Gene for regulating and controlling malic acid accumulation of loquat fruits and application of gene
By cloning and overexpressing the EjTCP4 gene in loquat fruit, the problem of unclear regulation of malic acidity in loquat fruit was solved, significantly increasing malic acid content and improving fruit flavor quality, providing theoretical guidance for loquat fruit breeding.
Patent Information
- Application Number
- CN202510539359.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-27
- Publication Date
- 2025-10-31
AI Technical Summary
The existing technology does not understand the mechanism of acidity regulation in loquat fruit and lacks targeted genetic improvement targets, resulting in a lack of effective methods for regulating the acidity of loquat fruit.
The key gene EjTCP4 involved in malic acid accumulation in loquat fruit was cloned and verified. By overexpressing EjTCP4 in tobacco through genetic engineering, the malic acid content was significantly increased, verifying its positive regulatory function.
The function of the EjTCP4 gene in regulating malic acid accumulation in loquat fruit was clarified, which significantly increased the malic acid content in the fruit, improved the flavor quality of the fruit, and provided a theoretical basis for loquat fruit breeding.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and in particular relates to a gene that regulates the accumulation of malic acid in loquat fruit and its application. Background Technology
[0002] Malic acid, an important metabolite in plants, involves several key metabolic pathways in its biosynthesis, including the tricarboxylic acid cycle, glycolysis, gluconeogenesis, glyoxylate cycle, and the synthesis of some secondary metabolites. In loquat (Eriobotrya japonica) fruit, malic acid is the most abundant organic acid component, not only determining the fruit's nutritional value but also serving as a key indicator of its flavor and quality.
[0003] Studies have shown that relying solely on the content of soluble sugars or organic acids is insufficient to comprehensively assess the flavor characteristics of fruits, while an appropriate ratio of soluble sugars to organic acids (i.e., the sugar-acid ratio) significantly affects the taste quality of fruits. The TCP family of transcription factors is a class of important regulatory factors widely involved in plant growth, development, and responses to abiotic stresses. Recent studies have found that members of this family play a crucial role in the regulation of soluble sugar metabolism in fruits.
[0004] Loquat fruit is the fruit of the loquat tree, and it has a sweet taste. Loquat fruit is soft and juicy, with a sweet and sour flavor. With the development and application of biotechnology, today's loquats are even sweeter and have a delicate texture. Loquat fruit is rich in various nutrients needed by the human body, such as citric acid and malic acid, which can stimulate the secretion of digestive juices after consumption, thereby increasing appetite and promoting digestion. People with poor appetite or indigestion can eat loquat regularly, as it has stomach-strengthening and digestive-aiding effects.
[0005] Therefore, how to regulate the amount of malic acid in loquat fruit is extremely important for the improvement and breeding of loquat fruit. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention provides a gene for regulating malic acid accumulation in loquat fruit and its application, thus solving at least one of the technical problems raised in the background. The specific solution of this invention is as follows:
[0007] On the one hand, it provides a gene that regulates malic acid accumulation in loquat fruit and its application, which solves the technical problems of unclear acidity regulation mechanism and lack of targeted genetic improvement targets in existing technologies, and improves the breeding method for targeted improvement of loquat acidity.
[0008] This invention provides a gene that regulates the accumulation of malic acid in loquat fruit, the nucleotide sequence of which is shown in SEQ:NO.1.
[0009] Preferred primers for cloning the full-length EjTCP4 sequence are SEQ:NO.2 and SEQ:NO.3, and primers for gene expression analysis are SEQ:NO.4 and SEQ:NO.5.
[0010] This invention provides an application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit, in genetic engineering.
[0011] This invention provides the application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit, in increasing malic acid content in loquat fruit.
[0012] This invention provides an application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit, in loquat quality selection.
