Application of CsERF017 gene or protein coded by CsERF017 gene in regulating navel orange fruit sour rot resistance

Abstract

The invention discloses application of a CsERF017 gene or a protein coded by the CsERF017 gene to regulation and control of sour rot resistance of navel orange fruits, and belongs to the technical field of molecular biology and fruit and vegetable preservation. The nucleotide sequence of the CsERF017 gene is as shown in SEQ ID NO. 2; and the amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO.3. Experiments find that the CsERF017 gene is subjected to up-regulation expression after being infected by Geotrichum candidum citrus sour rot pathogenic bacteria, the resistance of navel orange fruits to sour rot can be enhanced by silencing or knocking out the CsERF017 gene, and the specific expression is that compared with a control group, transgenic fruits inoculated with Geotrichum candidum have the advantages that the sour rot symptoms are relieved, and the disease index and the TA content are reduced. According to the invention, an important gene pool and a new insight are provided for breeding of disease-resistant citrus varieties and development of molecular markers, and dependence of chemical synthetic pesticides in preservative and fresh-keeping treatment after citrus fruits are picked can be fundamentally reduced.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology and fruit and vegetable preservation technology, and particularly relates to the application of the CsERF017 gene or the protein encoded thereby in regulating the resistance of navel orange fruits to sour rot disease. Background Art

[0002] Citrus belongs to the arbor of the genus Citrus in the subfamily Aurantioideae of the family Rutaceae, including dozens of varieties such as mandarin, orange, navel orange, grapefruit, lemon, lime, citron, kumquat, etc., and is distributed in more than 140 countries and regions around the world. Its fruits are rich in nutrition and contain a variety of bioactive components beneficial to human health, such as vitamin C, polyphenols, flavonoids, limonoids, etc. Navel oranges are deeply favored by consumers due to their rich nutrition, delicious juice and unique taste. Compared with the common blue and green mold diseases in tangerines, the sour rot disease of citrus caused by the infection of Geotrichum citri-aurantii is more significant in the post-harvest storage and transportation of navel orange fruits. At the initial stage of the disease, the fruits soften and shrink and show a water-soaked phenomenon. After 3-5 days, a white frost-like mold layer grows on the surface of the lesion, which quickly expands into a white near-circular mold spot. The diseased fruits will crack and emit a pungent rancid smell, and the whole fruit becomes water-soaked and cannot be picked up.

[0003] At present, the control of post-harvest sour rot disease of citrus fruits mainly relies on chemically synthesized pesticides, but many chemically synthesized substances are extremely likely to cause adverse problems such as pesticide residues, the generation of drug-resistant strains, harm to human health, and pollution of the ecological environment. The research on the molecular mechanism of citrus fruit resistance to sour rot disease at home and abroad is relatively slow, and there is no report on the functional analysis and regulatory mechanism of genes related to sour rot disease resistance at present.

[0004] When protecting plant tissues from pathogen infection, plants have evolved a highly sophisticated immune defense mechanism through precise regulation of the expression of disease resistance or defense-related genes, thereby achieving effective regulation of the response to pathogenic microorganisms. In this mechanism, transcription factors play a key regulatory role in plant disease resistance responses. They can 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 promoter region of downstream interacting genes through the GCC-box cis-acting element, activating or inhibiting the expression of interacting genes and participating in the regulation of biological processes such as plant growth and development, maturation and senescence, and stress responses. Overexpression of the SlERF01 gene in tomatoes can significantly enhance the defense ability of leaves against Stemphylium lycopersici, specifically manifested as an increase in the content of resistance substances such as lignin and callose, and upregulation of the expression of the pathogenesis-related protein SlPR1 gene, thereby enhancing the hypersensitive response of tomato leaves to S. lycopersici infection (Yang et al, 2020). In citrus peels, transient overexpression of the CsERF1B gene promotes the accumulation of lignin content while mediating jasmonic acid signals by directly activating the CsOPR11 and CsAOS3L genes in the jasmonic acid signaling pathway and the expression of lignin synthesis genes, significantly improving the resistance of fruits to green mold disease (Li et al, 2023). In addition, Wang et al (2020) found an ERF transcription factor located in the nucleus, MdERF11, in 'Royal Gala' apples. Overexpression of this gene in callus significantly increased the SA content and induced the expression of related genes such as EDS1, PAL, PR1, and NPR1 in the SA synthesis pathway, thereby enhancing the resistance of fruits to Botryosphaeria dothidea. Research in the past 20 years has shown that ERF transcription factors mainly improve plant resistance to biotic (bacteria, fungi, oomycetes, viruses, etc.) stresses by mediating secondary cell wall metabolism, plant hormone (jasmonic acid, salicylic acid, melatonin, etc.) signals, and the activity of pathogenesis-related proteins. However, there are few reports on the research of citrus ERF transcription factors in response to the disease resistance function of G. citri-aurantii. Summary of the Invention

[0005] The object of the present invention is to provide the application of the CsERF017 gene or the protein encoded thereby in regulating the resistance of navel orange fruits to sour rot disease, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the application of the CsERF017 gene or the protein encoded thereby in regulating the resistance of navel orange fruits to sour rot. The nucleotide sequence of the CsERF017 gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO.3.

