Application of CitCesA8 gene in regulation and control of biosynthesis of citrus juice sac cellulose

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to application of a CitCesA8 gene in regulation and control of citrus juice sac cellulose biosynthesis. After grapefruit juice sacs are treated at different temperatures, the CitCesA8 gene expression level and the cellulose content of the juice sacs show an obvious positive correlation trend. The content of cellulose in leaves can be effectively increased by overexpressing the gene in the leaves of the Huyou. By constructing the recombinant plasmid pBI121-CitCesA8, the stable overexpression of the CitCesA8 gene in the arabidopsis thaliana is realized, and the cellulose content and spatial distribution of the arabidopsis thaliana stalk are obviously increased. The results show that the CitCesA8 gene can be used for regulating and controlling the cellulose content of citrus juice sacs and the occurrence of dry water, can also be applied to breeding citrus varieties with low juice sac cellulose content and good slag melting property in molecular marker-assisted breeding, and has wide application prospects in fresh keeping and quality improvement of picked citrus fruits.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically involving the application of the CitCesA8 gene in regulating cellulose biosynthesis in citrus juice sacs. Background Technology

[0002] Citrus fruits, belonging to the Rutaceae family and the Citrus genus, are the world's largest fruit production. Citrus fruits have a unique flavor, rich in soluble sugars, citric acid, ascorbic acid, and other nutrients and physiologically active substances, making them highly popular with consumers. However, citrus fruits are susceptible to various diseases during the later stages of growth and post-harvest storage, such as water loss disease, anthracnose, Penicillium mold, and sour rot. Post-harvest diseases account for more than 30% of total citrus production losses annually, making it a primary factor affecting the sustainable and healthy development of the citrus industry. Water loss disease, in particular, is a unique physiological disease of citrus, often occurring during fruit ripening and post-harvest storage. It significantly reduces the juice content, nutrients, and aroma components of citrus fruits, severely impacting their edible quality and commercial value. Citrus water loss is an extremely complex physiological process. Current research focuses primarily on physiological and biochemical aspects, metabolism, and histological morphology. The causes and specific regulatory mechanisms of water loss remain unclear, and effective measures to delay water loss are still lacking. Therefore, clarifying the mechanism of citrus dehydration is crucial for mitigating postharvest dehydration and improving fruit quality.

[0003] Secondary cell wall formation and thickening of citrus juice sacs are closely related to the occurrence of drought. Studies have shown that the morphological structure of granulated juice sacs in 'Guanxi Honey Pomelo' is significantly different from that of normal juice sacs, with thickened cell walls and a significant increase in the distribution of cellulose and hemicellulose. Similar phenomena have been observed in mandarin oranges; compared to normal juice sacs, drought-stricken juice sacs of Satsuma mandarins have higher contents of alcohol-insoluble matter, pectin, cellulose, and lignin. Research has found that the content of structural polysaccharides, such as pectin, cellulose, and hemicellulose, in drought-stricken juice sacs of Maxu grapefruit is more than twice that in normal juice sacs. This is because the activity of cell wall degrading enzymes such as polygalacturonase and cellulase decreases during drought, while the activity of key enzymes in the lignin synthesis pathway, PAL, CAD, and POD, is upregulated, leading to a significant accumulation of cell wall components. Previous studies on drought have focused on lignin metabolism; however, key genes involved in regulating cellulose synthesis in citrus have not yet been reported. Therefore, identifying key genes involved in citrus cellulose synthesis is of great significance for elucidating the mechanism of citrus drought and mitigating its occurrence. By targeting and regulating the gene expression of cellulose synthase, the synthesis and metabolism of cellulose in fruits can be controlled, thereby achieving precise improvement of fruit texture and storage quality. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the CitCesA8 gene in regulating cellulose biosynthesis in citrus juice sacs. This invention demonstrates that the CitCesA8 gene can be used to regulate cellulose content and water loss in citrus juice sacs, and can also be applied in marker-assisted breeding to select citrus varieties with low cellulose content and good pulpiness. It has broad application prospects in postharvest preservation and quality improvement of citrus fruits.

[0005] This invention provides the application of the CitCesA8 gene in regulating cellulose biosynthesis in citrus juice sacs, and the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0006] As a preferred embodiment, the regulation includes overexpressing the CitCesA8 gene to promote citrus juice sac cellulose biosynthesis; and knocking out or silencing the CitCesA8 gene to reduce citrus juice sac cellulose biosynthesis.

[0007] This invention also provides primer pairs for amplifying the CitCesA8 gene, the primer pairs comprising a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO:2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3;

[0008] The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0009] The present invention also provides a biological material for regulating the expression level of the CitCesA8 gene, including biological material overexpressing the CitCesA8 gene and biological material knocking out or silencing the CitCesA8 gene;

[0010] The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0011] As a preferred embodiment, the biological material overexpressing the CitCesA8 gene includes one or more of the following: a recombinant expression vector containing the CitCesA8 gene, a recombinant microorganism containing the CitCesA8 gene, and a recombinant microorganism containing the recombinant expression vector.

[0012] As a preferred embodiment, the biological material for knocking out or silencing the CitCesA8 gene includes one or more of the following: a knockout vector containing the CitCesA8 gene knockout target, a silencing vector containing the CitCesA8 gene silencing target, and a recombinant microorganism containing the knockout vector or the silencing vector.

