CsbHLH118 gene and its application in regulating citrus sour rot

Abstract

The present invention discloses CsbHLH118 The invention discloses a gene and its application in regulating citrus acid rot, belonging to the field of genetic engineering technology. The nucleotide sequence thereof is shown in SEQ ID NO.1. The invention provides a transcription factor CsbHLH118 , it was found through experiments that transient overexpression CsbHLH118 It can delay the occurrence of navel orange sour rot and instantly silence CsbHLH118 Loss of resistance to acid rot pathogens CsbHLH118 It plays a positive regulatory role in the resistance of postharvest sour rot of navel orange. The application provides a target gene for the development of effective fungicides.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to CsbHLH118 Genes and their use in regulating citrus sour rot. Background Art

[0002] Sour rot bacteria ( Geotrichum citri-aurantii ) infection is sour rot, which is one of the main fungal diseases of navel oranges after harvest, causing significant economic losses to navel oranges during storage, transportation and sales. At present, the prevention and control of navel orange sour rot is still mainly based on chemical synthetic fungicides, but the environmental pollution and drug resistance problems caused by the long-term use of chemical fungicides are becoming increasingly prominent. Therefore, it is particularly important to find sour rot resistance genes and study their disease resistance mechanisms for the development of effective preservation technologies.

[0003] bHLH transcription factors are one of the largest transcription factor families in plants and are classified according to their typical structural domains of about 60 amino acids, namely the basic amino acid region and the α-helix-loop-α-helix region (HLH region). The basic amino acid region has DNA recognition and binding sites that can recognize the E-box sequence of downstream target genes, thereby regulating the transcription of target genes and affecting the response of plants to biotic and abiotic stresses. This has been widely reported in model plants such as tobacco, Arabidopsis, and woody plants such as apples and citrus fruits. bHLH transcription factors are involved in a variety of physiological processes and are related to plant growth and development, environmental stress, and biotic stress. Related studies have reported that overexpression of bHLH genes can improve the resistance of crops such as tomatoes, apples, Arabidopsis, and poplars to pathogens.

[0004] Existing technologies have reported on the role of bHLH in regulating citrus bacterial canker, flavor quality such as color, sugar and organic acids, and secondary metabolism, but there are few reports on the role of bHLH in the prevention and control of citrus postharvest sour rot. Summary of the invention

[0005] The object of the present invention is to provide CsbHLH118 Genes and their application in regulating citrus acid rot are used 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 a method for regulating citrus acid rot CsbHLH118 The gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is a CsbHLH118 protein that regulates citrus sour rot, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] The third technical solution of the present invention comprises the CsbHLH118 Recombinant vector, expression cassette, transgenic cell line or recombinant bacteria of the gene.

[0010] The fourth technical solution of the present invention is: CsbHLH118 Application of the gene, the CsbHLH118 protein or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in regulating citrus sour rot.

[0011] The fifth technical solution of the present invention is a method for regulating citrus acid rot, overexpressing CsbHLH118 The gene may upregulate the level of CsbHLH118 protein, delaying the occurrence of citrus fruit sour rot; silence CsbHLH118 The gene may downregulate CsbHLH118 protein levels and promote disease in citrus fruits.

[0012] The sixth technical solution of the present invention is: CsbHLH118 Application of the gene, the CsbHLH118 protein or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in cultivating transgenic citrus plants resistant to sour rot.

[0013] The seventh technical solution of the present invention is a method for cultivating a transgenic citrus plant resistant to acid rot, comprising the following steps: CsbHLH118 The gene is introduced into the target plant.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention provides a transcription factor capable of positively regulating the resistance of postharvest sour rot pathogens of navel oranges CsbHLH118 Transient overexpression of this gene can improve the resistance of navel orange to acid rot pathogens, while silencing this gene will result in a larger lesion area than the control group. CsbHLH118 The gene is involved in regulating the occurrence of postharvest sour rot in navel oranges. This gene supplements and improves the mechanism of bHLH transcriptional regulation of postharvest diseases in navel oranges, provides a theoretical basis for analyzing the occurrence mechanism of navel orange sour rot, and provides a candidate gene for the molecular breeding industry to delay or inhibit postharvest sour rot in navel oranges through gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 for CsbHLH118 Full-length amplification and phylogenetic tree analysis of the gene.CsbHLH118 Gel electrophoresis of genes, CsbHLH118 A is the target gene obtained by amplification; Marker is the DNA molecular weight standard; B is CsbHLH118 Phylogenetic tree analysis between homologous genes.

