Grape VvSPX-MFS3 gene and application thereof

Abstract

The invention relates to a grape VvSPX-MFS3 gene and application of the grape VvSPX-MFS3 gene. The grape VvSPX-MFS3 is subjected to cloning and functional analysis through physiology, molecules and other methods, and the VvSPX-MFS3 is found to participate in grape fruit ripening and soluble sugar accumulation as a positive regulatory factor.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology technology, and specifically relates to a grape VvSPX-MFS3 gene and its application. Background Art

[0002] my country is a major grape producer in the world, with a grape cultivation area of ​​11.985 million mu. The output of table grapes has firmly ranked first in the world, and the grape cultivation area has jumped to second place in the world (Liu Fengzhi, 2017). As of 2015, my country's strawberry planting area has reached 2 million mu, making it the world's largest strawberry producer and consumer (Zhang Yuntao, 2015). Grape and strawberry sightseeing and picking has become a fashionable leisure activity for Beijing citizens, and an important way to increase the income of farmers in the suburbs of Beijing, becoming a new highlight of Beijing's modern agriculture and urban agriculture.

[0003] However, the cultivation of grapes and strawberries is affected by low phosphorus stress, which is prone to uneven fruit quality, poor market competitiveness, and low selling prices. Fruit yield, edible quality centered on sugar content, and appearance quality centered on color and size are greatly limited by the effective phosphorus content in the soil. Studies have shown that when phosphorus is deficient, the content of vitamin C, malic acid, galactosonic acid, proline, lysine, sorbitol-6-phosphate, malic acid, and citric acid is also inhibited (Afroz et al., 2016; Valentinuzzi et al., 2015b; Zhang et al., 2020), and the anthocyanin content is high (Valentinuzzi et al., 2015a). However, other field experiments have shown that the application of phosphite (an absorption transfer salt similar to inorganic phosphorus) can increase the anthocyanin content of strawberries (Estrada-Ortiz et al., 2013). Therefore, different types and timings of phosphorus application have different effects on fruit quality formation (Jezek et al., 2018). In addition, long-term excessive application of phosphorus fertilizer will not only cause a waste of resources, but also deteriorate the physical and chemical properties of the soil (soil acidification or increased alkalinity), thereby affecting the normal growth and development of fruit trees, and even causing "premature aging" and "phosphorus-deficient stiffness" in fruit trees, resulting in reduced production (Zheng Xiaoneng et al., 2018). Therefore, the application of phosphorus fertilizer needs to be precisely controlled, and revealing the specific mechanism of phosphorus in fruit development and quality regulation is the basis for precision fertilization.

[0004] Since fruits are mainly eaten fresh, improving fruit quality has become a key breakthrough in the development of the modern fruit industry. Sugar content is the core indicator of fruit quality traits, which affects the taste and flavor of the fruit and ultimately affects the commercial value of the fruit. Sugar content is affected by factors such as genetics, environment and cultivation technology. Among many environmental conditions, the reasonable supply of fertilizer is a key factor in determining fruit quality. As one of the essential macroelements for fruit trees, phosphate fertilizer plays an important role in the formation of fruit quality, especially sugar accumulation and metabolism. Since the soluble sugar accumulated in the fruit is mainly stored in the vacuole, the transportation, metabolism and accumulation of sugar in the vacuole determine the sugar content of the fruit.

