Gene participating in waxberry fruit sugar transport and application thereof
By cloning and overexpressing the bayberry fruit sugar transport gene MrSWEET5a, the problem of sugar accumulation regulation in bayberry fruit was solved, the sugar content and quality of the fruit were significantly improved, and a molecular basis was provided for bayberry quality improvement.
Patent Information
- Application Number
- CN202510785113.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-09-26
AI Technical Summary
Existing technologies make it difficult to effectively regulate the accumulation and transport of sugar in bayberry fruit, which affects the quality of the fruit and its post-harvest storage performance.
The gene MrSWEET5a involved in sugar transport in bayberry fruit was cloned and identified. Through genetic engineering, this gene was overexpressed in tomato fruit, significantly promoting sugar accumulation.
By positively regulating the sugar transport process in bayberry fruit, the sugar content in the fruit is significantly increased, the flavor quality of the fruit is improved, and a molecular basis is provided for the selection and breeding of high-quality varieties.
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Figure CN120699982A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant molecular biotechnology and genetic engineering, and in particular relates to a gene involved in sugar transport in bayberry fruit and an application thereof. Background Art
[0002] In plant physiological metabolism, carbohydrates not only serve as core substrates for energy metabolism and major components of cell wall structure, but also play important signaling roles, regulating both primary and secondary metabolism. As the primary material basis for fruit sweetness, sugars and their derivatives, sugar alcohols, play a crucial role in fruit quality. Research has shown that the main soluble sugars accumulated in fruit are sucrose, fructose, and glucose, along with smaller amounts of sugar alcohols such as sorbitol and inositol. These carbohydrates, as important substrates for respiratory metabolism, can be converted into each other through the sugar metabolism pathway, directly influencing the fruit's taste, flavor, nutritional quality, and postharvest storage properties. During postharvest fruit physiological processes, sugars, as metabolic substrates, are closely linked to the ripening and aging processes of fruit, and their accumulation levels are meticulously regulated by sugar metabolism and transport processes. Specifically, soluble sugars are transported from the sieve tube-companion cell complex to the converging cells via specific sugar transporters or plasmodesmata. They are ultimately stored in the vacuole via sucrose transporters and monosaccharide transporters located on the tonoplast.
[0003] Sugar efflux transporters (SWEETs) are an important family of sugar transporters that play multiple roles in plant growth and development: regulating the growth and development of tissues and organs by mediating the distribution and utilization of nutrients; maintaining intracellular sugar homeostasis; and participating in plant responses to biotic stresses (such as pathogen infection) and abiotic stresses. Analysis of the Myrica rubra genome and transcriptome sequencing data revealed that Myrica rubra contains 16 SWEET gene family members. The goal was to screen and identify SWEET members involved in fruit sugar transport to enrich the regulatory network for sugar accumulation.
[0004] Therefore, regulating the soluble sugar content of bayberry fruit through molecular means will provide a new genetic improvement strategy for the breeding of high-quality varieties. Summary of the Invention
[0005] Based on the above technical problems, the present invention provides a gene involved in sugar transport in bayberry fruit and its application, which solves at least one technical problem raised by the background technology. The specific solutions of the present invention are as follows:
[0006] One object of the present invention is to provide a gene involved in sugar transport in bayberry fruit, the nucleotide sequence of the gene is shown in SEQ: NO.1.
[0007] Preferably, the primers for cloning the full-length MrSWEET5a sequence are SEQ: NO.2 and SEQ: NO.3, and the primers for conducting gene expression analysis are SEQ: NO.4 and SEQ: NO.5.
[0008] Another object of the present invention is to provide an application of a gene MrSWEET5a involved in sugar transport in bayberry fruit in genetic engineering.
[0009] Another object of the present invention is to provide an application of MrSWEET5a, a gene involved in sugar transport in bayberry fruit, in increasing the sugar content in the fruit.
[0010] Another object of the present invention is to provide an application of a gene MrSWEET5a involved in sugar transport in bayberry fruit in bayberry quality breeding.