[0008] Another technical solution of the present invention is the application of the CsERF017 gene in cultivating transgenic citrus plants resistant to sour rot.

[0009] A third technical solution of the present invention is the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the CsERF017 gene in enhancing the resistance of navel orange fruits to sour rot.

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

[0011] A fifth technical solution of the present invention is a method for enhancing the resistance of navel orange fruits to sour rot, by silencing or knocking out the CsERF017 gene or down-regulating the level of the protein encoded thereby, to enhance the resistance of navel orange fruits to sour rot.

[0012] A sixth technical solution of the present invention is a method for cultivating transgenic citrus plants resistant to sour rot, by silencing or knocking out the CsERF017 gene or down-regulating the level of the protein encoded thereby, to enhance the resistance of transgenic plants to sour rot.

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

[0014] The present invention realizes the first identification and cloning of the ethylene response transcription factor CsERF017 gene, which helps to reveal the interaction mechanism between Geotrichum citri-aurantii and citrus fruits, and at the same time provides new ideas and methods for the application of genetic transformation technology in the innovation of disease-resistant navel orange germplasm. The present invention provides an innovative strategy for controlling citrus sour rot at the molecular level, which can fundamentally reduce the use of chemical pesticides and thus effectively break through the existing technical bottlenecks.

[0015] The CsERF017 gene is closely related to the resistance of navel oranges to sour rot and is up-regulated after being infected by Geotrichum citri-aurantii. In view of this, through genetic transformation experiments, the present invention finds that silencing or knocking out the CsERF017 gene can significantly enhance the resistance of navel orange fruits to sour rot, while overexpressing this gene reduces the defense ability of fruits against Geotrichum citri-aurantii infection, thus clarifying the specific function of the CsERF017 gene in the mechanism of navel orange resistance to sour rot.

[0016] The present invention provides a method for breeding and creating navel orange germplasm materials resistant to sour rot disease, providing important theoretical support and practical guidance for disease-resistant breeding and molecular marker development of citrus fruits. By precisely regulating the expression level of the navel orange CsERF017 gene, the present invention effectively improves the disease-resistant traits of navel orange fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a graph showing the disease development of navel orange fruits after inoculation with Geotrichum candidum. Among them, A is the phenotypic graph of fruit lesions, B is the graph of the determination result of the disease index, C is the graph of the change in peel hardness, D is the graph of the change in protopectin in the peel, E is the graph of the change in lignin in the peel, F is the graph of the change in cellulose content in the peel, and G is the graph of the expression of the CsERF017 gene.

[0019] Figure 2 It is a gel electrophoresis gel image of the ethylene response factor CsERF017 gene of the navel orange described in the present invention.

[0020] Figure 3 It is a maximum likelihood method (ML) phylogenetic tree of the homologous genes of the CsERF017 gene described in the present invention.

[0021] Figure 4 It is a subcellular localization map of the CsERF017 gene described in the present invention in the leaves of Nicotiana benthamiana.

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

[0023] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms described in this invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0028] The technical solutions described in this invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been publicly disclosed.

[0029] The embodiments of this invention provide the application of the CsERF017 gene or the protein encoded by it in regulating the resistance of navel orange fruits to sour rot. The nucleotide sequence of the CsERF017 gene is shown as SEQ ID NO.2; the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.3.

[0030] In some specific embodiments, overexpressing the CsERF017 gene or upregulating the level of the protein encoded by it reduces the resistance of navel orange fruits to sour rot, while silencing or knocking out the CsERF017 gene or downregulating the level of the protein encoded by it increases the resistance of navel orange fruits to sour rot.

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

[0032] The embodiments of the present invention also provide the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the CsERF017 gene in improving the resistance of navel orange fruits to sour rot disease.

[0033] The embodiments of the present invention also provide the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the CsERF017 gene in cultivating transgenic citrus plants resistant to sour rot disease.

[0034] The embodiments of the present invention also provide a method for improving the resistance of navel orange fruits to sour rot disease, by silencing or knocking out the CsERF017 gene or down-regulating the level of the protein encoded by it, to improve the resistance of navel orange fruits to sour rot disease.

[0035] The embodiments of the present invention also provide a method for cultivating transgenic citrus plants resistant to sour rot disease, by silencing or knocking out the CsERF017 gene or down-regulating the level of the protein encoded by it, to improve the resistance of transgenic plants to sour rot disease.

