Fruit organic acid content regulation gene MdNAC9 and application thereof

Abstract

The invention belongs to the technical field of biological genetic engineering, and relates to a fruit organic acid content regulation gene MdNAC9 and application thereof, and the nucleotide sequence of the MdNAC9 gene is shown as SEQ ID NO.1. It is found for the first time that MdNAC9 participates in regulation and control of malic acid accumulation in the fruit development process, along with continuous development of fruits, the expression level of MdNAC9 is remarkably positively correlated with the content of malic acid in the fruits, through overexpression of MdNAC9 and construction of MdNAC9 transgenic apples and MdNAC9 transgenic tomatoes, it is found that the accumulation amount of malic acid in the fruits of the MdNAC9 transgenic apples and MdNAC9 transgenic tomatoes is increased, and the accumulation amount of malic acid in the fruits of the MdNAC9 transgenic apples and MdNAC9 transgenic tomatoes is increased. The result shows that the MdNAC9 can positively regulate the accumulation of the organic acids in the fruits and improve the flavor of the fruits.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological genetic engineering and relates to a gene for regulating the organic acid content in fruits MdNAC9 and its application. Background Art

[0002] Apples are an important economic crop, and their quality is crucial for market value and consumer acceptance. The sugar and organic acid content of fruits are one of the key factors determining the overall quality of apples. Apples with high acidity usually have a more refreshing taste and a more distinct flavor, which are suitable for consumers who like a balance of sour and sweet. At the same time, they also perform well in processed foods (such as apple cider vinegar and apple juice) because acidity can enhance the flavor and preservation of the products. Apples with low acidity are usually sweeter and have a softer taste, which are suitable for most consumers, especially children and the elderly. The acidity of apples not only affects their flavor and market positioning but also determines their applicability in fresh and processed products. By regulating the acidity of apples, the needs of different consumers can be better met, and the market competitiveness of apples can be enhanced.

[0003] Fruit acids are organic acids naturally present in fruits. The fruit acidity belongs to a quantitative trait controlled by multiple genes, and the genetic mechanism is relatively complex. In recent years, researchers have identified a series of major genes affecting fruit acidity accumulation in fruits such as apples by means of genetic map construction and QTL mapping technology. Among them, the Ma gene located at the top of linkage group 16 accounts for 17% - 42% of the acidity variation in apple fruits, and has received extensive attention and research. In addition, the organic acid content in apples can also be regulated by a series of acid metabolism-related enzymes and proton pump genes. Although there have been some studies on the mapping of candidate genes related to apple fruit acidity, the accumulation of fruit acidity is a complex mechanism, and there are still many important genes and regulatory networks related to the regulation of fruit acidity that have not been explored. Summary of the Invention

[0004] To solve the above problems, the present invention provides a gene for regulating the organic acid content in fruits MdNAC9 and its application, MdNAC9 which can positively regulate the accumulation of malic acid in fruits.

[0005] To achieve the above object, the specific technical solutions of the present invention are as follows: The first aspect of the present invention provides an MdNAC9 application of a gene in regulating the organic acid content in plant fruits, and the MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, the application includes increasing the organic acid content in plant fruits.

[0007] Furthermore, the method for increasing the organic acid content in plant fruits is as follows: By using transgenic technology or gene editing technology to increase the expression level of the MdNAC9 gene in plants, so as to enhance the organic acid content in plant fruits.

[0008] The second aspect of the present invention provides an MdNAC9 application of the protein encoded by the gene in regulating the organic acid content in fruits, the MdNAC9 nucleotide sequence of the gene is as shown in SEQ ID NO.1; the MdNAC9 amino acid sequence of the protein encoded by the gene is as shown in SEQ ID NO.2.

[0009] The third aspect of the present invention provides an MdNAC9 application of the recombinant plasmid containing the MdNAC9 gene in regulating the organic acid content in fruits, the

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

[0010] The fourth aspect of the present invention provides an MdNAC9 application of the recombinant bacterium containing the MdNAC9 gene in regulating the organic acid content in fruits, the

[0011] nucleotide sequence of the gene is as shown in SEQ ID NO.1. Furthermore, the above-mentioned fruits are apples, tomatoes, pears or peaches.

[0012] Furthermore, the above-mentioned organic acid is malic acid.

