Application of csERF017 gene or the protein coded thereby in regulating resistance of navel orange fruit to sour rot

Abstract

The application discloses application of a CsERF017 gene or a protein coded by the same in regulation of resistance of navel orange fruits to sour rot, and belongs to the technical field of molecular biology and fruit and vegetable preservation. A nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 2, and an amino acid sequence of the protein coded by the same is shown in SEQ ID NO. 3. The application finds through experiments that the CsERF017 gene is up-regulated after infection of a citrus sour rot pathogen Geotrichum candidum, and silencing or knocking out the CsERF017 gene can enhance the resistance of navel orange fruits to sour rot, and the specific performance is that, compared with a control, the transgenic fruits after inoculation of the G. candidum have reduced symptoms of sour rot, and both a disease index and a TA content are reduced. The application provides an important gene bank and new insights for breeding of citrus disease-resistant varieties and development of molecular markers, and is helpful to fundamentally reduce the dependence on chemical synthetic pesticides in postharvest preservation treatment of citrus fruits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and fruit and vegetable preservation technology, in particular to the application of CsERF017 gene or the protein coded by the same in regulating the resistance of navel orange fruit to sour rot. BACKGROUND

[0002] Citrus is a tree belonging to Rutaceae Citrus, including dozens of varieties such as mandarin, orange, tangerine, pomelo, lemon, lime, lime, and gold orange, which are distributed in more than 140 countries and regions around the world. The fruit is rich in nutrients, rich in vitamin C, polyphenols, flavonoids, and other bioactive ingredients beneficial to human health. Navel oranges are favored by consumers due to their rich nutrition, delicious juice, and unique taste. Compared with the green mold disease commonly seen in broad-leaved oranges, the sour rot of citrus caused by Geotrichum citri-aurantii is more significant during postharvest storage and transportation of navel orange fruits. At the early stage of the disease, the fruit softens and shrinks, and shows water spots. After 3-5 days, a white mold layer appears on the diseased spot surface, which rapidly expands into a white, nearly circular mold spot. The diseased fruit may crack and emit a pungent rancid smell, and the whole fruit may become waterlogged and unable to be picked up.

[0003] Currently, the control of postharvest sour rot of citrus fruits mainly relies on chemical synthetic pesticides, but many chemical synthetic substances can easily cause pesticide residues, the emergence of drug-resistant strains, harm to human health, and pollution of the ecological environment. The molecular mechanism of citrus fruit resistance to sour rot has been relatively slow both at home and abroad, and there is no report on the functional analysis and regulatory mechanism of sour rot resistance-related genes.

[0004] Plants have evolved a set of highly sophisticated immune defense mechanisms to protect themselves from pathogenic infection. Through precise regulation of pathogenesis-related or defense-related gene expression, plants can effectively modulate their response to pathogenic microorganisms. Transcription factors play a key regulatory role in plant disease resistance response. They not only integrate upstream signals, but also coordinate the expression of a series of downstream defense-related genes. Ethylene-responsive transcription factor (ERF) is a core member of the AP2 / ERF transcription factor superfamily. It specifically binds to the GCC-box cis-acting element in the promoter region of the downstream interacting gene to activate or inhibit the expression of the interacting gene, and is involved in the regulation of plant growth and development, maturation and aging, and stress response. Overexpression of SlERF01 gene in tomato can significantly enhance the defense ability of tomato leaves against Stemphylium lycopersici. The specific performance is the increase of lignin, callose and other resistance substances, and the up-regulated expression of pathogenesis-related protein SlPR1 gene, thereby enhancing the hypersensitive response of tomato leaves to S. lycopersici infection (Yang et al, 2020). In citrus peel, transient overexpression of CsERF1B gene activates CsOPR11 and CsAOS3L genes in the jasmonic acid signaling pathway, as well as the expression of lignin synthesis genes, mediates jasmonic acid signaling, and promotes the accumulation of lignin content, thereby significantly improving the resistance of fruits to green mold (Li et al, 2023). In addition, Wang et al. (2020) found an ERF transcription factor located in the nucleus, MdERF11, in 'Royal Gala' apple. Overexpression of the gene in callus significantly increased the content of SA and induced the expression of EDS1, PAL, PR1, NPR1 and other related genes in the SA synthesis pathway, thereby enhancing the resistance of fruits to V. inaequalis. Research in the past 20 years has shown that ERF transcription factors mainly mediate cell wall secondary metabolism, plant hormones (jasmonic acid, salicylic acid, melatonin, etc.) signaling and pathogenesis-related protein activity to improve plant resistance to biological (bacteria, fungi, oomycetes, viruses, etc.) stress. However, there are few reports on the role of citrus ERF transcription factors in responding to G. citri-aurantii disease resistance. SUMMARY

[0005] The purpose of the present application is to provide the application of CsERF017 gene or the protein coded by the gene in regulating the resistance of navel orange fruits to sour rot, so as to solve the problems existing in the prior art.