[0005] Phosphorus (P) is one of the three major mineral elements necessary for plant growth and is an important component of nucleic acids, phospholipids, and coenzymes in cells. The effect of phosphorus on fruit quality involves the synergistic accumulation of P and sugar (Bai et al., 2021). The phosphorus content in mature strawberry fruit is positively correlated with the sugar content. The soluble solid content (SSC) of phosphorus-deficient strawberry fruit is lower, indicating that phosphorus deficiency will have a negative impact on sugar biosynthesis (Valentinuzzi et al., 2015a). Appropriately increasing phosphorus levels can increase strawberry yield, increase the soluble solid content (SSC) of strawberry fruit, reduce the organic acid content of the fruit, and improve fruit quality (Cao et al., 2015; Valentinuzzi et al., 2015b). When phosphorus is deficient, the sugar-acid ratio of strawberries decreases, and the vitamin C content of the fruit decreases significantly (Afroz et al., 2016; Li et al., 2014; Zhang et al., 2020). After appropriate phosphorus supplementation, the weight of strawberry fruit increases significantly (Afroz et al., 2016). Increased phosphorus application is beneficial to the formation, accumulation and transport of carbohydrates in grapes, which can increase the sugar content of grapes (Zhang Xiaojuan et al., 2012). Overexpression of FvmiR399, an important regulatory factor in the phosphorus nutrition signaling pathway, increases the soluble solid content of the fruit, and significantly increases the fructose and glucose content (Wang et al., 2017a; Wang et al., 2019). In addition, phosphorus content, water use efficiency (WUE) and photosynthetic rate (Pn) in strawberry fruits treated with phosphoric acid were all increased (Cao et al., 2015). A study in 2016 found that exogenous application of phosphorus fertilizer increased the soluble solids SSC in the fruit. The mechanism of action may be mainly divided into two aspects: the first possibility is that exogenous application of P fertilizer increases the P content of strawberry plants, thereby enhancing the photosynthetic efficiency of strawberries. Previous literature reported that the photosynthetic rate is closely related to the P content in the leaves (Johnson, 1984). Another possibility is that the increase in P content affects the overall sugar transport and distribution of strawberries. Fredeen et al. (1989) found that the P content in plants can not only affect the transport and distribution of sugars between the root system and the stem, but also affect the root configuration, such as changing some physiological characteristics of the root system and affecting the density and elongation of root hairs. Therefore, the higher P content in the fruit may promote the transportation and storage of sugars from leaves to fruits during the growth and ripening period of the fruit (Cao Fei, 2016).

[0006] Plants mainly use inorganic phosphorus (Pi) in the form of phosphate (H2PO4- and HPO42-), but these inorganic phosphorus are easily converted into organic phosphorus and difficult to use (Raghothama, 1999). In order to adapt to the phosphorus-deficient environment, plants have evolved many proteins related to phosphorus absorption and transport. The plant inorganic phosphorus transporters (phosphate transporters) reported so far are mainly divided into 6 families: PHT1, PHT2, PHT3, PHT4, PHT5 and PHO1 families.

[0007] In 2015, researchers isolated Arabidopsis vacuoles for voltage clamp experiments and found that VPT1 is a vacuolar phosphorus transporter responsible for the transport of Pi from the cytoplasm to the vacuole (Liu et al., 2015). In a 2016 article, VPT1 was also named PHT5;1. They used 31P-MRS (magnetic resonance spectroscopy) to determine the distribution of Pi in the cytoplasm and vacuole in different materials (Liu et al., 2016). The knockout mutant of pht5;1 showed toxic concentrations of Pi in the cytoplasm.

[0008] Although studies have found that phosphorus nutrition can affect the formation of grape fruit quality, the genes that directly regulate grape vacuolar phosphorus transport and thereby affect fruit sugar accumulation have yet to be identified. Summary of the invention

[0009] In order to solve the above technical problems, the present application provides a VvSPX-MFS3 protein, characterized in that the amino acid sequence is as shown in SEQ ID NO.1.

[0010] The present application also provides a nucleic acid molecule encoding the protein of claim 1. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1.

[0011] The present application also provides a biomaterial, characterized in that the biomaterial is a recombinant vector or a recombinant microorganism, and contains the above-mentioned nucleic acid molecule.

[0012] The present application also provides the use of the above-mentioned protein or nucleic acid molecular biological material in regulating the development of grape fruit.

[0013] In some embodiments, fruit development is one or more of fruit coloration, fruit soluble solids content, fruit phosphorus content, fruit ripening, or fruit sugar accumulation.

[0014] In some embodiments, the regulation is positive regulation or negative regulation.

[0015] In some embodiments, the fruit develops into fruit firmness.

[0016] On the one hand, this application can fill the theoretical gaps in the study of the molecular mechanism of vacuolar phosphorus transporters regulating sugar accumulation, and provide a scientific basis for the study of the ripening and quality formation mechanism of non-respiratory climacteric fruits. On the other hand, it can provide a basis for the precise application technology of phosphate fertilizers, lay the foundation for improving the utilization efficiency of plant phosphate fertilizers and improving fruit quality, so as to reduce the application of chemical fertilizers, improve fertilizer utilization, reduce production costs, protect the environment, and achieve healthy and sustainable development of the capital's fruit industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide further understanding of the present application and constitute a part of the specification. They are used to explain the present application together with the embodiments of the present application and do not constitute a limitation of the present application.