[0013] The fifth aspect of the present invention provides a method for cultivating plants with high malic acid content, including the following steps: Connect the above-mentioned MdNAC9 gene to an expression vector to obtain a recombinant plasmid; Heterologously express the recombinant plasmid in plant tissues through the Agrobacterium rhizogenes-mediated transformation method, and after cultivation, obtain plants with high malic acid content.

[0014] Furthermore, the expression vector is pCAMBIA1300 or pCAMBIA2300.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention discovers for the first time that MdNAC9 is involved in the regulation of malic acid accumulation during fruit development. As the fruit develops continuously, MdNAC9 the expression level is significantly positively correlated with the malic acid content in the fruit. By overexpressing MdNAC9 , constructing MdNAC9 transgenic apples and MdNAC9 transgenic tomatoes, it is found that MdNAC9 transgenic apples andMdNAC9 The accumulation of malic acid in the fruits of transgenic tomatoes increases, indicating that MdNAC9 it can positively regulate the accumulation of malic acid in fruits, improve fruit flavor, and play a potentially important role in optimizing fruit quality and directional selection breeding for fruit acidity regulation. Brief Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is a graph of malic acid content at different stages of apple fruit development.

[0018] Figure 2 For different stages of apple fruit development MdNAC9 Measurement results of the expression level.

[0019] Figure 3 For the process of apple fruit development MdNAC9 Schematic diagram of the correlation analysis between the expression level and the malic acid content; the abscissa is the relative content of malic acid in the metabolome, and the ordinate is MdNAC9 The relative expression level.

[0020] Figure 4 It is a subcellular localization map of MdNAC9. In the upper row of pictures in the figure, the leaf cell localization transformed with the empty vector; the lower row of pictures is MdNAC9 The leaf cell localization transformed; from left to right in each row of pictures are the green fluorescent protein picture, the red fluorescent protein picture, the bright field picture, the merged and superimposed picture of the green fluorescent protein picture, the red fluorescent protein picture and the bright field picture.

[0021] Figure 5 Is MdNAC9 The relative expression level of the gene in the control group (transformed with the empty vector), MdNAC9 The relative expression level and the phenotypic map of transgenic apple fruits; Figure 5 Figure A of Figure 5 is a physical picture of an apple fruit, MdNAC9 Figure B of MdNAC9 After overexpression of the gene in MdNAC9 The relative expression level in transgenic apple fruits; Control represents the control group, OE-1, OE-2 and OE-3 are p Three parallel samples of transgenic apples; ** indicates p <0.01, * indicates

[0022] Figure 6 For MdNAC9 The relative expression levels of the MdNAC9 gene in the control group (transformed with empty vector), MdNAC9 and the phenotypic diagram of the transgenic tomato fruits; Figure 6 Figure A of Figure 6 is a physical picture of the tomato fruit, MdNAC9 and Figure B of MdNAC9 is the relative expression level of the MdNAC9 gene in the transgenic tomato fruits after overexpression; Control represents the control group, and OE-4, OE-5, and OE-6 are p three parallel samples of transgenic tomatoes; * indicates

[0023] Figure 7 For MdNAC9 the effect of overexpressing the MdNAC9 gene on malic acid content in apple fruits; Control represents the control group, and OE-1, OE-2, and OE-3 are p three parallel samples of transgenic apples; * indicates

[0024] Figure 8 For MdNAC9 the effect of overexpressing the MdNAC9 gene on malic acid content in tomato fruits; Control represents the control group, and OE-4, OE-5, and OE-6 are p three parallel samples of transgenic tomatoes; ** indicates p <0.01, and * indicates Specific implementation manners

[0025] The following describes the specific implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] Apple is an important economic crop, and its quality is crucial for market value and consumer acceptance. The sugar and organic acid contents of fruits are one of the key factors determining the overall quality of apples. Fruit acid is a natural organic acid present in fruits, and fruit acidity is a quantitative trait controlled by multiple genes with a relatively complex genetic mechanism. Although there have been some studies on the mapping of candidate genes related to apple fruit acidity, the accumulation of fruit acidity is a complex mechanism, and many important genes and regulatory networks related to the regulation of fruit acidity have not been explored.

