Application of citrus CsERF017 gene in repelling diaphorina citri

Abstract

The invention discloses an application of a citrus CsERF017 gene in repelling diaphorina citri. The nucleotide sequence of the CsERF017 gene is as shown in SEQ ID NO. 1. The diaphorina citri has relatively weak tropism on lemon plants over-expressed by CsERF017, so that the CsERF017 is a gene with the capability of repelling the diaphorina citri, and the gene can be used as an important candidate gene for cultivating diaphorina citri varieties. The invention can provide important gene resources and powerful theoretical support for cultivating phylloxera-resistant citrus varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of the citrus CsERF017 gene in repelling citrus psyllids. Background Art

[0002] Citrus is a very important economic crop, widely distributed throughout the world. Citrus Huanglongbing (HLB) is a major global citrus disease caused by the bacterium Candidatus Liberibacter and primarily transmitted in the field by the citrus psyllid, Diaphorina citri. Current methods for controlling citrus psyllids in citrus production primarily focus on physical, chemical, and biological control. However, these methods are far from ideal in terms of practicality or economic efficiency. Therefore, breeding psyllid-resistant citrus varieties is considered the optimal solution for controlling psyllids.

[0003] In previous research, the applicant used RNA-Seq technology to assess global transcriptome changes in sweet orange leaves before and after feeding by the citrus psyllid. The study identified significant differentially expressed genes before and after psyllid feeding. By identifying functional genes involved in psyllid repellency in citrus and clarifying their regulatory mechanisms for psyllid behavior, the study provides important genetic resources and strong theoretical support for the development of psyllid-resistant citrus varieties. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a citrus CsERF017 gene and its use in repelling citrus psyllids. The present invention identified a transcription factor CsERF017 from the citrus variety sweet orange (Citrus sinensis). The expression of this gene was strongly induced and downregulated by the feeding of citrus psyllids. By utilizing gene overexpression technology to enhance the expression of the CsERF017 gene, the gene exhibited a phenotype with enhanced expression levels of the CsERF017 gene. The results showed that compared with the control lemon plants, the citrus psyllids had a very weak tendency to transiently overexpress lemon plants of CsERF017, indicating that CsERF017 is a gene with the ability to repel citrus psyllids. The research results reveal that CsERF017 can participate in the defense response induced by citrus psyllids and can be used as a candidate gene for breeding psyllid-resistant citrus varieties.

[0005] Therefore, the first object of the present invention is to provide a use of the citrus CsERF017 gene or a biological material comprising the gene in repelling citrus psyllids. The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.1.

[0006] The amino acid sequence of the protein encoded by the CsERF017 gene is shown in SEQ ID NO. 2. It should be understood that, without affecting the structure and activity of the CsERF017 transcription factor, those skilled in the art can make various substitutions, additions, and / or deletions of one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2 to obtain a protein with equivalent function.

[0007] A second objective of the present invention is to provide the use of the citrus CsERF017 gene or a biomaterial containing the gene in cultivating insect-resistant plants. The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 1. Preferably, the use is in cultivating plants resistant to the citrus psyllid. Preferably, the plant is a citrus plant.

[0008] A third object of the present invention is to provide the use of the citrus CsERF017 gene or biological materials containing the gene in plant germplasm improvement. The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 1. Preferably, the use is in improving plant germplasm resources to enhance resistance to citrus psyllids. Preferably, the plant is a citrus plant.

[0009] The present invention demonstrates through experiments that overexpressing the CsERF017 gene reduces the tropism of citrus psyllids toward lemon plants. Therefore, a fourth object of the present invention is to provide a method for reducing the tropism of citrus psyllids toward plants, by overexpressing the citrus CsERF017 gene through transgenic technology. The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 1. Preferably, the plant is a citrus plant.

[0010] The present invention discloses the application of the CsERF017 gene from the sweet orange citrus variety in citrus psyllid-resistant cultivar breeding. The authors successfully cloned the sweet orange CsERF017 gene and found that citrus psyllids have a very weak tropism for lemon plants transiently overexpressing CsERF017. This indicates that CsERF017 is a gene with the ability to repel citrus psyllids and, therefore, could serve as an important candidate gene for breeding psyllid-resistant citrus varieties. This invention provides important genetic resources and strong theoretical support for breeding psyllid-resistant citrus varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the result of the CsERF017 gene's response to feeding by citrus psyllids.