[0018] Figure 1 VvSPX-MFS3 gene sequence analysis.

[0019] Figure 2 Detection of VvSPX-MFS3 gene expression pattern.

[0020] Figure 3 VvSPX-MFS3 expression regulates grape fruit ripening and sugar accumulation. DETAILED DESCRIPTION

[0021] In order to specifically illustrate the universal design concept of the present application, specific experimental parameters are used as an example below, but this should not be used as a reason to limit the scope of protection of the present application.

[0022] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0023] Example 1 Acquisition of grape VvSPX-MFS3 gene

[0024] 1.1 Total RNA extraction and reverse transcription

[0025] According to the morphological changes of grape fruit development and maturation (Coombe, 1995), the pulp of grape fruit at 7 stages of growth and development was taken and quickly frozen in liquid nitrogen. The sampling periods include: EL31 (fruit diameter is about 4mm), EL32 (fruit diameter is greater than 7mm, and the bunch begins to tighten), EL33 (fruit becomes larger but hard and green), EL34 (fruit begins to soften), EL35 (fruit begins to color), EL36 (fruit color is about 50%), and EL37 (fruit is almost completely colored). In addition, 3-5g of fresh plant samples of roots, stems, leaves, and buds of three grape plants, strawberry fruits, and aerial parts of Arabidopsis plants were taken and quickly frozen in liquid nitrogen. RNA was extracted from the above samples using a rapid RNA extraction kit (Huayueyang, product number 0416-50, GK type), and all steps were carried out according to the instructions.

[0026] Using Invitrogen's SuperScript TM II Reverse Transcriptase Kit for reverse transcription. Add 7 μL of premixed enzyme to each reverse transcription sample, mix thoroughly, react at 42℃ for 50 min, denature at 75℃ for 15 min, and immediately take out after the reaction and store at -20℃ for later use or directly perform fluorescence quantitative experiments.

[0027] 1.2 Real-time PCR experiment

[0028] This experiment uses 96 Real Time PCR System (Roche, Germany) instrument and Premix ExTaq TM Quantitative PCR reaction was performed using the PCR kit (Qian Jin). VvEF1-α was used as the internal reference gene (Zha Qian et al., 2016).

[0029] 1.3 Target gene connected to T vector and transformed

[0030] Recovery of target fragments: Use Axygen DNA gel recovery kit to recover target fragments.

[0031] Connect the T vector: connect at 16℃ for more than 4 hours or at 4℃ overnight, then transform the competent E. coli.

[0032] Heat shock method to transform E. coli:

[0033] 1. Take out the competent E. coli cells and place them on ice to slowly thaw;

[0034] 2. Add the ligation system to the competent medium and let it stand on ice for 30 minutes;

[0035] 3. Heat shock in a 42℃ water bath for 90 seconds, then quickly take out and place on ice for 1 minute;

[0036] 4. Add 1 mL of LB recovery liquid medium and restore the culture at 37°C for 1 hour;

[0037] 5. Spread it on LB solid culture medium containing corresponding antibiotics, dry it in a sterile clean bench, and then invert it in a 37℃ incubator until a single clone grows.

[0038] Identification of positive clones by colony PCR: Pick a single clone with a sterile toothpick, streak it on a solid LB medium containing the same antibiotics, and then place it in the above PCR reaction system and gently stir it. The streaked LB medium is inverted and incubated in a 37°C incubator for 8-16 hours. After the PCR reaction is completed, agarose gel electrophoresis is performed to determine the positive clone.

[0039] 1.4 Plasmid extraction and enzyme digestion identification

[0040] Plasmid extraction using Axygen Plasmid Miniprep Kit:

[0041] 1.5 Target gene and target vector

[0042] The extracted plasmid was digested and identified by enzyme digestion. After reacting at the optimal temperature of the restriction endonuclease for 1-3 hours, agarose gel electrophoresis was performed to determine the correct plasmid based on the size of the target fragment. At the same time, sequencing was used to further determine the correctness of the target fragment sequence.