[0006] To achieve the above object, the present application provides the following scheme:

[0007] In one of the technical solutions of the present application, the application of CsERF017 gene or the protein coded thereby in regulating the resistance of navel orange fruit to sour rot disease, the nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 2, and the amino acid sequence of the protein coded thereby is shown in SEQ ID NO. 3.

[0008] In another technical solution of the present application, the application of CsERF017 gene in cultivating citrus transgenic plants resistant to sour rot disease.

[0009] In a third technical solution of the present application, the application of recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing CsERF017 gene in improving the resistance of navel orange fruit to sour rot disease.

[0010] In a fourth technical solution of the present application, the application of recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing CsERF017 gene in cultivating citrus transgenic plants resistant to sour rot disease.

[0011] In a fifth technical solution of the present application, a method for improving the resistance of navel orange fruit to sour rot disease, the CsERF017 gene is silenced or knocked out, or the level of the protein coded thereby is down-regulated, thereby improving the resistance of navel orange fruit to sour rot disease.

[0012] In a sixth technical solution of the present application, a method for cultivating citrus transgenic plants resistant to sour rot disease, the CsERF017 gene is silenced or knocked out, or the level of the protein coded thereby is down-regulated, thereby improving the resistance of the transgenic plants to sour rot disease.

[0013] Based on the above technical solutions, the present application has the following technical effects:

[0014] The present application realizes the first identification and cloning of ethylene response transcription factor CsERF017 gene, helps to reveal the interaction mechanism between G.citri-aurantii and citrus fruit, and provides a new idea and method for applying genetic transformation technology to disease-resistant navel orange germplasm innovation.

[0015] The CsERF017 gene is closely related to the resistance of navel orange to sour rot disease, and is up-regulated after being infected by G.citri-aurantii.

[0016] The application provides a method for breeding and creating an acid rot disease-resistant navel orange germplasm material, and provides important theoretical support and practical guidance for disease-resistant breeding and molecular marker development of citrus fruits. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 Figure A is a fruit lesion phenotype chart, figure B is a determination result chart of disease index, figure C is a change chart of fruit peel hardness, figure D is a change chart of protopectin in fruit peel, figure E is a change chart of lignin in fruit peel, figure F is a change chart of cellulose content in fruit peel, and figure G is a CsERF017 gene expression chart.

[0019] Figure 2 Figure is a gel electrophoresis gel chart of the navel orange ethylene response factor CsERF017 gene according to the present application.

[0020] Figure 3 Figure is a maximum likelihood method (ML) phylogenetic tree of the CsERF017 gene according to the present application.

[0021] Figure 4 Figure is a subcellular localization chart of the CsERF017 gene according to the present application in Nicotiana benthamiana leaves.

[0022] Figure 5 Figure is a chart showing the effects of transient expression of the CsERF017 recombinant vector according to the present application in navel orange fruit peel on fruit phenotype (A), fruit peel hardness (B), lesion diameter (C), disease index (D), protopectin content (E), lignin content (F), and cellulose content (G). DETAILED DESCRIPTION

[0023] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In case of conflict, the content of the present specification will control.

[0026] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples given are exemplary only.

[0027] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0028] The technical solutions described in the present application are all conventional solutions in the art unless specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed.

[0029] The application provides application of a CsERF017 gene or a protein coded by the CsERF017 gene in regulating resistance of navel orange fruits to sour rot, the nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO. 2, and the amino acid sequence of the protein coded by the CsERF017 gene is shown in SEQ ID NO. 3.

[0030] In some specific embodiments, overexpression of the CsERF017 gene or upregulation of the protein coded by the CsERF017 gene reduces the resistance of navel orange fruits to sour rot, and silencing or knocking out the CsERF017 gene or downregulation of the protein coded by the CsERF017 gene improves the resistance of navel orange fruits to sour rot.

[0031] The application also provides application of the CsERF017 gene in cultivating citrus transgenic plants resistant to sour rot.

[0032] The application also provides application of the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacteria containing the CsERF017 gene in improving resistance of navel orange fruits to sour rot.

[0033] The application also provides application of the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacteria containing the CsERF017 gene in cultivating citrus transgenic plants resistant to sour rot.

[0034] The application also provides a method for improving resistance of navel orange fruits to sour rot, wherein the CsERF017 gene is silenced or knocked out or the protein encoded by the CsERF017 gene is down-regulated, so that the resistance of the navel orange fruits to sour rot is improved.

[0035] The application also provides a method for cultivating citrus transgenic plants resistant to sour rot, wherein the CsERF017 gene is silenced or knocked out or the protein encoded by the CsERF017 gene is down-regulated, so that the resistance of the transgenic plants to sour rot is improved.

[0036] In some specific embodiments, the CsERF017 gene is silenced or knocked out or the protein encoded by the CsERF017 gene is down-regulated by using the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacteria containing the CsERF017 gene.