[0013] This invention also provides the application of the primer pairs or the biomaterials described herein in regulating citrus juice cell cellulose biosynthesis and / or creating new germplasm.

[0014] The present invention also provides a method for reducing citrus juice sac cellulose biosynthesis by knocking down or removing the CitCesA8 gene in the target citrus genome to obtain citrus with reduced juice sac cellulose biosynthesis.

[0015] The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0016] The present invention also provides a citrus model for creating increased juice sac cellulose content by overexpressing the CitCesA8 gene in target citrus fruits to obtain citrus fruits with increased juice sac cellulose biomass; the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0017] This invention also provides the application of the CitCesA8 gene in regulating the occurrence of citrus waterlogging, wherein the regulation includes overexpressing the CitCesA8 gene to promote the occurrence of citrus waterlogging; and knocking out or silencing the CitCesA8 gene to reduce the occurrence of citrus waterlogging.

[0018] The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0019] Beneficial Effects: This invention provides the application of the CitCesA8 gene in regulating cellulose biosynthesis in citrus juice sacs. After treating pomelo juice sacs with different temperatures, this invention found a significant positive correlation between the expression level of the CitCesA8 gene and the cellulose content of the juice sacs. Overexpression of the gene in pomelo leaves effectively increased the cellulose content in the leaves. By constructing the recombinant plasmid pBI121-CitCesA8, stable overexpression of the CitCesA8 gene was achieved in Arabidopsis thaliana, significantly increasing the cellulose content and spatial distribution in Arabidopsis stems. These results indicate that the CitCesA8 gene can be used to regulate cellulose content and waterlogging in citrus juice sacs, and can also be applied in marker-assisted breeding to select citrus varieties with low cellulose content and good pulpiness. It has broad application prospects in postharvest preservation and quality improvement of citrus fruits.

[0020] This invention demonstrates through experiments that the CitCesA8 gene is a key gene involved in citrus cellulose synthesis. The discovery of the CitCesA8 gene is of great significance for elucidating the mechanism of citrus drought and mitigating its occurrence. By targeting and regulating the expression of the CitCesA8 gene, the synthesis and metabolism of cellulose in the fruit can be regulated, thereby achieving precise improvement in fruit texture and storage quality. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 Phylogenetic analysis of the cellulose synthase CesA8 protein in citrus and other species in Example 1, where red text indicates candidate CesA8 proteins in citrus, blue text indicates CesA8 proteins in Arabidopsis thaliana, and the gene number encoding the protein is in parentheses;

[0023] Figure 2 Subcellular localization of CitCesA8 in nuclear-localized tobacco in Example 2, scale bar = 50 μm;

[0024] Figure 3 This is a schematic diagram of the grapefruit juice cell culture and temperature treatment in Example 3;

[0025] Figure 4 This section describes the changes in cellulose metabolism during the high-temperature-induced dehydration process of citrus juice sacs in Example 3. A represents toluidine blue staining and Karl von Fluhr staining of juice sac cellulose; scale bar = 200 μm; B represents the determination of juice sac cellulose content; C represents the detection of CitCesA8 gene expression. Data are expressed as mean ± standard error (n = 3). Student's t-test was used to analyze the significance of differences between groups at a confidence level of 95% (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0026] Figure 5 Transient overexpression of the CitCesA8 gene in pomelo leaves in Example 4; where A is GUS staining of leaves after injection, scale bar = 500 μm; B and C are the relative expression levels and cellulose content of CitCesA8 after transient overexpression. Data are expressed as mean ± standard error (n = 6). Student's t-test was used to analyze the significance of differences between groups, with a confidence level of 95% (*, P < 0.05; **, P < 0.01).

[0027] Figure 6The effect of heterologous overexpression of CitCesA8 on cellulose synthesis in Arabidopsis thaliana plants in Example 5 is shown in Figure 5. A represents the identification of stably overexpressing CitCesA8 lines; B represents the relative expression levels of CitCesA8 and AtCesA8 after stable overexpression (n=5), with ND indicating no detection; C represents the cellulose content determination of WT and overexpressing plants (n=5); D represents toluidine blue and Carcorflul staining of paraffin sections of Arabidopsis thaliana stems (scale bar = 100 μm); E represents the average gray value of the cell wall fluorescent label (n=12). Student's t-test was used to analyze the significant differences between each group and WT (*, P<0.05; ***, P<0.001). Detailed Implementation

[0028]

