Application of citrus CsAPD2-2 gene in reducing citric acid content of citrus fruits

Abstract

The invention belongs to the technical field of molecular breeding, and discloses application of a citrus CsAPD2-2 gene in reducing the citric acid content of citrus fruits. The invention discloses the application of the CsAPD2-2 gene in regulating and controlling the citric acid content of plant leaves / fruits for the first time. According to the invention, the sugar orange CsAPD2-2 gene highly associated with the citric acid content of the fruits is successfully cloned, and the gene is specifically and highly expressed in the citrus fruits. The gene is over-expressed in citrus and tomato, or the gene is knocked out in citrus by virtue of a CRISPR / Cas9 gene editing technology, so that the content of citric acid in fruits is correspondingly reduced and increased, and meanwhile, the gene does not influence the properties of flowering and fruiting time, pulp color, single fruit weight, soluble solid content and the like of plants; therefore, the CsAPD2-2 gene is a fruit citric acid content negative regulation gene with strong pertinence.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to citrus. CsAPD2-2 Application of genes in reducing citric acid content in citrus fruits. Background Technology

[0002] Acidity is a key determinant of citrus flavor quality, caused by the accumulation of citric acid in the vacuoles of juice sacs. The citric acid content in citrus fruits varies greatly among different varieties: high-acid varieties, such as citron and lemon, can contain up to 60 mg / mL of citric acid. -1 For acid-free varieties such as sugar oranges, the content is less than 1 mg / mL. -1 (He et al., Transcriptome analysis reveals the common and specific pathways of citric acid accumulation in different citrus species, Horticultural Plant Journal, 2025, 11: 520-534). Excessively high or low citric acid content significantly impacts the flavor and sensory quality of citrus fruits, ultimately affecting consumer preference and the development of the entire industry. Molecular breeding using gene resources related to citric acid metabolism can target and efficiently improve existing breeding challenges, such as the problem of high acid accumulation in mature fruits during the selection of extra-early and early-maturing citrus varieties, the problem of excellent pulp color but excessive acidity, and the decline in acidity and flavor during post-harvest storage. This will better promote the improvement of citrus quality and the healthy development of the citrus industry. Currently, the mining of gene resources related to citric acid metabolism mainly utilizes citrus varieties with significant differences in citric acid content, identifying relevant regulatory genes through a combination of genetic and multi-omics methods. However, these are mostly transcriptional regulatory genes, which can affect many other important traits of citrus (such as flower and fruit color), thus exhibiting poor specificity for citric acid regulation and limiting their widespread applicability (Rao et al., 2021, 2: 100138). Therefore, the gene resources for regulating citric acid in citrus fruit remain scarce, and research on their functions and mechanisms of action is not yet in-depth, hindering the rapid development of molecular breeding in this field.

[0003] The RING-type E3 ubiquitin ligase family of genes widely influences fruit development and various stress responses in plants, such as bananas. MaBAH1 Promotes chlorophyll degradation and fruit ripening; apples MdMIEL1 Negative regulation of anthocyanin biosynthesis and cold stress resistance; tomato SlCOP1-1Monoubiquitination modification enhances fruit resistance to gray mold; RING-type E3 ubiquitin ligases in Arabidopsis regulate pollen mitosis. In citrus, RING-type E3 ubiquitin ligases were previously cloned from Dark Orange and Red Dark Orange. CsAPD2-2 The author renamed it CsAPD2 (Cs6g08410; CDS coding sequence 1431 bp; amino acid number 476); it was found to be mainly expressed in fruit juice vesicles. Further correlation analysis showed that... CsAPD2 The transcription level was positively correlated with the changes in citric acid content in six citrus varieties, therefore it is speculated that... CsAPD2 It may have the function of positively regulating citric acid metabolism in fruit (Bai Yingxin. Screening and preliminary functional verification of key genes for differential accumulation of citric acid in citrus [D]. Huazhong Agricultural University, 2020. DOI:10.27158 / d.cnki.ghznu.2020.000856.).

[0004] By identifying genes highly associated with citric acid metabolism in citrus fruits and clarifying their functions and mechanisms, we can provide important genetic resources and strong theoretical support for the breeding of new germplasm with diversified acidity and the improvement of fruit quality. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art described above. It provides a citrus... CsAPD2-2 Genes and their application in regulating citric acid content in citrus fruits. The applicant uses acid-free tangerines (…). Citrus reticulata Based on cv.) mutant material, a ring-type E3 ubiquitin ligase was identified through transcriptomic and metabolomic WGCNA (Weighted Correlation Network Analysis) with normal Satsuma mandarins, and named according to the annotation. CsAPD2-2 This gene is specifically expressed in citrus fruits, and its expression level is the opposite of the low citric acid content phenotype of mutant fruits. It maintains a high expression level throughout the entire fruit development period, and exogenous citric acid treatment can reduce its transcriptional expression. Using transgenic technology and CRISPR / Cas9 gene editing technology, this gene was stably overexpressed or inactivated in kumquat. The results showed that in citrus leaves, overexpression of this gene significantly reduced citric acid content, while inactivation of the gene significantly increased citric acid content. Further transgenic overexpression of this gene in tomatoes also caused a significant decrease in citric acid content in the fruit, without significantly affecting the appearance, flesh color, or soluble solids content of the tomato fruit. In summary, CsAPD2-2 Negative regulation of citric acid content in fruit can serve as a candidate gene for molecular improvement of acidity and flavor in citrus fruits.

