Application of citrus transcription factor CsAGL9 gene in regulation and control of citric acid content in citrus fruits

Abstract

The invention provides an application of a citrus transcription factor CsAGL9 gene and a protein coded by the citrus transcription factor CsAGL9 gene in regulating and controlling the content of citric acid in citrus fruits, the nucleotide sequence of the CsAGL9 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO.2. The content of citric acid in the fruits can be reduced through overexpression in the oranges; the content of citric acid in fruits can be increased by silencing the gene. It is further verified that the gene promotes the activity of the citrus citric acid transporter CsTCT1. The discovery provides a new gene resource for citrus quality improvement and molecular design breeding, and provides an efficient tool for fruit quality improvement.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology and specifically relates to a citrus transcription factor CsAGL Application of 9 genes in regulating citric acid content in citrus fruits. Background Art

[0002] Citrus is one of the world's most important fruits and a major cash crop widely cultivated in southern China, playing a key role in promoting my country's economic development. Citrus pulp is rich in sugars, organic acids, vitamins, and carotenoids, possessing significant nutritional value and antioxidant properties. Citrus fruit quality determines its market value and is primarily categorized into intrinsic and external qualities. External qualities are primarily reflected in fruit size, shape, color uniformity, and peel smoothness; internal qualities are more important, encompassing flesh texture, aroma, nutritional content, and flavor characteristics. With economic development, people's demand for fruit quality is increasing. Research shows that consumers prioritize the flavor characteristics of citrus fruit when purchasing. Organic acids (citric acid and malic acid) and soluble sugars (sucrose, fructose, and glucose) are the core factors determining the flavor and taste of citrus fruit. While sugar content in citrus is relatively stable, organic acid content varies more significantly. Citric acid, the primary organic acid component, is the primary factor in citrus flavor quality. Therefore, identifying key genes that regulate citric acid content is crucial for improving citric acid content in citrus fruit.

[0003] MADS-box transcription factors are one of the most extensively studied families in plants. Early research on their functions focused primarily on floral organs. Recent studies have shown that MADS-box transcription factors are involved in regulating fruit ripening and quality. MADS1 and MADS2 are involved in banana fruit ripening, and RNAi inhibition of these factors inhibits fruit ripening and delays fruit softening. However, few studies have examined the regulation of citric acid content in fruit by MADS-box transcription factors.

[0004] Therefore, it is necessary to conduct further research on related transcript genes and provide a gene related to the citric acid content of citrus fruits. Further genetic engineering technology can provide a feasible solution for the subsequent cultivation of excellent flavor citrus varieties. Summary of the Invention

[0005] In view of this, the present invention provides a citrus transcription factor CsAGL Application of 9 genes in regulating citric acid content in citrus fruits.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide a citrus transcription factor CsAGL Application of 9 genes in regulating citric acid content in citrus fruits, theCsAGL The nucleotide sequence of the 9 gene is shown in SEQ ID NO.1; SEQ ID NO.1:.

[0007] The second object of the present invention is to provide the above CsAGL Application of the protein encoded by gene 9 in regulating the citric acid content in citrus fruits, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2; EQ ID NO.2: MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSNRGKLYEFCSSSSMLKTLERYQKCNYGAPEPNVSAREALELSSQQEYLKLKARYEALQRSQRNLLGEELGPLNSKEL ESLERQLDMSLKQIRSTRTQYMLDTLTELQHKEQLLSEANKTLKQRTMTLRHADFAGLQLMEGYQVNTLQLNPSAEDCGYGLKPAQPQGDTFFHALECEPTLQIGYQPADPISVVTAGPSLNNYMQGWLPC.

[0008] A third object of the present invention is to provide a method for regulating the citric acid content in citrus fruits, wherein: Reducing citric acid content in citrus fruit by overexpression CsAGL 9 genes achieved; Increasing the citric acid content in citrus fruits by silencing CsAGL 9 genes achieved.

[0009] Furthermore, through overexpression CsAGL The specific steps of obtaining citrus with low citric acid content by 9 genes are as follows: construct an overexpression vector and infect citrus with Agrobacterium to obtain overexpression plants, thereby obtaining citrus with low citric acid content.