[0029] As one specific implementation, the amino acid sequence of the CitCesA8 protein is as shown in SEQ ID.As shown in NO:4: MMQSGAPVCNTCGDQVGFTANGEVFVACSECNFPICKSCFDDEIKEGRKACLRCASPYDENLLDDVGTKESGNRSTVAAQLSNSENTGIHARHVSNVSTVDSEYNDESGNPIWKNRVESWKDKKNKKKKTAAKAEKEAEVPPAQQMEENQQSPEAALPLSTLIPVPRSKLGPYRTVIIVRLIILGLFFHYRVTHPVDSALGLWLTSVICEIWFAFSWVLDQFPKWSPVDRETYIDRLSARFEREGEPSELAAVDFFVSTVDPLKEPPLITANTVLSILALDYPVDKVSCYVSDDGAAMLTFETLVQTADFARKWVPFCKKFSIEPRAPEFYFSQKIDYLKDKIQPSFVKERRAMKRDYEEYKVRINALVAKAQKTPEEGWTMQDGTSWPGNNTRDHPGMIQVFLGHSGACDIEGNELPRLVYVSREKRPGYQHHKKAGAENALVRVSAVLTNAPYILNLDCDHYVNNSKAVREAMCFMMDPQVGRDVCYVQFPQRFDGIDKSDRYANRNIVFFDVNMKGLDGIQGPMYVGTGCMFNRQALYGYGPPTMPTLPKTSSSCTWCGCCSCCCPSKKPSKDLSEAYRDAKREELDAAIFNLKEIDNYDDYERSMLISQMSFEKTFGLSSVFIESTLMENGGVPDSANPSTLIKEAIHVISCGYEEKTEWGKEIGWIYGSVTEDILTGFKMHCRGWRSLYCMPLRPAFKGSAPINLSDRLHQVLRWALGSVEIFLSRHCPLWYGFGGGRLKLLQRLAYINTIVYPFTSLPLIAYCSLPAICLLTGKFIIPTLSNLASVLFLGLFLSIIATSVLELRWSGVTIEDLWRNEQFWVIGGVSAHLFAVFQGFLKMLAGLDTNFTVTSKAADDLEFGELYIIKWTTLLIPPTSLIIVNMVGVVAGFSDALNKGYEAWGPLFGKVFFAFWVIVHLYPFLKGLMGRQNRTPTIVVLWSVLLASVFSLVWVKIDPFVEKTNSATLGQTCISIDC。

[0030] In one specific implementation, the regulation includes overexpressing the CitCesA8 gene to promote cellulose biosynthesis in citrus juice sacs; and knocking out or silencing the CitCesA8 gene to reduce cellulose biosynthesis in citrus juice sacs. In a specific embodiment of the present invention, by transiently overexpressing the CitCesA8 gene in pomelo leaves, it was found that the cellulose content in the EV control was 82.04 mg / g FW, while the cellulose content in the leaves after overexpressing CitCesA8 was 129.92 mg / g FW, an upregulation of 0.58 times compared to the EV control, proving that CitCesA8 is a cellulose synthase gene in citrus.

[0031] The present invention also provides primer pairs for amplifying the CitCesA8 gene, the primer pairs comprising a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO:2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3; and the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0032] The primer pairs used in this invention to amplify the CitCesA8 gene are shown below:

[0033] Forward primer (SEQ ID NO:2): 5'-ATGATGCAATCTGGGGCTCCTGTAT-3';

[0034] Reverse primer (SEQ ID NO:3): 5'-GCAATCTATGGAGATGCAGGTTTGG-3'.

[0035] The present invention also provides a biological material for regulating the expression level of the CitCesA8 gene, including biological material overexpressing the CitCesA8 gene and biological material knocking out or silencing the CitCesA8 gene; the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0036] In one specific embodiment, the biological material overexpressing the CitCesA8 gene includes one or more of the following: a recombinant expression vector containing the CitCesA8 gene, a recombinant microorganism containing the CitCesA8 gene, and a recombinant microorganism containing the recombinant expression vector. In one specific embodiment, the recombinant expression vector containing the CitCesA8 gene comprises inserting the CitCesA8 gene into the pBI121 vector to obtain the pBI121-CitCesA8 overexpression vector. In one specific embodiment, the recombinant microorganism containing the CitCesA8 gene includes Agrobacterium GV3101 containing the CitCesA8 gene.

[0037] As one specific implementation, the biological material for knocking out or silencing the CitCesA8 gene includes one or more of the following: a knockout vector containing the CitCesA8 gene knockout target, a silencing vector containing the CitCesA8 gene silencing target, and a recombinant microorganism containing the knockout vector or the silencing vector.

[0038] This invention also provides the application of the primer pairs or the biomaterials described herein in regulating citrus juice cell cellulose biosynthesis and / or creating new germplasm.

[0039] This invention also provides a method for reducing cellulose biosynthesis in citrus juice sacs by knocking down or removing the CitCesA8 gene from the genome of a target citrus fruit to obtain citrus fruit with reduced cellulose biosynthesis in juice sacs; the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

[0040] This invention also provides a citrus model for increasing sap cellulose content. The CitCesA8 gene is overexpressed in target citrus fruits to obtain citrus fruits with increased sap cellulose content. The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1. In a specific embodiment, the pBI121-CitCesA8 overexpression vector is transformed into Agrobacterium GV3101 strain, and then the overexpression vector is transformed into target citrus fruits using an Agrobacterium-mediated transgenic method to obtain citrus fruits with increased sap cellulose content.

[0041] In a specific embodiment of the present invention, heterologous overexpression of the CitCesA8 gene in Arabidopsis thaliana plants revealed that the cellulose contents of Line 3 and Line 6 in the overexpressing plants were 37.00 mg / g FW and 38.32 mg / g FW, respectively, both higher than those in the WT line (31.61 mg / g FW). The content and distribution of cellulose marked in purple in the CitCesA8 overexpressing lines were significantly increased compared to the WT line; the intensity of the blue fluorescence signal marked by Karl Fischer in the CitCesA8 overexpressing lines was also significantly higher than that in the WT line. These results indicate that heterologous overexpression of CitCesA8 significantly increased the cellulose content and distribution in Arabidopsis thaliana plants, suggesting that the CitCesA8 gene is a key gene involved in citrus cellulose synthesis.