[0006] The first aspect of the present invention is to provide CsAPD2-2 Applications of genes.

[0007] The second aspect of the present invention aims to provide a... CsAPD2-2 Applications of gene-related biomaterials.

[0008] A third aspect of the present invention aims to provide targeted upregulation. CsAPD2-2 Application of reagents for gene expression levels.

[0009] The fourth aspect of this invention aims to provide a method.

[0010] The fifth aspect of this invention aims to provide a method for increasing the citric acid content in citrus fruits.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides CsAPD2-2 Application of genes in any of (1)-(5): (1) Regulate the citric acid content in plant leaves; (2) Prepare products that regulate the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

[0012] In some embodiments of the present invention, improving the quality of plant fruits means increasing or decreasing the citric acid content in plant fruits.

[0013] In some embodiments of the present invention, the regulation of citric acid content in plant leaves includes increasing or decreasing the citric acid content in plant leaves.

[0014] CsAPD2-2 Genes negatively regulate the citric acid content in plant leaves or fruits.

[0015] The present invention CsAPD2-2 Compared with the background art mentioned CsAPD2 They are different. Although both belong to the RING type E3 ubiquitin ligase, they differ in coding sequence length, amino acid number and similarity, and physiological function.

[0016] A second aspect of the invention provides a connection with CsAPD2-2 Application of gene-related biomaterials in any one of (1)-(5): (1) Regulate the citric acid content in plant leaves; (2) Prepare products that regulate the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The biomaterial carries CsAPD2-2 Genes, the ones mentioned CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

[0017] In some embodiments of the present invention, improving the quality of plant fruits means increasing or decreasing the citric acid content in plant fruits.

[0018] In some embodiments of the present invention, the biomaterial is any one of a1)-a8): a1) CsAPD2-2 Nucleic acid molecules of genes; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4).

[0019] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0020] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0021] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0022] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0023] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0024] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0025] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0026] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0027] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0028] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0029] In some embodiments of the present invention, the regulation of citric acid content in plant leaves includes increasing or decreasing the citric acid content in plant leaves.

[0030] A third aspect of the invention provides targeted upregulation CsAPD2-2 The application of the reagent for gene expression levels in any one of (1)-(5): (1) Reduce the citric acid content in plant leaves; (2) Prepare products that reduce the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

[0031] This invention utilizes transgenic technology to overexpress [the gene] in citrus and tomatoes. CsAPD2-2 All of these can reduce the citric acid content in citrus leaves and tomato fruits, without affecting the appearance of the fruit, the color of the pulp, or the content of soluble solids; conversely, knocking out citrus (kumquat material) using CRISPR / Cas9 can reduce the citric acid content. CsAPD2-2 The gene significantly increases the citric acid content in the leaves. This indicates that... CsAPD2-2Genes specifically negatively regulate citric acid accumulation in fruits.

[0032] In some embodiments of the present invention, improving the quality of plant fruits means reducing the citric acid content in plant fruits.

[0033] In some embodiments of the present invention, the high-quality plant fruit is a plant fruit containing low citric acid content.

[0034] A fourth aspect of the invention provides a method for overexpressing in a target plant. CsAPD2-2 Gene; The method includes any one of 1)-3): 1) A method for reducing the citric acid content in plant leaves; 2) A method for improving the quality of plant fruits; 3) A method for cultivating genetically modified plants that produce high-quality fruit; CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

[0035] In some embodiments of the present invention, improving the quality of plant fruits means reducing the citric acid content in plant fruits.

[0036] In some embodiments of the present invention, the method includes the following steps: Build CsAPD2-2 Gene overexpression vectors; The overexpression vector was transferred into Agrobacterium and then used to infect plants.

[0037] In some embodiments of the present invention, the method for constructing the overexpression vector includes cloning. CsAPD2-2 The genetic steps will recycle the products and Kg2E-35SA - The FLAG linking step is used to construct the overexpression vector (i.e., Kg2E- CsAPD2-2 (overexpression vector).

[0038] In some embodiments of the present invention, when the plant is tomato, the overexpression vector is transferred into Agrobacterium and then used to infect tomato explants. After screening the infected tomato explants, callus tissue grows, which is then further cultivated to obtain... CsAPD2-2 Stable overexpression tomato plants (i.e. tomato plants with low citric acid content in their fruit).

[0039] Right now CsAPD2-2 Gene overexpression vector Kg2E-CsAPD2-2 Genetic transformation was performed on tomato explants using Agrobacterium-mediated transformation, and molecular identification was conducted to obtain... CsAPD2-2 Stable overexpression tomato lines.

[0040] For early-maturing and early-ripening plant varieties, or plants with excellent fruit color and high citric acid content, overexpression can be achieved through molecular breeding techniques. CsAPD2-2 Genes, and then cultivate new plant germplasm with excellent color, acidity and flavor.

[0041] A fifth aspect of the present invention provides a method for increasing the citric acid content in citrus fruits, the method comprising reducing the citric acid content in citrus plants. CsAPD2-2 Gene expression levels.

