PpWRKY4 gene regulating peach aphid resistance and its application

Abstract

The present invention discloses a PpWRKY4 gene for regulating peach aphid resistance and its application, wherein the nucleotide sequence of the PpWRKY4 gene is shown in SEQ ID NO.1. The amino acid sequence encoded by the PpWRKY4 gene is shown in SEQ ID NO.2. The present invention uses leaves of aphid-resistant peach varieties before and after aphid inoculation as materials for transcriptome sequencing, and for the first time discovers the effect of the PpWRKY4 gene on aphid resistance. At the same time, the present invention verifies the PpWRKY4 overexpression vector by transforming it into Arabidopsis thaliana, and the results show that the number of aphids on Arabidopsis thaliana overexpressing the PpWRKY4 gene is significantly less than that of the wild type. The present invention lays a foundation for breeding aphid-resistant peach varieties.

Description

Technical Field

[0001] The invention relates to a PpWRKY4 gene for regulating peach aphid resistance and application thereof, and belongs to the field of agricultural biotechnology. Background Art

[0002] Peach aphid (Myzuspersicae Sülzer), also known as peach red aphid, greasy insect, tobacco aphid, oil aphid, belongs to the order Homoptera, family Aphididae. Peach aphid has a wide distribution range and occurs in Korea, Japan, Indonesia, India, North America, Europe, Africa and other places. It is one of the most widely distributed aphids in the world and is distributed in major peach producing areas in my country. Peach aphid has a wide host range and can feed on more than 400 plants in more than 50 families, mainly including Rosaceae plants such as peach and plum, cruciferous plants such as rapeseed, and Solanaceae plants such as tobacco and potato. In addition, peach aphid has a strong ability to transmit viruses and can transmit 115 plant viruses, accounting for 67.7% of the plant viruses transmitted by all aphids. Peach aphid can cause serious damage to crops. After being eaten by peach aphids, the young leaves of the plants curl horizontally or irregularly to the reverse side, which hinders the nutrient absorption of the leaves, deteriorates the nutrition of the leaves, and even turns yellow. The honeydew excreted by aphids on leaves can easily induce mildew, interfere with plant photosynthesis, and seriously affect the yield and quality of crops. The peach aphid has a strong reproductive capacity, prominent overlapping of generations, parthenogenetic reproduction, and strong adaptability, making it one of the most difficult pests to control.

[0003] At present, the prevention and control of peach aphids mainly relies on pesticides. However, due to the widespread use of nicotine insecticides in recent years, peach aphids have developed resistance to them, making prevention and control more difficult; in addition, the large-scale use of broad-spectrum insecticides also causes serious harm to pollinating insects and natural enemy insects, and damages the ecological environment of orchards. Therefore, mining aphid-resistant genes from resistant resources and cultivating new peach aphid-resistant varieties are important links in peach resource and breeding research. The present invention uses aphid-resistant resources to mine a WRKY gene involved in the peach aphid resistance process, laying a foundation for cultivating aphid-resistant peach varieties. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a PpWRKY4 gene obtained from peach for the first time, which has the function of regulating aphid resistance.

[0005] In order to achieve the above object, one of the technical solutions of the present invention is to provide:

[0006] The PpWRKY4 gene for regulating peach aphid resistance, the nucleotide sequence of the PpWRKY4 gene is shown in SEQ ID NO.1.

[0007] The PpWRKY4 gene is a complete ORF encoding 547 amino acids.

[0008] The amino acid sequence encoded by the PpWRKY4 gene is as shown in SEQ ID NO.2.

[0009] One of the technical solutions of the present invention is to provide: application of the PpWRKY4 gene in regulating peach aphid resistance.

[0010] Specifically, after aphid infection, the PpWRKY4 gene is induced to express in peach leaves.

[0011] The specific method is as follows: the PpWRKY4 gene is forwardly connected with the vector pRI101-AN to obtain a pRI101-PpWRKY4 recombinant expression vector, the pRI101-PpWRKY4 recombinant expression vector is transformed into DH5α Escherichia coli, positive bacteria are screened to extract plasmids, the plasmid with the correct PpWRKY4 gene sequence in the expression vector verified by sequencing is transformed into the Agrobacterium strain GV3101, an infection solution is prepared, the above-ground part of the Arabidopsis plant is inverted in the infection solution, cultured in the dark overnight, and then the plant can be cultivated and managed normally.

[0012] Beneficial effects of the present invention:

[0013] The present invention uses the leaves of aphid-resistant peach varieties before and after aphid inoculation as materials for transcriptome sequencing, and for the first time discovers the effect of the PpWRKY4 gene on aphid resistance. At the same time, the present invention verifies the PpWRKY4 overexpression vector by transforming it into Arabidopsis thaliana, and the results show that the number of aphids on Arabidopsis thaliana overexpressing the PpWRKY4 gene is significantly less than that of the wild type. The present invention lays a foundation for breeding aphid-resistant peach varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the phylogenetic tree of peach PpWRKY4 gene and homologous gene proteins in other species.