[0042] This invention also provides the application of the CitCesA8 gene in regulating citrus dehydration, wherein the regulation includes overexpressing the CitCesA8 gene to promote citrus dehydration; and knocking out or silencing the CitCesA8 gene to reduce citrus dehydration. The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1. In a specific embodiment of this invention, by analyzing the changes in CesA8 gene expression and cellulose content during the dehydration process of pomelo juice cells, it was found that in normal juice cells (treated at 15℃), the cellulose components labeled with toluidine blue and Karl Kovrull were concentrated on the outer wall of the juice cells; while in dehydrated juice cells (treated at 35℃), the accumulation of cellulose components inside the juice cells increased rapidly. 49 days after treatment, the cellulose content in the 35℃ group was 11.5 times that of the 15℃ control group. Compared with the 15℃ control group, the expression level of the CitCesA8 gene was significantly upregulated in the 35℃ treatment group. The results showed that during the dry season, the gene expression level of CitCesA8 was significantly positively correlated with the cellulose content in juice cells.

[0043] To further illustrate the present invention, the application of the CitCesA8 gene provided by the present invention in regulating citrus juice sac cellulose biosynthesis is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0044] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0045] Example 1: Cloning and Evolutionary Analysis of the CitCesA8 Gene

[0046] (I) Experimental Methods

[0047] Based on the reference sequence of the CitCesA8 gene obtained from the Citrus pan-genome database (http: / / www.citrusgenomedb.org / ), gene-specific primer pairs were designed. The upstream primer is shown in SEQ ID NO:2: 5'-ATGATGCAATCTGGGGCTCCTGTAT-3', and the downstream primer is shown in SEQ ID NO:3: 5'-GCAATCTATGGAGATGCAGGTTTGG-3'. PCR amplification was performed using pomelo fruit cDNA as a template. The PCR reaction volume was 50 μL, including 25 μL of 2×Phanta Max Master Mix (Vizan, China), 2 μL each of the upstream and downstream primers (10 μM), 1 μL of cDNA template, and 20 μL of DEPC H2O. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, 37 cycles; 72℃ for 5 min, 4℃ hold. After agarose gel electrophoresis analysis and PCR product recovery and purification, it was ligated into the target vector pEASY[ The pEASY-CitCesA8 recombinant plasmid was obtained by heat shock transfection into competent E. coli DH5α cells using the Blunt Cloning Kit (Chemical, China); catalog number CB101-01). Positive colonies were picked and sequenced for verification.

[0048] (II) Experimental Results

[0049] 1. The CDS sequence of the CitCesA8 gene, which matches the Citrus genome database, was obtained by sequencing verification. It is shown in SEQ ID NO:1 and contains 2943 nucleotides, encoding a protein of 980 amino acids, as shown in SEQ ID NO:4.

[0050] 2. Multiple sequence alignment was performed between SEQ ID NO:1 and the amino acid sequence of the CesA8 gene, which has been reported to have functions related to cellulose synthesis. The results are as follows: Figure 1 As shown, CitCesA8 clusters closely with PpCesA8 in peaches and PbrCesA8a / b in pears, providing preliminary evidence that CitCesA8 is involved in cellulose synthesis in citrus.

[0051] Example 2: Subcellular localization analysis of the CitCesA8 gene

[0052] (I) Experimental Methods

[0053] 1. The cDNA-specific fragment of the CitCesA8 gene obtained in Example 1 (with the terminator removed) was amplified using primer sequences with BamHI restriction sites [upstream primer: 5'-CGGTACCCGGGGATCCATGATGCAATCTGGGGCTCC-3' (SEQ ID NO: 5); downstream primer: 5'-CGA CTCTAGAGGATCCGCAATCTATGGAGATGCAGGTTTGG-3' (SEQ ID NO: 6)]. The CDS sequence of the CitCesA8 gene was ligated into the pCAMBIA1300-eGFP vector using the ClonExpress II One Step Cloning Kit homologous recombination ligase (Vizan, China). (Zhao Chenning, Liu Xiaojuan, Gong Qin, Cao Jinping, Shen Wanxia, ​​Yin Xueren, Grieson Donald, Zhang Bo, Xu Changjie, Li Xian, Chen Kunsong, Sun Chongde*. Three AP2 / ERF family members modulate flavonoid synthesis by regulating type IVchalcone isomerase in citrus. Plant Biotechnology Journal. 2020, DOI:10.1111 / pbi.13494.)

[0054] 2. The recombinant plasmids that were correctly sequenced were transformed into GV3101 Agrobacterium competent cells (Weidi, China) by heat shock method, plated on LB solid medium containing 50 mg / L kanamycin and 25 mg / L gentamicin, and the correct positive clones were picked, amplified and shaken, and then glycerol was added and stored at -80℃.