[0010] In some specific embodiments, preferably, the primers used to construct the overexpression vector are as follows: pENTER1A- CsAGL 9-F: 5'-AAAGGAACCAATTCAGTCGACATGGGAAGGGGTAGGGTTGA-3'; pENTER1A- CsAGL 9-R: 5'-TGGAAAAGGGAATTCGGTACCGCATGGTAGCCATCCTTGCAT-3'.

[0011] Furthermore, through silence CsAGL The specific steps of obtaining citrus with high citric acid content by 9 genes are as follows: construct a silencing vector and infect citrus with Agrobacterium to obtain mutant plants, thereby obtaining citrus with high citric acid content.

[0012] In some specific embodiments, preferably, the primers used to construct the silencing vector are as follows: GW- CsAGL9-F: 5'-GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGGGAAGGGGTAGGGTTGA-3'; GW- CsAGL 9-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCGGACACATTCGGTTCTGGT-3'.

[0013] The fourth object of the present invention is to provide a gene expression cassette, the gene expression cassette comprising the above CsAGL 9 genes.

[0014] A fifth object of the present invention is to provide a recombinant expression vector comprising the above-mentioned gene expression cassette.

[0015] A sixth object of the present invention is to provide an engineered bacterium comprising the above-mentioned recombinant expression vector.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a new citrus transcription factor CsAGL9 Overexpression of this gene in citrus fruits reduced citric acid content, while silencing it increased fruit content. Further verification confirmed that this gene promoted the activity of the citric acid transporter CsTCT1 in citrus. This discovery provides a new genetic resource for citrus quality improvement and molecular design breeding, and offers a highly effective tool for fruit quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a technical flow chart of the present invention;

[0018] Figure 2 The citrus development stage in Example 1 of the present invention CsAGL 9. Expression trend result graph; Figure 3 In Example 1 of the present invention CsAGL 9. Subcellular localization detection results;

[0019] Figure 4 In Example 2 of the present invention CsAGL Schematic diagram of the analysis of relative expression levels and citric acid content in 9-gene transgenic overexpression calli; A is real-time fluorescence quantitative detection CsAGL 9 Overexpression in wound healing CsAGL 9 expression level; B is real-time fluorescence quantitative detection CsAGL 9. Expression level of CsTCT1 in overexpression callus; C is CsAGL 9Citrate content in overexpression callus;

[0020] Figure 5 In Example 3 of the present invention CsAGL Schematic diagram of the analysis of relative expression levels and citric acid content in 9-gene transgenic silenced calli; A is real-time fluorescence quantitative detection CsAGL 9Silent Healing CsAGL 9 expression level; B is real-time fluorescence quantitative detection CsAGL 9. Expression level of CsTCT1 in silenced callus; C is CsAGL 9Citrate content in silent callus;

[0021] Figure 6 This is the Y1H system verification in Example 4 of the present invention CsAGL 9 can bind to the promoter of the CsTCT1 gene;

[0022] Figure 7 The dual luciferase assay in Example 5 of the present invention is verified CsAGL 9 Promotes the transcriptional activity of CsTCT1; A is a schematic diagram of LUC; B is LUC imaging and enzyme activity verification CsAGL 9 promotes the transcriptional activity of CsTCT1. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0024] Example 1 This embodiment provides citrus CsAGL The cloning and subcellular localization of the full-length cDNAs of the 9 genes are as follows: 1.1 [[ID=3P]]CsAGL Cloning of full-length cDNA of 9 genes Citrus RNA was extracted and amplified using citrus cDNA as a template using high-fidelity enzymes. The amplification primers are as follows: CsAGL 9. Forward primer for gene amplification: 5′-AAAGGAACCAATTCAGTCGACATGGGAAGGGGTAGGGTTGA-3′; CsAGL 9. Reverse amplification primer for gene amplification: 5'-TGGAAAAGGGAATTCGGTACCGCATGGTAGCCATCCTTGCAT-3'.

[0025] The PCR amplification system is as follows:

[0026] The PCR reaction procedure is as follows:

[0027] After amplification, the amplified product was purified and recovered using the AxyPrep-96 DNA gel recovery kit (Axygene, USA). The purified product was then ligated into the pENTER1A vector and transformed into competent Escherichia coli DH5α plate. The plate was incubated at 37°C for 12 h. After PCR positive identification, the bacteria were shaken and the positive clones were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were used to identify the clones. CsAGL 9 full-length gene sequence.

