Application of citrus CsbZIP5b gene in regulation and control of sugar acid metabolism of fruits

Abstract

The invention discloses application and a method for regulating and controlling the acidity and sweetness of citrus fruits by a saccharic acid metabolism regulation and control factor, and relates to the technical field of plant transgenosis. According to the key points of the technical scheme, the invention provides applications of the citrus CsbZIP5b gene in regulation and control of sugar or acid metabolism of plant fruits, preparation of products for regulation and control of sugar or acid metabolism of plant fruits, cultivation of high-sugar and low-acid citrus varieties and preparation of products for cultivation of high-sugar and low-acid citrus varieties, and provides a recombinant vector containing the citrus CsbZIP5b gene. By adjusting the expression of the citrus CsbZIP5b gene, when the expression of a saccharic acid metabolism regulation factor is improved, the sucrose content in the fruit is remarkably increased, and the citric acid content is reduced; when the expression of the saccharic acid metabolism regulation factor is inhibited, the sucrose content in the fruit is obviously reduced, and the citric acid content is increased; the purposes of regulating and controlling the acidity and sweetness of the citrus fruits and improving the taste are achieved, the life quality of people is improved, and the method has important significance for promoting high-quality agricultural development in China.

Description

Technical Field

[0001] The present invention relates to the technical field of plant transgenic technology, and more particularly to the application of citrus CsbZIP5b gene in regulating sugar and acid metabolism in fruits. Background Art

[0002] Fruit ripening is a highly coordinated and complex physiological process involving a series of metabolic, biochemical and structural changes that ultimately shape fruit quality, including flavor, aroma, texture and nutritional content. This process differs greatly between climacteric and non-climacteric fruits. Climacteric fruits, such as bananas, apples, mangoes and tomatoes, undergo a burst of respiration and ethylene production at the onset of ripening. Meanwhile, non-climacteric fruits, including citrus, grapes and strawberries, lack a substantial burst in both respiration and ethylene production. Citrus fruit development can be divided into three stages: cell division, expansion and ripening, and the dynamic balance between sugar accumulation and organic acid degradation plays a central role in defining taste characteristics. Therefore, understanding the molecular regulation of sugar and acid metabolism during fruit ripening is crucial for genetic improvement of citrus and improving fruit quality, and is the problem we are currently trying to address. Summary of the Invention

[0003] To address the deficiencies in the prior art, the present invention aims to provide an application of the citrus CsbZIP5b gene in regulating the sugar and acid metabolism of fruits, thereby achieving the purpose of regulating the sourness and sweetness of citrus fruits, improving the taste, and enhancing the fruit quality.

[0004] The above technical objectives of the present invention are achieved through the following technical solutions: In one aspect, the present invention provides a use of a citrus CsbZIP5b gene in regulating sugar or acid metabolism in fruit, wherein the sequence of the CsbZIP5b gene is as shown in SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence as shown in SEQ ID NO: 1.

[0005] In another aspect, the present invention also provides the use of the citrus CsbZIP5b gene in preparing products for regulating fruit sugar or acid metabolism, wherein the sequence of the CsbZIP5b gene is as shown in SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence as shown in SEQ ID NO: 1.

[0006] In another aspect, the present invention also provides the use of the citrus CsbZIP5b gene in cultivating high-sugar, low-acid citrus varieties, wherein the sequence of the CsbZIP5b gene is as shown in SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown in SEQ ID NO: 1.

[0007] On the other hand, the present invention also provides the use of the citrus CsbZIP5b gene in preparing products for cultivating high-sugar, low-acid citrus varieties, wherein the sequence of the CsbZIP5b gene is as shown in SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown in SEQ ID NO: 1.

[0008] In another aspect, the present invention also provides use of a recombinant vector comprising any of the above-mentioned citrus CsbZIP5b genes in regulating fruit sugar and acid metabolism, wherein the recombinant vector is an overexpression vector or an RNA interference vector.

[0009] Furthermore, the overexpression vector is constructed through the GoldenGate system, including cloning the sequence of the citrus CsbZIP5b gene into a first vector, and inserting it into the overexpression vector of the GoldenGate system after sequencing to obtain the overexpression vector.