[0018] Figure 2 Figure 2 shows the effect of transient overexpression of the CsbHLH118-pBI121 recombinant vector in navel orange fruit on the growth of sour rot pathogens and gene expression. A shows the disease on the 6th day after inoculation, B shows the GUS staining on the 1st day after inoculation, and C shows the lesion diameter and CsbHLH118 Relative gene expression (* P <0.05,** P <0.01,*** P <0.001).

[0019] Figure 3 For a moment of silence CsbHLH118 Effects of genes on the progression of navel orange fruit sour rot. A is the disease status of navel orange fruit after inoculation, and B is the diameter of the lesion at different inoculation times (* P <0.05,** P <0.01,*** P <0.001). DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0023] The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

[0024] In addition, in order to have a more intuitive understanding of the technical solution of the present invention, some professional terms involved in the present invention are explained as follows:

[0025] A "mutant" refers to an individual that has undergone a mutation and has phenotypic characteristics that are different from the wild type.

[0026] "Expression vector" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0029] The embodiment of the present invention provides a method for regulating citrus acid rot CsbHLH118 The gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0030] The embodiment of the present invention also provides a CsbHLH118 protein for regulating citrus sour rot, and its amino acid sequence is shown in SEQ ID NO.2.

[0031] The embodiment of the present invention also provides a CsbHLH118 Recombinant vector, expression cassette, transgenic cell line or recombinant bacteria of the gene.

[0032] In some specific embodiments, the recombinant vector is CsbHLH118-pBI121.

[0033] In some specific embodiments, the recombinant bacterium is GV3101 Agrobacterium, and the recombinant vector is CsbHLH118-pBI121. Recombinant vector for transient overexpression CsbHLH118 When the gene is expressed, it can inhibit the growth of navel orange sour rot bacteria.

[0034] In some specific embodiments, the recombinant expression vector is CsbHLH118-pTRV2. CsbHLH118 When the gene is expressed, the growth of navel orange sour rot bacteria cannot be inhibited.

[0035] The embodiment of the present invention also provides the CsbHLH118 Application of the gene, the CsbHLH118 protein or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in regulating citrus sour rot.

[0036] The present invention also provides a method for regulating citrus sour rot, by overexpressing CsbHLH118 The gene may upregulate the level of CsbHLH118 protein, delaying the occurrence of citrus fruit sour rot; silence CsbHLH118 The gene may downregulate CsbHLH118 protein levels and promote disease in citrus fruits.

[0037] The embodiment of the present invention also provides the CsbHLH118 Application of the gene, the CsbHLH118 protein or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in cultivating transgenic citrus plants resistant to sour rot.

[0038] The present invention also provides a method for cultivating a transgenic citrus plant resistant to acid rot, comprising the following steps: CsbHLH118 The gene is introduced into the target plant.

[0039] In some specific embodiments, the CsbHLH118 The gene is introduced into the target plant through the recombinant vector, expression box, transgenic cell line or recombinant bacteria.

[0040] The citrus acid rot pathogen in the embodiments of the present invention comes from the Jiangxi Key Laboratory of Fruit and Vegetable Storage and Preservation. Example 1

[0041] Gene CsbHLH118 Sequence acquisition and analysis

[0042] This implementation case provides CsbHLH118 The acquisition and identification of genes specifically include the following steps:

[0043] 1. Gene CsbHLH118 Obtaining the full-length cDNA sequence.

