sequence information obtained at NCBI5.0 software design full-length amplification primer SEQ ID NO:1 and SEQ ID NO:2, the primer sequences are respectively as follows:
PpERF98-Full length-F:5’–ATGCACTATATATCTTGCATGTCACAA--3’
PpERF98-Full-length-R:5’--CTAATGGGTTGGTTTCCCCTGTCTA--3’
the amplified product was subjected to DNA gel recovery kit (Sangon Biotech, shanghai) to recover the desired fragment, pEASY-Blunt Zero Cloning Kit (Transgene, beijing) to join the vector to obtain a positive clone, and then was subjected to sequencing by TSINGKE corporation (TSINGKE, wuhan) to obtain a sequence, and the full-length sequence SEQ ID NO of PpERF 98: 5 is:
ATGCACTATATATCTTGCATGTCACAAGCACAACCCATATTTTTGCCACCAAATTTGAATAAAGACCTGTTTCTGCTTAACATGGAAGGGAAGGGAGTGGAGAACCAGCAGAAGGAGCAGACTAAGGTAAGAGATCAAACCCGGTATCGAGGGATTCGGAGGCGACCGTGGGGCAAGTTTGCTGCTGAAATACGTGACCCTTCAAGAAATGGGGCACGCCTATGGCTAGGCACATTTGAGACAGCTGAAGAGGCAGCTAGGGCTTATGATCGAGCTGCTTTCGGCTTCCGGGGTCATTTGGCCATCCTCAACTTCCCTAATGACTACCAGTATCATAACCCATCAAGCTCTTTGATCAGCACTTCATCCTCTTCATCATCATCTCCATTTTCTGCTGCTGATATTGGAAAGAGTACTAATTTTGGCAGAGGCCAAGAAGAAGAAGAAGTTATAGAGTTTGAGTACCTGGACAACATGGTTTTGGAGGAGCTTCTTGACACAAAAGAGGATCATCATAGACAGGGGAAACCAACCCATTAG
the total length of the sequence is 540bp, and an electrophoresis photograph is shown in figure 1. The protein sequence of ppenf 98 was aligned with the ERF98 protein sequences of species like arabidopsis, prune, apricot, apple, and citrus using MEGA7, and the AP2 domain comprising ERF family characteristics was found at the N-terminus and the acid activation domain of EDLL was found at the C-terminus (fig. 2).
2. Analysis of relative expression level of PpERF98
The peach branch inoculation method refers to high epitaxy (2016). Observing the disease symptoms of the branches at 1, 2 and 3d time points after inoculation; phloem tissue at 0.5-1cm around the lesion was taken at time points 0, 6, 12, 24, 48, 72h and frozen at-80 ℃ for later use. Total plant RNA was extracted using EASY spin Plus plant RNA rapid extraction kit (Aidlab, beijin), nanodrop one (Thermo, USA) to detect RNA concentration and quality; cDNA synthesis using reverse transcription kitRT Reagent Kit with gDNA Eraser (TaKaRa, dalia, china). qRT-PCR Using HieffTM qPCR SYBR&Green Master Mix (Low Rox Plus) (YEASEN, shanghai) instrument was Quantum studio 6 (ABI, USA). The candidate gene was designed with Primer5 software to quantify the Primer, and housekeeping gene pptref 2 (Translation enlongation factor 2) (Tong et al 2009) was used as a peach reference gene, for calculation methods reference Livak and Schmittgen (2001). Sequence specific primers were as follows:
ppenf 98 fluorescent quantitative primer sequence:
PpERF98-F:5’--TGATCAGCACTTCATCCTCTTCA--3’
PpERF98-R:5’--CTCCTCCAAAACCATGTTGTCC--3’
primer sequence of peach internal reference gene PpTEF2 (Tong et al 2009):
PpTEF2-F:5’--AGCAAGTCACCCAACAAGCATA--3’
PpTEF2-R:5’--CCAACCAAACTCTTCAGCCAAT--3’
the peach gummosis bacteria L.theobromae are used for respectively inoculating the current annual branches of the peach 'spring snow' and the 'Dahongpao', the results show that the sensitivity of the 'spring snow' and the 'Dahongpao' to the gummosis bacteria is obviously different, the disease spots of the 'spring snow' are obviously larger than those of the 'Dahongpao', and the sensitivity of the 'spring snow' is larger than that of the 'Dahongpao' (figure 3). Analysis of the relative expression level of ppearf 98 in two peach variety shoots after inoculation of gummosis bacteria revealed a positive correlation with the sensitivity of the peach variety to gummosis bacteria (fig. 4).