[0027] The present invention provides a gene for regulating the content of organic acids in fruits MdNAC9 and its application. Based on the results of transcriptome analysis of the genes of fruits of the cold-region small apple 'Jinhong' at different developmental stages, a candidate gene that may be involved in regulating the content of malic acid in fruits was obtained MdNAC9 . It was found through experiments MdNAC9 is involved in the regulation of malic acid accumulation during fruit development, and as the fruit develops continuously, MdNAC9 the expression level is significantly positively correlated with the content of malic acid in the fruit. By overexpressing MdNAC9 , transgenic apples MdNAC9 and MdNAC9 transgenic tomatoes were constructed. It was found that MdNAC9 the content of malic acid in the fruits of transgenic apples MdNAC9 and MdNAC9 transgenic tomatoes increased, indicating that

[0028] Example 1: MdNAC9 Relationship between gene expression pattern and malic acid accumulation in fruits Fruit samples of the cold-region 'Jinhong' small apple plants at 30 days, 60 days, 90 days, and 120 days after full bloom were collected respectively. The MdNAC9 gene expression levels of fruits at different stages and the content of malic acid in fruits at different stages were measured by qRT-PCR method, and three biological replicates were performed for each sample.

[0029] MdNAC9 The nucleotide sequence of the

[0030] gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. Atgagcggcagcgtcgcacctgaagaaggagatgcaatgcgtgtgaatgtgagccgactacctgggttcggattctcccccactgatgagttactggtcagctactacctgaagaacaagatagaggggacggactcccatttccgccacgtcatccctgaaatcgatgtctgcaagtacgagccctgcgatattcctgcattcttcccagaagttcatgggaaggagtggttcttctttagccggctcgattacaagtacaacggcactcgctgcaaccggaccacgggtcaaggcttttacaagatcacaggaatggatcgtaagatcagggctgaagaatccaaagctgtgattgggaagaagaggattctgacattctacgaaggtcgtctgccgaaatcaaagaagaccaattgggtactccatgagtataatctcacagaaactaaggtgagttctaaacccaccaagcagatgaactttgtcctctggcgcctgaagaacatgtcagctagttataagaagccgaagggtgatccaatccacggcgaattagctgatacaggtgctacctcggaagattatcaagctgctgtaagtgacgtgattgcagagccagtggaacagctgggataa。

[0031] SEQ ID NO.2: MSGSVAPEEGDAMRVNVSRLPGFGFSPTDELLVSYYLKNKIEGTDSHFRHVIPEIDVCKYEPCDIPAFFPEVHGKEWFFFSRLDYKYNGTRCNRTTGQGFYKITGMDRKIRAEESKAVIGKKRILTFYEGRLPKSKKTNWVLHEYNLTETKVSSKPTKQMNFVLWRLKNMSASYKKPKGDPIHGELADTGATSEDYQAAVSDVIAEPVEQLG*。

[0032] The malic acid content was determined by gas chromatography - mass spectrometry (GC - MS), and the specific determination method is as follows.

[0033] 1. Sample extraction: Take 0.1 g of the sample and extract malic acid with 1.4 mL of 75% (v / v) methanol solution, adding 400 ppm of libitol as the internal standard.

[0034] 2. Sample treatment: Shake the sample at 70 °C and 900 rpm for 30 min to ensure sufficient extraction; after centrifugation, separate the supernatant and transfer the supernatant into a mixture containing 750 μL of chloroform (CHCl3) and 1.4 mL of ddH2O, and mix again.

[0035] 3. Sample drying: After centrifuging the mixed liquid, separate the supernatant, and take 2 μL and 50 μL of the supernatant samples respectively for drying treatment.

[0036] 4. Derivatization treatment: Derivatize the dried sample with 40 μL of methoxyamine hydrochloride and 60 μL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA).

[0037] 5. GC-MS analysis: Use a GCMS-2010SE instrument to analyze the derivatized sample and determine the content of malic acid.

[0038] Figure 1 It is the diagram of malic acid content at different stages of apple fruit development. Figure 2 It is for the genes regulating the accumulation of fruit acids at different stages of apple fruit development. MdNAC9 Expression diagram. From Figure 1 and Figure 2 it can be seen that MdNAC9 has a relatively high expression level in the fruits 90 days after full bloom.

[0039] Figure 3 It is the schematic diagram of the correlation analysis between the expression level of MdNAC9 and the malic acid content during apple fruit development. From Figure 3 it can be seen that with the fruit development, the malic acid content shows a significant positive correlation with the MdNAC9 expression level.

[0040] The above results indicate that MdNAC9 is involved in the regulation of malic acid accumulation during apple fruit development.

[0041] Example 2: Subcellular localization of MdNAC9 Using the pCAMBIA1300-GFP vector, by the method of homologous recombination, select Kpn I and BamH I two restriction enzyme sites to construct the recombinant plasmid pCAMBIA1300- MdNAC9 to explore the subcellular localization of MdNAC9. The specific steps are as follows.