[0055] 3. Subcellular localization analysis was performed using transgenic *Nicotiana benthamiana* with nucleus-localized mCherry (Zhao Chenning, Liu Xiaojuan, Gong Qin, Cao Jinping, Shen Wanxia, ​​Yin Xueren, Grieson Donald, Zhang Bo, Xu Changjie, Li Xian, Chen Kunsong, Sun Chongde*. Three AP2 / ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase incitrus. Plant Biotechnology Journal. 2020, DOI:10.1111 / pbi.13494.). After two activations by inoculating the preserved *Agrobacterium* culture onto LB solid medium, the cells were scraped and resuspended in MES osmotic buffer (10 mM MES, 10 mM MgCl2, 150 μM MAS, pH 5.6) and adjusted to OD500. 600 The 1.0 gene was injected into 4-week-old tobacco leaves. The pCAMBIA1300-eGFP empty vector was used as a control. The subcellular localization of the gene was observed and photographed using an LSM780 confocal microscope (Zeiss, Germany) 48 h after injection.

[0056] (II) Experimental Results

[0057] like Figure 2 As shown, the distribution of the empty-carrying eGFP fluorescent protein in the cytoplasm is non-targeted, while the fluorescent protein carried by CitCesA8 is mainly distributed in the cytoplasm. This is consistent with the previous reports on the localization of cellulose synthase gene family members in other species, indicating that CitCesA8 may function as a cellulose synthase in citrus.

[0058] Example 3: Analysis of changes in CesA8 gene expression and cellulose content during the dehydration process of pomelo juice sacs

[0059] (I) Experimental Methods

[0060] 1. Construction of the drought model: Grapefruit was used as the experimental material, collected from Changshan County, Quzhou City, Zhejiang Province. Grapefruit with a diameter of 90-100 mm, uniform maturity, and free from pests, diseases, and mechanical damage were selected for in vitro juice cell culture. Fruit disinfection procedures: After rinsing the fruit surface with clean water, the fruit was immersed in 75% ethanol and 1% sodium hypochlorite solution for 10 min and 30 min respectively for disinfection. The fruit was cut open in a clean bench, and the outer peel and membranes were removed. The fruit was then placed in a sealed bottle containing MT medium. The MT medium was prepared as follows: 1 L of medium contained 80 g sucrose, 7.5 g agar powder, and 4.43 g Murashige & Tucker powder (Phytotech, USA), pH 5.8. The experiment set up a control group at 15℃ and a treatment group at 35℃. The fruits were placed in a constant temperature and humidity incubator (Yiheng, Shanghai) in the dark. Samples were taken at 7, 14, 21, 35, and 49 days after treatment. The treatment process is as follows. Figure 3 As shown in the figure. Each group had 3 biological replicates, and each replicate included at least 5 flasks of juice cells. After sampling, the samples were flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0061] 2. Determination of cellulose content in juice sacs:

[0062] (1) Place the juice cells in liquid nitrogen and crush them. Weigh 1g of juice cell powder into a 50mL centrifuge tube, add 25mL of 95% ethanol, heat in a 90℃ water bath for 30min, cool, centrifuge at 3,000rpm for 5min and discard the supernatant. Repeat this extraction step 3 times. Place the precipitate in a 65℃ oven and dry it to constant weight.

[0063] (2) Take 100mg of precipitate, add 15mL ddH2O, keep warm in a 50℃ water bath for 30min, and repeat the extraction 3 times to remove the original pectin.

[0064] (3) Add 25 mL of 0.5 mol / L sulfuric acid to the above precipitate, heat in a water bath at 85°C for 10 min, cool and centrifuge at 3,000 rpm for 5 min to remove the original pectin.

[0065] (4) Add 10 mL of 4 mM NaOH (containing 100 mM NaBH4) to the precipitate extracted in the previous step, and extract with ultrasonic assistance for 6 h. Then centrifuge at 10,000 rpm for 10 min to remove hemicellulose. This precipitate is the cellulose substance.

[0066] (5) Add 3 mL of 80% sulfuric acid solution to the precipitate, incubate on ice for 2 h, then add 6 mL of ddH2O, hydrolyze in a boiling water bath at 100℃ for 5 h, take 100 μL and add 3 mL of 0.25% anthrone-sulfuric acid solution, incubate in a water bath at 90℃ for 15 min, cool and measure the OD value at 620 nm, use D-glucose solution as a standard curve for quantification, and express the result as mg / g DW.

[0067] 3. Immunofluorescence labeling of cytocellulose in juice cells:

[0068] (1) Preparation of paraffin wax blocks:

[0069] ① Sampling and fixation: Fresh juice cells were soaked in FAA fixative (containing 45% ethanol) for 48 hours, the top of the juice cells was cut off, and they were placed in an embedding cassette;

[0070] ② Dehydration: Dehydrate the sacs using a gradient of ethanol: 75% ethanol for 1 h → 80% ethanol for 1 h → 90% ethanol for 1 h → 95% ethanol for 1 h → anhydrous ethanol for 1 h → anhydrous ethanol overnight;

[0071] ③Transparency: Place the juice cells in xylene solution at room temperature for 30 minutes, repeat 3 times;

[0072] ④ Degassing: Place the crushed wax in a xylene solution and allow it to dissolve naturally in a 37°C oven to obtain a saturated paraffin-xylene solution. Place the embedding box in the saturated paraffin-xylene solution, put it into a vacuum bottle, and evacuate the air. Gently tap the bottle wall to help the air bubbles escape. After evacuation, place it in a 37°C oven overnight.