[0037] The full-length of the CsERF017 gene of navel orange is 3960 bp, the full-length of the cDNA sequence thereof is 600 bp, and the CsERF017 gene encodes 199 amino acids; the full-length of the nucleotide sequence of the CsERF017 gene is shown as SEQ ID NO. 1, the nucleotide sequence of the coding region of the CsERF017 gene is shown as SEQ ID NO. 2, and the amino acid sequence of the CsERF017 gene is shown as SEQ ID NO. 3.

[0038] The application clones the CsERF017 gene and constructs a corresponding action vector. The subcellular localization experiment shows that the transcription factor is consistent with the nuclear marker localization and has a nuclear localization signal. The application finds through experiments that the CsERF017 gene is up-regulated after the citrus sour rot pathogen Geotrichum candidum is infected, silencing or knocking out the CsERF017 gene can enhance the resistance of navel orange fruits to sour rot, and the transgenic fruits inoculated with the Geotrichum candidum have reduced symptoms of sour rot, and the disease index and TA content are reduced compared with the control. The application provides an important gene bank and new insights for breeding of citrus disease-resistant varieties and development of molecular markers, and helps to fundamentally reduce the dependence on chemical synthetic pesticides in postharvest preservation treatment of citrus fruits.

[0039] The application discloses application of a citrus transcription factor CsERF017 gene in resistance regulation of navel orange sour rot, and finds that silencing or knocking out the CsERF017 gene can significantly improve the resistance of navel orange fruits to sour rot, and overexpression of the gene reduces the defense ability of the fruits to Geotrichum candidum infection.

[0040] The CsERF017 gene is an ERF transcription factor closely related to the resistance of navel orange fruits to sour rot, and overexpression of the CsERF017 gene reduces the resistance of navel orange fruits to sour rot.

[0041] Meanwhile, the application further discloses that the CsERF017 gene has a nuclear localization signal in Nicotiana benthamiana.

[0042] The CsERF017 gene is a novel ERF transcription factor and is closely related to the disease resistance regulation of navel oranges. Based on the functional analysis of the CsERF017 gene and in combination with the prior art, a navel orange plant or germplasm material resistant to sour rot can be obtained by those skilled in the art. Therefore, the application of the ERF transcription factor in variety breeding and creation of navel orange germplasm resources resistant to sour rot is protected. The CsERF017 gene can be used for molecular-assisted breeding to breed disease-resistant materials and create new genetic disease-resistant materials, so as to achieve the purpose of green and sustainable prevention and control of citrus sour rot. In addition, the application also protects a breeding and creation method of a citrus germplasm resource material resistant to sour rot, in which the CsERF017 gene is silenced or knocked out, so as to realize genetic improvement of citrus disease-resistant varieties.

[0043] In order to completely and unambiguously understand the technical solutions of the application, the citrus sour rot, navel orange sour rot and canker disease all express the same technical meaning, and the pathogenic bacteria thereof is Geotrichum candidum. The meaning of "citrus" or "navel orange" in the application refers to fruits that have reached the mature stage and can be eaten, and does not include plants (seedlings) or tissue culture seedlings obtained by tissue culture.

[0044] The present application screens out an ERF transcription factor CsERF017 gene related to resistance to sour rot by transcriptome sequencing analysis of navel orange fruits under Geotrichum candidum infection stress, and confirms that the expression amount has a positive regulation relationship with fruit softening; subcellular localization research shows that the gene is located in the nucleus and has transcriptional self-activation activity. With the help of molecular biology means and genetic transformation technology, silencing or knocking out CsERF017 gene can enhance the resistance of navel orange fruits to sour rot, which is specifically manifested that, compared with the control, the transgenic fruits after inoculation of Geotrichum candidum have reduced sour rot symptoms, reduced disease index and maintained a relatively complete cell wall structure. Therefore, the present application not only provides important gene resources for the breeding of navel orange resistant varieties, but also has important guiding significance and application prospect for citrus breeding.

[0045] The fruit test material involved in the present application is Newhall navel orange (Citrus sinensis L.cv 'Newhall'), which is collected from a standard navel orange planting garden in Junping Fruit Industry, Nankang District, Ganzhou City, Jiangxi Province.

[0046] The citrus Geotrichum candidum strain involved in the present application is purchased from China Agricultural Microbial Culture Collection Center (ACCC21171).

