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
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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.
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