[0044] RNA was extracted from navel orange peel using an RNA extraction kit (purchased from Tiangen Biochemical Technology Beijing Co., Ltd., catalog number: DP441), and cDNA was obtained using a reverse transcription kit (purchased from Yisheng Biotechnology Shanghai Co., Ltd., catalog number: 11141ES60). CsbHLH118 The complete CDS sequence of the gene was used to design upstream and downstream primers for full-length amplification using Primer 3 online software:

[0045] CsbHLH118-FP (SEQ ID NO.3): 5'-ATGGGAAAGGCCAGCA-3';

[0046] CsbHLH118-RP (SEQ ID NO. 4): 5'-TCACTTAATTGAATGATCAAGTTTCAGCTGC-3'.

[0047] PCR amplification was performed using the cDNA from navel orange peel as a template. The 50 μL PCR reaction system included: 20 μL double distilled water, 1 μL cDNA template, 2 μL upstream primer, 2 μL downstream primer, and 25 μL high-fidelity enzyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number: 10154ES03).

[0048] The PCR amplification program was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 45 s, for 30 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0049] The target gene amplified by PCR CsbHLH118 The full-length cDNA was verified by agarose gel electrophoresis ( Figure 1 A), which was sequenced CsbHLH118 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0050] SEQ ID NO.1:

[0051] SEQ ID NO.2: MGKGQQHKLLFVATTKVNDGNSDNNNNKKMILRKDIERQRRQHLSMLHASLRSLLPLQSITGKRSISDHINEEARNYINYLNKNIQELGLKRDKLKN LSELGAVGHGSSNWDNCLQNCVTIHPYSGGVEIVINSVHKEENLKLSKVMEAVLEEGLDVIRCASTQTDEGFFHTIQAEVGDLTCVDLRVLQLKLDHSIK*.

[0052] Depend on Figure 1 The results of A and sequencing show that the target gene obtained by PCR amplification CsbHLH118 and CsbHLH118 The genomic coding region sequences of the genes were consistent in length, with a total length of 591 bp, encoding 196 amino acids.

[0053] 2. GenesCsbHLH118 Sequence analysis and homologous evolutionary relationships.

[0054] Using the NCBI (National Center for Biotechnology Information) database, we compared genes CsbHLH118 For homologous genes in different species, CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw) was used to align multiple sequences and construct evolutionary trees ( Figure 1 The conserved domains were analyzed by NCBI CD-Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and Gendoc32 software was used to analyze the conserved domains. CsbHLH118 Multiple sequence alignment of the homologous genes.

[0055] Depend on Figure 1 As shown in B, the target gene was obtained by PCR amplification CsbHLH118 Clementine CcbHLH118 Gene, mango MibHLH118 Genes and pistachios PvbHLH118 The genes are closely related in evolution.

[0056] Gendoc32 software CsbHLH118 The results of multiple sequence alignment of its homologous genes showed that CsbHLH118 The gene is widely present in different species, relatively conservative in evolution, and has a typical bHLH conserved domain. Related studies have shown that the HLH conserved domain can participate in the regulation of plant biotic and abiotic stresses. Example 2

[0057] Recombinant expression vector construction and transformation

[0058] This example provides the construction and transformation of a recombinant expression vector, and its application method is as follows:

[0059] (I) Construction of CsbHLH118-pBI121 overexpression vector:

[0060] According to the function of pBI121 vector, CsbHLH118 After the gene is inserted into the 35S promoter, the vector pBI121 comes with a GUS marker gene, which satisfies the function of studying gene overexpression. Based on the available restriction sites (XbaI and BamHI) of pBI121, the homology arm primers were designed using the Primer 3 online software:

[0061] CsbHLH118-XbaI-FP (SEQ ID NO.5): 5'-TTGGAGAGAACACGGGGGACTCTAGAATGGGAAAGGGCCAGCAACATAAATTATTATTT-3';

[0062] CsbHLH118-BamHI-RP (SEQ ID NO. 6): 5'-ATAAGGGACTGACCACCCGGGGATCCCTTAATTGAATGATCAAGTTTCAGCTGCAGCAC-3'.

[0063] PCR amplification was performed using primers with restriction sites to obtain the residues with the stop codon removed. CsbHLH118 For the gene, the PCR amplification reaction system was 1 μL each of the upstream and downstream primers, 1 μL of cDNA template, 1 μL of Pfu DNA polymerase (Lifetechnology), 5 μL of 10×Pfu buffer, and finally added with deionized water to 50 μL.