[0011] The beneficial effects of the present invention are:
[0012] The present invention clarifies the key gene MrSWEET5a involved in sugar transport in bayberry (Myrica rubra) fruit, and successfully amplifies the full-length sequence of the gene using PCR technology. In view of the technical bottlenecks in genetic transformation of bayberry as a perennial woody plant, the present invention uses the transgenic system of the model plant tomato to perform heterologous verification of bayberry gene function. MrSWEET5a was overexpressed in tomato fruit, and the results showed that the soluble sugar content in the transgenic tomato fruit was significantly increased. Further studies have shown that MrSWEET5a affects the sugar accumulation of the fruit by positively regulating the sugar transport process during bayberry fruit development. The findings of this study not only enrich the functional research of the SWEET gene family in fruit quality regulation, provide a new theoretical basis for the fruit sugar metabolism regulatory network, but also have important application value in bayberry quality improvement breeding, and provide a molecular basis for cultivating new high-quality bayberry varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 : Expression changes of MrSWEET5a during the development and ripening of bayberry.
[0014] Figure 2 : Changes of sucrose content during the development and ripening of bayberry.
[0015] Figure 3 : Correlation between MrSWEET5a expression and sucrose content in Bayberry.
[0016] Figure 4 : Changes in MrSWEET5a expression in transgenic tomato fruits.
[0017] Figure 5 : Changes in sucrose content in transgenic tomato fruits. DETAILED DESCRIPTION
[0018] The technical solutions of the present invention are described in detail below through specific embodiments. It is clearly stated here that these embodiments are only used for illustration and are not to be construed as limiting the scope of the present invention.
[0019] An embodiment of the present invention provides a gene MrSWEET5a for sugar transport in bayberry fruit, the nucleotide sequence of which is shown in SEQ: NO.1.
[0020] The primers used to clone the full-length MrSWEET5a sequence were SEQ: NO.2 and SEQ: NO.3, and the primers used for gene expression analysis were SEQ: NO.4 and SEQ: NO.5.
[0021] The characteristic functions of this gene are as follows:
[0022] 1. The nucleotide sequence of the gene is shown in SEQ: NO.1.
[0023] 2. Gene expression characteristics: As bayberry fruit matures, MrSWEET5a expression continues to increase, and gene expression is positively correlated with sugar accumulation in the fruit.
[0024] 3. Overexpression of MrSWEET5a in tomato fruits significantly promoted sugar accumulation.
[0025] Another object of the present invention is to provide the use of the MrSWEET5a gene in genetic engineering. Specifically, SEQ: NO. 6 and SEQ: NO. 7 sequences are used to construct a transgenic vector. Overexpression of MrSWEET5a in tomato fruit significantly increases sugar content and improves the flavor quality of the fruit. Transgenic tomato plants overexpressing MrSWEET5a were obtained using genetic engineering techniques. Overexpression of MrSWEET5a significantly promotes sugar accumulation.
[0026] Another object of the present invention is to provide an application of MrSWEET5a, a gene involved in sugar transport in bayberry fruit, in increasing the sugar content in the fruit.
[0027] Another object of the present invention is to provide an application of a gene MrSWEET5a involved in sugar transport in bayberry fruit in bayberry quality breeding.
[0028] The present invention will be further described below with reference to specific embodiments and accompanying drawings:
[0029] Example 1: MrSWEET5a gene cloning
[0030] (1) Experimental methods
[0031] The gene MrSWEET5a was identified through transcriptome screening. Primer pairs SEQ:NO.2 and SEQ:NO.3 were designed and amplified using Myrica rubra cDNA as a template. The MrSWEET5a sequence SEQ:NO.1 was obtained by PCR and verified by sequencing. The PCR reaction system was 25 μL and consisted of: 12.5 μL P510 enzyme, 9.5 μL ddH2O, 1 μL each of the upstream and downstream primers (10 μM), and 1 μL cDNA. The PCR protocol was 98°C for 30 seconds, followed by 35 cycles of 98°C for 10 seconds, 58°C for 5 seconds, and an extension at 72°C for 15 seconds. Finally, an extension at 72°C for 1 minute was performed, and the cells were stored at 4°C.
[0032] (2) Experimental results
[0033] After sequencing verification, the MrSWEET5a sequence SEQ: NO.1 was obtained, which matched the bayberry fruit genome database.