[0047] Example 1

[0048] Expression analysis of navel orange CsERF017 in response to Geotrichum candidum infection

[0049] Referring to the in vivo efficacy treatment method of Zheng Jie (Zheng Jie. Response mechanism of Geotrichum candidum to environmental pH [D]. Xiangtan University, 2023) and Weng Tian (Weng Tian, Wang Yuqing, Long Chaoan. Antifungal mechanism of geranial on Geotrichum candidum [J]. Food Science, 2023, 44(1): 14-21), the specific experimental steps after slight modification are as follows: select navel orange fruits with consistent size / maturity, uniform coloring and no mechanical damage, and sequentially perform water washing, 1.0%-2.0% sodium hypochlorite solution disinfection for 2 min and sterile water washing; then the samples are randomly divided into a treatment group and a control group, and treated with 200 mg / L cinnamyl alcohol solution and distilled water for 2 min respectively, and placed on a sterile clean bench for ventilation and drying. A 3mm-diameter and 3mm-deep circular hole is punched on the equator of the navel orange fruit with a sterilized needle, 15 μL of Geotrichum candidum spore suspension is inoculated, and then the fruit is transferred to a preservative box disinfected with 75% ethanol and placed in a constant temperature incubator (temperature 27±1℃, relative humidity 90%-95%) for culture for 6 days. The fruit disease condition is observed every 1 day, the fruit skin hardness at a distance of 15 mm from the wound is measured, and the fruit skin tissue samples within a range of 10-20 mm from the wound are taken and quickly frozen in liquid nitrogen, and stored at-80℃ for subsequent analysis.

[0050] (I) Measurement of lesion diameter and disease index

[0051] Lesion diameter: The diameter of the lesion was measured by the cross method.

[0052] Lesion index: The fruit was defined as diseased when the lesion diameter was greater than 3 mm. The sour rot disease index is shown in Table 1.

[0053] Table 1. Sour rot disease index and lesion diameter

[0054]

[0055] The sour rot disease index of navel orange (%) was calculated according to the following formula:

[0056]

[0057] (II) Measurement of fruit peel hardness

[0058] The fruit peel hardness at 15 mm from the hole was measured by TA.XT Plus texture analyzer (SMS, UK) and was defined as the maximum force obtained during the penetration process. The results were expressed in N.

[0059] (III) Measurement of the contents of protopectin, lignin and cellulose in the fruit peel

[0060] Measurement of protopectin content: 2.0 g of navel orange peel sample was placed in 10 mL of 95% ethanol and boiled in a water bath for 30 min, then cooled and centrifuged (8000 rpm, 15 min). The supernatant was discarded and the above steps were repeated four times. The obtained precipitate was mixed with 20 mL of distilled water and placed in a 50°C water bath for 30 min. After cooling to room temperature, it was centrifuged again at 8000 rpm for 15 min and the supernatant was discarded. 25 mL of 0.5 mM H2SO4 solution was added to the centrifuge tube, boiled in a water bath for 1 h, then cooled and centrifuged at the same speed for 15 min. The supernatant was collected and diluted to 50 mL with distilled water. The absorbance at 530 nm was measured by the carbazole colorimetric method, and the protopectin content was calculated according to the galacturonic acid standard curve, with the results expressed in mg / g.

[0061] Measurement of lignin content: 5.0 g of fruit peel powder was added to 15 mL of 95% ethanol and centrifuged for 10 min. The obtained precipitate was washed three times with 95% ethanol and ethanol-n-hexane (1:2 by volume), respectively, and the precipitate was collected and dried to constant weight at 65°C. The results were expressed in mg / g.

[0062] Determination of cellulose content: 2.0 g of peel powder was placed in a 50 mL centrifuge tube, 30 mL of 3% neutral detergent was added, and hydrolysis was performed in a boiling water bath for 1 h. After cooling to room temperature, centrifugation was performed at 8000 rpm for 10 min, and the supernatant was discarded. The obtained precipitate was washed with distilled water and acetone three times in turn. The residue was transferred to a 50 mL centrifuge tube, 30 mL of 2M HCl was added, and hydrolysis was performed at 100°C for 50 min. Centrifugation was performed again at 8000 rpm for 10 min. The precipitate was washed with distilled water until the pH value reached 6.5-7.0, and then washed with acetone twice and dried in a constant temperature drying oven at 60°C until the weight was constant. The dried residue was placed in a 50 mL centrifuge tube, 5 mL of 72% H2SO4 was added, and hydrolysis was performed in a 35°C water bath for 1 h. Then 25 mL (4-5 times the volume) of distilled water was added, and hydrolysis was performed at 100°C for 1 h. After centrifugation, the supernatant was transferred to a 50 mL volumetric flask, and the volume was adjusted to 50 mL with distilled water. The absorbance was determined at 620 nm by anthrone colorimetry, and the cellulose content was calculated according to the glucose standard curve, and the result was expressed in mg / g.