[0013] The beneficial effects of this invention are:
[0014] This invention identifies for the first time the key gene EjTCP4 involved in malic acid accumulation in loquat fruit. Bioinformatics analysis shows that EjTCP4 belongs to the CIN subfamily of the plant TCP transcription factor family. Real-time quantitative PCR analysis of EjTCP4 expression patterns in different loquat varieties revealed a significant positive correlation between its expression level and malic acid content (p<0.05). To further verify the function of EjTCP4, the complete coding sequence of this gene was obtained by PCR amplification, and a plant overexpression vector was constructed. Heterologous overexpression of EjTCP4 in tobacco (Nicotiana tabacum) showed that the malic acid content in overexpressing tobacco plants was significantly higher than that in the control (p<0.01), fully demonstrating the positive regulatory function of EjTCP4 on malic acid accumulation. The expression of the EjTCP4 gene not only significantly affects malic acid metabolism in loquat fruit but also improves fruit flavor quality by regulating the sugar-acid ratio. This invention provides a new theoretical basis for the functional study of the TCP gene family in fruit quality regulation, and has important theoretical and practical guiding significance for the breeding of loquat fruit flavor quality improvement. Attached Figure Description
[0015] Figure 1 Malic acid content in different varieties of loquat fruit.
[0016] Figure 2 Expression levels of EjTCP4 in different varieties of loquat fruit.
[0017] Figure 3 Correlation analysis of malic acid content and EjTCP4 expression in loquat fruits of different varieties.
[0018] Figure 4 Tobacco plants that overexpress EjTCP4.
[0019] Figure 5 Overexpression of EjTCP4 significantly increased the malic acid content in tobacco plants. Detailed Implementation
[0020] The technical solution of the present invention will be described in detail below through specific embodiments. It is clearly stated here that these embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention.
[0021] This invention provides a gene EjTCP4 that regulates malic acid accumulation in loquat fruit, the nucleotide sequence of which is shown in SEQ:NO.1.
[0022] The primers used to clone the full-length EjTCP4 sequence are SEQ:NO.2 and SEQ:NO.3, and the primers used for gene expression analysis are SEQ:NO.4 and SEQ:NO.5.
[0023] The gene's functional characteristics are as follows:
[0024] 1. The nucleotide sequence of the gene is shown in SEQ:NO.1.
[0025] 2. Gene expression characteristics: The higher the expression of EjTCP4, the higher the malic acid content of loquat fruit. Gene expression is significantly positively correlated with the accumulation of malic acid in loquat fruit.
[0026] 3. Overexpression of EjTCP4 in tobacco significantly increased the accumulation of malic acid in tobacco plants.
[0027] Another objective of this invention is to provide a method for screening relevant functional target genes and clarifying the function of target genes through gene function verification. TCP members related to malic acid accumulation were screened through gene family analysis and correlation analysis. Then, transient expression vectors were constructed using the sequences SEQ:NO.2 and SEQ:NO.3. Using genetic engineering techniques, tobacco plants overexpressing EjTCP4 were obtained, significantly increasing the malic acid content in the tobacco plants, thus verifying that EjTCP4 positively regulates malic acid accumulation in loquat fruit.
[0028] Another objective of this invention is to provide an application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit, in increasing malic acid levels in loquat fruit.
[0029] Another objective of this invention is to apply the gene EjTCP4, which regulates malic acid accumulation in loquat fruit, to loquat quality selection.
[0030] The present invention will be further described below with reference to specific embodiments and accompanying drawings:
[0031] Example 1: Cloning of the EjTCP4 gene
[0032] (I) Experimental Methods
[0033] EjTCP4, a TCP family member significantly positively correlated with malic acid accumulation, was screened through gene family analysis and correlation analysis. Primer pairs SEQ:NO.2 and SEQ:NO.3 were designed, and PCR amplification was performed using loquat cDNA as a template to obtain the EjTCP4 sequence SEQ:NO.1, which was then verified by agarose gel electrophoresis. The PCR reaction system consisted of 25 μl of the following components: 12.5 μl P510 enzyme, 9.5 μl ddH2O, 1 μl each of forward and reverse primers (10 μM), and 1 μl cDNA. The PCR reaction program was as follows: 98℃ for 30 s; 98℃ for 10 s, 58℃ for 5 s, 72℃ for 15 s extension, for 35 cycles; final extension at 72℃ for 1 min, and storage at 4℃.
[0034] (II) Experimental Results
[0035] The EjTCP4 sequence SEQ:NO.1, which matches the loquat fruit genome, was obtained by agarose gel electrophoresis.