[0042] (1)Total RNA extraction and first-strand cDNA synthesis: The All-in-One Plant RNA Extraction Kit from ComWin Biotech Co., Ltd. (product number: CW2598S) was used to extract RNA from apple plants. First, the plant materials were washed clean with distilled water, blotted dry with filter paper, weighed according to the requirements in the kit, wrapped with tinfoil, and stored in an ultra-low temperature freezer at -80 °C. When needed, take them out and extract RNA according to the steps in the kit instructions. The reverse transcription kit from TransGen Biotech (product number: AE311-02) was used to synthesize the first-strand cDNA, and the specific steps refer to the instructions.

[0043] (2)PCR amplification: Using the synthesized first-strand cDNA as a template, design a specific primer combination for PCR amplification. Add each component required for the reaction into a PCR tube according to the system in Table 1 and flick the bottom of the tube gently to mix well. Set the PCR reaction conditions according to Table 2 to perform PCR amplification.

[0044] The specific primer combination includes MdNAC9-F and MdNAC9-R. The nucleotide sequence of MdNAC9-F is shown as SEQ ID NO.3, and the nucleotide sequence of MdNAC9-R is shown as SEQ ID NO.4; SEQ ID NO.3: 5’-ACGGGGGACGAGCTCGGTACCATGGCTGCTCCTACAACCCCAA-3’; SEQ ID NO.4: 5’-GGTGTCGACTCTAGAGGATCCCGGCCCATCCATGTTCC-3’.

[0045] Table 1 PCR reaction system Table 2 PCR reaction conditions Note: “-” in the table indicates no cycle.

[0046] (3)Electrophoresis: Perform 1wt% agarose gel electrophoresis on the PCR amplification products, select a single band with higher brightness for gel cutting and gel extraction. The gel extraction was carried out using the DNA Product Purification Kit from ComWin Biotech Co., Ltd. (product number: CW2301M).

[0047] (4)Gel extraction: The recovered DNA fragment and the pCAMBIA1300-GFP vector were digested with two restriction enzymes respectively. After digestion, the digestion products were placed in a water bath at 80 °C for 15 min to inactivate the enzymes thermally and stored at 4 °C for later use; The double digestion systems of the pCAMBIA1300-GFP vector and the DNA fragment are shown in Table 3 and Table 4 respectively.

[0048] Table 3 Double digestion system of pCAMBIA1300-GFP vector Table 4 Double digestion system of DNA fragment (5) Ligation: The digested DNA fragment and the digested pCAMBIA1300-GFP vector were ligated according to the ligation system shown in Table 5 at a temperature of 22°C.

[0049] Table 5 Ligation system of digestion products (6) Verification: Use PCR technology to detect MdNAC9 whether it is ligated to the pCAMBIA1300-GFP vector. When the amplified target band size is consistent with the MdNAC9 CDS size, it proves that the vector construction is successful, and finally the successfully ligated MdNAC9 recombinant plasmid pCAMBIA1300- MdNAC9 is screened.

[0050] The obtained recombinant plasmid pCAMBIA1300- MdNAC9 was injected into tobacco leaf cells. After culturing for 3 days, the fluorescence of leaf epidermal cells was observed using a confocal microscope, with tobacco leaf cells injected with the pCAMBIA1300-GFP vector (leaves transformed with the empty vector) as the control.

[0051] As Figure 4 shown, the leaf cells transformed with the empty vector were covered with green fluorescence, indicating that the empty vector can be used, while the leaf cells transformed with the target protein MdNAC9 were localized in the nucleus, indicating that MdNAC9 is a nuclear localization protein.

[0052] Example 3: Analysis of the expression level of MdNAC9 in transgenic apples and transgenic tomato fruits To explore the function of MdNAC9 in apple growth and development, the transient expression technology was used to transiently activate gene expression in apple fruits to analyze its effects on gene expression levels and organic acids.

[0053] At the same time, to clarify the specific biological function of MdNAC9 during the malic acid accumulation process, the present invention used the recombinant plasmid pCAMBIA1300- MdNAC9It was heterologously expressed in "Little Tom" tomato plants through Agrobacterium rhizogenes-mediated transformation, and three stable overexpressing transgenic tomato lines, OE-4, OE-5, and OE-6, were obtained.

[0054] Fruit samples of transgenic apples and transgenic tomatoes were collected and determined by qRT-PCR MdNAC9 gene expression levels.

