Effect of tomato leaf miner oral secretion effector gene REPAT38 in inhibition of host plant defense

Abstract

The invention relates to the technical field of agricultural biology, in particular to an effect of a tomato leaf miner oral secretion effector gene REPAT38 in inhibition of host plant defense. The gene REPAT38 encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2. The effector induces pore closing within 0.5 h and inhibits synthesis of JA, ET and ABA within 1 h. In addition, seven plant hormone response genes, namely AOC, MYC2, ACS1A, PAL, PR1, EIL2 and SRK2E, are inhibited at different time points. Therefore, the oral secretion effector REPAT38 can weaken the defense capability of tomato hosts, so that the feeding performance of insects is improved. After the gene is interfered, the plant defense inhibition capability of insects is weakened.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to the role of oral secretion effector gene REPAT38 of tomato leafminer in inhibiting host plant defense. Background Art

[0002] In the long-term co-evolution with host plants, herbivorous insects have evolved a variety of feeding methods in order to better adapt to and utilize their hosts. At the same time, in order to cope with the feeding of herbivorous insects, plants have evolved a variety of defense strategies, including direct defense and indirect defense. Insects release saliva during feeding, and their saliva comes into direct contact with plants. The components of saliva will be recognized by the host plant and induce or inhibit defense responses. Among them, the components that can be recognized by the plant and induce the plant's defense response are called elicitors, while the components that inhibit the plant's defense response are called effectors.

[0003] The tomato leafminer, Phthorimaea absoluta, is an omnivorous pest that can harm nearly 40 plant species. Therefore, it is necessary to study the interaction between the effectors of oral secretions of the tomato leafminer and the defense response of tomato plants. Summary of the invention

[0004] The invention aims to provide an effector of oral secretions of tomato leafminer.

[0005] Another object of the present invention is to provide the above-mentioned tomato leafminer oral secretion effector gene REPAT38.

[0006] Another object of the present invention is to provide the application of the above gene in the defense of tomato leafminer against host plants.

[0007] Another object of the present invention is to provide a method for improving the defense capability of a host plant of tomato leafminer.

[0008] According to a specific embodiment of the present invention, the oral secretion effector gene REPAT38 of the tomato leafminer was cloned for the first time, and the full-length nucleotide sequence of its cDNA is shown in SEQ ID No:1.

[0009] SEQ ID No: 1:

[0010] GAACTGTGATAAGTTGCTAACATTTTAAAACATAAAGATGAAGGCCATCCTAGTACTATGCATGTTGGCAGCGCTGGCTCTTACCGAAGCATCAGTTGCTCGTGCCCCGGCAACACGAAGCAATCCATCTTTGGGATACATTCAGTATGGAGATAGACTCCTAGCAAGGCAGTATCTTGCCAAGGGAGCGCGACCAAACGCCATCCA GTATCGAGACCTGATTTACCGCGGGAACGCTACCACGCGAATCAGTGCCATTCAAGCCATGGAAGTGGGCTACACGCAGTGGGCCAGCGCGTGGTCGTAGCTGGTGGAATTGGTTGGAACTCGGCCACAGTGCGCGTGCAATCCGCGCGCGGCTACGGCTATAACTATCAGATCGACATTTGGGGACGATGAGTTACTACATATT.

[0011] The amino acid sequence of the oral secretion effector receptor REPAT38 of the tomato leafminer is shown in SEQ ID NO:2.

[0012] SEQ ID NO: 2:

[0013] MKAILVLCMLAALALTEASVARAPATRSNPSLGYIQYGDRLLARQYLAKGAR PNAIQYRDLIYRGNATTRISAIQAMEVGYTQWASAWVVAGGIGWNSATVRVQSAR GYGYNYQIDIWGR.

[0014] According to the method for improving the defense ability of the host plant of tomato leafminer, the method comprises the step of interfering with the expression of the oral secretion effector gene REPAT38 of tomato leafminer, wherein the oral secretion effector gene REPAT38 of tomato leafminer encodes a protein having an amino acid sequence as shown in SEQ ID NO:2.

[0015] According to the method for improving the defense ability of the host plant of the tomato leafminer of the present application, the tomato leafminer is fed with the dsRNA of the oral secretion effector gene REPAT38.

[0016] According to the method for improving the defense ability of the host plant of the tomato leafminer of the present application, the dsRNA is obtained by amplifying the oral secretion effector gene REPAT38 of the tomato leafminer using the following primers:

[0017] dsREPAT38F:5′ TAATACGACTCACTATAGGG GGCAACACGAAGCAATCCAT3′;

[0018] dsREPAT38R:5′ TAATACGACTCACTATAGGG ATTCCACCAGCTACGACCCA3′.