[0015] Figure 2 The expression levels of PpWRKY4 gene in peach leaves of aphid-resistant plants (R32) and aphid-susceptible plants (S27) before and after aphid infestation. In the figure, R0, R6, R12, R24, and R48 represent the expression levels of PpWRKY4 gene at 0, 6, 12, 24, and 48 hours after aphid inoculation in R32, respectively. S0, S6, S12, S24, and S48 represent the expression levels of PpWRKY4 gene at 0, 6, 12, 24, and 48 hours after aphid inoculation in S27, respectively. Different lowercase letters indicate significant differences (p<0.05).

[0016] Figure 3 The expression levels of PpWRKY4 gene in transgenic Arabidopsis and wild-type Arabidopsis. In the figure, different lowercase letters indicate significant differences (p<0.05).

[0017] Figure 4 The figure shows the changes in the number of aphids on transgenic Arabidopsis and wild-type Arabidopsis. DETAILED DESCRIPTION

[0018] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0019] Example

[0020] 1. Genetic Screening

[0021] The leaves of aphid-resistant peach from the F2 population of Prunus truncatula hybrids before and after aphid inoculation were used as materials. Total RNA was extracted according to the instructions of the Universal Plant RNA Extraction Kit (Beijing Huayueyang Biological). The RNA concentration and OD were measured using a Nanodrop 1000 micro-volume UV-visible spectrophotometer (Thermo Fisher Scientific). 260 / 280 The value was then reverse transcribed into cDNA using the FastKing cDNA First-Strand Synthesis Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The reaction system and procedures are described in the instruction manual.

[0022] The transcriptome sequencing was performed on the leaves of aphid-resistant peach from the F2 population of the hybrid of Prunus cerasifera before and after aphid inoculation. By comparing the gene expression levels before and after aphid resistance, the PpWRKY4 gene related to aphid resistance was screened out. The protein sequence encoded by the peach PpWRKY4 gene and the phylogenetic relationship of homologous gene proteins in other species are shown in Figure 1 .

[0023] 2. Real-time fluorescence quantitative PCR verification

[0024] The peach leaves of the aphid-resistant individual (R32) and the aphid-susceptible individual (S27) in the F2 population of Prunus persica hybrid at 0, 6, 12, 24, and 48 hours after aphid infection were used as materials, and cDNA was synthesized according to the method in step 1.

[0025] Using cDNA as a template, primers were designed and Real-time PCR (fluorescence quantitative PCR) amplification was performed using the SYBR Green I method to quantitatively analyze the expression level of the PpWRKY4 gene in the leaves of peach cultivars that were resistant to aphids and susceptible to aphids before and after aphid infection.

[0026] The primers for fluorescence quantitative PCR were designed by NCBI, and the sequences are as follows:

[0027] Upstream primer: 5'-ATTTGATGGTGGCTCGGTCG-3' (SEQ ID NO.3)

[0028] Downstream primer: 5'-TTCCAAATGGACTCGGAGGTG-3' (SEQ ID NO.4)

[0029] The peach Actin gene was used as the internal reference gene, and the primer sequences were as follows:

[0030] Upstream primer: 5'-GATTCCGGTGCCCAGAAGT-3' (SEQ ID NO.5)

[0031] Downstream primer: 5'-CCAGCAGCTTCCATTCCAA-3' (SEQ ID NO.6)

[0032] The PCR amplification system is shown in Table 1:

[0033] Table 1 Real-time PCR amplification system

[0034]

[0035] The instrument used was the Roche 480II real-time fluorescence quantitative PCR system, and the amplification program was shown in Table 2.

[0036] Table 2 Real-time PCR amplification procedure

[0037]

[0038] The relative expression was calculated using 2 -ΔΔCt SPSS software was used to perform variance analysis on the expression of PpWRKY4 gene in peach leaves of aphid-resistant and aphid-susceptible varieties before and after aphid infestation. The specific results are shown in Figure 2 .from Figure 2 It can be seen that at 0, 6, 12, 24, and 48 hours after aphid inoculation, the expression level of the PpWRKY4 gene in the aphid-resistant variety was significantly higher than that in the aphid-susceptible variety.

[0039] 3. Cloning of full-length gene CDS sequence

[0040] Homologous recombination was used for cloning. Cloning primers with homology arms were designed based on the reference sequence of the PpWRKY4 gene in the peach Lovell genome (Prunus persica Genome v2.0.a1). The restriction enzyme cutting site NdeI / BamHI was selected, and its position in the primer was underlined.

[0041] Refer to the method in step 1 to clone the cDNA of R32, use cDNA as template, use primers, and use Phanta Max Super-Fidelity DNA Polymerase (Novozyme) ) for PCR amplification.

[0042] The primer sequences are as follows:

[0043] Upstream primer: 5'-gggaattc catatg ATGACAGAAAAAGAAAAAAGGTGCT-3'(SEQ ID NO.7)

[0044] Downstream primer: 5'-cgc ggatcc TTACACAAAGATTTGCTTCTTCTTTTAGC-3'(SEQ ID NO.8)

[0045] The PCR amplification system is shown in Table 3.