[0012] Figure 2Overexpressing the CsERF017 gene in lemons reduces the tropism of the citrus psyllid. Figure a shows the positive plant test results for lemon plants transiently overexpressing the CsERF017 gene; and figure b shows the tropism of the citrus psyllid toward lemon plants transiently overexpressing the CsERF017 gene. DETAILED DESCRIPTION

[0013] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0014] Example 1: Obtaining the sweet orange CsERF017 gene

[0015] The present invention utilizes bioinformatics methods combined with PCR amplification to clone genes. Specifically, a search was performed in the sweet orange genome database (http: / / citrus.hzau.edu.cn / index.php), revealing the gene ID for CsERF017, Cs4g07040. Using sweet orange leaf cDNA as a template, upstream primer F: ATGGTGAAGCAC GTAGTC; downstream primer R: TTAAAAATTCCAAAGAAACGAATCTTG were designed based on the CsERF017 sequence. PCR amplification of the product yielded a 600-bp cDNA fragment encoding the CsERF017 gene (nucleotide sequence shown in SEQ ID NO. 1). This gene encodes 199 amino acids, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0016] The cDNA sequence of the CsERF017 gene is shown in SEQ ID NO. 1, specifically:

[0017] ATGGTGAAGCACGTAGTCGAAAAGCCTGCTGAGAGAAGTGACTCTCGTTACAAGGGT

[0018] GTCCGAAAGCGAAAGTGGGGGAAATATGTGTCTGAAATCAGACTACCCAACAGCCGT

[0019] GCCCGTATCTGGCTGGGCTCCTACGACACAGCAGAAAAAAGCAGCGCGTGCTTTCGAC

[0020] GCTGCTTTGTTTTGCTTACGTGGCCGATCAGCCAAGTTTAATTTCCCGGACAACCCACC

[0021] CGACATTCAGGCGGGCGCTCACTTAAGCCATCTGAGATTCAAGCTTGTGGCGGCTCAG

[0022] TTCGCGAATTCGGAGCCGCTGAGGAGTCAATCGCCAGAACAGTCGGTGTCCGAATTG

[0023] CAAACGGAGTACTTATCGCCGTCGATTTCTGAGGCAACGGTTCAGTTGGACAGCGATG

[0024] GGGTTTTTGACGGGTCTTTATTGGATCTTTTAACGGCATCGAGTTCCAGTAACTACCCT

[0025] TCCGAATATGGGATATTTCCTGGGTTTGATGACCTGTCTAATGACATTTTTGCTCCGCAA

[0026] CTCCCAGCTGTTGATTTTGCAGACGAGAATTTTGATGGGCTATTGTTGAATCAAGATTC

[0027] GTTTCTTTGGAATTTTTAA

[0028] The amino acid sequence of the protein encoded by the CsERF017 gene is shown in SEQ ID NO. 2, specifically: MVKHVVEKPAERSDSRYKGVRKRKWGKYVSEIRLPNSRARIWLGSYDTAEKAARAFDAALFCLRGRSAKFNFPDNPPDISGGRSLKPSEIQAVAAQFANSEPLRSQSPEQSVSELQTEYLSPSISEATVQLDSDGVFDGSLLDLLTASSSSNYPSEYGIFPGFDDLSNDIFAPQLPAVDFADENFDGLLLNQDSFLWNF*

[0029] Example 2: Expression of the CsERF017 gene is strongly downregulated by feeding by citrus psyllids

[0030] In an experiment involving citrus psyllids feeding on sweet orange seedlings, adults were starved for 12 hours and then placed in a glass container containing three-month-old sweet orange seedlings. Orange leaves were collected after feeding for 12, 24, and 48 hours, with 0 hours serving as a control. Leaf RNA was extracted and expression of the CsERF017 gene was detected using real-time quantitative PCR. The primer pair for CsERF017 real-time PCR is as follows: CsERF017-qRT-F: TCAATCGCCAGAACAGTCGG; CsERF017-qRT-R: AAGACCCGTCAAAAACCCCA.

[0031] The results showed that the expression of CsERF017 gene was strongly down-regulated by citrus psyllid feeding ( Figure 1 ), indicating that the CsERF017 gene may be involved in the defense response induced by citrus psyllids.

[0032] Example 3: Lemons overexpressing the CsERF017 gene reduce the tropism of citrus psyllids

[0033] (1) Vector construction

[0034] Using sweet orange leaf cDNA as a template, primers were designed to amplify the ORF of the CsERF017 gene in sweet orange. The length of the ORF was 600 bp and ligated into the POX-GFP vector (kindly provided by Professor Peng Xinxiang's laboratory at South China Agricultural University) between the KpnI and HindIII restriction sites via homologous recombination. The primers were designed as follows:

[0035] CsERF017-OE-F:GAACGATAGGGTACCATGGTGAAGCACGTAGTC;

[0036] CsERF017-OE-R:CGTACTAGTAAGCTTAAAATTCCAAAGAAACGAATCTTG.