[0019] The present invention provides the use of the oral secretion effector REPAT38 of the tomato leafminer in inhibiting the defense of the host plant. The research results show that when the REPAT38 gene is interfered with, the stomatal conductance of the tomato leaves is significantly increased compared with the control group, indicating that the effector REPAT38 gene can induce stomatal closure; when the REPAT38 gene is interfered with, the synthesis of JA, ET and SA is higher than that of the control, indicating that the REPAT38 gene can inhibit the synthesis of JA, ET and SA; when the REPAT38 gene is interfered with, the expression levels of 7 plant hormone response genes AOC, MYC2, ACS1A, PAL, PR1, EIL2 and SRK2E at different time points are higher than those of the control, indicating that the REPAT38 gene can inhibit the expression of the gene. Therefore, the oral secretion effector REPAT38 can weaken the defense ability of the tomato host, thereby improving the feeding performance of the insect. When the gene is interfered with, the ability of the insect to inhibit the defense of the plant will be weakened. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The structure of the oral secretion effector REPAT38 from the tomato leafminer is shown;

[0021] Figure 2 It shows the changes in the expression level of the REPAT38 gene after the tomato leafminer feeds on the dsRNA;

[0022] Figure 3 Shows the effect of REPAT38 on stomatal opening and closing in the leaves of the host plant tomato;

[0023] Figure 4 Showing the impact of REPAT38 in regulating plant hormone synthesis;

[0024] Figure 5 Showing the effects of REPAT38 on regulating the expression of plant response genes. DETAILED DESCRIPTION

[0025] Example 1: Cloning of the full-length cDNA sequence of the REPAT38 gene of the tomato leafminer

[0026] Take 4 larvae of tomato leafminer and put them into a 1.5mL centrifuge tube. After freezing in liquid nitrogen, grind them into a slurry using a grinding rod, then extract RNA and store at -80℃ for later use. According to the instructions of the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for PCR, the extracted RNA was reverse transcribed to synthesize cDNA. Using cDNA as a template, primers were designed and PCR amplification was performed. The designed primers are shown in Table 1.

[0027] Table 1 Primer sequences for cloning the full-length cDNA of REPAT38 gene

[0028]

[0029] Using the sequence in Table 1, a cDNA sequence of the REPAT38 gene was obtained by PCR amplification with a total length of 413 bp. The obtained gene has a nucleotide sequence as shown in SEQ ID No: 1, and the gene encodes 120 amino acid sequences as shown in SEQ ID No: 2. Analysis of the amino acid sequence encoded by the cloned gene showed that its calculated molecular weight was 13.17 kDa and the theoretical isoelectric point (pI) was 10.07. Figure 1 As shown, REPAT38 is a small secreted protein of 120 amino acid residues, whose amino terminal region has a signal peptide that is cleaved at the position of 18-19 amino acids and contains the transcription activator MBF2.

[0030] Example 2: Analysis of the response of the REPAT38 gene to host plant defense against tomato leafminer

[0031] 2.1 Synthesis of target gene dsRNA and related gene primers

[0032] (1) Design the primer sequences of REPAT38 gene dsRNA, 12 plant hormone response genes (AOC, ACO1, MYC2, ACS1A, ACS1B, AOS, OPR3, PAL, PR1, EIL2, ERF1 and SRK2E) quantitative PCR, and the internal reference gene β-actin, as shown in Table 2 below.

[0033] Table 2 Related primer sequences

[0034]

[0035]

[0036] The extraction of total RNA from tomato leafminer and the synthesis of cDNA were the same as in Example 1. T7 primer PCR amplification and product purification were performed, and the purified PCR product was used as a template for synthesizing dsRNA. dsRNA was synthesized and purified using a kit according to the instructions of the kit. Plant total RNA extraction and cDNA synthesis were performed according to the instructions of the plant RNA extraction and cDNA synthesis kit.

[0037] 2.2 dsRNA feeding

[0038] Fresh leaves were dried for 2 hours, and second-instar larvae of tomato leafminers were picked in advance and starved for 2 hours. The petioles fully absorbed dsREPAT38 at a concentration of 25 μg / mL, and the larvae were picked back to the leaves and allowed to feed for 48 hours before sampling. Tomato leafminers fed with an aqueous solution containing dsEGFP (final dsEGFP concentration of 25 μg / mL) were used as controls. The control group and the treatment group were replicated 5 times, with 30 larvae in each replicate. The quantitative PCR detection kit was used according to the instructions. -ΔΔCT Methods The relative expression of dsREPAT38 gene after RNAi interference was calculated. The silencing efficiency of REPAT38 was detected after 60 hours of feeding, and it was found that the expression of REPAT38 gene in the dsREPAT38 group was significantly downregulated, which was significantly reduced by 56.7% compared with the control group (t=2.571, df=8, P<0.05).