[0063] (iv) Extraction of RNA from navel orange peel

[0064] Extraction of RNA from navel orange peel was based on the Trizol method optimized in this experiment, and the specific operation steps were as follows: 0.5 g of peel sample powder was mixed with 5 mL of Buffer, immediately vortexed for 2 min, and then stood for 10 min. Then centrifugation was performed at 12000 rpm for 20 min at 4°C, the supernatant was transferred to a new tube, 5 mL of chloroform was added, vortexed for 2 min again, and then stood for 10 min. After repeating the above centrifugation step, the supernatant was transferred to a new tube, an equal amount of frozen isopropyl alcohol was added, and the two liquids were mixed thoroughly by gently inverting, and then stood at room temperature for 10 min. After centrifugation (4°C, 12000 rpm) for 20 min, the precipitate was collected and immersed in 3 mL of pre-cooled 75% ethanol at -20°C for 2 h. After discarding the ethanol, the precipitate was air-dried, 800 μL of TESAR was added for dissolution, then 800 μL of Bu / CTAB and Aq / CTAB were added respectively, and vortexed vigorously for 2-5 min. Centrifugation was performed at 4°C and 12000 rpm for 20 min, the supernatant was transferred to a 1.5 mL centrifuge tube, 350 μL of 3M sodium chloride was added, and vortexed for 1 min. After centrifugation (4°C, 8000 rpm) for 6 min, the lower liquid was transferred to a new tube, 50 μL of 3M NaAc (pH 5.2) and 1 mL of anhydrous ethanol were added, mixed, and then placed at -20°C overnight. The next day, the sample was centrifuged at 4°C and 12000 rpm for 10 min, the precipitate was dissolved in 50 μL of DEPC water, and stored at 4°C for 1 h to obtain the navel orange RNA solution, which was stored at -80°C.

[0065] (v) Real-time fluorescent quantitative PCR (q-PCR) detection and analysis

[0066] q-PCR primers were designed online using NCBI Primer-BLAST based on sequencing results.

[0067] CsERF017-qF: 5'-AGGAGTC AATCGCCAGAACA-3';

[0068] CsERF017-qR: 5'-AACCGTTGCCTCAGAAATCG-3'.

[0069] q-PCR detection was performed using a quantitative real-time PCR instrument (T100 Thermal Cycle) to validate relevant genes. The reaction system consisted of a total volume of 10 μL, including 1 μL cDNA, 0.3 μL forward and reverse primers, 3.4 μL ddH2O, and 5 μL LTB Green. The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 95℃ incubation for 15 s, repeated 39 times; 60℃ annealing for 30 s (melting curve temperature); and 95℃ extension for 5 s. The relative gene expression levels were calculated using -2... △△Ct Law.

[0070] The inhibitory effect of juniper alcohol treatment on postharvest sour rot of navel orange fruit is as follows: Figure 1 As shown. Two days after inoculation with G. citri-aurantii, the control group fruits showed water-soaked rot at the wound site. As the infection progressed, the rotten area expanded, and the degree of rot worsened. Figure 1 (A and B). In contrast, juniper alcohol treatment significantly delayed the onset of disease to 3 days after inoculation and significantly reduced the degree of rot, indicating that juniper alcohol treatment can significantly delay the disease progression of navel orange fruit after inoculation with G. citri-aurantii and reduce its incidence. Figure 1 The results of C-F assays showed that juniper alcohol treatment helped maintain the structural integrity of the cell walls in navel orange peel, mainly by delaying the decline in peel firmness, protopectin, lignin, and cellulose content; simultaneously, juniper alcohol effectively inhibited the upregulation of the CsERF017 gene. Figure 1 (G). This suggests that the expression level of the CsERF017 gene may play an important regulatory role in the postharvest resistance mechanism of navel orange to acid rot.

[0071] Example 2

[0072] Cloning and Homology Analysis of the CsERF017 Gene in Navel Orange

[0073] (I) Reverse transcription to synthesize cDNA

[0074] Use Hifair Ⅲ1 stStrand cDNA Synthesis SuperMix for qPCR (Yisheng Biotech Co., Ltd.) reverse transcription kit was used to reverse transcribe the RNA obtained in Example 1 to synthesize 1st Strand cDNA, which was used as a template for amplification of the target gene.

[0075] (II) Cloning of CsERF017 gene

[0076] The complete sequence of the coding region (CDS) of CsERF017 gene was obtained according to the genome of sweet orange (C. sinensis v2.0), and the cloning primers were designed using Premier 5.0 software. The primer pair for amplifying the gene was as follows:

[0077] CsERF017-F1: 5'-ATGGTGAAGCACGTAGTCG-3';

[0078] CsERF017-R1: 5'-AAAATTCCAAAGAAACGAATCTTG ATTC-3'.

[0079] The CDS full-length of CsERF017 gene was amplified by PCR using cDNA in navel orange peel as a template, and the PCR reaction system is shown in Table 2.

[0080] Table 2 PCR reaction system for amplifying CsERF017 gene

[0081]

[0082]

[0083] The PCR amplification program was as follows: pre-denaturation at 98℃ for 30 s; denaturation at 98℃ for 30 s, annealing at 55℃ for 5 s, extension at 72℃ for 5 s, 34 cycles of thermal cycling; extension at 72℃ for 1 min, and storage at 4℃.