[0042] In some embodiments of the present invention, the reduction of citrus plants CsAPD2-2 The steps involved in regulating gene expression include using gene editing technology to knock out / lower the expression level in citrus plants. CsAPD2-2 The steps of gene generation.

[0043] In some embodiments of the present invention, the gene editing technology includes ZFNs, TALENs, or CRISPR / Cas9 technology.

[0044] In some embodiments of the present invention, CRISPR / Cas9 technology is used for knockout / knockdown. CsAPD2-2 Gene.

[0045] In some embodiments of the present invention, CRISPR / Cas9 technology is used for knockout / knockdown. CsAPD2-2 The gene involves the following steps: introducing a vector containing sgRNA and Cas9 protein into citrus plants, and then screening to obtain citrus varieties with high citric acid content.

[0046] The beneficial effects of this invention are: This invention is disclosed for the first time. CsAPD2-2 Application of genes in regulating citric acid content in plant leaves / fruits. This invention successfully cloned a citric acid content highly correlated with that of the Satsuma mandarin orange. CsAPD2-2 This gene is specifically highly expressed in acid-free mutant citrus fruits. Overexpression of this gene in citrus and tomatoes, or knockout of this gene in citrus using CRISPR / Cas9 gene editing technology, resulted in corresponding decreases and increases in citric acid content in the fruit, respectively. Simultaneously, this gene did not affect traits such as flowering and fruiting time, pulp color, single fruit weight, and soluble solids content, indicating that... CsAPD2-2 This gene is a highly targeted negative regulator of citric acid content in fruits. Therefore, it can serve as an important candidate gene for regulating fruit acidity, providing crucial genetic resources and strong theoretical support for the breeding of new germplasm and the improvement of citrus quality, ultimately aiming to meet the taste preferences of different market segments. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a comparison chart of citric acid content throughout the entire fruit development period of normal Satsuma mandarins (CK) and mutant Satsuma mandarins (DS).

[0048] Figure 2 As in Embodiment 2 of the present invention CsAPD2-2 A graph showing the response to changes in citric acid content; where (a) is... CsAPD2-2 Expression levels throughout the fruit development of the mutant Satsuma mandarin; (b)-(c) show the effects of high-concentration exogenous citric acid treatment on the expression levels of the mutant Satsuma mandarin. CsAPD2-2 The effect of transcription. (See figure.) .

[0049] Figure 3 As in Embodiment 3 of the present invention CsAPD2-2 Molecular characteristics and tissue expression specificity diagrams; where (a) is... CsAPD2-2 with citrus CsAPD2 (a) shows the results of homologous gene comparison; (b) shows the results of homologous gene comparison. CsAPD2-2 with citrus CsAPD2 Phylogenetic tree of homologous genes; (c) is CsAPD2-2 Expression of citrus in different tissues (root, stem, leaf, flower, and pulp) was analyzed using a one-way Duncan ANOVA. Different letters represent different expression levels. p The difference was significant at the <0.05 level.

[0050] Figure 4 As in Embodiment 4 of the present invention CsAPD2-2 Diagrams showing the construction of overexpression vectors and CRISPR / Cas9 gene knockout vectors, along with their electrophoretic identification images; where (a) CsAPD2-2 (a) Construction diagram of the overexpression vector; (b) Construction diagram of the CRISPR / Cas9 gene knockout vector; (c) Construction diagram of the CRISPR / Cas9 gene knockout vector. CsAPD2-2 Electrophoretic identification diagram of overexpression vector; (d) Electrophoretic identification diagram of CRISPR / Cas9 gene knockout vector.

[0051] Figure 5 As in Embodiment 5 of the present invention CsAPD2-2 Molecular identification diagrams of citrus / tomato overexpression or knockout materials; where (a) shows 3 plants. CsAPD2-2 (a) shows citrus overexpression lines that fluoresce green; the scale bar is 2 cm. (b) shows 3 lines. CsAPD2-2 Citrus overexpression lines CsAPD2-2 Expression level; (c) 2 citrus trees CsAPD2-2 Gene knockout lines; (d) are two citrus CsAPD2-2 gene knockout lines. CsAPD2-2Expression levels; (e) shows three CsAPD2-2 overexpressing tomato lines, with fruits exhibiting green fluorescence; the scale bar is 3 cm. (f) shows the expression levels of the three CsAPD2-2 overexpressing tomato lines. CsAPD2-2 Expression level; (g) represents the target site mutation type in two citrus CsAPD2-2 gene knockout lines. In the figure, ns indicates no significant difference. .

[0052] Figure 6 As in Embodiment 6 of the present invention CsAPD2-2 Graph showing the determination of citric acid content in citrus / tomato overexpression or knockout materials; where (a) is... CsAPD2-2 (a) Graph showing the determination of citric acid content in citrus overexpression plants; (b) is... CsAPD2-2 Graph showing the determination of citric acid content in knockout citrus plants; (c) is... CsAPD2-2 A graph showing the citric acid content in tomato fruits from tomato plants overexpressing the gene. In the graph, .