[0073] ⑤Wax impregnation: Transfer the vacuum bottle to a 62℃ oven and degas it again. After 30 minutes, place the embedding box in 62℃ paraffin wax for 4 hours.

[0074] ⑥ Embedding: Select well-shaped cells during embedding, remove air bubbles, fix the bottom of the cells to the bottom of the wax box after waxing, cover with the embedding box, and place on a cold table to solidify for 30 minutes.

[0075] (2) Paraffin sectioning and dewaxing:

[0076] ① Paraffin sections: Sections were prepared using a semi-automatic rotary microtome (Leica, Germany), with a section thickness of 5μm and a spreading temperature of 49℃. The sections were then placed on adhesive glass slides (CITOTEST, Jiangsu) and baked in an oven at 62℃ for 12 hours.

[0077] ② Dewaxing: Dewax the paraffin sections three times in xylene solution, 5 minutes each time; wash them in sequence with anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol and distilled water, 2 minutes each time.

[0078] (3) In situ staining of cell wall:

[0079] ① Toluidine blue staining: Toluidine blue was used to label cellulose and lignin in the tissue, resulting in purple and blue staining, respectively. A 0.1% toluidine blue staining solution (Solarbio, Beijing) was prepared. The sections were placed in a staining jar and allowed to stand at room temperature for 5 minutes. They were then washed three times with distilled water for 5 minutes each time, allowed to dry slightly, and then mounted with neutral resin. The sections were observed and photographed using a VS200 digital slide scanner (Olympus, Japan).

[0080] ②Carl Cofluell fluorescence staining: Dilute Carl Cofluell staining solution (Coolaber, Beijing) 5 times and add it to the tissue sample. Then add 100 μL of 10% potassium hydroxide solution. After incubation in the dark for 5 min, wash three times with 1×PBS buffer (pH 7.4) for 5 min each time. After drying slightly, cover with a coverslip and observe and photograph using a digital slide scanner. Channel DAPI, excitation / emission wavelength is 405 nm / 454-488 nm.

[0081] 4. RNA extraction, cDNA synthesis, and gene expression analysis:

[0082] (1) RNA extraction: Total RNA was extracted from juice cells using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing). The sample volume was approximately 200 mg. The total RNA concentration was determined by NanoDrop (ThermoFisher Scientific, USA), and the RNA quality was identified using an Agilent 2100 bioanalyzer (Agilent Technology, USA).

[0083] (2) cDNA synthesis: Following the instructions of the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing) reverse transcription kit, 1 μg of total RNA was used to remove gDNA and then reverse transcribed to synthesize cDNA.

[0084] (3) Gene expression analysis: RT-qPCR primers were designed using NCBI / Primer-BLAST online software (https: / / www.ncbi.nlm.nih.gov / ), with CitACTIN as the internal reference gene. The primers are shown in SEQ ID NO:7: 5'-CATCCCTCAGCACCTTCC-3' and SEQ ID NO:8: 5'-CCAA CCTTAGCACTTCTCC-3'. The CitCesA8 primers are shown in SEQ ID NO:9: 5'-GTTTTCC TTGGACACAGCGG-3' and SEQ ID NO:10: 5'-GCAGATACCCTCACCAGA GC-3'. The primer specificity was verified by melting curve analysis and RT-qPCR product sequencing. RT-qPCR was performed in a 20 μL system: 10 μL ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing), 0.6 μL each of forward and reverse primers (10 μM), 2 μL cDNA, and DEPC-treated... 6.8 μL of H2O. The reaction program was 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles; 95℃ for 10 s, with the melting curve increasing by 0.5℃ every 5 s from 65℃ to 95℃. RT-qPCR was performed using a CFX96Touch real-time quantitative PCR instrument (BIO-RAD, USA), employing 2... -ΔCt The relative expression level of CitCesA8 in citrus juice cells was calculated.

[0085] (II) Experimental Results

[0086] like Figure 4 As shown in Tables 1 and 2, in normal juice cells (treated at 15℃), the cellulose components labeled with toluidine blue and Karl von Fluhr were concentrated on the outer wall of the juice cells; while in the dehydrated juice cells (treated at 35℃), the accumulation of cellulose components inside the juice cells increased rapidly. At 49 days after treatment, the cellulose content in the 35℃ group was 11.5 times that of the 15℃ control group. Compared with the 15℃ control group, the expression level of the CitCesA8 gene was significantly upregulated in the 35℃ treatment group. These results indicate that during the dehydrated process, the gene expression level of CitCesA8 is significantly positively correlated with the cellulose content in juice cells.