[0084] The results of agarose gel electrophoresis of the PCR amplification product are shown in Figure 2 The size of the CDS sequence of the gene CsERF017 was consistent with the expected value. A clear DNA band of about 600 bp was observed in the electrophoretogram in the cloning experiment using navel orange cDNA as a template. Figure 2 Through multiple sequence alignment and Conserved Domains analysis in NCBI, it was found that the full-length nucleotide sequence of CsERF017 gene was as shown in SEQ ID NO. 1, which had a typical AP2 / EREBP domain. The nucleotide sequence of the coding region of the gene obtained by sequencing analysis was as shown in SEQ ID NO. 2, and the amino acid sequence of the encoded protein was as shown in SEQ ID NO. 3.

[0085]

[0086] SEQ ID NO. 2: ATGGTGAAGCACGTAGTCGAAAAGCCTGCTGAGAGAAGTGACTCTCGTTACAAGGGTGTCCGAAAGCGAAAGTGGGGGAAATATGTGTCTGAAATCAGACTACCCAACAGCCGTGCCCGTATCTGGCTGGGCTCCTACGACACAGCAGAAAAAGCAGCGCGTGCTTTCGACGCTGCTTTGTTTTGCTTACGTGGCCGATCAGCCAAGTTTAATTTCCCGGACAACCCACCCGACATATCAGGCGGGCGCTCACTTAAGCCATCTGAGATTCAAGCTGTGGCGGCTCAGTTCGCGAATTCGGAGCCGCTGAGGAGTCAATCGCCAGAACAGTCGGTGTCCGAATTGCAAACGGAGTACTTATCGCCGTCGATTTCTGAGGCAACGGTTCAGTTGGACAGCGATGGGGTTTTTGACGGGTCTTTATTGGATCTTTTAACGGCATCGAGTTCCAGTAACTACCCTTCCGAATATGGGATATTTCCTGGGTTTGATGACCTGTCTAATGACATTTTTGCTCCGCAACTCCCAGCTGTTGATTTTGCAGACGAGAATTTTGATGGGCTATTGTTGAATCAAGATTCGTTTCTTTGGAATTTTTAA;

[0087] SEQ ID NO. 3: MVKHVVEKPAERSDSRYKGVRKRKWGKYVSEIRLPNSRARIWLGSYDTAEKAARAFDAALFCLRGRSAKFNFPDNPPDISGGRSLKPSEIQAVAAQFANSEPLRSQSPEQSVSELQTEYLSPSISEATVQLDSDGVFDGSLLDLLTASSSSNYPSEYGIFPGFDDLSNDIFAPQLPAVDFADENFDGLLLNQDSFLWNF*.

[0088] (iii) Homology analysis of CsERF017 gene

[0089] Phytozome platform (https: / / phytozome.jgi.doe.gov / ) was used to analyze the cross-species homologous genes, and phylogenetic analysis was carried out on CsERF017 gene. The MEGAX software was used to construct the phylogenetic tree based on the maximum likelihood method (Maximum Likelihood, ML), and it was found that the CsERF017 gene was far from the reported Arabidopsis thaliana ERF017 evolutionary relationship Figure 3 ), and it was inferred that the gene was a new member of ERF transcription factor in Citrus sinensis.

[0090] Example 3

[0091] Subcellular localization of CsERF017 gene in Citrus sinensis

[0092] (I) Recombinant vector construction

[0093] The amplification primer pair was designed with the PRI101-eGFP as the starting vector, and the insertion site was between NdeI and BamHI:

[0094] F: 5'-GTTCTTCACTGTTGATACATATG ATGGTGAAGCACGTAGTCG-3';

[0095] R: 5'-CTTGCTCACCATGGATCC AAAATTCCAAAGAAACGAATCTTGATTC-3'.

[0096] The CsERF017 gene without stop codon was obtained by PCR technology, and the constructed DNA fragment was purified and recovered. Then, according to the method of one-step cloning kit (Novagen company), the product was prepared with double enzyme digested PRI101-eGFP for recombination reaction. Finally, 10 μL of recombination product was added to 50 μL of DH5α competent cells, and the mixture was placed on ice for 30 min, then heated at 42℃ for 45 s. After transformation of E. coli, the plate was cultured in a 37℃ incubator for 16 h. The positive single colony was picked and sent to Genescript Biotech Co., Ltd. for sequencing verification to obtain the accurate target gene sequence.

[0097] (II) Agrobacterium transformation

[0098] 3 μL of CsERF017-PRI101-eGFP plasmid DNA with correct sequence was mixed with 50 μL of GV1301 Agrobacterium competent cells, and then placed in ice, liquid nitrogen, 37℃ water and ice bath for 5 min respectively. After resuspension in 700 μL of LB without antibiotics for 2 h, the plate was cultured in a 28℃ incubator for 2 d. The positive single colony was picked and PCR amplified to show a single and clear band, and the band position was consistent with the plasmid DNA. The bacteria were stored in a -80℃ refrigerator.