[0010] Furthermore, the RNA interference vector is constructed through the Gateway system, including cloning the sequence of the citrus CsbZIP5b gene into an interference vector to obtain the RNA interference vector.

[0011] In another aspect, the present invention also provides a method for regulating sugar and acid metabolism in fruit, comprising: overexpressing the CsbZIP5b gene in the fruit to increase sucrose content and reduce citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO: 1.

[0012] In another aspect, the present invention also provides a method for regulating sugar and acid metabolism in fruit, comprising: inhibiting the expression of CsbZIP5b gene in fruit to reduce sucrose content and increase citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO: 1.

[0013] In another aspect, the present invention further provides a method for cultivating high-sugar, low-acid transgenic citrus, comprising: Constructing an Agrobacterium engineering bacterium containing a CsbZIP5b gene overexpression vector; introducing the vector into citrus fruit tissues by transient transformation; Screen transgenic plants and verify phenotypes by measuring sucrose or citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO: 1.

[0014] The citrus CsbZIP5b gene encodes an amino acid sequence of SEQ ID NO: 2, with a sequence length of 267 aa.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention discovers the role of the citrus CsbZIP5b gene in regulating the sweet and sour metabolism of fruit, provides uses of the citrus CsbZIP5b gene in regulating fruit sugar or acid metabolism, in preparing products for regulating fruit sugar or acid metabolism, in cultivating high-sugar, low-acid citrus varieties, in preparing products for cultivating high-sugar, low-acid citrus varieties, and provides a recombinant vector containing the citrus CsbZIP5b gene; 2. The present invention regulates the expression of the citrus CsbZIP5b gene: when the expression of the sugar-acid metabolism regulatory factor is increased, the sucrose content in the fruit significantly increases and the citric acid content decreases; when the expression of the sugar-acid metabolism regulatory factor is inhibited, the sucrose content in the fruit significantly decreases and the citric acid content increases; thereby achieving the purpose of regulating the sourness and sweetness of citrus fruit to improve the taste, and ultimately obtaining citrus fruit with high-quality flavor, thereby improving people's quality of life and having important significance for promoting the high-quality development of my country's agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings: Figure 1 This is a schematic diagram of the dual luciferase gene experiment principle in Example 2 of the present invention; Figure 2 This is the LUC fluorescence image of the CsSPSpro+OE-CsbZIP5b combination in Example 2 of the present invention; Figure 3 This is the LUC fluorescence image of the CsHSFA6Bpro+OE-CsbZIP5b combination in Example 2 of the present invention; Figure 4 This is a graph showing the results of sucrose content determination in citrus fruits of the control group, overexpression group, and interference group in Example 2 of the present invention; Figure 5 This is a graph showing the results of measuring citric acid content in citrus fruits of the control group, overexpression group, and interference group in Example 2 of the present invention; Figure 6 This is a schematic diagram of the principle of CsbZIP5b in Example 2 of the present invention regulating sugar increase and acid reduction in citrus. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0018] The following are some of the experimental materials and reagents involved in the examples: (1) Reverse transcription kit: HiScript II Q RT SuperMix for qPCR purchased from Novozymes, China; (2) High-fidelity enzyme: Phanta Max Super-Fidelity DNA Polymerase purchased from Novozymes, China; (3) Cloning vector: pTOPO-BLUNT Simple Vector purchased from Aidlab, China; (4) The first vector: pTOPO purchased from Aidlab, China, model CV17; (5) Overexpression vector: pGK1300 OE (6) Interference vector: pK7WIWG2D (RNAi) (7) Escherichia coli DH5α competent cells: purchased from Weidi Biotechnology, China; (8) Sequencing verification was completed by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.

[0019] Example 1: Isolation and cloning of the citrus CsbZIP5b gene Step 1: The citrus CsbZIP5b gene sequence was extracted from the sweet orange genome sequence (Citrus sinensis, version 3) downloaded from the Huazhong Agricultural University Citrus Genome website (http: / / citrus.hzau.edu.cn / index.php). The nucleotide sequence is shown in SEQ ID NO: 1. PCR amplification primers for cloning the CsbZIP5 gene were designed using Primer5 software (forward primer: 5'-ATGTTGTCCGCTTGTCCG-3', reverse primer: 5'-TTATGTGATTAATGATGAGGTTTGTTCAT').