[0028] Sequencing results showed that the gene contained an open reading frame (ORF) of 774 bp, encoding 257 amino acids. Molecular weight prediction showed that the protein had a molecular weight of 29.26 kDa and an isoelectric point of 8.058. The gene was named [[ID=4P]]CsAGL 9, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0029] 1.2 CsAGL9 Gene expression analysis during development RNA was extracted from citrus fruit juice cells during the development period, and real-time fluorescence quantitative PCR (qRT-PCR) was used to analyze the CsAGL9 The expression pattern of genes during the developmental period was analyzed. Real-time fluorescence quantitative PCR was performed using AceQ qPCR SYBR Green Master Mix reagent according to the instructions. The prepared reaction system was reacted using QuantStudio™ 7 Flex Real-Time PCR Fluorescence Quantitative Analyzer. Actin As an internal reference gene (forward primer: 5'-CCGACCGTATGAGCAAGGAAA-3'; reverse primer: 5'-TTCCTGTGGACAATGGATGGA-3'), 2 -ΔΔCt The algorithm calculates gene expression. CtrAGL91 Real-time quantification primers (forward primer: 5′-GGAACAGTTGCTGAGCGAAG-3′; reverse primer: 5′-GTTCACATTCCAAGGCGTGA-3′).

[0030] Figure 2 The results show that: CsAGL9 The expression level showed an increasing trend with the developmental stage.

[0031] 1.3 CsAGL 9 gene subcellular localization Amplification CsAGL9 ORF region (excluding the stop codon, PCR amplification system and reaction procedure refer to Example 1.1), and designed primers as follows: p101YFP- CsAGL 9-F: 5'-ATATGGGATCTACTAGTGAATTCATGGGAAGGGGTAGGGTTGA-3'; p101YFP- CsAGL 9-R: 5'-GGGGGTACCGTCGACGGATCCGCATGGTAGCCATCCTTGCAT-3'.

[0032] The target gene was constructed into the YFP101 vector, with the YFP protein located at the 3' end of the gene and its expression driven by the CaMV35S promoter ( Figure 2 Middle A). 35S: CsAGL 9-YFP and the control 35S:YFP were transiently transformed into the epidermal cells of Nicotiana benthamiana leaves, and the protoplasts were isolated and the vacuoles were extracted. Laser confocal fluorescence observation showed that the fluorescence of the control filled the entire epidermal cells, while the transformed 35S: CsAGL 9-YFP fluorescence was only detected in the cytoplasm and nucleus. Figure 2 B) confirmed CsAGL 9 is located in the cytoplasm and nucleus.

[0033] Example 2 This example provides the construction of an overexpression gene vector, genetic transformation of callus, positive identification, and citric acid content determination, as follows: 2.1 Construction of overexpression vector Extract citrus RNA and use citrus cDNA as template to design primers for cloning CsAGL 9 Obtain the full-length gene (the method is the same as Example 1).

[0034] Linearize the pENTER1A vector and clone CsAGL The 9 fragments were ligated into the pENTER1A vector in one step using the One Step Cloning Kit (Novozymes, China).

[0035] The pENTER1A vector was connected to CsAGL 9. Perform LR reaction to connect the plasmid with the pK7GW2D vector. For instructions, refer to Gateway. ® The BP ClonaseTM Ⅱ kit instructions were followed and the positive clones that were sequenced correctly were shaken. Then the plasmid was extracted using the AxyPrep plasmid DNA miniprep reagent (Axygen, USA) to obtain the final overexpression vector pK7GW2D- CsAGL9. Transform the vector into competent Agrobacterium EHA105 for later use. The steps of amplified fragment recovery, positive clone identification and sequencing are similar to those in Example 1.

[0036] 2.2 Genetic transformation of callus (1) Preparation for wound healing The wild-type callus of National Day No. 1 was suspended in MT suspension in a clean bench and cultured with shaking suspension at room temperature for 48 hours.

[0037] (2) Preparation of Agrobacterium infection solution In the clean bench, use the sterilized inoculation loop to pick up the pK7GW2D- [[ID=6P]]CsAGL 9 Agrobacterium, streak on the culture medium containing 50mg / L Spec+75mg / L Rif antibiotics, turn the culture medium upside down and place it in a 28℃ incubator for dark culture for 2 days. Pick a single clone and inoculate it on a new culture medium containing 50mg / L Spec+75mg / L Rif antibiotics, then turn it upside down in the incubator and continue to culture for 2 days. Take a sterilized 100mL small triangular flask in the clean bench and pour 50mL of MT liquid culture medium containing 20mg / L AS (Acetosyringone). Scrape the grown Agrobacterium and dissolve it in MT liquid culture medium containing 20mg / L AS. Under the condition of 28℃, shake at 200r / min for 20min and adjust the bacterial concentration to OD 600 The value is 0.4-0.6.