[0046] Table 3 PCR amplification system

[0047]

[0048] Transfer the prepared system above into a PCR tube or plate, place it into a PCR instrument, and perform PCR amplification. The amplification program is shown in Table 4.

[0049] Table 4 PCR amplification program

[0050]

[0051] The PCR products were electrophoresed on 1.2% agarose gel and purified using a DNA gel recovery kit (Novagen ) for recycling.

[0052] The recovered PCR product was sent to Shanghai Biotech Co., Ltd. for Sanger sequencing, and the nucleotide sequence was shown in SEQ ID NO. 1. The amino acid sequence was translated by software BioXM (v2.7) to obtain SEQ ID NO. 2.

[0053] IV. Construction of overexpression vector

[0054] The pRI101-AN vector was digested with restriction endonucleases NdeI / BamHI, and then the recovered PCR product of the PpWRKY4 gene was ligated with T4 ligase to obtain the pRI101-PpWRKY4 recombinant expression vector, which was then transformed into DH5α Escherichia coli and spread on LB medium containing kanamycin for positive screening. The positive colonies were cultured in LB liquid medium containing kanamycin, and the plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0055] 5. Agrobacterium-mediated transgenic

[0056] 1. Agrobacterium transformation

[0057] The plasmid with the correct PpWRKY4 gene sequence in the expression vector verified by sequencing was used to transform Agrobacterium GV3101 competent cells. The transformed Agrobacterium GV3101 competent cells were spread on LB screening medium containing kanamycin and rifampicin, cultured at 28°C for 2-3 days for positive screening, and the positive single colony was placed in LB liquid medium containing kanamycin and rifampicin, and cultured at 28°C, 220rpm for 1 day to obtain Agrobacterium GV3101 bacterial liquid containing the target plasmid.

[0058] 2. Prepare infection solution

[0059] Agrobacterium GV3101 containing the target plasmid was cultured in LB liquid medium containing kanamycin and rifampicin (10 μL of the above Agrobacterium GV3101 bacterial solution was added to each 5 mL of liquid medium), and cultured overnight at 28°C and 220 rpm; 2-3 mL of the liquid culture was added to 50 mL of LB liquid medium containing kanamycin and rifampicin, and cultured with shaking at 28°C and 220 rpm for 4-5 h, centrifuged at 4000 rpm for 20 min, the supernatant was discarded, the precipitated cells were collected, and the cells were resuspended in infection medium (1 / 2 MS medium) to a concentration of OD 600 = about 0.8, add 0.02% surfactant silweetl-77 to prepare the infection solution.

[0060] 3. Infection

[0061] Invert the above-ground part of the wild-type Arabidopsis plant in the infection solution for 1-2 minutes, culture in the dark overnight, and culture in a light incubator (darkness 23°C, 14h / light 18°C, 10h) to observe its growth phenotype. Infect again after one week and wait for the harvest of T0 generation transgenic seeds.

[0062] VI. Phenotypic Identification of Transgenic Arabidopsis

[0063] Arabidopsis T0 transgenic seeds were harvested, disinfected and planted on 1 / 2MS medium containing 100 mg / L kanamycin to select positive plants. After 8 days, it was observed that the positive plants had strong growth potential, dark green color, and vigorous root system, while the non-positive plants turned yellow and withered until they died. RNA was extracted from the leaves of positive seedlings (transgenic Arabidopsis) that grew well on 1 / 2MS medium containing kanamycin and reverse transcribed into cDNA. Real-time PCR verification was performed using the primers used for fluorescence quantification. SPSS software was used to perform variance analysis on the expression of the PpWRKY4 gene in transgenic Arabidopsis and wild-type Arabidopsis. The results are shown in Figure 3 .from Figure 3 It can be seen that the expression level of the PpWRKY4 gene in the Arabidopsis strains overexpressing the PpWRKY4 gene (OE1, OE2, OE3) is significantly higher than that in the wild type (WT).

[0064] When the transgenic Arabidopsis and wild-type Arabidopsis grew to bolting, 10 aphids were inoculated on each Arabidopsis plant, and the number of aphids was recorded every day for 7 consecutive days. SPSS software was used to perform variance analysis on the number of aphids on transgenic Arabidopsis and wild-type Arabidopsis. The results are as follows: Figure 4 .from Figure 4 It can be seen that 7 days after aphid inoculation, the number of aphids on the Arabidopsis lines overexpressing the PpWRKY4 gene (WRKY-OE1, WRKY-OE2, and WRKY-OE3) was significantly less than that on the wild type (WT).

Claims

1. Overexpression PpWRKY4 The application of the gene in improving peach aphid resistance is characterized in that: Said PpWRKY4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Said PpWRKY4 The gene is a complete ORF encoding 547 amino acids.

3. The use according to claim 1, characterized in that: Said PpWRKY4 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: After aphid infestation, the PpWRKY4 The gene was induced to express in peach leaves.

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