[0043] The correctly sequenced plasmid and the target vector were subjected to restriction digestion reaction, followed by agarose gel electrophoresis, the target band was cut and the target fragment was recovered, and a ligase was used for ligation reaction, and then the competent E. coli was transformed. Similarly, the positive clones were identified by colony PCR reaction and restriction digestion. At the same time, the plasmids identified as positive by PCR were sent for sequencing for further confirmation, and finally the positive plasmids containing the target fragments were obtained.

[0044] 1.6 Transformation of Agrobacterium with target gene

[0045] Prepare Agrobacterium competent cells, transform Agrobacterium competent cells by electroporation, and pick positive single clones for PCR identification.

[0046] 1.7 Cloning and sequence analysis of VvSPX-MFS3 gene

[0047] The CDS coding region of VvSPX-MFS3 (VIT_215s0048g01090) was amplified using cDNA of 'Kyoho' grape fruit as template. Sequence alignment of VvSPX-MFS3 and Arabidopsis thaliana vacuolar phosphate transporter AtVPT1 was performed using DNAMAN software. The sequence identity between VvSPX-MFS3 and AtVPT1 was 69.6% ( Figure 1 A). Sequence analysis of Vv VvSPX-MFS3 revealed that the VvSPX-MFS3 gene is 2091 bp (SEQ.ID.NO.2), encoding 696 amino acids (SEQ.ID.NO.1), belonging to the SPX-MFS family protein, and containing the conserved SPX and MFS domains ( Figure 1 B).

[0048] Example 2 Obtaining Grape Transient Transformation Material

[0049] Establishment of the in vitro transient transformation system of grape fruit: The constructed vector was transferred into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method. After plaques grew, single clone plaques were picked and placed in 5 mL of LB liquid culture medium containing corresponding resistance and rifampicin, and the bacterial solution was activated by shaking at 28°C.

[0050] The activated bacterial suspension was transferred to 50 mL LB liquid medium [with kanamycin and rifampicin, 10 mM MES (pH 5.7) and 20 μM acetosyringone] for large-scale propagation, and the bacteria were shaken at 28°C and 220 rpm overnight.

[0051] On the second day, the cells were enriched by centrifugation at 5,000 g for 10 min and the bacterial suspension was resuspended in infection buffer to a OD600 value of 0.8-1.0.

[0052] Activate the bacteria at 28℃ for 2h and prepare for injection.

[0053] VvSPX-MFS3 OE, VvSPX-MFS3 RNAi and empty Agrobacterium were injected into EL34 grape berries, with 30 berries injected for each treatment. The fruit phenotypes were observed and recorded every day after injection and photographed. 1 g of pulp from the injected part was taken 6 days after injection to extract RNA for gene expression detection (n=3). 7 days after injection, the seeds and peels of every 3 berries were removed and mixed as a sample, which was quick-frozen in liquid nitrogen and analyzed for its physiological indices (fresh samples were taken for total phosphorus determination).

[0054] Example 3 Determination of total phosphorus content in grape fruit

[0055] Weigh about 1g of fresh grape pulp and put it into a 50mL porcelain crucible. Use a 1 / 10,000 balance to weigh the crucible and the crucible plus the fresh sample. Use a pencil to number the bottom of the crucible.

[0056] The crucible was dried in an oven at 120°C for 30 min, then dried at 80°C for 10 h to constant weight. After cooling to room temperature, the crucible plus the dry sample was weighed.

[0057] The sample was placed in a muffle furnace and incinerated at 560℃ for 6h.

[0058] Add 2 mL of 6 M HCl (HCl:H2O=1:1) to a porcelain crucible to dissolve the ash, and dry it on a low heat in an electric furnace (pay attention to low heat, high heat may cause explosion, do not dry it completely, remove it when it is almost dry).

[0059] The inner wall of the porcelain crucible was repeatedly cleaned with 0.1M HCL (9mL HCL plus water to 1L), and the resulting solution was transferred into a 50mL volumetric flask and fixed to volume.

[0060] The solution was filtered into a 10 ml centrifuge tube using a funnel and filter paper, and 0.1 M HCl was used as a blank control. The sample was then measured using an inductively coupled plasma mass spectrometer (ICAP6300 Radial, Thermo Fisher, Waltham, MA, USA).