[0020] Step 2: Extract total RNA from citrus fruit using the TRIzol® method. The specific experimental procedures are as follows: Step 3: Then use a reverse transcription kit to synthesize cDNA. The specific experimental steps are as follows: Step 4: Using citrus fruit cDNA as a template, amplify the gene sequence using PCR primers for the cloned CsbZIP5b gene and a high-fidelity enzyme from Novagen. The specific experimental procedure is as follows: 1 μL of citrus fruit cDNA template, 2 μL of the CsbZIP5b gene forward primer and 2 μL of the reverse primer, add 25 μL of Novagen high-fidelity enzyme buffer and 1 μL of high-fidelity enzyme, and amplify using the recommended protocol in the manufacturer's instructions, with an annealing temperature of 56°C and an extension time of 30 seconds.

[0021] Step 5: Recover the target band from the PCR product by agarose gel electrophoresis, and connect the recovered product containing the target band to the cloning vector pTOPO-BLUNT Simple Vector.

[0022] Step 6: Transform E. coli DH5α competent cells. The E. coli DH5α competent cells were from the Weidi Bio brand. After positive colonies were detected, Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. was commissioned to use Sanger sequencing (dideoxy sequencing method) for further sequencing verification.

[0023] The sequencing results showed that the cDNA sequence of the citrus CsbZIP5b gene was shown in SEQ ID NO: 1, with a sequence length of 558 bp; the encoded amino acid sequence was shown in SEQ ID NO: 2, with a sequence length of 267 aa.

[0024] SEQ ID NO: 1 ATGTTTGTCCGCTTGTCCGGCCATTTTCTCATCGGAGATGATGTTCGGAAACCCTTTTCCcGATTTCGAAAGTGAGTTCACGCCGTGGGACTTACCGGACCCTTTTCCAGCCCCAAACCAATCACCGATCCCTGCGGTAT CAAGTTCCgGTTCCGATGAACCGAACCAGATCCAAACCAACTCAAACTCTGGTTCAGATGAGCCGAggCAGACGGTTTCTGTCATCGACGAGCGTAAGCGTAGACGCATGATATCGAACCGcGAATCGGCCCGGAGGTCA CGCATGCGTAAACAGAAGCATTTAGAAAACCTAAGGAACCAGCTGAACCGCCTTAGAATGGAGAACCGGGAACTATCGAACCGGTTACGTTTGCCTTGCATCACTGTCAACGTGTAAGGACaGACAATGATCGGCTCC GGTCCGAACATACTATTCTCCGGCGAAGACTGTCGGAAATACGTCAAATTTTGCTTTACAGGCAGCTcCAACAGGTCACGTCTGCATGGCCATGCAATTCCGCTGTCACGAATGAACAAACCTCATCATTAATCACATAA SEQ ID NO: 2 MLSACPAIFSSEMMFGNPFPDFESEFTPWDLPDPFPAPNQSPIPAVSSSGSDEPNQIQTNSNSGSDEPRQTVSVIDERKRRRMISNRESARRSRMRKQKHLENLRNQLNRLRMENRELSNRLRFALHHCQRVRTDNDRLRSEHTILRRRLSEIRQILLYRQLQQVTSAWPCNSAVTNEQTSSLIT* Example 2: Expression and effect verification of citrus CsbZIP5b gene Step 1: Enhancing Citrus CsbZIP5b Gene Expression: An overexpression vector (OE-CsbZIP5b) containing the CsbZIP5b gene was constructed. The CsbZIP5b gene sequence was cloned into the pTOPO vector and, after sequencing, inserted into the Goldengate system overexpression vector pGK1300 OE. This resulted in the OE-CsbZIP5b overexpression vector for enhancing CsbZIP5b gene expression. The overexpression vector was then transiently transformed into kumquat fruit via Agrobacterium-mediated transformation for transient expression. Transient overexpression experiments were conducted. Seven days after injection, samples were photographed for phenotype observation and immediately frozen in liquid nitrogen at -80°C until use. Transiently overexpressed fruit were divided into three groups, with 10 independent fruits in each group serving as biological replicates for quantitative analysis of citric acid content, sucrose content, and gene expression levels.