[0038] (3) Infection and pre-cultivation In the clean bench, pour out the MT liquid culture medium in the room temperature suspended callus, transfer it to the Agrobacterium infection solution, vacuum it for 15 minutes, take it out and let it stand at room temperature for 10 minutes. After pouring out the infection solution in the clean bench, use sterilized filter paper to absorb the infection solution from the callus and place it on MT solid culture medium (MT + 30g / L sucrose + 20mg / L AS). After leaving it at room temperature for 48 hours, transfer it to the screening culture medium (MT + 30g / L sucrose + 50mg / L Kan).

[0039] (4) Positive callus screening The calli cultured in the screening medium for 30 days were placed under ultraviolet light to search for calli with GFP fluorescence signals. The calli containing GFP signals were transferred to a new screening medium and cultured for 30 days to obtain overexpression plants.

[0040] 2.3 Identification of positive callus The callus lines containing fluorescent signals and the control group were analyzed by qPCR. CsAGL The expression of 9 was analyzed by fluorescence quantitative analysis, and the results showed that compared with the wild type WT,CsAGL 9 The relative expression level of CsAGL9 gene in transgenic callus was reduced. The positive callus was subcultured and its citric acid content was analyzed by GC.

[0041] The qPCR detection system is as follows:

[0042] The qPCR reaction procedure is as follows:

[0043] 2.4 Determination of citric acid content After quick freezing of callus in liquid nitrogen, 0.2 g of the ground sample was weighed into a tube. After adding 1.4 mL of 75% methanol, the sample and methanol were vortexed for 10 s. 100 μL of Ribitol (1.2 mg / mL) was added and vortexed for 10 s. The sample was shaken in a shaker at 70°C, 950 rpm / s for 30 min, and then centrifuged at 10,000 rpm for 15 min at room temperature. 750 μL of the supernatant was collected and placed in a new centrifuge tube, and 400 μL of chloroform was added. Add 750 μL of ultrapure water, vortex for 10 seconds, centrifuge at 10,000 rpm at room temperature for 10 minutes, take 200 μL of supernatant into a 2 mL centrifuge tube, evaporate to dryness under vacuum at room temperature for 4 hours until it becomes a transparent gel, then add 40 μL of methoxyamine hydrochloride (20 mg / mL dissolved in pyridine) and shake at 950 rpm in a 37°C metal bath for 2 hours. Take out the centrifuge tube, centrifuge at 10,000 rpm for 1 minute, add 60 μL of MSTFA and shake at 300 rpm in a 37°C metal bath for 30 minutes, centrifuge at 10,000 rpm for 1 minute, and then transfer to a vial containing an inner liner tube for GC analysis for subsequent GC determination of citric acid content. Figure 4 The results showed that: equivalent to the control group, overexpression ​ The citric acid content of the 9-gene positive callus was significantly lower than that of the control group.

[0044] Example 3 This example provides the construction of a silencing vector, genetic transformation of callus, positive identification, and citric acid content determination, as follows: 3.1 Construction of transformation vector Using citrus cDNA as template, specific primers were designed to amplify ​ 9 gene 3' end non-conserved region fragment (PCR amplification system and reaction procedure see Example 1.1), the primer sequence is: GW- ​ 9-F: 5'-GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGGGAAGGGGTAGGGTTGA-3'; GW- ​9-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCGGACACATTCGGTTCTGGT-3'.

[0045] pDonor 221 vector and clone ​ The 9 fragments were connected using the Bp reaction. For instructions, see Gateway ® According to the instructions of BPClonaseTM Ⅱ kit, shake the positive clones that have been sequenced correctly.