[0053] Figure 7 It is in embodiment 7 of the present invention CsAPD2-2 The effect of overexpression on flowering and fruit development in tomatoes; where (a)-(d) are plotted. CsAPD2-2 The graph shows the effect of CsAPD2-2 overexpression on tomato flowering (ab) and fruiting time (cd), with scale bars of 1 cm for (a) and (c); (e)-(h) show the effect of CsAPD2-2 overexpression on tomato fruit shape (e), single fruit weight (f), soluble solids content (g), fruit color, and seed color (h), with a scale bar of 3 cm for (h). In the graph, ns indicates no significant difference. . Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1: Obtaining Satsuma mandarins CsAPD2-2 Gene The applicant previously obtained acid-free tangerines lacking citric acid. Citrus reticulata The cv. mutant exhibits the same pattern of changes in the content of major soluble sugars and organic acids throughout the entire fruit development period as normal Satsuma mandarins, except that the citric acid content is at an extremely low level. Figure 1 (Li et al., Dynamic Analysis of the Fruit Sugar-Acid Profile in a Fresh-Sweet Mutant and Wild Type in 'Shatangju' (Citrus reticulata cv.), 2024, 13: 2722). Using this acid-free mutant (DS) material, the applicant conducted transcriptomic and metabolomic WGCNA (Weighted Correlation Network Analysis) analyses with normal satsuma mandarins (CK), uncovering a high correlation between citric acid content and other factors. CsAPD2-2 The gene (ID: LOC102612248) was obtained by searching the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and the citrus database (http: / / citrus.hzau.edu.cn / ). Then, using cDNA from the mutant Satsuma mandarin fruit as a template, the gene was cloned and sequenced by PCR amplification (using the following PCR primers: F: 5'-atgttgctgcctctctctctgg-3' (SEQ ID NO:3), R: 5'-ccagagagagaggcagcaacat-3' (SEQ ID NO:4)). CsAPD2-2 The CDS coding sequence (SEQ ID NO:1) is 1227 bp in length and encodes 408 amino acids (SEQ ID NO:2).

[0058] CsAPD2-2 The CDS sequence of the gene is shown in SEQ ID NO:1, specifically:

[0059] CsAPD2-2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2, specifically: MLLPLSLALLLPSYLVDMFRPVVLSPPTGHPQRWHKTWARLLAPLTIWICVSVSLRYGYYGDCRMVLGPGSSRLMKASSLFVRQVEVRSGSEKGFLLYGFSEK PQLTHETNWTVSNFLMVGSYSRKGISLWLNNGSAIRMRWETRGSKLNETQLVIIKGEREFQTLLPKFTSSSDPPALTDPIDGKNAEYDVEEDDMYYFGVTNKN PRSITMTMNVNVTSKIYDLTKAKNMCSSSNGSCRLKLLFPNSQYVILTTPSNGDLDGWHVEVSFAARLIIYIAILGFITLIIFLVSKYLGACDGNSDNTDSTAAREVTETDPLVHEKPVQFTYGTTNNDEDDDAGFSSTSSDDLYDAKLCVICYDDQRNCFFVPCGHCATCYDCGQRIMKDDSKVCPICRTLIHKVRRLFTP (SEQ ID NO:2).

[0060] Example 2 CsAPD2-2 Transcription is induced by citric acid and is negatively correlated with the citric acid content of the fruit. Further investigation CsAPD2-2 To investigate the relationship between gene expression and the low-acidity trait in mutant fruits, pulp samples were collected from both normal and mutant mandarin oranges at 100, 130, 160, 190, 220, 250, and 280 days after flowering. RNA was extracted from the pulp and detected using quantitative real-time PCR. CsAPD2-2 Gene expression status.

[0061] At the same time, carry out CsAPD2-2 Experiment on gene expression response to exogenous citric acid: Citrus seedlings germinated from seed were hydroponically cultured (photoperiod 14 h light / 10 h dark, temperature 30°C, light intensity 450 μmol / m²). 2After 28 days (at a relative humidity of 50%–60%), exogenous citric acid treatment experiments were conducted. In the concentration gradient response experiment, 0 mM–6 mM citric acid was added to the Hoagland nutrient solution of the treatment groups, while the control group received nutrient solution without citric acid. Leaves were collected 6 h after treatment for subsequent experiments. In the time gradient response experiment, 4 mM citric acid was added to the Hoagland nutrient solution of the treatment groups, while the control group also received nutrient solution without citric acid. Leaves were collected at 0 h, 2 h, 4 h, and 6 h. Finally, RNA was extracted from the samples and detected using quantitative real-time PCR. CsAPD2-2 Gene expression status.

[0062] The primers used for real-time PCR are shown below: CsAPD2-2-qRT-F: 5'-AGTGACAACACAGATAGCACAGCAG-3' (SEQ ID NO: 5); CsAPD2-2-qRT-R: 5'-TAGGTGAATTGAACTGGCTTCTCGTG-3' (SEQ ID NO: 6).

[0063] Experimental results showed that, contrary to the low-acidity trait of the mutant, CsAPD2-2 Its expression level remained high throughout the entire fruit development period. Figure 2 a) Treatment with high concentrations of exogenous citric acid caused CsAPD2-2 Transcription downregulation ( Figure 2 (bc). The above results indicate that exogenous citric acid induces... CsAPD2-2 Transcription downregulated, and CsAPD2-2 The expression was negatively correlated with the citric acid content of the fruit.