[0025] Step 2: Inhibition of Citrus CsbZIP5b Gene Expression: An RNAi vector, RNAi-CsbZIP5b, containing the citrus CsbZIP5b gene was constructed by cloning the citrus CsbZIP5b gene sequence into the interference vector pK7WIWG2D. This RNAi vector, RNAi-CsbZIP5b, was then transiently transformed into kumquat fruits via Agrobacterium-mediated transformation for transient expression. Transient overexpression experiments were performed. Seven days after injection, samples were photographed to observe phenotypes and immediately frozen in liquid nitrogen at -80°C until use. Fruits undergoing transient interference expression were divided into three groups, with 10 independent fruits in each group serving as biological replicates for quantitative analysis of citric acid content, sucrose content, and gene expression levels.

[0026] Step 3: The present invention uses a dual luciferase gene experiment to determine the regulation of CsbZIP5b on the target gene promoter activity, such as Figure 1As shown, in the Reporter vector, REN is Renilla fluorescence, which serves as an internal reference fluorescence; LUC is firefly fluorescence; Protarget gene is the target gene promoter, used to drive the expression of the LUC firefly fluorescence gene, and the promoter activity can be judged according to the firefly fluorescence intensity; p35S in the Effector vector is a strong promoter, used to drive gene expression; T35S is a terminator, used to terminate gene expression; CsbZIP5b is the target gene.

[0027] like Figure 2 As shown in the figure, the fluorescence values ​​of the control group CsSPSpro and the combination of CsSPSpro+OE-CsbZIP5b were measured, wherein the control group CsSPSpro used P35S to drive the empty gene (no gene sequence) and the CsSPS promoter to drive the co-expression of the LUC gene, which was used as the control group; CsSPSpro+OE-CsbZIP5b used P35S to drive CsbZIP5b and the CsSPS promoter to drive the co-expression of the LUC gene, which was used as the experimental group. As can be seen from the figure, compared with the control group CsSPSpro, the LUC fluorescence value of the CsSPSpro+OE-CsbZIP5b combination was significantly increased, indicating that the CsbZIP5b gene binds to and enhances the expression of the downstream target gene CsSPS. Figure 3 As shown, the fluorescence values ​​of the control group CsHSFA6Bpro and the combination of CsHSFA6Bpro+OE-CsbZIP5b were measured. The control group CsHSFA6Bpro was co-expressed with the empty gene (no gene sequence) driven by P35S and the LUC gene driven by the promoter of CsHSFA6B, which served as the control group; CsHSFA6Bpro+OE-CsbZIP5b means that CsbZIP5b can bind to the CsHSFA6B promoter region and activate the expression of CsHSFA6B, which means that CsHSFA6B is downstream of CsbZIP5b and is its target gene. As can be seen from the figure, compared with the control group CsHSFA6Bpro, the LUC fluorescence value of the combination of CsHSFA6Bpro+OE-CsbZIP5b is significantly increased, indicating that the CsbZIP5b gene binds to and enhances the expression of the downstream target gene CsHSFA6B.

[0028] Step 4: Detect the contents of sucrose and citric acid in transgenic and control materials by ultra-high performance gas chromatography.

[0029] During the test, a control group, an overexpression group, and an interference group were set up. The control group was labeled EV-1. The overexpression group was the three groups of transiently overexpressed fruits in step 1 labeled as overexpression group 1 (OE-1), expression group 2 (OE-2), and expression group 3 (OE-3); the interference group was the three groups of transiently interfered expression fruits in step 2 labeled as interference group 1 (RNAi-1), interference group 2 (RNAi-2), and interference group 3 (RNAi-3).