[0046] The pDonor 221 vector was connected to ​ 9 plasmid was ligated with pK7GWIWG2D vector by LR reaction. The usage method was as per the instructions of Gateway® BP ClonaseTM Ⅱ kit. The positive clones with correct sequencing were shaken. Then the plasmid was extracted with AxyPrep plasmid DNA miniprep reagent (Axygen, USA) to obtain the final silencing vector pK7GWIWG2D- ​ 9. The vector was transformed into competent Agrobacterium EHA105 for later use. The steps of amplified fragment recovery, positive clone identification and sequencing were as described in Example 1.

[0047] 3.2 The steps of genetic transformation of callus were the same as those in Example 2.

[0048] 3.3 The steps for identifying positive callus are the same as those in Example 2.

[0049] 3.4 The steps for determining the citric acid content are the same as those in Example 2. ​ The results showed that: equivalent to the control group, silent ​ The citric acid content of the 9-gene positive callus was significantly higher than that of the control group.

[0050] Example 4 This example provides a yeast one-hybrid system for the verification of transcription factors ​ The interactions of the 9 genes with the citrus citrate transporter CsTCT1 are as follows: 4.1 Vector Construction Based on the promoter prediction results from the PLANTCARE website, select a fragment of appropriate length and design amplification primers based on the multiple cloning site on the pAbAi vector. Using citrus DNA as a template, design specific primers to amplify the non-conserved region at the 3' end of the TCT1 gene (see Example 1.1 for the PCR amplification system and reaction procedure). The primer sequences are: pAbAi-CsTCT1-F: 5'-GAAAAGCTTGAATTCGAGCTCATGCAAACGCTGGGATTGTC-3'; pAbAi-CsTCT1-R: 5'-AGCACATGCCTCGAGGTCGACTTTAGATAAGTCTAAAACGATGTTGTTTCA-3'.

[0051] The amplified product was inserted into the pAbAi vector by homologous recombination and then transformed into DH5α Escherichia coli competent cells. The homologous recombination and E. coli transformation operations were the same as above.

[0052] Will ​ The 9 genes were constructed into pGADT7 (see Example 1.1 for the PCR amplification system and reaction procedure). The primer sequences were: AD-CsTCT1-F: 5'-GTACCAGATTACGCTcatatg ATGGGAAGGGGTAGGGTTGA-3'; AD-CsTCT1-R: 5'-CAGCTCGAGCTCGATggatcc TTAGCATGGTAGCCATCCTTGC-3'.

[0053] 4.2 Yeast strain construction The recombinant positive plasmid was linearized by BstBI single enzyme digestion. The enzyme digestion system is shown in the table below.

[0054]

[0055] Integrate 1 µg of linearized plasmid into the Y1H Gold strain as follows: (1) Take Y1H Gold yeast cells stored at -80℃, streak them on YPDA plates (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, 0.2% adenine sulfate 50 mL / L, adjust pH to 6.0 with 1 mol / L KOH, add 20 g / L agar, and sterilize at high pressure at 121℃ for 20 min), and incubate at 30℃ for about 3 days to grow 2 mm to 3 mm plaques; (2) Pick a single clone and culture it in 5 mL YPDA (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, 0.2% adenine sulfate 50 mL / L, 1 mol / L KOH to adjust pH to 6.0, autoclave at 121°C for 20 min) liquid culture medium, at 30°C, 220 rpm, overnight. (3) Take 50 μL of the culture from the previous step, add 50 mL of YPDA liquid medium, and culture at 30°C, 220 rpm, and shake for 5 h until the OD 600 About 0.6; (4) Centrifuge at 2400 rpm for 5 min, discard the supernatant, collect the cells, and gently suspend the cells in 20 mL of 1×TE Buffer; (5) Centrifuge at 2400 rpm for 5 min, discard the supernatant, collect the cells, gently suspend the cells with 1 mL of 1×LiAC / 0.5×TE Buffer, and incubate at room temperature for 5 min to prepare the competent yeast. (6) Denature the carrier DNA at 95°C for 10 min and quickly cool it in ice. (7) Take 5 μL of linearized plasmid, add 10 μL of denatured carrier DNA, mix well with a pipette, add 100 μL of yeast competent cells, and finally add 500 μL of 1×PEG / LiAc and mix gently; (8) Place in a 30°C water bath for 30 minutes, taking the tube out and mixing it upside down every 10 minutes; (9) Add 50 μL of dimethyl sulfoxide to the centrifuge tube, invert and mix, and place in a 42°C water bath for 15 min. Invert and mix every 5 min. (10) Centrifuge at 2500 r / min for 15 s, discard the supernatant, resuspend with 1 mL of ddH2O, centrifuge at 2400 r / min for 1 min, discard the supernatant, add 100 μL of ddH2O and spread on SD / -Ura plates; (12) Culture at 30°C for about 3 days, and pick out the positive clones for plaque detection.