[0064] Example 3 CsAPD2-2 It is mainly expressed in citrus pulp and belongs to the RING-type E3 ubiquitin ligase family. Reports indicate that it was cloned from dark orange and red dark orange. CsAPD2 (Cs6g08410 or LOC102608051; CDS coding sequence 1431bp; amino acid number 476) is a RING-type E3 ubiquitin ligase, mainly expressed in fruit juice vesicles, and it is speculated that it may positively regulate the function of citric acid metabolism in fruit (Bai Yingxin. Screening and preliminary functional verification of key genes for differential accumulation of citric acid in citrus [D]. Huazhong Agricultural University, 2020. DOI:10.27158 / d.cnki.ghznu.2020.000856.).

[0065] The aforementioned citrus fruits CsAPD2 The present invention cloned from mandarin oranges CsAPD2-2The gene was compared with the protein sequence using BLAST in the NCBI database, and the similarity was only 24.06%; further analysis was conducted using the NCBI database... CsAPD2-2 Homologous genes from species such as rice, Arabidopsis thaliana, and tomato were obtained, and then these homologous gene sequences were compared with those of citrus using DNAMAN software. CsAPD2 We jointly performed conserved amino acids and functional domains analysis of the protein sequence; simultaneously, we used MEGA7 software to construct a phylogenetic tree using the neighbor-joining method to clarify... CsAPD2-2 The degree of kinship between a gene and its homologous genes.

[0066] In addition, RNA was extracted from the roots, stems, leaves, flowers, and pulp of normal tangerines and then detected using quantitative real-time PCR technology. CsAPD2-2 Gene-specific expression in different tissues of citrus. CsAPD2-2 The quantitative primers are shown in Example 2.

[0067] The results showed that CsAPD2-2 It is mainly expressed in the pulp ( Figure 3 c), is an E3 ubiquitin ligase containing the C3HC4 type RING motif ( Figure 3 (a) belongs to citrus CsAPD2 They are homologous genes, but their protein similarity is low, only 24.06%, and they are evolutionarily distantly related. Figure 3 (b) The above results also indicate CsAPD2-2 For different CsAPD2 Another homologous gene.

[0068] Example 4 CsAPD2-2 Citrus / Tomato Overexpression and Construction of Citrus CRISPR / Cas9 Gene Knockout Materials (1) CsAPD2-2 Construction of Citrus / Tomato Overexpression Vectors Using cDNA from the mutant Satsuma mandarin fruit as a template, a reaction system was prepared according to the instructions of the high-fidelity enzyme Phanta Flash Super-Fidelity DNA Polymerase (Vazyme; CAT: P521-d1) for PCR amplification of the CDS coding sequence of CsAPD2-2 (CsAPD2-2-F: 5'-TAGGTCTCAAATGatgttgctgcctctctctctg-3' (SEQ ID NO:7), CsAPD2-2-R: 5'-gaagattgtttaccccttagTTCGTGAGACCGC-3' (SEQ ID NO:8)). The electrophoresis image of the amplification product is shown below. Figure 4As shown in c; then, following the instructions, using the recombinase (Vazyme; CAT: C117), the recovered product fragment was combined with the vector Kg2E-35SA-FLAG (digested with BsaI (NEB; CAT: R3733V)). Figure 4 In step a), a recombination reaction was performed to obtain the recombinant plasmid Kg2E-CsAPD2-2. Sequencing confirmation of the recombinant plasmid was completed by Shanghai Sangon Biotech Co., Ltd.

[0069] (2) CsAPD2-2 Construction of Citrus CRISPR / Cas9 Gene Knockout Vector refer to CsAPD2-2 The genome sequence information (SEQ ID NO:9) was obtained, and a specific target site with a 20bp+PAM sequence was designed on the first exon of CsAPD2-2 using the CRISPR / Cas9 target site design analysis website (http: / / skl.scau.edu.cn / ). Target primers CsAPD22-sgRNA-F (5'-gttgCCACTGGGCATCCTCAGCGG-3', SEQ ID NO:10) and CsAPD22-sgRNA-R (5'-aaacCCGCTGAGGATGCCCAGTGG-3', SEQ ID NO:11) were synthesized, denatured at 95°C, and annealed to a double-stranded structure at room temperature. Finally, using Golden Gate technology, the annealed target primers were ligated to the Kg2E-cas9 vector with the restriction endonuclease BpiI (NEB; CAT: R0539V) and T4 DNA ligase (NEB; CAT: M0202V). Figure 4 (b) Then, the bacterial culture was detected by PCR using characteristic fragments (838 bp in length) (Cas9-CX-F: 5'-GGGCCAAATGCCATAAGAGG-3' (SEQ ID NO:12), Cas9-CX-R: 5'-CGGATAAACCTTTTCACGCCC-3' (SEQ ID NO:13)) and sequencing. Figure 4 (d) The recombinant plasmid Kg2E-cas9-CsAPD2-2 was obtained. Primer synthesis and sequencing were performed by Shanghai Sangon Biotech Co., Ltd.

[0070] Golden Gate technology system: plasmid Kg2E-cas9 30 ng, annealed target primers (CsAPD22-sgRNA-F and CsAPD22-sgRNA-R) 0.1 μM, T4 DNA ligase buffer 1 μL, T4 DNA ligase 1 μL, BpiI 2 μL, and deionized water to a final volume of 10 μL; then react at 37°C for 5 minutes, then at 16°C for 5 minutes, repeating this temperature cycle 30 times, and finally denature at 60°C for 5 minutes to terminate the reaction.