[0034] Example 2: Correlation between MrSWEET5a expression in bayberry fruit and fruit sugar content
[0035] (1) Experimental methods
[0036] 1. Bayberry fruit materials
[0037] Bayberry fruits were collected from the Lin'ge Family Ecological Farm in Lin'an District, Hangzhou, Zhejiang Province. Samples were collected at four key developmental stages (S1-S4) at 58, 65, 72, and 80 days after full bloom. To ensure the reliability of the experimental results, three biological replicates were set up at each sampling point, with at least six fruits per replicate. Samples were immediately flash-frozen in liquid nitrogen after collection and stored in a -80°C ultra-low temperature freezer until subsequent analysis.
[0038] 2. RNA Extraction and cDNA Synthesis
[0039] Total RNA was extracted from bayberry fruit using the CTAB method and analyzed using PrimeScript TM cDNA was synthesized using the RT reagent Kit with gDNAEraser (Perfect Real Time) according to the manufacturer's instructions. RNA quality and concentration were determined using the A260 / A280 absorbance ratio using a NanoDrop One spectrophotometer.
[0040] 3. Gene Expression
[0041] The MrActin (SEQ: NO.11) of Myrica rubra was used as the internal reference gene, the primers were SEQ: NO.12 and SEQ: NO.13, and the primers for MrSWEET5a were SEQ: NO.4 and SEQ: NO.5. The real-time quantitative PCR (RT-qPCR) reaction system included 10 μl TBGreen Primix Ex Taq TM II (Tli RNaseH Plus), 1 μL each of upstream and downstream primers (10 μM), 2 μL cDNA, and 6 μL ddH₂O. The reaction procedure was 95°C for 30 s, followed by 95°C for 10 s and 60°C for 30 s, for 40 cycles. The instrument used was a BLOER-LineGene 9600 series real-time fluorescence quantitative PCR instrument. A negative control using ddH₂O as the reaction template was included in each assay.
[0042] RNA from bayberry fruits at different maturity stages was used as material for transcriptome sequencing to analyze the expression patterns of SWEET gene family members in bayberry fruits.
[0043] 4. Detection and analysis of soluble sugar content
[0044] Soluble sugar extraction: Weigh 0.1 g of ground fruit into a 1.5 mL spiral centrifuge tube, add 1.4 mL of chromatographic methanol (pre-cooled at -20°C), seal the tube, vortex for 1 minute, shortly, place in a 70°C metal bath with constant temperature shaking at 950 rpm for 15 minutes. Centrifuge at 10,000 g for 10 minutes at 4°C, transfer the supernatant to a 10 mL centrifuge tube, add 750 μL of chloroform (pre-cooled at -20°C) and 1.5 mL of double-distilled water (pre-cooled at 4°C), vortex for 1 minute, and centrifuge at 2,200 g for 10 minutes at room temperature. Aspirate 1 mL of the supernatant for organic acid component analysis.
[0045] 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 evaporate briefly at 30°C under vacuum for 5 h. Dissolve the precipitate thoroughly with 60 μL of methoxyamine hydrochloride (20 mg / mL dissolved in pyridine), seal the tube, vortex, and incubate briefly at 37°C for 1.5 h, then shake in a metal bath at 950 rpm. Add 40 μL of BSTFA, vortex, seal the tube, and incubate briefly at 37°C for 30 min, then shake in a metal bath at 950 rpm. Transfer the tube to a sample vial and use for gas chromatography (GC) analysis.
[0046] Gas chromatography (GC) detection: Rear injector: 10 μL syringe, 1 μL injection volume; pre-injection sample washes three times with anhydrous ethanol / 95% ethanol and chromatographic methanol; post-injection washes three times with anhydrous ethanol / 95% ethanol and chromatographic methanol; sample aspirations six times, sampling depth 6 mm. Rear inlet: heater settings 250°C, pressure 13.5 psi, N2 total flow 13 mL / min, septum purge flow 3 mL / min, split ratio 10:1, gas saver on: flow 20 mL / min, time 3 min. Column: pressure 13.5 psi, flow 1 mL / min. Column oven temperature: Initial value 100°C, hold for 1 min; increase to 185°C at 2.5°C / min; increase to 190°C at 0.35°C / min; increase to 250°C at 8°C / min, hold for 5 min; increase to 280°C at 5°C / min, hold for 3 min (total run time 70.7 min), then run at 100°C for 1 min (the maximum tolerance of the HP-5 column is 325°C). Front detector: heater 280°C, H2 flow rate 40 mL / min, air flow rate 400 mL / min, N2 flow rate 40 mL / min.