[0037] Amplification system: 50 μL system, 2 μL each of upstream and downstream primers, 1 μL cDNA, 25 μL 2× Phanta Max Master Mix (Dye Plus), 20 μL H O. After the vector is sequenced correctly, the plasmid is extracted and transformed into Agrobacterium competent GV3101 (pSoup-p19).

[0038] The plasmids POX-GFP and POX-CsERF017-GFP were transformed into Agrobacterium competent cell GV3101 (pSoup-p19) to obtain POX-GFP and POX-CsERF017-GFP Agrobacterium, respectively.

[0039] (2) Preparation of Agrobacterium infection solution

[0040] POX-GFP and POX-CsERF017-GFP Agrobacterium monoclones were picked and activated overnight in 5 mL of LB liquid medium containing the corresponding antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin). The activated Agrobacterium liquid was inoculated into fresh LB medium containing antibiotics at a volume ratio of 1:100 and cultured at 28°C overnight. Centrifuge at 4000 r / min for 10 minutes, collect the cells, add infection buffer (10 mmol / L MES, 10 mmol / L MgCl2, 200 μmol / L AS, pH = 5.6-5.7) to suspend the cells and adjust the OD 600 To 1.0. After standing in the dark for 2-3 hours, it can be used to inject lemon leaves.

[0041] (3) Transient transformation of lemon leaves with Agrobacterium

[0042] Fresh lemon seeds were removed intact from the fruit, soaked in 1 mol / L NaOH solution for 15 minutes to remove pectin, rinsed with sterile water, spread on clean gauze, and placed in a 30°C incubator for germination. After germination, the seeds were transferred to a mixture of nutrient soil and vermiculite in a 2:1 mass ratio and cultured in an artificial plant culture chamber (L:D = 16:8h, 28°C, RH 60±5%). Watering was carried out regularly to promote germination. When the lemon plants reached the 6-8 leaf stage, they were used for Agrobacterium injection. Plants injected with POX-GFP Agrobacterium served as controls.

[0043] (4) Identification of CsERF017 transient overexpression-positive plants and citrus psyllid tropism experiments

[0044] The expression of CsERF017 gene in lemon leaves with transient overexpression of CsERF017 was analyzed. The real-time quantitative primers for CsERF017 were: CsERF017-qRT-F: TCAATCGCCAGAACAGTCGG; CsERF017-qRT-R: AAGACCCGTCAAAAACCCCA.

[0045] The citrus psyllid tropism experiment was conducted on three lemon lines (CsERF017-OE#2, CsERF017-OE#3 and CsERF017-OE#5) that transiently overexpressed CsERF017. The number of fallen insects on the leaves of the overexpressing lemon plants and the control plants was counted at 4, 6, 12, 24 and 48 hours.

[0046] The results showed that the expression of CsERF017 gene in CsERF017-OE#2, CsERF017-OE#3 and CsERF017-OE#5 plants was 2.5, 3 and 4.5 times that in the control plants, respectively. Figure 2 a). The number of insects falling on the leaves of the three CsERF017-OE lemon plants was significantly less than that of the control plants ( Figure 2 b) Citrus psyllids have a weaker tropism for lemon plants overexpressing CsERF017, indicating that CsERF017 is a gene with the ability to repel citrus psyllids.

[0047] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of the citrus CsERF017 gene or a biological material containing the gene in repelling citrus psyllids, characterized in that: The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the CsERF017 gene is shown in SEQ ID NO.

2.

3. Use of the citrus CsERF017 gene or a biological material containing the gene in cultivating insect-resistant plants, characterized in that: The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The breeding of insect-resistant plants is breeding of plants resistant to citrus psyllids.

5. Application of the citrus CsERF017 gene or biological materials containing the gene in plant germplasm resource improvement, characterized in that: The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that: The plant germplasm resource improvement is the germplasm resource improvement that improves the plant's resistance to citrus psyllids.

7. The use according to claim 1, 3 or 5, characterized in that: The biological material is an expression box, a recombinant vector, a recombinant bacterium or a transgenic cell line.

8. The use according to claim 3 or 5, characterized in that: The plant is a citrus plant.

9. A method for reducing the tropism of citrus psyllids to plants, characterized in that: The citrus CsERF017 gene is overexpressed by transgenic technology, and the nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that The plant is a citrus plant.

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