[0071] CsAPD2-2 The genomic sequence information is shown in SEQ ID NO:9. The underlined regions are exons, the italicized regions are target sites, and the bolded parts are PAM sequences. CAGCAGAGCTACTGGTTGAGTTGAAGCTACTGATAGTACTATGTTTGGCCACACACATGCTTTAGTAAACCTACTAGTCCTCTTCCAATTTCAAATCTTCACCGCAAATTTAGTAAT ATGTTGCTGCCTCTCTCTCTCTGGCTC TCCTACTCCCTAGTTACCTAGTAGACATGTTCAGGCCAGTCGTCTTATCTCCTC CCACTGGGCATCCTCAGCGGTG G CATAAAACTTGGGCTCGCCTGCTTGCTCCTCTTACCATATGGATATGTG GCATGTAATTTTGCATCTTCTGTTGCAATTCCTCTTGATGAAGTCATCACCATTCAATCTAACTTTTTTTTTTTTTTTCCTTTTTCAAAATTTGCATGTTGCAG TTTCGGTAAGTCTGCGATACGGGTACTATGGAGATTGCCGCATGGTGCTTGGACCAGGGTCGTCAAGACT GATGAAGGCAAGTTCTTTGTTCGTTCGCCAAGTCGAAGTCAGGTCTGGTAGCGAAAAAGGGTTTCTGCTTTATGGG TTCTCTGAGAAGCCTCAGTTAACTCATGAAACTAATTGGACTGTCTCGAACTTTTTGATGGTTGGATCTTATAGTC GCAAG GTGGCATTTCAATTTCGTCCTTTGAATTAAATTCAGCTACACTACTTCAGCACCTTAGCTTTCTATAGTTAAAAATGATTAAATTAATCTTTCAG GGAATTTCTTTGTGGTTGAACAACGGCTCTGCTATCCGTATGAGATGGGAAA CTCGGGGCAGCAAATTAAATGAAACCCAATTGGTCATAATTAAAG GTTAGTACGGTTTATTAAAGTTCTTTGTGGGCTAAACTTGTATCATTTGTGATTTCATTGGTTGACAAAATGTACGCATGTGTCTACTTGCTCTAG GAGAACGAGAA TTTCAGACATTACTGCCAAAATTTACAAGTTCCTCTGATCCCCCAGCTCTGACCGATCCCATCGACG GTATGATTTTTGTTTCTCTTGTCTCCTCTTTTCAAATGAATAAAAAATCGAAAATAGGACGCGCCAGATAGGGGTCGAACCTATGACCTTCTGCTTAGGAAACAGACGCTCTATCCACTGAGCTACAGGCGCTCTGTTGAAAACCTTGCATACTTAAGAGATTATATCTGATTTAATCTCAGTATACTTAATTTTTTATTTTTCAG GTAAAAATGCAGAATACGATGTTGAGGAAGA CGACATGTACTATTTTGGTGTCACAAACAAGAATCCTCGAAGCATAACAATGACAATGAATGTAAATGTCACATCG AAAATATATGATCTGACAAAAGCGAAGAACATGTGCTCATCGTCAAACGGATCTTGCCGGCTCAAACTCTTGTTTC CCAATAGTCAATATGTCATACTTACAACACCCAGCAAT GTAAGCGGCCCTCATTCTTTTCCCACCTTTGTACCGTGCTCAACAAGTTAACCAATTCCCTGAGGGCTGAGACTTGAATCGGCCTCAATCAATAGTAATTTATTCAATTGCAG G GAGATCTAGATGGCTGGCACGTTGAGGTATCATTTGCGGCTCGTTTGATAATTTACATTGCAATTTTAG GTACGATATCATTCATAAAACTCATTTGATGATTCTTCTTTTTACAACATCTTTTCACACCCACTCTCATTAACCGGATTTTAATTCGTGCTGTCTTTAG GATTTATCACCTTGATTATCTTTCTGGTATCAAAATACCTTGGAGCCTGTGATGGTAAT AGTGACAACACAGATAGCACAGCAGCTAGAGAAGTAACTGAGACTGACCCTCTAGTCCACGAGAAGCCAGTTCAAT TCACCTATGGAACAACCAATAATGACGAAGACGATGATGCAGGATTTTCTAGTACCTCTTCAGATGATCTCTATGA CGCAAAATTATGTGTAATTTGTTACGACGATCAACGAAACTGCTTCTTCGTTCCATGTGGCCATTGTGCAACATGT TATGACTGTGGTCAGAG GTATACGTTAAAAATCGACCTTAATGTTTATTCTCATGAGACATAGTAAATCTGTGTTAGCGACTTTATTGATGAATTGTTTTAATTTGCAG GATTATGAAGGATGATAGCAAGGTGTGTCCAATATGTCGTACG CTTATTCATAAGGTTAGAAGATTGTTTACCCCTTAG AGGTAAACTGAGGTAGGAAAACTTGTGTGATTACATAAGAGAAAGATATTTTATGTGTATAGGGAGTAATAGATCTTGCTTTCTCGGTTAGGATAAGTGAGTCTTTAATTGTTTGCACTTCGAATCCATGGCTTAGCTTCTTAATGTGCTGCAAGATATTTGCTCCTCGGATTAAGCTACAAGGTCATGGAAGCATGAACACATAAGGCTGAATTTCTTGTTAGCACGAGCAAAAATACAATTCCTTAGAAGTCTGTTTATGCCGAAAATGGCTGAATCACGTTGCCTGTTAATGACT(SEQ ID NO:9)。