[0039] 2.3 Observation of stomata on the lower epidermis of leaves

[0040] The lower leaves of tomato plants were selected, and the larvae treated with dsEGFP and dsREPAT38 fed on the leaves for 0.5h, 1h, 2h, and 4h, respectively. The blank control group was leaves that were not fed by larvae. Use a blade to take about 1 square centimeter of the lower epidermis from the wound of the leaf. Place the lower epidermis on a slide, cover it with a coverslip, and immediately observe it under a biological microscope (BX63). Three fields of view were randomly selected from each sample, the number of stomata opened and closed, and the proportion of stomatal closure was calculated. The research results showed that at 0.5h, the stomatal conductance of tomato leaves increased significantly by 31.07% (OS dsREPAT38 vs.OS dsEGFP ), while at 1 h, the stomatal conductance decreased significantly by 22.36%. This result indicates that the oral secretion effector REPAT38 of tomato leafminer regulates the stomatal conductance of tomato leaves. Figure 3 shown.

[0041] 2.4 Detection of plant hormone content

[0042] A double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used. The specimen, standard, and HRP-labeled detection antibody were added to the coated microwells pre-coated with antibodies to jasmonic acid (JA), jasmonic acid L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA), in sequence, and incubated and thoroughly washed. The substrate TMB was used for color development. TMB was converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of color is positively correlated with jasmonic acid (JA), jasmonic acid L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration. Figure 4 As shown, this effector inhibited the synthesis of JA, ET and SA at 1 h.

[0043] 2.5 Detection of plant response gene expression

[0044] Follow the instructions of the quantitative PCR detection kit to operate. -ΔΔCT The relative expression of genes was calculated by this method. Figure 5 As shown, seven plant hormone response genes, AOC, MYC2, ACS1A, PAL, PR1, EIL2 and SRK2E, were inhibited at different time points.

[0045] The above research results indicate that REPAT38, as an oral secretion effector, can weaken the defense ability of the tomato host and play an important role in the process of tomato leafminers feeding on tomato leaves.

[0046] The present invention cloned the full-length cDNA of the oral secretion effector REPAT38 gene from the tomato leafminer, used RNAi technology, took the oral secretion effector REPAT38 gene as the target, monitored the changes in the leaf stomata, plant hormone (jasmonic acid (JA), jasmonoyl L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET) and abscisic acid (ABA)) concentrations and the relative expression levels of 12 plant hormone response genes (AOC, ACO1, MYC2, ACS1A, ACS1B, AOS, OPR3, PAL, PR1, EIL2, ERF1 and SRK2E) at 0.5, 1, 2 and 4 hours, explored the molecular mechanism of plant-insect interaction mediated by REPAT38, and showed that:

[0047] When the REPAT38 gene was interfered, the stomatal conductance of tomato leaves increased significantly compared with the control group at 0.5h, indicating that the effector REPAT38 gene can induce stomatal closure;

[0048] When the REPAT38 gene was interfered, the synthesis of JA, ET and SA was higher than that of the control, indicating that the REPAT38 gene can inhibit the synthesis of JA, ET and SA;

[0049] When the REPAT38 gene was interfered with, the expression levels of seven plant hormone response genes, AOC, MYC2, ACS1A, PAL, PR1, EIL2 and SRK2E, were higher than those of the control at different time points, indicating that the REPAT38 gene can inhibit the expression of this gene.

[0050] The results showed that the oral secretion effector REPAT38 can weaken the defense ability of tomato hosts, thereby improving the feeding performance of insects. When the gene is interfered with, the ability of insects to suppress plant defense will be weakened, which is reflected in the changes of plant defense hormones and response genes. Plant hormones such as jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) are involved in the regulation of defense against herbivores. In the reprogramming of biotic stress, transcription factors coordinate the biosynthesis of defense genes and the dynamic interactions between metabolites in biosynthesis.

[0051] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. REPAT38, an effector gene of oral secretions of tomato leafminer, characterized in that The tomato leafminer oral secretion effector gene REPAT38 encodes a protein with an amino acid sequence as shown in SEQ ID NO:

2.

2. The tomato leafminer oral secretion effector gene REPAT38 according to claim 1, characterized in that: The nucleotide sequence of the gene REPAT38 is shown in SEQ ID No:

1.

3. Use of the oral secretion effector gene REPAT38 of the tomato leafminer according to claim 1 in inhibiting the defense of host plants.

4. A method for improving the defense capability of a host plant of tomato leafminer, characterized in that: Interfering with the expression of the oral secretion effector gene REPAT38 of the tomato leafminer, wherein the oral secretion effector gene REPAT38 of the tomato leafminer encodes a protein with an amino acid sequence as shown in SEQ ID NO:

2.

5. The method for improving the defense capability of a host plant of tomato leafminer according to claim 4, characterized in that: Tomato leafminers were fed with dsRNA of the oral secretion effector gene REPAT38.

6. The method for improving the defense capability of a host plant of tomato leafminer according to claim 4, characterized in that: The dsRNA is obtained by amplifying the oral secretion effector gene REPAT38 of the tomato leafminer using the following primers: dsREPAT38 F:5′ TAATACGACTCACTATAGGG GGCAACACGAAGCAATCCAT3′; dsREPAT38 R:5′ TAATACGACTCACTATAGGG ATTCCACCAGCTACGACCCA3′。

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