3. VIGS-mediated gene silencing of peach seedlings pprf 98
Selecting a specific target fragment with the length of 100bp from the full-length sequence of PpERF98, wherein the sequence of the target fragment is SEQ ID NO:6:
AAGAAGAAGAAGAAGTTATAGAGTTTGAGTACCTGGACAACATGGTTTTGGAGGAGCTTCTTGACACAAAAGAGGATCATCATAGACAGGGGAAACCAAC
specific primers are designed, and a target fragment of the VIGS-PpERF98 is amplified, and is connected with a viral vector pCaRNA3 through FastDigestXbal fast cutting enzyme of Thermo company and T4 DNA Ligase of TaKaRa company, so as to obtain a recombinant vector PNRSV-PpERF98 of the VIGS. The primer sequence for amplifying the VIGS-PpERF98 fragment is SEQ ID NO:3 and SEQ ID NO:4:
VIGS-PpERF98-F:5’--GCTCTAGACAAGAAGAAGAAGAAG--3’
VIGS-PpERF98-R:5’--GCTCTAGAGTTGGTTTCCCCTGTCTAT--3’
peach seedlings were subjected to VIGS transient transformation by the method referenced Cui and Wang (2017). PNRSV-PpERF98 and pCaRNA1&2 are respectively transferred into agrobacterium GV3101 to be competent, and PNRSV-PpERF98 and pCaRNA1 are respectively carried after positive detection&2, and then re-using MMA (10 mmol/L MES,10mmol/L MgCl) 2 200 mu mol/L acetosyringone, pH=5.6-5.7) is regulated to OD about 1.0, and after being uniformly mixed according to the volume ratio of 1:1, bacterial liquid is injected into peach seedling leaves with 6-8 leaves of seedling age, normal culture is carried out in a dark place for 2d, and pCaRNA3 and pCaRNA1 are used&2 as negative control, PNRSV-PpPDS and pCaRNA1&2 is a positive control. qRT-PCR analysis was performed on the uninjected functional leaves of the silenced seedlings after 4 weeks, and the results showed that the method can effectively silence the expression level of the PPERF98 gene of the seedlings (FIG. 5).
4. Identification of resistance to the Coptoterma strain L.theobromae by silenced peach plants
Leaf inoculation of the peach gummosis bacterial strain l.theobromae was performed with the method of reference Zhao Lina (2012) using the silenced peach plants and control in vitro leaves as material. The disease symptoms were observed after 1 and 2d inoculation, and the results showed that the area of the lesions of the silencing pprf 98 peach plants was significantly smaller than that of the control, and the resistance to gummosis bacteria was significantly enhanced (fig. 6).
5. qRT-PCR analysis of disease course related genes in silenced wild peach plants
The relative expression amounts of the genes related to the disease course proteins were analyzed by taking 5mm of tissues around the inoculation points of the silenced wild peaches and the control leaves at the time points 1 and 2d after the inoculation. The results showed that silencing of the leaves of wild peach plants significantly upregulated expression of both salicylic acid disease-course-dependent genes PpPR1, ppPR2 (fig. 7). Silencing the pprf 98 in peach seedlings may enhance the resistance of the silenced peach plants to gummosis bacteria by increasing the expression of the PpPR1, ppPR2 disease course related protein genes.
Peach disease course protein related gene primer:
PpPR1-F:5’--TGACAAGGTGTGTGGGCATT--3’
PpPR1-R:5’--CGGATCATAGTTGCACCCGA--3’
PpPR2-F:5’--ACAGGAGGACCATTGGCTTG--3’
PpPR2-R:5’--ACGGCCATGGTATGAAGCTC--3’
6. construction of the overexpression vector
Through Gateway vector homologous recombination technology, two rounds of PCR reactions are performed to add a linker to a target fragment by utilizing Phanta Max Super-Fidelity DNA Polymerase (Vazyme, nanjing), and the template of the first round of PCR is plasmid containing target genes, and the primer sequences are as follows:
attB-PpERF98-F:
5’--AAAAAGCAGGCTCCATGCACTATATATCTTGCATGTCACAAG--3’
attB-PpERF98-R:
5’–AGAAAGCTGGGTTCTAATGGGTTGGTTTCCCCTGTCTA--3’
template of second round PCR, one round of product, primer sequence:
attB-F:5’--GGGGACAAGTTTGTACAAAAAAGCAGGCT--3’
attB-R:5’--GGGGACCACTTTGTACAAGAAAGCTGGGT--3’
and then performing BP reaction and LP reaction by using a Gateway kit of Thermo company, and connecting the PpERF98 full-length fragment to an entry vector pDONR207 and an over-expression vector pK7WG2D in sequence to complete construction of the over-expression vector pK7WG2D-PpERF98.
The overexpression vector pK7WG2D-PpERF98 was transferred into E.coli DH 5. Alpha. By thermal excitation, and PCR positive detection was performed by 2 XEs Taq Master mix (Dye) which was a century company. Plasmids were extracted by full formula Jin Sheng company EASY pure Plasmid MiniPrep Kit and finally transferred into the transformed Agrobacterium GV3101 (WEIDI, shanghai) by thermal excitation.