[0056] 4.3 Screening of positive clones (1) Place the single colony identified on the SD / -Ura plate in 5 mL of defective Ura liquid medium sterilized at 121°C, culture at 30°C, 220 rpm, overnight; (2) Take 50 μL of the culture from the previous step, add 50 mL of YPDA liquid medium, and culture at 30°C, 220 rpm, and shake for 5 h until the OD 600 About 0.6; (3) Centrifuge at 2400 rpm for 5 min, discard the supernatant, collect the cells, and gently suspend the cells in 20 mL of 1×TE Buffer; (4) Centrifuge at 2400 rpm for 5 min, discard the supernatant, collect the bacteria, gently suspend the bacteria in 1 mL of 1×LiAC / 0.5×TE Buffer, and incubate at room temperature for 5 min to prepare the competent yeast. (5) Denature the carrier DNA at 95°C for 10 min and quickly cool it in ice. (6) Take 5 μL of AD-CsAGL9 and AD empty plasmids, add 10 μL of denatured carrier DNA, mix by pipetting, add 100 μL of yeast competent cells, and finally add 500 mL of 1×PEG / LiAc and mix gently; (7) Place in a 30°C water bath for 30 minutes, taking the tube out and mixing it upside down every 10 minutes; (8) Add 50 μL of dimethyl sulfoxide to the centrifuge tube, mix thoroughly by inversion, and place in a 42°C water bath for 15 min. Take the tube out and mix thoroughly by inversion every 5 min. (9) Centrifuge at 2500 r / min for 15 s, discard the supernatant, resuspend with 1 mL of ddH2O, centrifuge at 2400 r / min for 1 min, discard the supernatant, add 100 μL of ddH2O and spread on SD / -Leu plates; (10) Culture at 30°C for about 3 days and pick out the positive clones for plaque detection.

[0057] (11) The AD empty load and AD-CsAGL9 yeast single colonies were dissolved in ddH2O and diluted 10-fold and 100-fold, respectively, and then dropped onto SD / -Leu and SD / -Leu+AbA100-500 plates to observe the yeast growth.

[0058] ​ The results showed that the combination of AD-AGL9 and TCT1 could still grow in the defective culture medium (-L) with an AbA concentration of 100 ng / ml, but the growth of the combination of AD empty vector and TCT1 was inhibited, indicating that AGL9 can bind to the TCT1 promoter.

[0059] Example 5 This example provides a dual luciferase system for verification ​ 9 genes promote the transcriptional activity of CsTCT1, as follows: 5.1 Vector Construction Design primers containing BamHI and KpnⅠ restriction site sequences for amplification ​ 9 full-length, and its amplified fragment was inserted into the pGreenⅡ 62SK vector by homologous recombination to obtain Effector. Primers containing KpnⅠ and BamHI restriction site sequences were designed to amplify the CsTCT1 promoter sequence, and its amplified fragment was inserted into the pGreenⅡ 0800-LUC vector by homologous recombination to obtain Reporter. The specific construction steps were the same as above.

[0060] The primer sequences are as follows: 62SK- ​ 9-F: 5'-CGCTCTAGAACTAGTGGATCCATGGGAAGGGGTAGGGTTGA-3'; 62SK- ​ 9-R: 5'-TCAGCGTACCGAATTGGTACCTTAGCATGGTAGCCATCCTTGC-3'; 0800- ​ 1-F: 5'-CTATAGGGCGAATTGGGTACCATGCAAACGCTGGGATTGTC-3'; 0800- ​ 1-R: 5'-CGCTCTAGAACTAGTGGATCCTAGATAAGTCTAAAACGATTGTTGTTTCAA-3'.