[0099] (Three) Tobacco leaf infection

[0100] Several tobacco seeds were sown and cultured for a month under 12h light, and then used for experiments. Agrobacterium containing the CsERF017-PRI101-eGFP vector was resuspended with a 10mM MgCl2(120μM AS) suspension, and the suspension OD 600 was adjusted to about 0.6, and well-growing tobacco plants were selected, injected from the lower epidermis of the tobacco leaves with a 1mL syringe without a needle, and labeled. The PRI101-eGFP empty vector was used as a control. The injected tobacco plants were cultured for 2d under weak light, and the labeled Agrobacterium-injected tobacco leaves were made into slides, observed under a laser confocal microscope, and photographed.

[0101] The PRI101-eGFP empty vector was distributed in the nuclei and cell membranes of the tobacco leaf epidermal cells, while the CsERF017-PRI101-eGFP was only detected for green fluorescence in the nuclei Figure 4 , thus indicating that the CsERF017 transcription factor is located in the nuclei, which is consistent with the transcriptional regulation characteristics of transcription factors.

[0102] Example 4

[0103] Transient overexpression of navel orange CsERF017 gene

[0104] (One) Construction of CsERF017-PBI121 transient overexpression vector

[0105] When amplifying the CsERF017 gene, XbaI and BamHI restriction enzyme recognition sequences were inserted into the 5' ends of the forward and reverse primers, respectively, and the amplification primer pair was:

[0106] CsERF017-F3 forward primer: 5'-AGAACACGGGGGACTCTAGA ATGGTGAAGCAC GTAGTCG-3';

[0107] CsERF017-R3 reverse primer: 5'-GACTGACCACCCGGGGATCC AAAATTCCAAA GAAACGAATCTTGATTC-3'.

[0108] The enzyme digestion reaction used a 50μL system: 1.5μL XbaI, 1.5μL BamHI, 5μL 10×M Buffer, 20μL plasmid DNA, and 22μL deionized H2O.

[0109] The system was mixed gently and centrifuged briefly, and then placed in a 37°C environment for 3h of reaction. After confirming success by agarose gel electrophoresis detection of 50μL of the enzyme digestion product, the remaining product was recovered and connected to the PBI121 vector after purification.

[0110] (II) Transient transformation of Newhall navel orange peel

[0111] (1) IM (500mL) solution preparation (freshly prepared): 4-morpholine ethanesulfonic acid (MES) 5.137g, glucose 2.632g, sodium dihydrogen phosphate 0.164g, make up to 500ml, adjust pH to 5.6-5.7, sterilize and cool, then add 20x AB Salts 26.316ml.

[0112] (2) 20x AB Salts (500mL, vortex if precipitate occurs): ammonium chloride 10g, magnesium sulfate heptahydrate 3g, potassium chloride 1.5g, calcium chloride 0.1g, ferrous sulfate heptahydrate 0.025g.

[0113] (3) MES (10mM MgCl2, 10mM MES 500mL) solution: magnesium chloride 0.475g, MES 1.066g, make up to 500mL, adjust pH to 5.5-5.6, autoclave.

[0114] (4) Preparation of MMA suspension: first prepare 0.1mol / L AS solution, 1mol / L MgCl2solution and 0.5mol / L MES (fatty acid methyl ester sulfonate) solution, then take 2mL of the prepared MES solution, 1mL of the MgCl2solution and 0.1mL of the AS solution, and then make up to 100mL with ultrapure water to prepare the MMA solution, so that the final concentrations of MES, MgCl2and AS are 100mmol / L, 10mmol / L and 10μmol / L, respectively.

[0115] (5) Preparation of infection solution: use the Agrobacterium liquid obtained in the transformation test in Example 3 (containing the CsERF017-PBI121 recombinant plasmid and the PBI121 empty vector) to inoculate LB liquid medium containing kanamycin and rifampicin, and place in a 28°C, 200rpm shaker. When the OD 600 value of the bacterial solution reaches 0.6-0.8, centrifuge at 4°C, 5000rpm for 10min, then resuspend the precipitated bacteria with an equal volume of MMA solution, and repeat twice.

[0116] (6) Peeling injection

[0117] The navel orange fruits were washed with clean water and then drained. The fruits were then treated with 1.0% sodium hypochlorite solution for 2 min, and then washed with flowing clean water to remove the residual disinfectant. The fruits were then placed on a clean bench to dry. A hole (3 mm in diameter and 3 mm in depth) was punched on the equator of each fruit with a sterilized needle. Then, 0.5 mL of Agrobacterium infection solution was injected into the hole using a syringe without a needle. After the fruit was completely penetrated, a new hole was punched 1 cm to the right of the original hole, and 15 μL of white Geotrichum spore suspension with a concentration of 1 x 10 5 CFU·mL -1

[0118] (7) Storage and sampling

[0119] After the bacteria solution was absorbed, the fruits were placed in a box (10 fruits per box), sealed with a polyethylene plastic bag, and placed in a constant temperature incubator (temperature: 27 ± 1 ℃, relative humidity: 90% to 95%) for dark culture. The appearance of the fruits was observed at regular time intervals for 1 to 5 days, and the diameter of the disease spot was measured. Meanwhile, the peel tissue samples were taken from 10 to 20 mm outside the fruit wound, chopped, and then subjected to liquid nitrogen quick-freezing treatment. The samples were stored at -80 ℃ for the determination of the resistance characteristics of the fruits.