[0036] In some specific embodiments, a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the CsERF017 gene is used to silence or knock out the CsERF017 gene or down-regulate the level of the protein encoded by it.

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

[0038] The present invention clones the CsERF017 gene and constructs a corresponding functional vector. Subcellular localization experiments show that this transcription factor is consistent with the nuclear marker localization and has a nuclear localization signal. The present invention discovers through experiments that the CsERF017 gene is up-regulated after being infected by Geotrichum candidum, the pathogenic bacterium of citrus sour rot disease. Silencing or knocking out the CsERF017 gene can enhance the resistance of navel orange fruits to sour rot disease. Specifically, compared with the control, the transgenic fruits after inoculation with Geotrichum candidum show reduced symptoms of sour rot disease, and both the disease index and TA content are decreased. The present invention provides an important gene pool and new insights for the breeding of disease-resistant citrus varieties and the development of molecular markers, and helps to fundamentally reduce the dependence on chemically synthesized pesticides in the post-harvest preservation and anti-corrosion treatment of citrus fruits.

[0039] The present invention discloses the application of the citrus transcription factor CsERF017 gene in regulating the resistance to sour rot of navel oranges. Through genetic transformation experiments, it is known that silencing or knocking out the CsERF017 gene can significantly improve the resistance of navel orange fruits to sour rot, while overexpressing this gene reduces the defense ability of the fruits against Geotrichum candidum infection. The Agrobacterium infection experiment on navel orange fruits shows that the CsERF017 gene has a significant negative regulatory effect on citrus sour rot, and this gene has a relatively distant evolutionary relationship with the AP2 / ERF transcription factors in reported plants. It is a newly discovered gene in citrus and can be developed as a potential resistance molecular marker.

[0040] The CsERF017 gene is an ERF transcription factor closely related to the resistance of navel orange fruits to sour rot. Overexpressing the CsERF017 gene reduces the resistance of navel orange fruits to sour rot. Through RNA-seq and RT-qPCR techniques, the present invention finds that the expression of the CsERF017 gene is significantly different at different stages of the interaction between navel oranges and Geotrichum candidum when the navel orange fruits are infected with Geotrichum candidum. The CsERF017 gene is induced by Geotrichum candidum, and its expression level increases with the prolongation of the time when navel orange fruits are infected with Geotrichum candidum.

[0041] Meanwhile, the present invention further reveals that the CsERF017 gene has a nuclear localization signal in Nicotiana benthamiana.

[0042] The CsERF017 gene, as a novel ERF transcription factor, is closely related to the regulation of navel orange disease resistance. Based on the functional analysis of the CsERF017 gene and in combination with the existing technology, those of ordinary skill in the art can obtain navel orange plants or germplasm materials resistant to sour rot. Therefore, the present invention requests protection for the application of the ERF transcription factor in variety breeding and creating navel orange germplasm resource materials resistant to sour rot. The CsERF017 gene can be used for molecular-assisted breeding to select disease-resistant materials and create new genetically disease-resistant materials, so as to achieve the purpose of green and sustainable prevention and control of citrus sour rot. In addition, the present invention also requests protection for a method for breeding and creating citrus germplasm resource materials resistant to sour rot, by silencing or knocking out the CsERF017 gene to achieve the genetic improvement of citrus disease-resistant varieties.

[0043] To understand the technical solution of the present invention completely and without any objection, the citrus sour rot, navel orange sour rot, and citrus canker described in the present invention all express the same technical meaning, and their pathogen is Geotrichum candidum. The "citrus" or "navel orange" described in the present invention refers to the mature and edible fruits, excluding the plants (seedlings) or the tissue culture seedlings obtained by tissue culture.

[0044] In this invention, transcriptome sequencing analysis was performed on navel orange fruits under the infection stress of Geotrichum candidum, and the ERF transcription factor CsERF017 gene related to resistance to sour rot was screened out, and it was confirmed that its expression level has a positive regulatory relationship with fruit lesion softening; subcellular localization studies showed that this gene is located in the nucleus and has transcriptional self-activation activity. By means of molecular biology methods and genetic transformation techniques, silencing or knocking out the CsERF017 gene can enhance the resistance of navel orange fruits to sour rot. Specifically, compared with the control, the transgenic fruits after inoculation with Geotrichum candidum showed reduced symptoms of sour rot, a decreased disease index, and maintained a relatively intact cell wall structure. Therefore, this invention not only provides important gene resources for the breeding of resistant navel orange varieties, but also has important guiding significance and application prospects for citrus breeding.

[0045] The fruit test materials involved in this invention are Newhall navel oranges (Citrus sinensis L. cv 'Newhall'), which were collected from the standard navel orange orchard of Junping Fruit Industry in Nankang District, Ganzhou City, Jiangxi Province.