[0064] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, extension at 72°C for 1 min, 25 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0065] The amplified DNA fragment was purified and recovered (Tiangen, catalog number: D6492-01), and the pBI121 empty plasmid was double-digested (XbaI and BamHI restriction endonucleases, NEB). The amplified DNA fragment and the digested plasmid were connected using Fermentas' T4 DNA ligase. The recombinant plasmid was then transformed into TOP10 competent cells and sent to Wuhan Jinkairui Bioengineering Co., Ltd. for platform sequencing to obtain accurate recombinant plasmids.

[0066] (II) Construction of pTRV-CsbHLH118 silencing vector:

[0067] Based on the gene sequence obtained above (SEQ ID NO.1) and the available restriction sites (XbaI and SmaI) of pTRV2, the VIGS primer homology arm primers were designed using the VIGS tool (https: / / vigs.solgenomics.net / ) online software on the Solanaceae genome website:

[0068] CsbHLH118-XbaI-FP (SEQ ID NO.7): 5'-TGAGTAAGGTTACCGAATTCTCTAGATGGAAATTGTGATAAATAGTGTCCATAA-3';

[0069] CsbHLH118-SmaI-RP (SEQ ID NO. 8): 5'-TTTAATGTCTTCGGGACATGCCCGGGCTTAATTGAATGATCAAGTTTCAGCTG-3'.

[0070] PCR amplification was performed using primers with restriction sites to obtain the residues with the stop codon removed. CsbHLH118 For the gene, the 50 μL PCR amplification reaction system consisted of 2 μL of upstream and downstream primers, 1 μL of cDNA template, 25 μL of high-fidelity enzyme premix (Yisen Company, catalog number: 10154ES03), and 20 μL of deionized water.

[0071] The PCR amplification program was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, for 30 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0072] The amplified DNA fragment was purified and recovered (OMEGA, catalog number: D2500-01), and the pTRV2 empty plasmid was double-digested (XbaI and SmaI restriction endonucleases, Thermo Scientific). ExnaseII (purchased from Vazyme, catalog number: C112-01) was used to connect the amplified DNA fragment and the linear plasmid vector that had been digested. The recombinant plasmid was then transformed into DH5α competent cells (purchased from Qingke) and sent to Hunan Youkanglai Biotechnology Co., Ltd. for sequencing to obtain the accurate recombinant plasmid.

[0073] 3. Agrobacterium transformation

[0074] Take the CsbHLH118-pBI121 and CsbHLH118-pTRV2 plasmids with correct sequences confirmed by sequencing, take 3 μL plasmids respectively and add them to 50 μL GV3101 Agrobacterium competent cells, mix thoroughly, place on ice for 5 min, place in liquid nitrogen for 5 min, transfer to a 37°C water bath for 5 min, and then place on ice for 5 min to cool. Add 700 μL LB liquid medium and mix thoroughly, shake and culture on a shaker at 28°C and 200 rpm for 30 min, and inoculate the transformed Agrobacterium on LB solid medium and culture at 28°C for 2 d. Further take a single clone for sequencing verification, select 20 μL of positive Agrobacterium, add it to 3 mL LB liquid medium (containing antibiotics) and culture overnight (28°C, 200 rpm), and then store glycerol bacteria (50% glycerol: bacterial liquid = 1:1) in a negative 80°C refrigerator for use. Example 3

[0075] Transient overexpression CsbHLH118 Effects of Genes on the Growth of Acid Rot Fungi in Navel Orange

[0076] 1. Picking and processing of navel orange fruits

[0077] 1. Fruit picking and disinfection. The mature fruits of the navel orange variety 'Newhall' (Ganzhou, Jiangxi) were picked from the navel orange orchard in Ganzhou City, Jiangxi Province on November 19, 2023, and transported to the Jiangxi Key Laboratory of Fruit and Vegetable Storage and Preservation on the same day. Select fruits with uniform shape and size, without mechanical injuries and pests and diseases, wash with tap water, soak in 0.5% sodium hypochlorite solution for 2 min, and finally rinse with distilled water, dry naturally for use. Use an inoculation needle to inoculate two wounds 1 cm apart on the equatorial surface of each fruit. The wound diameter is about 3 mm and the depth is about 3 mm.