[0072] Example 5 CsAPD2-2 Construction and Molecular Identification of Transgenic Citrus / Tomato Materials (1) Stable genetic transformation of citrus and tomato In situ genetic transformation of citrus seedlings: Referring to the previously established authorized patent for citrus genetic transformation (patent number: ZL201410555974.6) and the method of Zhang et al. (Zhang et al., A simple and efficient inplanta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens, Scientia Horticulture, 2017, 214:174-179), specifically, the recombinant plasmids Kg2E-CsAPD2-2 and Kg2E-cas9-CsAPD2-2 were transformed into Agrobacterium tumefaciens EHA105. After two activation plasmid coatings on resistance plates, the bacterial cells were scraped off, and the bacterial solution was diluted with sterile deionized water. 600 Adjust the pH to 0.6-0.8, add 20 μM acetylsyl syringone, and incubate in the dark for 2 hours for later use. Take healthy kumquat seedlings that are about 26 days old and have good growth, make a horizontal cut with a blade 2 cm below the first true leaf, and quickly place a 10 μL white pipette tip saturated with bacterial solution on the cut surface. After standing for 30 minutes, put a new pipette tip on the cut surface, incubate in the dark with moisture, and after 3 days, replace it with a pipette tip containing kanamycin for selection culture. After 5 days, remove the pipette tip, incubate in the light, and use a portable handheld fluorescent lamp (LUYOR, 3415RG) to detect GFP fluorescence and select positive buds in time. Finally, after the positive buds have grown for 1-2 months, graft them for propagation to obtain citrus overexpression and gene knockout lines.

[0073] Tomato genetic transformation: Six days after tomato seed germination, cotyledons were used for genetic transformation; the Kg2E-CsAPD2-2 plasmid was transformed into Agrobacterium GV3101, activated twice on resistant plates, and then co-cultured with tomato explants to complete infection and transformation; in regeneration culture, subculture was performed every 15 days until regenerated shoots appeared, with the antibiotic Kan concentration adjusted to 70 mg / L. -1The specific method followed that of Jones et al. (Jones et al., Down-regulation of DR12, an auxin-response-factorhomolog, in the tomato results in a pleiotropic phenotype including darkgreen and blotchy ripening fruit, Plant Journal, 2002, 32: 603-613), to obtain the tomato overexpression lines.

[0074] (2) Molecular detection of transgenic citrus and tomato plants After obtaining the above-mentioned positive citrus / tomato lines through transgenic methods, a suitable amount of leaf tissue was taken for RNA extraction, and quantitative PCR was used to further clarify the RNA content. CsAPD2-2 The expression of .

[0075] for CsAPD2-2 The overexpression level of citrus / tomato materials was further clarified by quantitative PCR detection of cDNA. The primers used for quantitative PCR detection were the same as in Example 2.

[0076] For citrus CRISPR / Cas9 gene knockout materials, there are cases where quantitative PCR cannot detect gene transcription downregulation. Therefore, genomic DNA needs to be extracted separately. A DNA fragment of approximately 200 bp (specifically 237 bp in length) containing the target site is amplified by PCR (CsAPD2-2-Cas9-JC-F: 5'-CCTAGTAGACATGTTCAGGCC-3' (SEQ ID NO:14), CsAPD2-2-Cas9-JC-R: 5'-TATCGCAGACTTACCGAAACTGC-3' (SEQ ID NO:15)). After agarose gel electrophoresis and gel extraction, the fragment is sent to Xi'an Qingxue Biotechnology Co., Ltd. for sequencing, and further confirmation is achieved using the Hi-TOM high-throughput mutation analysis method. CsAPD2-2 Types of gene mutations.

[0077] The results showed that 3 plants were obtained. CsAPD2-2 Citrus overexpression strains, CsAPD2-2 The expression level increased by more than 60 times. Figure 5 (ab); and obtained 3 plants at the same time. CsAPD2-2 Tomato overexpression lines, their CsAPD2-2 The expression level increased by more than 2000 times. Figure 5 (in the middle of the ef); and also obtained 2 citrus trees. CsAPD2-2 Gene knockout lines, with target site mutations of C insertion or polybase deletion ( Figure 5 c, d, and g).

[0078] In summary, this invention successfully constructed and obtained... CsAPD2-2 Citrus / tomato overexpression materials and citrus CRISPR / Cas9 gene knockout materials.

[0079] Example 6 CsAPD2-2 Negative regulation of citric acid content in plant fruits The results obtained in Example 5 CsAPD2-2 Citrus / tomato overexpression or gene knockout materials were cultured in a plant culture room under the same conditions as in Example 2. Citric acid content was determined using ultrapure water extraction: Citrus leaves and tomato fruits at the green-ripe stage were ground and crushed with liquid nitrogen, then mixed at a ratio of 1 g sample to 5 mL of ultrapure water; extraction was then performed following the procedure of 70℃ water bath for 15 min, sonication for 15 min, and centrifugation at 12000 g for 15 min; finally, the obtained extract was filtered through a 0.22 μm filter to obtain the citric acid assay solution.