[0046] The Geotrichum candidum strain of citrus involved in this invention was purchased from the China Center for Agricultural Culture Collection (ACCC21171).

[0047] Example 1

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

[0049] Referring to the in vivo control efficacy treatment methods of Zheng Jie (Zheng Jie. Research on the response mechanism of Geotrichum citri-aurantii to environmental pH [D]. Xiangtan University, 2023) and Weng Tian (Weng Tian, Wang Yuqing, Long Chaoan. Antibacterial mechanism of geraniol against Geotrichum citri-aurantii [J]. Food Science, 2023, 44(1): 14-21), the specific experimental steps after slight modification are as follows: Select navel orange fruits with the same size / maturity, uniform coloring and no mechanical damage, and rinse them with water, disinfect them with 1.0%-2.0% sodium hypochlorite solution for 2 min and then rinse with sterile water; Subsequently, randomly divide the samples into a treatment group and a control group, and treat them with 200 mg / L hinokitiol solution and distilled water for 2 min respectively, and place them in a sterile ultra-clean workbench to air dry. Use a sterilized needle to make a round hole with a diameter of 3 mm and a depth of 3 mm at the equator of the navel orange fruit, inoculate 15 μL of Geotrichum candidum spore suspension, and transfer it to a fresh-keeping box disinfected with 75% ethanol after complete absorption, and place it in a constant temperature incubator (temperature 27±1 °C, relative humidity 90%-95%) for 6 d. Observe the fruit disease situation every 1 d, measure the peel hardness at 15 mm outside the wound, and at the same time take peel tissue samples within the range of 10-20 mm outside the wound, quickly freeze them with liquid nitrogen, and store them at -80 °C for subsequent analysis.

[0050] (1) Determination of lesion diameter and disease index

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

[0052] Disease index: When the lesion diameter was greater than 3 mm, the fruit was defined as an infected fruit, and the sour rot disease grade is shown in Table 1.

[0053] Table 1 Sour rot disease grade and lesion diameter

[0054]

[0055] The calculation formula for the sour rot disease index (%) of navel oranges is as follows:

[0056]

[0057] (II) Determination of peel hardness

[0058] The TA.XT Plus texture analyzer (SMS, UK) was used to measure the peel hardness at 15 mm outside the hole, and the maximum force obtained during the penetration process was determined, and the result was expressed as N.

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

[0060] Determination of protopectin content: Weigh 2.0 g of navel orange peel sample and place it in 10 mL of 95% ethanol. After boiling water bath for 30 min, cool it and then centrifuge (8000 rpm, 15 min). Discard the supernatant. Repeat the above steps 4 times. Mix the obtained precipitate with 20 mL of distilled water, place it in a water bath at 50 °C for 30 min, cool it to room temperature and then centrifuge at 8000 rpm for 15 min again. Discard the supernatant. Add 25 mL of 0.5 mM H2SO4 solution to the centrifuge tube, hydrolyze it in a boiling water bath for 1 h and then cool it, centrifuge at the same speed for 15 min again, collect the supernatant, and make up the volume to 50 mL with distilled water. Measure the absorbance at 530 nm by the carbazole colorimetric method, and calculate the protopectin content according to the galacturonic acid standard curve. The result is expressed as mg / g.

[0061] Determination of lignin content: Weigh 5.0 g of peel powder, add 15 mL of 95% ethanol, centrifuge for 10 min, and wash the obtained precipitate three times with 95% ethanol and ethanol-n-hexane (volume ratio 1:2) in turn. Collect the precipitate and dry it to constant weight at 65 °C. The result is expressed as mg / g.

[0062] Determination of cellulose content: Take 2.0g peel powder and place it in a 50mL centrifuge tube, add 30mL 3% neutral detergent, hydrolyze in a boiling water bath for 1h, cool to room temperature, centrifuge at 8000rpm for 10min, and discard the supernatant. The obtained precipitate is washed three times with distilled water and acetone respectively. The residue is transferred to a 50mL centrifuge tube, 30mL 2M HCl is added, and it is kept at 100℃ for 50min, and centrifuged again at 8000rpm for 10min. The precipitate is washed with distilled water until the pH value reaches 6.5-7.0, and then washed twice with acetone, and placed in a 60℃ constant temperature drying oven to dry to constant weight. The dried residue is placed in a 50mL centrifuge tube, 5mL 72% H2SO4 is added, and it is bathed in a 35℃ water bath for 1h, and then 25mL (4-5 times the volume) of distilled water is added, and then hydrolyzed at 100℃ for 1h. After centrifugation, transfer all the supernatant to a 50 mL volumetric flask and dilute to 50 mL with distilled water. The absorbance was measured at a wavelength of 620 nm using the anthrone colorimetric method, and the cellulose content was calculated based on the glucose standard curve, and the result was expressed in mg / g.