[0047] (2) Experimental results
[0048] The results of real-time quantitative PCR and gas chromatography (GC) analysis showed that the expression of MrSWEET5a gene showed specific changes: as the fruit matured, the gene expression showed a significant upward trend (see Appendix Figure 1 The dynamic change pattern of sucrose content was highly consistent with the expression trend of MrSWEET5a (Appendix Figure 2 Correlation analysis based on the Pearson correlation coefficient further confirmed that there was a significant positive correlation between the expression level of MrSWEET5a and sucrose content (Appendix Figure 3 ).
[0049] Example 3: Transgenic tomatoes overexpressing MrSWEET5a promote sugar accumulation
[0050] (1) Experimental methods
[0051] 1. Vector Construction
[0052] MrSWEET5a was amplified using PCR with primer pair SEQ:NO.6 and SEQ:NO.7. The PCR system and reaction procedure were the same as in Example 1. The PCR product and target vector (pBinGFP2) were digested with restriction endonucleases Kpn I and Xba I, ligated, and transformed into Escherichia coli. Positive colonies were selected and sequenced for verification. The plasmid with confirmed correct sequence was transformed into the Agrobacterium tumefaciens strain GV3101::pSoup using the freeze-thaw method.
[0053] 2. Gene Expression in Transgenic Plants
[0054] After the common tomato was transformed by genetic engineering technology to obtain the transformed plant, the mature fruit was taken. After the samples were collected, they were frozen with liquid nitrogen in a -80°C refrigerator. The total RNA of the tomato was extracted using the Polysaccharides&Polyphenolics-rich Plant Total RNA Kit, and the cDNA synthesis process was as described in Example 2. In the RT-qPCR detection, the primer pair for MrSWEET5a was SEQ: NO.4 and SEQ NO.5, and the common tomato SlACT-7 gene (SEQ: NO.8) was used as the internal reference gene, and the primers were SEQ: NO.9 and SEQ: NO.10. The real-time quantitative PCR (RT-qPCR) reaction system included 10 μL TB Green Primix Ex Taq TM II (Tli RNaseH Plus), 1 μL each of upstream and downstream primers (10 μM), 2 μL cDNA, and 6 μL ddH₂O. The reaction procedure was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The instrument used was a BLOER-LineGene 9600 series real-time fluorescence quantitative PCR instrument. A negative control using ddH₂O as the reaction template was included in each assay.
[0055] 3. Detection and analysis of soluble sugar content
[0056] The fully ground tomato fruit was used to detect the soluble sugar content, and the extraction and detection process was the same as in Example 2.
[0057] 100 μL of the supernatant extract was placed in a 1.5 mL spiral centrifuge tube, and 10 μL of ribitol (2 mg / mL) was added as an internal standard for derivatization. The organic acid content was determined by gas chromatography (GC) using an HP-5 column. The detection method parameters were the same as in Example 2.
[0058] (2) Experimental results
[0059] The results of real-time quantitative PCR and gas chromatography (GC) analysis showed that there were significant differences in the expression level of SWEET5a and sucrose content between transgenic tomatoes and wild-type plants. The relative expression level of SWEET5a in transgenic tomatoes was significantly higher than that in wild-type controls (see Appendix). Figure 4 At the same time, the sucrose content of the transgenic plants also showed significant differences compared with the wild type (Appendix Figure 5 ).
[0060] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A gene involved in sugar transport in bayberry fruit, characterized in that The nucleotide sequence of the gene is shown in SEQ: NO.
1.
2. A gene involved in sugar transport in bayberry fruit according to claim 1, characterized in that The primers used to clone the full-length MrSWEET5a sequence were SEQ: NO.2 and SEQ: NO.3, and the primers used for gene expression analysis were SEQ: NO.4 and SEQ: NO.
5.
3. Use of the gene MrSWEET5a involved in sugar transport in bayberry fruit as claimed in claim 1 or 2 in genetic engineering.
4. Use of the gene MrSWEET5a involved in sugar transport in bayberry fruit as claimed in claim 1 or 2 in increasing the sugar content in the fruit.
5. Use of the gene MrSWEET5a involved in sugar transport in bayberry fruit as claimed in claim 1 or 2 in bayberry quality breeding.