[0036] Example 2: There is a significant positive correlation between EjTCP4 expression and malic acid content in loquat fruit.
[0037] (I) Experimental Methods
[0038] 1. Loquat fruit material
[0039] Different varieties of loquat were harvested at maturity, including white-fleshed varieties 'A010' and 'Yingshuang', and red-fleshed varieties 'Algeria', 'Zhehong 16', and 'Longmen'. The peel and pit were removed from the fruits, and the pulp tissue was frozen in liquid nitrogen and then stored at -80°C until use. Three biological replicates were set up, with at least five fruits in each replicate.
[0040] 2. RNA extraction and cDNA synthesis
[0041] Total RNA was extracted from loquat fruit using the CTAB method. 1.0 μg of RNA was then processed according to PrimeScript. TM The RT reagent kit with gDNA Eraser (Perfect Real Time) is used to synthesize cDNA according to the instructions.
[0042] 3. Gene expression
[0043] Loquat EjACT (SEQ:NO.6) was used as an internal reference gene, with primers SEQ:NO.7 and SEQ:NO.8, and EjTCP4 primers SEQ:NO.4 and SEQ:NO.5. The real-time quantitative PCR (RT-qPCR) reaction system included 10 μl TB Green Primix ExTaq. TM II (Tli RNaseH Plus), 1 μl each of forward and reverse primers (10 μM), 2 μl cDNA, 6 μl ddH2O. The reaction program was 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles. The instrument used was a BLOER-LineGene 9600 series real-time quantitative PCR instrument, and each assay included a negative control using H2O as a template.
[0044] 4. Extraction and analysis of malic acid content in loquat fruit
[0045] Malic acid extraction: Weigh 0.1g of the ground fruit into a 1.5mL spiral centrifuge tube, add 1.4mL of chromatographic methanol (pre-cooled at -20℃), seal the tube, vortex for 1min, short, and place at 70℃ for 15min in a 950rpm metal bath with constant temperature shaking. Centrifuge at 10000g for 10min at 4℃, aspirate the supernatant into a 10mL centrifuge tube, add 750μL of chloroform (pre-cooled at -20℃) and 1.5mL of double-distilled water (pre-cooled at 4℃), vortex for 1min, centrifuge at 2200g for 10min at room temperature, and collect 1mL of the supernatant for organic acid component analysis.
[0046] Derivatization: Pipette 100 μL of the extracted supernatant into a 1.5 mL spiral centrifuge tube, add 10 μL of ribitol (2 mg / mL) as an internal standard, and short-cycle at 30 °C under vacuum for 5 h. Dissolve the precipitate thoroughly with 60 μL (20 mg / mL dissolved in pyridine) of methoxyamine hydrochloride, seal the tube, vortex, short-cycle at 37 °C for 1.5 h, and then shake at 950 rpm in a metal bath. Add 40 μL of LSTFA, vortex, seal the tube, short-cycle at 37 °C for 30 min, and then shake at 950 rpm in a metal bath. Transfer the sample to a sample vial; it is ready for gas chromatography (GC) detection.
[0047] Gas chromatography (GC) detection: Post-injector: 10 μL syringe volume, 1 μL injection volume; pre-injection sample washing 3 times, followed by washing 3 times with anhydrous ethanol / 95% ethanol and chromatographic methanol; post-injection washing 3 times with anhydrous ethanol / 95% ethanol and chromatographic methanol; sample aspiration 6 times, sampling depth 6 mm. Post-injector: Heater setpoint 250℃, pressure 13.5 psi, total N2 flow rate 13 mL / min, septum purge flow rate 3 mL / min, split ratio 10:1, carrier gas saving on: flow rate 20 mL / min, time 3 min. Column: Pressure 13.5 psi, flow rate 1 mL / min. Column oven temperature: Initial value 100℃, hold for 1 min; increase to 185℃ at 2.5℃ / min; increase to 190℃ at 0.35℃ / min; increase to 250℃ at 8℃ / min, hold for 5 min; increase to 280℃ at 5℃ / min, hold for 3 min (total run time 70.7 min), then run at 100℃ for 1 min (HP-5 column can withstand a maximum temperature of 325℃). Pre-detector: Heater 280℃, H2 flow rate 40 mL / min, air flow rate 400 mL / min, N2 flow rate 40 mL / min.