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

[0064] RNA extraction from navel orange peel was based on the Trizol method optimized in this experiment. The specific steps were as follows: 0.5 g of peel sample powder was weighed and mixed with 5 mL of Buffer, immediately vortexed for 2 min and then allowed to stand for 10 min. Subsequently, the supernatant was transferred to a new tube at 12000 rpm for 20 min at 4 ° C, 5 mL of chloroform was added, vortexed again for 2 min and allowed to stand for 10 min. After repeating the above centrifugation steps, the supernatant was transferred to a new tube, an equal amount of frozen isopropanol was added, and the two liquids were mixed gently and allowed to stand at room temperature for 10 min. After centrifugation (4 ° C, 12000 rpm) for 20 min, the debris precipitate was collected and immersed in 3 mL of pre-cooled 75% ethanol and stored at -20 ° C for 2 h. After discarding the ethanol, the precipitate was air-dried, 800 μL of TESAR was added to dissolve, and then 800 μL of Bu / CTAB and Aq / CTAB were added respectively, and vortexed vigorously for 2 to 5 min. Centrifuge at 4°C, 12000rpm for 20min, transfer the supernatant to a 1.5mL centrifuge tube, add 350μL 3M sodium chloride, mix and vortex for 1min. After centrifugation (4°C, 8000rpm) for 6min, transfer the lower layer of liquid to a new tube, add 50μL 3M NaAc (pH5.2) and 1mL anhydrous ethanol, mix and place at -20°C overnight. The next day, centrifuge the sample at 4°C, 12000rpm for 10min, dissolve the precipitate in 50μL DEPC water, store at 4°C for 1h to obtain the navel orange RNA solution, and freeze at -80°C.

[0065] (V) Real-time fluorescence quantitative PCR (q-PCR) detection and analysis

[0066] Design q-PCR primers online using NCBI Primer-BLAST according to the sequencing results:

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

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

[0069] Verify the relevant genes using a fluorescence quantitative PCR instrument (T100 Thermal Cycle) for q-PCR detection. Reaction system: The total volume is 10 μL, including 1 μL cDNA, 0.3 μL forward and reverse primers, 3.4 μL ddH2O, and 5 μL TB Green; Reaction conditions are: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, incubation at 95 °C for 15 s, repeat 39 cycles, annealing at 60 °C for 30 s (dissolution curve temperature), extension at 95 °C for 5 °C. The relative expression level of the gene is calculated using the -2 △△Ct method.

[0070] The inhibitory effect of hinokitiol treatment on postharvest sour rot of navel orange fruits is as Figure 1 shown. At 2 d after inoculation with G. citri-aurantii, water-soaked rot appeared at the wound of the control group fruits. As the infection time progressed, the rotted area continued to expand, and the degree of fruit rot continued to increase ([[]] Figure 1 A and B in). In contrast, hinokitiol treatment significantly delayed the onset time of the disease to 3 d after inoculation, and the degree of rot was significantly reduced, indicating that hinokitiol treatment can significantly delay the disease progression of navel orange fruits after inoculation with G. citri-aurantii and can also reduce the incidence probability. Figure 1 The detection results in C–F in show that hinokitiol treatment helps to maintain the structural integrity of the navel orange peel cell wall, mainly manifested in delaying the decline of peel hardness, protopectin, lignin, and cellulose contents; At the same time, hinokitiol effectively inhibits the up-regulated expression of the CsERF017 gene ([[]] Figure 1 G in). It is thus speculated that the expression level of the CsERF017 gene may play an important regulatory role in the postharvest anti-sour rot mechanism of navel oranges.

[0071] Example 2

[0072] Cloning and homology analysis of the navel orange CsERF017 gene

[0073] (I) Reverse transcription to synthesize cDNA

[0074] Use HifairⅢ1 stThe RNA obtained in Example 1 was reverse-transcribed into 1st Strand cDNA using the Strand cDNA Synthesis SuperMix for qPCR (Shanghai Yeasen Biotechnology Co., Ltd.) reverse transcription kit, which was used as a template for the amplification of the target gene.

[0075] (II) Cloning of the CsERF017 gene

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

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

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

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

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

[0081]

[0082]

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

[0084] The agarose gel electrophoresis results of the PCR amplification products were as Figure 2 shown, and the size of the CDS sequence of the CsERF017 gene was consistent with the expectation. Using navel orange cDNA as a template for the cloning experiment, a clear DNA band with a size of approximately 600 bp was observed in the electrophoresis pattern ( Figure 2 ). Through multiple sequence alignment and Conserved Domains analysis in NCBI, it was found that the full-length nucleotide sequence of the CsERF017 gene was as shown in SEQ ID NO.1, with a typical AP2 / EREBP domain. The nucleotide sequence of the coding region of this gene obtained by sequencing analysis was as shown in SEQ ID NO.2, and the amino acid sequence of its encoded protein was as shown in SEQ ID NO.3.