[0120] (III) GUS staining after transient overexpression of juice sacs

[0121] The GUS staining kit provided by Coolaber Company was used. First, the X-Gluc solvent was melted in a 40 ℃ water bath. Then, 1 mL of the melted solvent was added to the X-Gluc dry powder tube, and the 50 x GUS staining concentrate was prepared after the solvent was completely dissolved. Then, 0.4 mL of the concentrate was mixed with 5 mL of GUS staining buffer. A 1 cm section of the peel of the fruit stored for 1 day was taken from the wound site and immersed in the GUS staining solution. The section was placed in a 28 ℃ incubator overnight. When blue spots appeared in the juice sacs, it was considered that the GUS expression site was obtained.

[0122] (IV) Determination of the resistance characteristics of the fruits

[0123] After the navel orange fruits were transiently overexpressed with the CsERF017 gene, the fruits were inoculated with the white Geotrichum spore suspension. The disease incidence of the fruits was observed every 1 day, and the samples were stored at -80 ℃ for the detection of the content of the disease resistance substances. The determination of the fruit disease spot diameter, disease index, peel hardness, and the contents of protopectin, lignin, and cellulose was performed according to the steps in Example 1.

[0124] After the fruits were inoculated with the white Geotrichum spore suspension, both the CsERF017 overexpression and the empty vector showed waterlogged disease spots. However, there were significant differences in the disease spot characteristics (hardness and disease spot diameter) Figure 5 ​The disease index of the empty vector control was 24.4, 29.7 and 53.7% at 3, 4 and 5 days after infection, respectively, while the disease index of the CsERF017 overexpression line was 35.7, 45.4 and 64.9%, respectively, which were significantly higher than that of the control PBI121 Figure 5 Middle D).

[0125] It was found through detection of the disease resistance related substances that the CsERF017 gene transiently overexpressed navel orange fruits had a significantly lower content of protopectin, lignin and cellulose in the peel than the PBI121 control group after inoculation with G. citri spores and storage for 3 days. Figure 5 E-G).

[0126] The inventors also constructed a CsERF017 gene silencing vector, and determined the resistance characteristics of the CsERF017 gene silenced fruits according to the above method, and the results showed that the CsERF017 gene silenced fruits had a significantly higher content of protopectin, lignin and cellulose in the peel than the control group after inoculation with G. citri spores and storage for 3 days.

[0127] The above results prove that overexpression of CsERF017 enhances the sensitivity of navel orange fruits to G. citri, and inhibition of the expression of CsERF017 improves the defense ability of navel orange fruits against sour rot.

[0128] In summary, the present application found that the peel hardness and the contents of protopectin, lignin and cellulose in the peel of navel orange fruits gradually decreased during the G. citri infection process, and the expression level of CsERF017 gene was positively correlated with the occurrence of sour rot, indicating that there was a negative regulatory relationship between the CsERF017 transcription factor and the content of disease resistance related substances in navel orange. Subcellular localization of the transcription factor in tobacco showed that it had a nuclear localization signal. After transient expression of the CsERF017-PBI121 recombinant vector in the peel of navel orange, the degree of sour rot infection was significantly aggravated, and the contents of disease resistance related substances such as protopectin, lignin and cellulose in the peel were significantly decreased, which fully verified that CsERF017 negatively regulated the resistance of navel orange fruits to sour rot.

[0129] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. CsERF017 The application of genes in regulating resistance to sour rot in navel oranges is characterized by, The CsERF017 The nucleotide sequence of the gene is shown in SEQ ID NO.2; Silence or Knockout CsERF017 Genes that enhance the resistance of navel oranges to sour rot disease.

2. CsERF017 The application of genes in the breeding of transgenic navel orange plants resistant to acid rot is characterized by, Silence or Knockout CsERF017 Genes that enhance the resistance of navel oranges to sour rot disease; CsERF017 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A method for improving the resistance of navel oranges to sour rot disease, characterized in that, Silence or Knockout CsERF017 Genes that enhance the resistance of navel oranges to sour rot disease; CsERF017 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A method for cultivating transgenic navel orange plants resistant to acid rot, characterized in that, Silence or Knockout CsERF017 Genes that enhance the resistance of transgenic plants to acid rot; CsERF017 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

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