[0087] Table 1 Statistical results of cellulose content

[0088]

[0089] Table 2 Relative expression levels of CitCesA8

[0090] CitCesA8 relative expression level 0d 7d 14d 21d 15℃ treatment 1.00±0.50 0.54±0.30 0.47±0.17 0.40±0.24 35℃ treatment 1.00±0.50 12.33±1.15 48.57±13.44 83.23±33.36

[0091] Example 4: Functional verification of transient overexpression of CitCesA8 in pomelo leaves

[0092] (I) Experimental Methods

[0093] 1. Verification of transient overexpression in leaf blades:

[0094] (1) Expression vector construction: The cDNA specific fragment of the CitCesA8 gene obtained in Example 1 (with the terminator removed) was amplified using primer sequences with BamHI restriction sites [upstream primer: 5'-GGACTCTAGAGGATCCATGATGCAATCTGGGGCTCC-3' (SEQ ID NO:11); downstream primer: 5'-GACCACCCGGGGATCCGCAATCTATGGAGATGCAGGTTTGG-3' (SEQ ID NO:12)]. The CDS sequence of the CitCesA8 gene was constructed into the pBI121 vector using the ClonExpress II One Step Cloning Kit homologous recombination ligase (Vizan, China). (Zheng Juan, Zhao Chenning, Liao Zhenkun, Liu Xiaojuan, Gong Qin, Zhou Chenwen, Liu Yilong, Wang Yue, Cao Jinping, Liu Lili, Wang Dengliang, Sun Chongde*. Functional characterization of two flavone synthase II members in citrus. Horticulture Research, 2023, 10: uhad113.) and sequenced to obtain the pBI121-CitCe sA8 recombinant plasmid.

[0095] The instantaneous method is as follows:

[0096] The pBI121-CitCesA8 recombinant plasmid and the pBI121 empty vector plasmid were heat-shocked and transformed into Agrobacterium EHA105 competent cells (WEIDI, Shanghai). A transient overexpression experiment was conducted on citrus leaves using 4-month-old pomelo seedlings. After activating the Agrobacterium carrying the recombinant plasmid twice, the bacteria were scraped and suspended in MES osmotic buffer and adjusted to OD0.05. 600 =0.75. After puncturing the leaves with a needle, the bacterial suspension containing the target gene and the empty vector were injected into the opposite leaves on both sides of the plant, respectively. After injection, the plants were placed in the dark for 1 day and then cultured normally. Samples were taken after 6 days to detect gene expression and substance content.

[0097] 2. GUS Staining Analysis: GUS tag expression analysis was performed on citrus leaves 6 days after transient transformation using a GUS staining kit (Coolaber, Beijing). The GUS staining solution was prepared according to the instructions. The leaves were placed in a staining jar and vacuum-treated. After incubation at 35℃ in the dark for 12 hours, the leaves were destained with 70% ethanol until the negative control turned white. The leaves were then observed and photographed using a SteREO Discovery.V8 stereomicroscope (Zeiss, Germany).

[0098] 3. Cellulose content determination: Same as in Example 3.

[0099] 4. RNA extraction, cDNA synthesis and gene expression analysis: Same as in Example 3.

[0100] (II) Experimental Results

[0101] like Figure 5 As shown, GUS staining was used to detect gene expression, and the results showed that the GUS tag was normally expressed in all overexpressing leaves. RT-qPCR results showed that the expression level of CitCesA8 in overexpressing leaves increased by 7.35 times compared to the EV control. Further analysis of leaf cellulose content showed that the cellulose content in the EV control was 82.04 mg / g FW, while the cellulose content in leaves overexpressing CitCesA8 was 129.92 mg / g FW, an upregulation of 0.58 times compared to the EV control. Combined with phylogenetic analysis and subcellular localization results, it was confirmed that CitCesA8 is a cellulose synthase gene in citrus.

[0102] Example 5: Functional Validation of Heterologous Overexpression of CitCesA8 in Arabidopsis thaliana Plants

[0103] (I) Experimental Methods

[0104] 1. Construction of expression vector: Same as in Example 4.

[0105] 2. Plant genetic transformation: Wild-type Arabidopsis thaliana Col-0 was genetically transformed using the flower immersion method to verify the function of the candidate gene CitCesA8. The constructed pBI121-CitCesA8 recombinant plasmid was transformed into Agrobacterium GV3101 strain (WEIDI, Shanghai). Positive clones were selected and cultured in LB liquid medium containing 50 mg / L Kan and 25 mg / L Get until OD. 600 =1.0, after centrifugation to collect bacterial cells, resuspend them in 1 / 2 MS liquid medium (containing 5% sucrose and 0.02% Silwet-L77), and set the OD value at 1.0. 600 Adjust the concentration to 0.6, repeat the infection of Arabidopsis inflorescences twice (one week apart), collect the mature pods and dry them at 37℃ to obtain T0 generation seeds.

[0106] 3. Screening and identification of positive lines: T0 generation seeds were sown in 1 / 2 MS medium containing 50 mg / L Kan and 50 mg / L Meropenem (MEM) for screening and identification by PCR to obtain T1 generation positive plants. After harvesting, the seeds were sown again in 1 / 2 MS screening medium, and T2 generation positive plants were obtained after PCR identification. After 5 weeks of soil culture, samples were taken for gene expression analysis and substance content determination. The PCR identification primers are shown in SEQ ID NO:13: 5'-AAGCAAGTGGATTGATGTGA-3' and SEQ ID NO:14: 5'-TCTTTTCATGGCCCTGCGCTCT-3', where the forward primer was taken from the vector fragment and the reverse primer was taken from the gene fragment. The PCR product length was approximately 1000 bp.