[0085]

[0086] SEQ ID NO.2: ATGGTGAAGCACGTAGTCGAAAAGCCTGCTGAGAGAAGTGACTCTCGTTACAAGGGTGTCCGAAAGCGAAAGTGGGGGAAATATGTGTCTGAAATCAGACTACCCAACAGCCGTGCCCGTATCTGGCTGGGCTCCTACGACACAGCAGAAAAAGCAGCGCGTGCTTTCGACGCTGCTTTGTTTTGCTTACGTGGCCGATCAGCCAAGTTTAATTTCCCGGACAACCCACCCGACATATCAGGCGGGCGCTCACTTAAGCCATCTGAGATTCAAGCTGTGGCGGCTCAGTTCGCGAATTCGGAGCCGCTGAGGAGTCAATCGCCAGAACAGTCGGTGTCCGAATTGCAAACGGAGTACTTATCGCCGTCGATTTCTGAGGCAACGGTTCAGTTGGACAGCGATGGGGTTTTTGACGGGTCTTTATTGGATCTTTTAACGGCATCGAGTTCCAGTAACTACCCTTCCGAATATGGGATATTTCCTGGGTTTGATGACCTGTCTAATGACATTTTTGCTCCGCAACTCCCAGCTGTTGATTTTGCAGACGAGAATTTTGATGGGCTATTGTTGAATCAAGATTCGTTTCTTTGGAATTTTTAA;

[0087] SEQ ID NO.3: MVKHVVEKPAERSDSRYKGVRKRKWGKYVSEIRLPNSRARIW LGSYDTAEKAARAFDAALFCLRGRSAKFNFPDNPPDISGGRSLKPSEIQAVAAQFAN SEPLRSQSPEQSVSELQTEYLSPSISEATVQLDSDGVFDGSLLDLLTASSSSNYPSEYGI FPGFDDLSNDIFAPQLPAVDFADENFDGLLLNQDSFLWNF*。

[0088] (3) Homology analysis of the CsERF017 gene

[0089] Cross-species homologous gene analysis was performed through the Phytozome platform (https: / / phytozome.jgi.doe.gov / ), and a phylogenetic study was carried out on the CsERF017 gene. The evolutionary tree was constructed using MEGAX software based on the Maximum Likelihood (ML) method, and it was found that the CsERF017 gene has a relatively distant evolutionary relationship with the reported Arabidopsis thaliana ERF017 ( Figure 3 ), inferring that this gene is a novel member of the ERF transcription factor in navel oranges.

[0090] Example 3

[0091] Subcellular localization of the navel orange CsERF017 gene

[0092] (I) Construction of recombinant vector

[0093] Using PRI101-eGFP as the starting vector, the insertion site is between NdeI and BamHI, and the amplification primer pair is designed as follows:

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

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

[0096] The CsERF017 without the stop codon was amplified by PCR technology, and the constructed DNA fragment was purified and recovered. Then, according to the instructions of the one-step cloning kit (Novoprotein), the product was formulated with the double-digested PRI101-eGFP for the recombinant reaction. Finally, 10 μL of the recombinant product was added to 50 μL of DH5α Escherichia coli competent cells, placed on ice for 30 min, heat-shocked in a 42 °C water bath for 45 s, transformed into Escherichia coli, and then plated. After culturing in an inverted position in a 37 °C incubator for 16 h, positive single colonies were picked and sent to Tsingke Biotechnology Co., Ltd. for sequencing verification to obtain the accurate target gene sequence.

[0097] (II) Agrobacterium transformation

[0098] Take 3 μL of the CsERF017-PRI101-eGFP plasmid DNA with correct sequence confirmation and mix it evenly with 50 μL of GV1301 Agrobacterium competent cells. Place it on ice, in liquid nitrogen, in 37 °C water, and in an ice bath for 5 min each in turn. Resuspend it with 700 μL of LB without antibiotics for 2 h and then plate it. Culture it in a 28 °C incubator for 2 d. Pick positive monoclonal colonies. After PCR amplification, there is a single and clear band, and the band position is consistent with the plasmid DNA. Preserve the bacteria in an -80 °C refrigerator.