7. Transient over-expression PpERF98 of peach seedlings and inoculation treatment
8-10 peach seedlings with fully developed leaves are selected for agrobacterium injection. The agrobacteria liquid containing pK7WG2D and pK7WG2D-PpERF98 is respectively injected into the peach leaf from the back of leaf by using a 1mL injector, 4-5 fully developed functional leaves are injected into each peach seedling, and the functional leaves are marked, and after the injection is completed, the functional leaves are placed in darkness at 22 ℃ for 2D, and then inoculation treatment is carried out. The results of observations of the disease symptoms after 1 and 2d inoculation show that agrobacterium-mediated transient overexpression can significantly increase the transcription level of ppenf 98 in peach leaves (fig. 8), the diameter of the lesions of the overexpressed peach plants is significantly larger than that of the control, the resistance to the gummosis bacterial strain is significantly reduced (fig. 9), and both PpPR1 and PpPR2 significantly down regulate the expression (fig. 10).
8. Positive screening of tomato genetic transformation and T2 generation plants
The agrobacterium-mediated genetic transformation of tomato cotyledons was performed by the method of Ouyang Bo et al (2003), and the relative amounts of the T0 generation plants were analyzed by qRT-PCR, as shown in fig. 11, and the transgenic lines with highest relative amounts of ppenf 98 expression were selected for further selection to obtain T2 generation seeds. After the T2 generation seeds are sown for 9d by spraying Shi Kana mould, shi Kana of the mould is sprayed on leaf surfaces in the morning and evening continuously for 4d, non-positive plants with yellowing deformity of the leaves are removed, single plant DNA is extracted by Edley company EASYspin Plus plant DNA Kit, PCR detection is carried out by using carrier primers, and the result shows that target bands with expected sizes appear on the positive plants.
The positive detection primer sequence of the overexpression vector pK7WG 2D:
pK7WG2D-F:5’--TTTCATTTGGAGAGGACTCC--3’
pK7WG2D-R:5’--TAACGTGACTCCCTTAATTC--3’
9. identification of T2-generation transgene and resistance of wild tomato to T.theobromae strain
And taking the isolated leaves of the second compound leaves of the T2 generation transgenic tomato and the wild tomato control strain as materials, and inoculating the peach gummosis bacterial strain L.theobromae on the back of the leaves. After inoculation for 3 and 5d, the disease symptoms are observed, and the results show that the resistance of the T2 generation transgenic strains OX#1 and OX#2 to the peach gummosis bacterial strain L.theobromae is obviously reduced, more infection points appear on the back of the leaf blades, and the disease spot area is increased (figure 12).
10. qRT-PCR analysis of disease course related protein genes in transgenic tomatoes
At 24 and 48 hours after inoculation, 5mm tissues around the inoculation points of T2 generation transgenic tomatoes and control leaves are taken and placed in liquid nitrogen at-80 ℃ for freezing, and qRT-PCR analysis of genes related to disease course proteins is carried out. The results showed that the expression of salicylic acid disease course-dependent genes SlPR1, slPR2, slPR5 and slprp 24 in T2-generation transgenic tomato leaves was inhibited after inoculation with gummosis peach (fig. 13). PpERF98 may attenuate the resistance of T2-generation transgenic tomato lines to T.necator by inhibiting the expression of these disease-associated protein genes.
Primer sequence of tomato disease course protein related gene:
SlPR1-F:5’--GATGTGGGACGATGAGAAGCAATG--3’
SlPR1-R:5’--GTTGCATCGAACCCTAGCACAACCT--3’
SlPR2-F:5’--CAGATTTCACTTCCGTATGCTCTT--3’
SlPR2-R:5’--CCATCCACTCTCTGACACAACAAT--3’
SlPRNP24-F:5’--GAGGGGAACTAAGATGGCACGTAT--3’
SlPRNP24-R:5’--CTCCACCACAATCACCAGTCTGAC--3’
SlPR5-F:5’--AACTGCCCCTACACCGTTTG--3’
SlPR5-R:5’--GCCCAAAACCACCAACTCTG--3’
tomato inner reference primer sequence:
SlActin-F:5’--ATGGCAGACGGAGAGGATATTCA--3’
SlActin-R:5’--GCCTTTGCAATCCACATCTGCTG--3’
11. PpERF98 overexpression or silencing affects salicylic acid accumulation in plants
The salicylic acid content was significantly increased after the peach seedlings were silenced with pprf 98, whereas the salicylic acid content was significantly lower than in the control group after overexpression, suggesting that pprf 98 might affect resistance of peach to gummosis bacteria by modulating the plant salicylic acid pathway (fig. 14).