[0055] Select MdNAC9 conserved regions to design specific primers for real-time fluorescence quantitative PCR. MdNAC9 The quantitative primer set for

[0056] contains a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.6. SEQ ID NO.5: 5’-CAAGTACAACGGCACTCGCT-3’;

[0057] Using the cDNA prepared in Example 2 as a template, BlazeTaq TM SYBR ® Green qPCRMix 2.0 real-time fluorescence quantitative PCR detection reagent (product number: QP031) from Guangzhou Yijin Biotechnology was used. Each component was added according to Table 6, and real-time fluorescence quantitative PCR analysis of the MdNAC9 gene was performed on a Bio-Rad CFX96 fluorescence quantitative PCR instrument. The PCR reaction conditions are shown in Table 7, and the data obtained were analyzed by the 2 -ΔΔCT method.

[0058] Table 6 qPCR component table Table 7 qPCR reaction conditions Note: "-" in the table indicates no cycle.

[0059] MdNAC9 The results of the expression levels of the Figure 5 and Figure 6 genes in transgenic apple and tomato fruits are shown in MdNAC9 and MdNAC9 Compared with the wild type, overexpression of the

[0060] gene can significantly increase the expression level of the MdNAC9 gene in apple and tomato plants. To clarifyMdNAC9 Regarding its specific biological functions during malic acid accumulation, the present invention further determined the malic acid content in the transgenic apples cultivated in Example 3 MdNAC9 and MdNAC9 the transgenic tomato fruits. The malic acid content was determined by gas chromatography-mass spectrometry (GC-MS). The specific determination method is as follows: 1. Sample extraction: Take 0.1 g of the sample, extract malic acid with 1.4 mL of 75% (v / v) methanol solution, and add 400 ppm ribitol as an internal standard.

[0061] 2. Sample treatment: Shake the sample at 70 °C and 900 rpm for 30 min to ensure sufficient extraction; after centrifugation, separate the supernatant, and transfer the supernatant into a mixture containing 750 μL of chloroform (CHCl3) and 1.4 mL of ddH2O, and mix again.

[0062] 3. Sample drying: After centrifuging the mixed liquid, separate the supernatant, and take 2 μL and 50 μL of the supernatant samples respectively for drying treatment.

[0063] 4. Derivatization treatment: Derivatize the dried sample with 40 μL of methoxyamine hydrochloride and 60 μL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA).

[0064] 5. GC-MS analysis: Use a GCMS-2010SE instrument to analyze the derivatized sample and determine the malic acid content.

[0065] The detection results are as Figure 7 and Figure 8 shown, Figure 7 and Figure 8 and the analysis results indicate that overexpression of the MdNAC9 gene can significantly increase the malic acid content in apple fruits and tomato fruits.

[0066] It should be noted that when the present invention involves numerical ranges, it should be understood that any value between the two endpoints of each numerical range and either of the two endpoints can be selected. Since the steps and methods adopted are the same as those in the examples, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. MdNAC9 Use of a gene in regulating the organic acid content of plant fruits, characterized in that, The said MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use according to claim 1 MdNAC9 Application of the gene in regulating the organic acid content of plant fruits, characterized in that The application includes increasing the organic acid content in plant fruits; the method for increasing the organic acid content in plant fruits is as follows: by transgenic technology or gene editing technology, the expression level of the MdNAC9 gene is increased to enhance the organic acid content in plant fruits.

3. An MdNAC9 application of a protein encoded by a gene in regulating the organic acid content of fruits, characterized in that The said MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.1; the said MdNAC9 amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. Application of a recombinant plasmid containing MdNAC9 gene in regulating fruit organic acid content, characterized in that The MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. Application of a recombinant bacterium containing MdNAC9 gene in regulating the organic acid content of fruits, characterized in that The said MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application according to claims 1 to 5, characterized in that The fruit is an apple, tomato, pear or peach.

7. The application according to claims 1 to 5, characterized in that The organic acid is malic acid.

8. A method for cultivating plants with high malic acid content, characterized in that, It includes the following steps: Connect the gene described in claim 1 MdNAC9 to an expression vector to obtain a recombinant plasmid; The recombinant plasmid is heterologously expressed in plant tissues by the Agrobacterium rhizogenes-mediated transformation method, and after cultivation, a plant with a high malic acid content is obtained.

9. The method according to claim 8, wherein The expression vector is pCAMBIA1300 or pCAMBIA2300.

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