[0099] (3) Infection of tobacco leaves

[0100] Sow a number of tobacco seeds. After one month of cultivation under 12 h light, they can be used for experiments. Resuspend the Agrobacterium containing the CsERF017 - PRI101 - eGFP vector with a suspension of 10 mM MgCl2 (containing 120 μM AS). Adjust the OD 600 of the suspension to about 0.6. Select tobacco plants with good growth conditions, inject from the lower epidermis of tobacco leaves using a 1 mL syringe without a tip, and make marks. Use the PRI101 - eGFP empty vector as a control. Incubate the injected tobacco plants under low light for 2 d. Take the marked tobacco leaves injected with Agrobacterium, prepare them into slides, observe under a laser confocal microscope, and take pictures.

[0101] In the nucleus and cell membrane of tobacco leaf epidermal cells, the PRI101 - eGFP empty vector is distributed, while green fluorescence of CsERF017 - PRI101 - eGFP is only detected in the nucleus ( Figure 4 ), indicating that the CsERF017 transcription factor is localized in the nucleus, which is consistent with the transcriptional regulation characteristics of transcription factors.

[0102] Example 4

[0103] Transient overexpression of the CsERF017 gene in navel oranges

[0104] (1) Construction of the CsERF017 - PBI121 transient overexpression vector

[0105] When amplifying the CsERF017 gene, recognition sequences of XbaI and BamHI restriction endonucleases are inserted at the 5' ends of the forward and reverse primers respectively. The amplification primer pairs are as follows:

[0106] Forward primer CsERF017 - F3: 5’ - AGAACACGGGGGACTCTAGA ATGGTGAAGCAC GTAGTCG - 3’;

[0107] Reverse primer CsERF017 - R3: 5’ - GACTGACCACCCGGGGATCC AAAATTCCAAA GAAACGAATCTTGATTC - 3’.

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

[0109] Vortex and mix the system gently, then centrifuge briefly. Incubate the mixture in an environment at 37°C for 3 h. Detect 50 μL of the digested product by agarose gel electrophoresis. After confirmation of success, recover the remaining product, purify it, and then ligate it to the PBI121 vector.

[0110] (2) Transient transformation of Newhall navel orange peel

[0111] (1) Preparation of IM (500 mL) solution (prepare fresh): 5.137 g of 4-morpholineethanesulfonic acid (MES), 2.632 g of glucose, 0.164 g of sodium dihydrogen phosphate. Make up to 500 ml, adjust the pH to 5.6 - 5.7. After autoclaving and cooling, add 26.316 ml of 20×AB Salts.

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

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

[0114] (4) Preparation of MMA suspension: First, prepare 0.1 mol / L AS solution, 1 mol / L MgCl2 solution, and 0.5 mol / L MES (fatty acid methyl ester sulfonate) solution. Take 2 mL of the prepared MES solution, 1 mL of the MgCl2 solution, and 0.1 mL of the AS solution respectively, and then make up to 100 mL with ultrapure water to prepare an MMA solution, so that the final concentrations of MES, MgCl2, and AS are 100 mmol / L, 10 mmol / L, and 10 μmol / L respectively.

[0115] (5) Preparation of the infiltration solution: Inoculate the Agrobacterium liquid (containing the CsERF017 - PBI121 recombinant plasmid and the PBI121 empty vector) obtained from the transformation experiment in Example 3 into an LB liquid medium containing kanamycin and rifampicin, and incubate it on a shaker at 28°C and 200 rpm. When the OD 600 value reaches 0.6 - 0.8, centrifuge at 4°C and 5000 rpm for 10 min, collect the precipitated cells, and resuspend them with an equal volume of MMA solution, repeating twice.

[0116] (6) Peel injection

[0117] After the navel orange fruits were washed with clear water and drained, they were disinfected with 1.0% sodium hypochlorite solution for 2 min, and then rinsed thoroughly with running water to remove the residual disinfectant water, and transferred to a clean bench for ventilation and drying. A hole (3 mm in diameter and 3 mm in depth) was made at the equator of the navel orange fruit with a sterilized needle, and 0.5 mL of Agrobacterium infection solution was injected into the hole with a syringe without a needle. After standing for 2 h to allow it to fully penetrate into the peel, a new hole was made 1 cm to the right of the original hole, and 15 μL of Geotrichum candidum spore suspension with a concentration of 1×10 5 CFU·mL -1 was inoculated.

[0118] (7) Storage and sampling

[0119] After the bacterial liquid was absorbed, they were packed in boxes (10 per box), and then sealed with polyethylene plastic bags and placed in an incubator (temperature 27±1 °C, relative humidity between 90% and 95%) for dark cultivation. Observe the appearance changes of the fruits regularly for 1 - 5 d, measure the diameter of the lesion, and at the same time take peel tissue samples within 10 - 20 mm outside the fruit wound, chop them up and carry out liquid nitrogen quick-freezing treatment, and store them at -80 °C for subsequent determination of fruit resistance characteristic indexes.