[0080] Citric acid content was determined using liquid chromatography (Agilent 1260 Infinity III HPLC): chromatographic grade citric acid standard (Yuan Ye; CAS#77-92-9) was dissolved in ultrapure water to prepare a stock solution with a concentration of 1 mg / mL. The stock solution was diluted to seven gradients: 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg / mL, and filtered through a 0.22 μm filter membrane before use. A C18 column (Agilent ZORBAX AB-Aq) was used for liquid chromatography separation of the standard and for constructing the citric acid standard curve. The mobile phase was potassium dihydrogen phosphate buffer (0.006 mol / L, pH 2.5), and the flow rate was 0.5 mL / min. -1 The column temperature was 40°C, the detection wavelength was 210 nm, and the measurement time was 20 min. Finally, under the same conditions, citric acid was separated from citrus and tomato samples by liquid chromatography, and the citric acid content (mg·g) in the samples was calculated using the plotted citric acid standard curve. -1 FW).

[0081] The results showed that CsAPD2-2 The citric acid content in the leaves of citrus overexpression materials decreased by approximately 30%-50%. Figure 6 (a) Conversely, citrus CsAPD2-2 In the knockout strains, the citric acid content increased by approximately 100%. Figure 6 (b) In addition, CsAPD2-2 The citric acid content in the fruit of tomato overexpression lines was also significantly reduced by approximately 28%-34%. Figure 6 (c)

[0082] In summary, this indicates CsAPD2-2 It negatively regulates citric acid accumulation in citrus leaves and tomato fruits.

[0083] Example 7 CsAPD2-2 It does not affect the main appearance and sweetness of the fruit. The tomato materials were grown under the same conditions as in Examples 2 and 6. For analysis... CsAPD2-2 To investigate the impact on plant fruit development, starting from the sowing date, the time when the first flower fully opens and the time when the first fruit forms were observed and recorded.

[0084] For analysis CsAPD2-2 To investigate the effects on the main appearance traits and sweetness of the fruit, the following indicators were measured after the fruit reached full maturity (at least 10 fruits were measured for each strain): Fruit shape index: Use vernier calipers to measure the longitudinal diameter and transverse diameter of the fruit, and calculate the longitudinal diameter / transverse diameter ratio; Single fruit weight: The fresh weight of a single fruit is measured using an electronic balance; Soluble solids content: The soluble solids content of the pulp juice was determined using a handheld saccharimeter (ATAGO, PAL-1).

[0085] The results showed that CsAPD2-2 After overexpression, there was no statistically significant difference in the flowering and fruiting time of tomatoes. Figure 7 (ad); at the same time CsAPD2-2 Overexpression had no significant effect on fruit color, seed color, fruit shape, single fruit weight, or soluble solids content. Figure 7 (eh). The above results indicate that... CsAPD2-2 It exhibits high specificity for regulating citric acid levels in fruits without affecting other important traits.

[0086] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, in the absence of conflict, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. CsAPD2-2 Application of genes in any of (1)-(5): (1) Regulate the citric acid content in plant leaves; (2) Prepare products that regulate the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

2. with CsAPD2-2 Application of gene-related biomaterials in any one of (1)-(5): (1) Regulate the citric acid content in plant leaves; (2) Prepare products that regulate the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The biomaterial carries CsAPD2-2 Genes, the ones mentioned CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

3. The application according to claim 2, characterized in that, The biomaterial is any one of a1)-a8): a1) CsAPD2-2 Nucleic acid molecules of genes; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4).

4. The application according to any one of claims 1-3, characterized in that, The regulation of citric acid content in plant leaves includes increasing or decreasing the citric acid content in plant leaves.

5. The application according to any one of claims 1-3, characterized in that, Improving the quality of plant fruits means increasing or decreasing the citric acid content in plant fruits.

6. Targeted upregulation CsAPD2-2 The application of the reagent for gene expression levels in any one of (1)-(5): (1) Reduce the citric acid content in plant leaves; (2) Prepare products that reduce the citric acid content in plant leaves; (3) Improve the quality of plant fruits; (4) Prepare products that improve the quality of plant fruits; (5) Genetically modified plants that produce high-quality fruit; The CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either citrus or tomato.

7. The application according to claim 6, characterized in that, Improving the quality of plant fruits involves reducing the citric acid content in the fruits.

8. A method, characterized in that, The method involves overexpression in the target plant. CsAPD2-2 Gene; The method includes any one of 1)-3): 1) A method for reducing the citric acid content in plant leaves; 2) A method for improving the quality of plant fruits; 3) A method for cultivating genetically modified plants that produce high-quality fruit; CsAPD2-2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant is either citrus or tomato; Improving the quality of plant fruits involves reducing the citric acid content in the fruits.

9. The method according to claim 8, characterized in that, The method includes the following steps: Build CsAPD2-2 Gene overexpression vectors; The overexpression vector was transferred into Agrobacterium and then used to infect plants.

10. A method for increasing the citric acid content in citrus fruits, characterized in that, The method includes reducing the concentration of [unclear] in citrus plants. CsAPD2-2 Gene expression levels.

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