[0078] 2. Activation of acid rot bacteria. Take out the navel orange acid rot bacteria strain stored in a -80℃ ultra-low temperature refrigerator, pipette 5 μL onto PDA medium, culture at 28℃ for 3 days, and subculture for later use.

[0079] 3. Activate Agrobacterium. Use a pipette to pipette 200 μL of Agrobacterium carrying plasmid stored at -80℃ into 8 mL of LB liquid medium. After shaking culture at 28℃ and 200 rpm for 12 h, pipette 6 mL of bacterial solution into 25 mL of liquid LB medium. Shake and culture at 28℃ and 200 rpm overnight for later use.

[0080] 4. Instantaneous transformation of navel orange peel. Prepare a suspension of acid rot fungi spores at a concentration of 1×10 7spores / mL, and the OD value of the Agrobacterium suspension was 0.8. On the clean bench, 0.5 mL of different Agrobacterium suspensions were inoculated into one of the wounds on the navel orange peel. The plasmids carried in the different Agrobacterium suspensions were: pBI121 (EV, Empty Vector, empty control group) and CsbHLH118-pBI121 (OE-CsbHLH118, overexpression genome). After the wound was dried naturally, 10 μL of the acid rot fungus spore suspension was inoculated into another wound 1 cm away. After the wound was dried naturally, the inoculated fruit was placed in a sterilized fresh-keeping box, the bottom of the fruit was supported by a plastic cover, and an appropriate amount of sterile water was added. It was cultured at 28°C for 6 days.

[0081] 2. Observation of lesion diameter

[0082] The diseased fruits were observed every day, the number of diseased fruits was counted and the diameter of the lesions was measured by the cross method. The experiment was repeated three times, with 8 fruits in each replicate.

[0083] (III) Transient overexpression of GUS staining in navel orange peel

[0084] Cut a 2 cm strip of peel with the center of the two inoculation holes as the midpoint for GUS staining observation. The GUS staining kit (Coolaber) was used for operation, and the steps were as follows: soak the prepared navel orange peel in GUS staining solution, keep it in a 28℃ incubator for 3 h, take it out to observe the GUS staining (blue dots) and take photos to record.

[0085] (Four) CsbHLH118 Gene expression analysis

[0086] The samples in the circular area with a radius of 1 cm and the center of the two inoculation holes were taken for gene expression analysis (QPCR). The total RNA extraction and cDNA acquisition of the samples were carried out according to the method in Example 1. CsbHLH118 The full-length cDNA sequence of the PCR product was used to design the upstream and downstream primers for real-time fluorescence quantitative PCR amplification and the upstream and downstream primers for the internal reference gene as follows:

[0087] CsbHLH118-FP (SEQ ID NO.9): 5'-GGGGGAGTGGAAATTGTGAT-3';

[0088] CsbHLH118-RP (SEQ ID NO. 10): 5'-ACACATGTCAGATCGCCAAC-3';

[0089] Actin-FP (SEQ ID NO. 11): 5'-CATCCCTCAGCACCTTCC-3';

[0090] Actin-RP (SEQ ID NO. 12): 5'-CCAACCTTAGCACTTCTCC-3'.

[0091] A real-time fluorescence quantitative PCR instrument (T100 Thermal Cycle) was used for QPCR detection. The QPCR reaction system was as follows: the total volume was 10 μL, of which 1 μL of cDNA was required, 0.3 μL of upstream and downstream primers, and 0.3 μL of ddH 2 3.4 µL of O and 5 µL of qPCR SYBRGreen Master Mix (purchased from Yisheng Biotechnology) are required.

[0092] The QPCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 58°C for 20 s, and extension at 72°C for 20 s, for 40 cycles; annealing at 60°C for 30 s (melting curve temperature), and extension at 95°C for 5 s.

[0093] CsbHLH118 The relative expression of genes was expressed using -2 △△Ct Method calculation.