[0107] 4. Determination of plant cellulose content and in situ staining analysis: Same as in Example 3, ImageJ software (National Institutes of Health, USA) was used to perform quantitative analysis of the fluorescent labeling results: First, the image was converted to 8-bit format, the measurement area was selected and the threshold range was adjusted to 12-255, and the fluorescence intensity of the area was measured by measuring the mean gray value of the area, with the unit being Arbitrary Units (AU).

[0108] 5. RNA extraction, cDNA synthesis and gene expression analysis: Same as in Example 3, using AtEF1α as the internal reference gene, primers as shown in SEQ ID NO:15: 5'-TGAGCACGCTCTTCTTGCTTTCA-3' and SEQ ID NO:16: 5'-GGTGGTGGCATCCATCTTGTTACA-3', AtCesA8 primers as shown in SEQ ID NO:17: 5'-GCTTTCGGTTGTGATTCCTATC-3' and SEQ ID NO:18: 5'-CACAGATGTTAGCCATAAACCG-3', and CitCesA8 primers as in Example 3.

[0109] (II) Experimental Results

[0110] The results are as follows Figure 6As shown in Table 3, PCR-specific band verification was performed, and specific bands were detected in all overexpression lines. RT-qPCR results showed that CitCesA8 transcripts were detected in all CitCesA8 overexpression lines, but not in the WT line. Simultaneously, the expression level of endogenous AtCesA8 in Arabidopsis was not significantly affected, confirming the successful construction of stable CitCesA8 overexpression lines. Cellulose content was measured, and the results showed that the cellulose content of Line 3 and Line 6 were 37.00 mg / g FW and 38.32 mg / g FW, respectively, both higher than that of the WT line (31.61 mg / g FW). Further preparation of Arabidopsis stem paraffin sections and labeling of cellulose components with toluidine blue showed that the content and distribution of purple-labeled cellulose in the CitCesA8 overexpression lines were significantly increased compared to the WT line; similarly, the intensity of the blue fluorescence signal labeled with Calcoflull in the CitCesA8 overexpression lines was significantly higher than that in the WT line (P<0.001). The above results indicate that heterologous overexpression of CitCesA8 significantly increased the cellulose content and distribution in Arabidopsis plants.

[0111] Table 3. Statistical results of average gray values

[0112]

[0113] Therefore, this invention demonstrates a significant positive correlation between the expression level of the CitCesA8 gene and the cellulose content of citrus juice sacs after treatment with different temperatures. Overexpression of this gene in citrus leaves effectively increases the cellulose content in the leaves. By constructing the recombinant plasmid pBI121-CitCesA8, stable overexpression of the CitCesA8 gene was achieved in Arabidopsis thaliana, significantly increasing the cellulose content and spatial distribution of Arabidopsis stems. These results indicate that the CitCesA8 gene can be used to regulate the cellulose content and waterlogging of citrus juice sacs, and can also be applied in marker-assisted breeding to select citrus varieties with low cellulose content and better pulpiness, showing broad application prospects in postharvest preservation and quality improvement of citrus fruits.

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the CitCesA8 gene in regulating citrus juice sac cellulose biosynthesis, characterized in that, The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The regulation includes overexpressing the CitCesA8 gene to promote citrus juice sac cellulose biosynthesis; and knocking out or silencing the CitCesA8 gene to reduce citrus juice sac cellulose biosynthesis.

3. Primer pairs for amplifying the CitCesA8 gene, characterized in that, The primer pair includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO:2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3; The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:

1.

4. A biomaterial for regulating the expression level of the CitCesA8 gene, characterized in that, This includes biological materials that overexpress the CitCesA8 gene and biological materials that knock out or silence the CitCesA8 gene; The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:

1.

5. The biomaterial according to claim 4, characterized in that, Biological materials that overexpress the CitCesA8 gene include one or more of the following: a recombinant expression vector containing the CitCesA8 gene, a recombinant microorganism containing the CitCesA8 gene, and a recombinant microorganism containing the recombinant expression vector.

6. The biomaterial according to claim 4, characterized in that, Biological materials that knock out or silence the CitCesA8 gene include one or more of the following: a knockout vector containing the CitCesA8 gene knockout target, a silencing vector containing the CitCesA8 gene silencing target, and a recombinant microorganism containing the knockout vector or the silencing vector.

7. The application of the primer pair of claim 3 or the biomaterial of any one of claims 4 to 6 in regulating citrus juice cell cellulose biosynthesis and / or creating new germplasm.

8. A method for reducing cellulose biosynthesis in citrus juice cells, characterized in that, By knocking down or removing the CitCesA8 gene in the genome of the target citrus, citrus fruits with reduced juice sac cellulose biomass were obtained. The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:

1.

9. A citrus model for creating increased cellulose content in juice sacs, characterized in that, Overexpression of the CitCesA8 gene in target citrus fruits yielded citrus fruits with increased juice sac cellulose biomass; the nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:

1.

10. The application of the CitCesA8 gene in regulating citrus drought, characterized by, The regulation includes overexpressing the CitCesA8 gene to promote citrus waterlogging; knocking out or silencing the CitCesA8 gene to reduce citrus waterlogging. The nucleotide sequence of the CitCesA8 gene is shown in SEQ ID NO:1.

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