[0030] The experimental results are as follows: Measured object control group Overexpression group 1 Overexpression group 2 Overexpression group 3 Interference Group 1 Interference Group 2 Interference Group 3 Sucrose content (mg / g) 22.2 37.78 36.82 30.48 18.95 19.38 16.45 Citric acid content (mg / g) 3.91 1.38 1.77 1.74 5.20 5.05 5.05 Experimental conclusion: Figure 4 、 Figure 5 As shown in the figure, it is a bar chart drawn based on the experimental results, from which we know that Figure 4 In the study, overexpression of CsbZIP5b significantly increased the sucrose content of citrus fruit compared with the control, while interference with CsbZIP5b reduced the sucrose content of citrus fruit; Figure 5 In the study, overexpression of CsbZIP5b significantly reduced the citric acid content in citrus fruit compared with the control, while interference with CsbZIP5b increased the citric acid content in citrus fruit, indicating that CsbZIP5b is a key regulatory factor in regulating sugar increase and acid reduction in citrus.

[0031] The regulatory mechanism of CsbZIP5b is as follows Figure 6 As shown, abscisic acid (ABA) can activate the expression of CsbZIP5b, which can positively regulate the expression of CsSPS1. CsSPS is one of the key enzymes in the sucrose synthesis process. Increased expression of CsSPS1 leads to increased sucrose content. Simultaneously, CsbZIP5b can also positively regulate the expression of CsHSFA6B, a regulatory gene regulated by the upstream CsbZIP5b. CsHSFA6B itself positively regulates the expression of downstream citric acid-related genes (CsACO1 and CsACO3). CsACO1 and CsACO3 are two aconitase enzymes. Increased expression of CsACO1 and CsACO3 leads to reduced citric acid content. They can convert citric acid into isocitrate, which is gradually decomposed through the GABA branch. Therefore, the present invention identifies CsbZIP5b as a key gene that can simultaneously regulate sugar accumulation and acid reduction in citrus.

[0032] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the citrus CsbZIP5b gene in regulating sugar and / or acid metabolism in fruit, characterized in that: The sequence of the CsbZIP5b gene is shown as SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown as SEQ ID NO:

1.

2. Application of the citrus CsbZIP5b gene in the preparation of products for regulating fruit sugar or acid metabolism, characterized in that: The sequence of the CsbZIP5b gene is shown as SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown as SEQ ID NO:

1.

3. Application of the citrus CsbZIP5b gene in cultivating high-sugar, low-acid citrus varieties, characterized in that: The sequence of the CsbZIP5b gene is shown as SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown as SEQ ID NO:

1.

4. Application of the citrus CsbZIP5b gene in the preparation and cultivation of products of high-sugar, low-acid citrus varieties, characterized in that: The sequence of the CsbZIP5b gene is shown as SEQ ID NO: 1 or a gene sequence comprising the nucleotide sequence shown as SEQ ID NO:

1.

5. Use of a recombinant vector comprising the citrus CsbZIP5b gene according to any one of claims 1 to 4 in regulating sugar and acid metabolism in fruit, characterized in that: The recombinant vector is an overexpression vector or an RNA interference vector.

6. The recombinant vector according to claim 5, characterized in that The overexpression vector is constructed through the GoldenGate system, comprising: cloning the sequence of the citrus CsbZIP5b gene into a first vector, and inserting the sequence into the overexpression vector of the GoldenGate system after sequencing to obtain the overexpression vector.

7. The recombinant vector according to claim 5, characterized in that The RNA interference vector is constructed through the Gateway system, comprising: cloning the sequence of the citrus CsbZIP5b gene into an interference vector to obtain the RNA interference vector.

8. A method for regulating sugar and acid metabolism in fruit, characterized in that: include: Overexpression of the CsbZIP5b gene in fruits increased sucrose content and decreased citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO:

1.

9. A method for regulating sugar and acid metabolism in fruit, characterized in that: include: Inhibiting CsbZIP5b gene expression in fruits reduces sucrose content and increases citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO:

1.

10. A method for cultivating high-sugar, low-acid transgenic citrus, characterized in that: include: Constructing an Agrobacterium engineering bacterium containing a CsbZIP5b gene overexpression vector; introducing the vector into citrus fruit tissues by transient transformation; Screen transgenic plants and verify phenotypes by measuring sucrose or citric acid content; The nucleotide sequence of the CsbZIP5b gene is shown in SEQ ID NO: 1.

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