[0061] The constructed recombinant plasmid was transformed into GV3101 competent Agrobacterium using the heat shock method, and transient expression transformation was performed using Nicotiana benthamiana as the plant material. The injection sites of the empty vector and the experimental group were symmetrical on both sides of the vein of the same leaf to ensure the reliability of the experimental results. After the injected tobacco plants were cultured in a growth chamber for 3 days, the same number of leaf discs were taken from the leaves injected with the bacterial solution, ground into a homogenate in 2mL of 0.1mol / L phosphate buffer under ice bath conditions, centrifuged at 10000 r / min for 10 minutes at 4℃, and the supernatant was aspirated and placed on ice for later use. The Dual-Luciferase® Reporter Assay System (Promega, USA) kit was selected to measure the activities of Firefly Lueiferase and Renilla luciferase. The operation method is referred to the instructions as follows: Reagent preparation: (1) Passive Lysis Buffer (PLB): Dilute 5 times the volume of PLB to 1 volume of PLB according to the usage each time; (2) Luciferase Assay Reagent II (Luc II): Add all the Luc substrate to 10 mL LucBuffer, dissolve and mix, then aliquot and store at -80°C for later use. (3) Stop&Glo® Reagent (Stop&Glo): Dissolve 50× Stop&Glo substrate in Stop&Glo Buffer according to the usage amount each time and use it immediately.

[0062] Experimental steps: (4) Add PLB to a 96-well white ELISA plate, 50 μL per well; (5) Take 50 μL of the collected sample supernatant and add it to the enzyme-labeled well containing PLB and let it stand for 10 minutes; (6) Add 50 μL of Luc II reagent to each well, shake gently to mix, and let it react at room temperature in the dark for 10 minutes; (7) Using the i-control software in the microplate reader, the luminescence assay program was used, with the detection time set to 10,000 ms and the pause time set to 1,000 ms, to detect the Luc fluorescence value; (8) Add Stop&Glo reagent to the ELISA plate, 50 μL per well, gently shake to mix, and allow to react for 0 min in the dark; (9) Detect the Ren fluorescence value using the same parameters; (10) Save and analyze the data.

[0063] ​ The results showed that the fluorescence value and luciferase activity of leaves co-transfected with AGL9 and TCT1 promoter were stronger than those of the combination of empty vector and TCT1 promoter, indicating that AGL9 activated TCT1 promoter and positively regulated TCT1 gene expression.

[0064] The above series of studies have proved that through silencing ​ The expression of 9 genes can increase the citric acid content in fruits; overexpression ​ Gene 9 reduces the citric acid content in the fruit.

[0065] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Citrus transcription factor CsAGL The application of 9 genes in regulating the citric acid content in citrus fruits is characterized by: described CsAGL The nucleotide sequence of the 9 gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 CsAGL The application of the protein encoded by gene 9 in regulating the citric acid content in citrus fruits is characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A method for regulating the citric acid content in citrus fruits, characterized in that: In the method: Reducing citric acid content in citrus fruit by overexpression CsAGL 9 genes achieved; Increasing the citric acid content in citrus fruits by silencing CsAGL 9 genes achieved.

4. The method according to claim 3, characterized in that Through overexpression CsAGL The specific steps of obtaining citrus with low citric acid content by 9 genes are as follows: construct an overexpression vector and infect citrus with Agrobacterium to obtain overexpression plants, thereby obtaining citrus with low citric acid content.

5. The method according to claim 4, characterized in that The primers used to construct the overexpression vector are as follows: pENTER1A- CsAGL 9-F:5'-AAAGGAACCAATTCAGTCGACATGGGAAGGGGTAGGGTTGA-3'; pENTER1A- CsAGL 9-R:5'-tggaaaagggaattcggtaccGCATGGTAGCCATCCTTGCAT-3'。 6. The method according to claim 3, characterized in that Through silence CsAGL The specific steps of obtaining citrus with high citric acid content by 9 genes are as follows: construct a silencing vector and infect citrus with Agrobacterium to obtain mutant plants, thereby obtaining citrus with high citric acid content.

7. The method according to claim 6, characterized in that The primers used to construct the silencing vector are as follows: GW- CsAGL 9-F:5'-GGGGACAAGTTTGTACAAAAAGCAGGCTTCATGGGAAGGGGTAGGGTTGA-3'; GW- CsAGL 9-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCGGACACATTCGGTTCTGGT-3’。 8. A gene expression cassette, characterized in that The gene expression cassette comprises the gene expression cassette according to claim 1 CsAGL 9 genes.

9. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene expression cassette according to claim 8.

10. An engineered bacterium, characterized in that: The engineered bacteria includes the recombinant expression vector described in claim 9.

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