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (4)
- An application of a ppearf 98 gene in regulating resistance to gummosis of peach, wherein the ppearf 98 gene has a sequence of SEQ ID NO:5.
- 2. the use of the pprf 98 gene according to claim 1 for regulating gummosis resistance of peach, wherein the gummosis resistance of peach is increased by silencing the pprf 98 gene by genetic engineering.
- 3. The application of the PpERF98 gene in regulating and controlling gummosis of peach, which is characterized in that the specific application method comprises the steps of selecting a target fragment from the full-length sequence of the PpERF98 gene, designing a primer of the target fragment to amplify the VIGS-PpERF98, connecting the target fragment with a virus-mediated silencing vector pCaRNA3 by an enzyme digestion method, constructing a VIGS vector, and introducing the VIGS vector into peach plants to improve the gummosis resistance of peach; the sequence of the target fragment is SEQ ID NO:6.
- 4. the use of the ppenf 98 gene according to claim 3 for controlling gummosis resistance of peach, wherein the primer pair VIGS-ppenf 98 sequence of the target fragment is SEQ ID NO:3 and SEQ ID NO:4.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211154241.2A CN116555321A (en) | 2022-09-21 | 2022-09-21 | Application of PpERF98 gene in regulation and control of gummosis resistance of peach |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211154241.2A CN116555321A (en) | 2022-09-21 | 2022-09-21 | Application of PpERF98 gene in regulation and control of gummosis resistance of peach |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116555321A true CN116555321A (en) | 2023-08-08 |
Family
ID=87500674
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211154241.2A Pending CN116555321A (en) | 2022-09-21 | 2022-09-21 | Application of PpERF98 gene in regulation and control of gummosis resistance of peach |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116555321A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113652447A (en) * | 2021-08-09 | 2021-11-16 | 华中农业大学 | VIGS-based efficient peach leaf gene silencing method |
-
2022
- 2022-09-21 CN CN202211154241.2A patent/CN116555321A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113652447A (en) * | 2021-08-09 | 2021-11-16 | 华中农业大学 | VIGS-based efficient peach leaf gene silencing method |
CN113652447B (en) * | 2021-08-09 | 2023-10-27 | 华中农业大学 | High-efficiency peach leaf gene silencing method based on VIGS |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111118005B (en) | MiRNA related to rice blast resistance, corresponding precursor and application | |
CN109825510B (en) | Lilium regale LrWRKY2 gene and application thereof | |
CN112029795B (en) | Application of MpICE1 transcription factor in improving plant disease resistance | |
CN101412751B (en) | Protein related to cold resistance of plant, coding genes and application thereof | |
CN117683109B (en) | Application of ScF3' H gene in improving cold resistance of potatoes | |
CN102021179B (en) | Application of rice gene KT484 to improvement of stress tolerance performance of plants | |
CN116555321A (en) | Application of PpERF98 gene in regulation and control of gummosis resistance of peach | |
CN109180791B (en) | Gene related to plant drought tolerance, and coding protein and application thereof | |
CN102719451B (en) | Poncirus trifoliata basic helix-loop-helix (PtrbHLH) and application in improving cold resistance of plant | |
CN111979251B (en) | Sequoia intermedia ThSHR3 gene for promoting plant growth and application thereof | |
CN104593383A (en) | Gene TaFBK1 with F-box structure field, and expression vector and application thereof | |
CN115976052A (en) | Wheat stem basal rot resistance gene TaHSP18.6, expression product, recombinant vector and application thereof | |
WO2022213453A1 (en) | Use of aluminum ion receptor alr1 gene or protein for regulating aluminum resistance of plant | |
CN112048507B (en) | Cloning and application of miRNA for enhancing rice blast resistance | |
CN114807165A (en) | Application of corn ZmNAC78 gene | |
CN113684217B (en) | Application of PpWRKY40a gene in regulation and control of gummosis resistance of peach | |
CN109554374B (en) | Application of PaMYB82 gene in regulating and controlling plant epidermal hair | |
CN112011550A (en) | Method for blocking exchange of petiole phloem identification signals | |
CN107326018B (en) | Method for cultivating plant low temperature resistant gene and transgenic low temperature resistant plant | |
CN101280008B (en) | Protein related to cold resistance of plant, coding genes and application thereof | |
CN112522296A (en) | WUSCHEL gene and application of encoding protein thereof in plant virus resistance | |
CN114606261B (en) | Method for establishing dangshen gene transient silencing system based on VIGS technology | |
CN104498504B (en) | Peanut AhRRS22 genes and its application in tobacco resistance to bacterial wilt | |
CN117126880B (en) | Application of NbKTI1 gene of Nicotiana benthamiana in regulation and control of plant virus resistance and transgenic plant cultivation method | |
CN116376964B (en) | Gene for regulating low-temperature germination of rice 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 |