[0048] (II) Experimental Results
[0049] Tests on mature fruits of five main loquat varieties showed (see attached) Figure 1 The malic acid content in the fruit of the 'Algeria' red-fleshed loquat variety reaches 13.35 ± 0.65 mg / g. -1 FW (fatty acid content) was significantly higher than other varieties (p<0.05). The white-fleshed variety 'Yingshuang' and the red-fleshed variety 'Zhehong 16' had the lowest malic acid content, at 5.68±0.19 mg g / kg, respectively. -1 FW and 5.93±0.19mg g -1 FW. Inter-varietal differences reached 2.3-fold, indicating a significant genetic regulatory basis. qPCR detection revealed (see attached...) Figure 2 The expression level of EjTCP4 in 'Algeria' was 2.62-4.27 times that of the low-acid varieties 'Yingshuang' and 'Zhehong 16' (p<0.05), and it showed a highly significant positive correlation with malic acid content (R=0.983**). (Appendix) Figure 3 ).
[0050] Example 4: Overexpression of EjTCP4 promotes malic acid accumulation in tobacco.
[0051] (I) Experimental Methods
[0052] 1. Carrier Construction
[0053] Using primer pairs SEQ:NO.2 and SEQ:NO.3, the CDS sequence of EjTCP4 was cloned. The PCR system and reaction procedure were the same as in Example 1. The expression vector pGreen 002962-SK was double-digested with restriction endonucleases Hind III and BamHI. The target PCR product was then ligated to the linearized vector via homologous recombination. The ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were screened on LB medium containing kanamycin (50 μg / mL), and the correctness of the recombinant plasmid was verified by colony PCR and sequencing. Positive clones with correct sequence verification were selected, and the recombinant plasmid was extracted and transformed into *Agrobacterium* GV3101::pSoup competent cells using electroporation. Finally, the recombinant plasmid was introduced into tobacco plants using the *Agrobacterium* injection method.
[0054] 2. Identification of plants with transient expression
[0055] Leaves from tobacco plants transformed with EjTCP4 were collected, and total RNA was extracted from the tobacco plants using TRIzol (Invitrogen). The cDNA synthesis process was as described in Examples 1 and 2. For RT-qPCR detection, the primer pair for EjTCP4 was SEQ:NO.4 and SEQ:NO.5, and the common tobacco NtACT-7 gene (SEQ:NO.9) was used as an internal reference gene, with primers SEQ:NO.10 and SEQ:NO.11. The RT-qPCR reaction system and procedure were the same as in Example 2.
[0056] 3. Detection and analysis of malic acid content
[0057] The thoroughly ground tobacco leaves were used for the analysis of organic acid content, and the extraction and detection process was the same as in Example 2.
[0058] Take 750 μL of supernatant extract into a 1.5 ml spiral centrifuge tube, add 10 μL of ribitol (2 mg / ml) as an internal standard for derivatization, and then determine the organic acid content by gas chromatography (GC). The chromatographic column is HP-5, and the HP-5 method parameters are the same as in Example 2.
[0059] (II) Experimental Results
[0060] Compared with the control (tobacco plants without EjTCP4), tobacco plants overexpressing EjTCP4 showed a significant increase in malic acid content, as shown in the instruction manual. Figure 4 and Figure 5 As shown.
[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
[0062]
[0063]
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[0065]
Claims
1. A gene regulating malic acid accumulation in loquat fruit, characterized in that, The nucleotide sequence of the gene is shown in SEQ:NO.
1.
2. The gene for regulating malic acid accumulation in loquat fruit according to claim 1, characterized in that, The primers used to clone the full-length EjTCP4 sequence are SEQ:NO.2 and SEQ:NO.3, and the primers used for gene expression analysis are SEQ:NO.4 and SEQ:NO.
5.
3. The application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit as described in claim 1 or 2, in genetic engineering.
4. The application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit as described in claim 1 or 2, in increasing malic acid content in loquat fruit.
5. The application of the gene EjTCP4, which regulates malic acid accumulation in loquat fruit as described in claim 1 or 2, in loquat quality selection.