[0120] (3) Transient overexpression of juice sac GUS staining

[0121] Using the GUS staining kit provided by Coolaber Company, first melt the X-Gluc solvent in a 40 °C water bath, and then take 1 mL of this solvent and add it to the X-Gluc dry powder tube. After fully dissolving, a 50×GUS staining concentrated solution was prepared. Then, take 0.4 mL of the above concentrated solution and mix it evenly with 5 mL of GUS staining buffer. Take a 1 cm segment of the peel at the wound site of the stored fruit for 1 day, immerse it in the GUS staining solution, and incubate it in an incubator at 28 °C overnight. When blue spots appear in the juice sacs, it is regarded as the GUS expression site.

[0122] (4) Determination of fruit resistance characteristic indexes

[0123] After the navel orange fruits were transiently overexpressed with the CsERF017 gene and inoculated with Geotrichum candidum spore suspension, observe the disease occurrence of the fruits every 1 day and sample and store them at -80 °C for detection of the content of disease-resistant substances. The operating steps for the determination of the fruit lesion diameter, disease index, peel hardness, and protopectin, lignin, and cellulose contents were the same as those in Example 1.

[0124] After inoculation with Geotrichum candidum spore suspension, both CsERF017 overexpression and the empty vector showed water-soaked lesions, but there were significant differences in the lesion characteristics (hardness, lesion diameter) ( Figure 5A-C). During the infection process of Geotrichum candidum, the disease indices of the empty vector control at 3, 4, and 5 days after infection were 24.4, 29.7, and 53.7%, respectively, while those of the CsERF017 overexpression lines reached 35.7, 45.4, and 64.9%, all significantly higher than that of the control PBI121( Figure 5 D).

[0125] By detecting disease-resistant related substances, it was found that the contents of protopectin, lignin, and cellulose in the pericarp of navel orange fruits with transient overexpression of the CsERF017 gene were significantly lower than those of the PBI121 control group after inoculating with Geotrichum candidum spores and storing for 3 days( Figure 5 E-G).

[0126] The inventors also constructed a CsERF017 gene silencing vector and determined the resistance characteristics of CsERF017 gene-silenced fruits according to the above method. The results showed that the contents of protopectin, lignin, and cellulose in the pericarp of CsERF017 gene-silenced fruits were significantly higher than those of the control group after inoculating with Geotrichum candidum spores and storing for 3 days.

[0127] The above results confirm that overexpression of CsERF017 enhances the sensitivity of navel orange fruits to Geotrichum candidum, while inhibiting its expression improves the defensive ability of navel orange fruits against sour rot.

[0128] In summary, the present invention found that during the infection process of Geotrichum candidum in navel orange fruits, the pericarp hardness and the contents of protopectin, lignin, and cellulose showed a gradually decreasing trend, while the expression level of the CsERF017 gene was positively correlated with the occurrence of sour rot, indicating that there is a negative regulatory relationship between the CsERF017 transcription factor of Newhall navel orange and the contents of disease-resistant substances. After subcellular localization of this transcription factor in tobacco, it was found that it has a nuclear localization signal. After transient expression of the CsERF017-PBI121 recombinant vector in the pericarp of Newhall navel orange, the infection degree of sour rot was significantly aggravated, and at the same time, the contents of disease-resistant related substances such as protopectin, lignin, and cellulose in the pericarp decreased significantly, which fully verified the negative regulation of CsERF017 on the resistance of navel orange fruits to sour rot.

[0129] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. CsERF017 The use of a gene or a protein encoded by the gene in regulating the resistance to sour rot of navel orange fruit is characterized in that: Said CsERF017 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: Overexpression CsERF017 Genes or upregulation of their encoded protein levels, reduced resistance to sour rot in navel orange fruit, silencing or knocking out CsERF017 The gene or down-regulation of the protein level it encodes can improve the resistance of navel orange fruit to sour rot.

3. CsERF017 Application of genes in breeding transgenic citrus plants resistant to acid rot.

4. Inclusion CsERF017 The invention relates to the application of a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium of a gene in improving the resistance of navel orange fruit to sour rot.

5. Inclusion CsERF017 The invention relates to the application of a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium of a gene in cultivating a transgenic citrus plant resistant to acid rot.

6. A method for improving the resistance of navel orange fruit to sour rot, characterized in that: Silence or knockout CsERF017 The gene or down-regulation of the protein level it encodes can improve the resistance of navel orange fruit to sour rot.

7. A method for cultivating transgenic citrus plants resistant to acid rot, characterized in that: Silence or knockout CsERF017 Genes or downregulating the levels of the proteins they encode can improve the acid rot resistance of transgenic plants.

8. The method according to claim 7, characterized in that Utilize the included CsERF017 Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria of genes, silencing or knocking out CsERF017 gene or downregulate the level of its encoded protein.

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