[0094] CsbHLH118 The results of transient overexpression of the gene in navel oranges showed that the navel orange fruit began to develop disease on the second day of storage after injury inoculation, and by the sixth day of storage, transient overexpression CsbHLH118 The diameter of the fruit lesions of the gene (OE-CsbHLH118) was 6.29 cm, which was significantly lower than that of the control group (EV) (7.29 cm) (P<0.001). Figure 2 The results of GUS staining showed that the fruit peel color of the OE-CsbHLH118 group was significantly darker than that of the EV group on the first day after inoculation, which was consistent with the results of gene expression, that is, on the first day of storage, the fruit peel color of the OE-CsbHLH118 group was significantly darker than that of the EV group. CsbHLH118 Gene expression was transiently and significantly induced upregulation ( Figure 2 The above results show that CsbHLH118 The gene can positively improve the resistance of navel oranges to acid rot fungi, thereby delaying the occurrence of post-harvest acid rot in navel oranges. Example 4

[0095] Momentary silence CsbHLH118 Effects of Genes on the Growth of Acid Rot Fungi in Navel Orange

[0096] 1. Picking and processing of navel orange fruits

[0097] 1. The harvesting and disinfection of navel orange fruits and the activation of the acid rot strains are all the same as in Example 3.

[0098] 2. Agrobacterium activation. The Agrobacterium carrying pTRV1, pTRV2, and pTRV2-CsbHLH118 was taken out from the -80°C refrigerator for activation. The specific activation process is as described in Example 3.

[0099] 3. Instantaneous transformation of navel orange peel. Prepare a suspension of acid rot fungi spores at a concentration of 1×10 7 spores / mL, and the OD value of the Agrobacterium suspension was 0.8. On the clean bench, different Agrobacterium suspensions were mixed in equal volumes, namely: pTRV2: pTRV1 = 1: 1 (pTRV; empty control group) and pTRV2-CsbHLH118: pTRV1 = 1: 1 (pTRV-CsbHLH118; overexpression genome). Take 0.5 mL of different Agrobacterium suspensions and inoculate one of the wounds on the navel orange peel. After the wound is naturally dried, inoculate 10 μL of the acid rot fungus spore suspension in another wound 1 cm away. After the wound is naturally dried, place the inoculated fruit in a sterilized fresh-keeping box, support the bottom of the fruit with a plastic cover, add an appropriate amount of sterile water, and culture at 28°C for 4 days.

[0100] (ii) Observation of lesion diameter is the same as in Example 3.

[0101] The results showed that the navel orange fruit began to develop disease on the first day of storage after injury inoculation, and became silent within 4 days of storage. CsbHLH118 The diameter of fruit lesions of the pTRV-CsbHLH118 gene was significantly higher than that of the control group (pTRV) (P<0.01). Figure 3 A, B). Indicates momentary silence CsbHLH118 Loss of resistance to acid rot pathogens.

[0102] Based on the descriptions in Examples 1, 2, 3, and 4, the present invention uses transient gene overexpression and silencing expression technology to identify CsbHLH118 The gene (SEQ ID NO.1) has an inhibitory effect on post-harvest sour rot pathogens of navel oranges, can delay the occurrence of post-harvest sour rot diseases of navel oranges, can be applied to genetic engineering for the prevention and control of post-harvest diseases of citrus, and can also be used as a target gene for the prevention and control of post-harvest diseases of navel oranges.

[0103] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. CsbHLH118 gene, CsbHLH118 protein or CsbHLH118 The application of gene recombinant vector, expression box, transgenic cell line or recombinant bacteria in delaying citrus acid rot.

2. A method for regulating citrus acid rot, characterized in that: Overexpression CsbHLH118 The gene may upregulate the level of CsbHLH118 protein and delay the occurrence of sour rot in citrus fruit.

3. CsbHLH118 gene, CsbHLH118 protein or CsbHLH118 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.

4. A method for cultivating transgenic citrus plants resistant to acid rot, characterized in that: The following steps are involved: Will CsbHLH118 The gene is introduced into the target plant.

5. The method according to claim 4, characterized in that CsbHLH118 Genes contain CsbHLH118 The recombinant vector, expression cassette, transgenic cell line or recombinant bacteria of the gene are introduced into the target plant.

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