[0061] Example 4 Determination of grape fruit quality related content

[0062] 4.1 Determination of fruit firmness and soluble solids content

[0063] Hardness: Use a FHM-5 fruit hardness meter (Takemura Electric Work Ltd., Japan) to press evenly perpendicularly on the surface of the grape fruit to read the value, and take the peak value as the standard reading. Soluble solids: Add the juice squeezed from the fresh fruit to the test hole of a handheld sugar meter (MASTER-100H; ATAGO, Japan) and press the measure key to read the value.

[0064] 4.2 Determination of sucrose, glucose and fructose content

[0065] The sample to be tested was ground into powder in liquid nitrogen, 0.5 g was accurately weighed, 10 mL of 80% ethanol was added, and the mixture was in a water bath at 80°C for 3 min. The mixture was centrifuged at 10,000 g for 10 min, and the supernatant was collected in a 100 mL conical flask.

[0066] The residue was treated with 10 mL of 80% ethanol in a water bath at 80°C for 20 min, centrifuged at 10,000 g for 10 min, repeated twice, and the supernatants were combined.

[0067] The remaining residue was washed and filtered with 1 mL of 80% ethanol. The filtrate was transferred into a 10 mL test tube and 2 drops of 5% α-naphthol were added. After mixing, concentrated sulfuric acid was slowly added along the wall of the tube. No purple ring appeared at the stratification point, indicating that the sugar in the sample was completely extracted.

[0068] The combined supernatant was evaporated in boiling water, washed twice with 20 mL of ultrapure water, fixed to 50 mL, 2 mL was passed through an LC-18 solid phase extraction column, the first 1 mL was discarded, the remaining 1 mL was collected and passed through a 0.45 μm filter membrane, and then loaded onto the machine.

[0069] The chromatographic conditions were as follows: Agilent Technologies 1200 Series HPLC, 6.5×300 mm Sugar-PakTM-1 column (Waters), ultrapure water as the mobile phase, flow rate of 0.4 mL / min, column temperature of 80.0°C, detector temperature of 70.0°C, and injection volume of 20 μL.

[0070] The standard samples used, glucose D-(+)Glucose, fructose D-(-)Fructose, and sucrose were purchased from Sigma.

[0071] Example 5 Detection of the expression pattern of VvSPX-MFS3 gene

[0072] VvSPX-MFS3 showed a decreasing trend during fruit development ( Figure 2 A). VvSPX-MFS3 was constructed into the Super1300:GFP vector to obtain the recombinant plasmid Super1300:VvSPX-MFS3-GFP. The plasmid and the vacuolar marker protein γ-TIP-mCherry were transiently transformed into tobacco leaves by Agrobacterium infection, and the protein localization was observed under a laser confocal microscope 3 days after infection. The results showed that VvSPX-MFS3-GFP (green light) and γ-TIP-mCherry (red light) proteins were normally expressed in tobacco leaves, and VvSPX-MFS3 and γ-TIP proteins were co-localized ( Figure 2 B).

[0073] Example 6 VvSPX-MFS3 gene expression regulates grape fruit ripening and sugar accumulation

[0074] In order to study the role of VvSPX-MFS3 in grape berry development, the VvSPX-MFS3 fragment was constructed into the pK7GWIWG2 vector to obtain VvSPX-MFS3 326 -pK7GWIWG2-RNAi recombinant plasmid, and the hairpin structure (intronsplicing hpRNA, ihpRNA)-mediated gene silencing technology was used to reduce the expression of VvSPX-MFS3. The VvSPX-MFS3 CDS was constructed into the Super1300 vector to obtain the VvSPX-MFS3OE recombinant plasmid, and the expression of VvSPX-MFS3 was increased by transient gene overexpression technology. Agrobacterium GV3101 strain transformed with VvSPX-MFS3RNAi or VvSPX-MFS3OE was injected into grape berries, and its phenotype was detected 7 days after injection. The results showed that in VvSPX-MFS3 RNAi fruits, fruit coloring was significantly inhibited, while in VvSPX-MFS3 OE fruits, coloring was accelerated ( Figure 3 A); Compared with the control, the expression of VvSPX-MFS3 was significantly increased in VvSPX-MFS3OE fruits, but significantly decreased in RNAi fruits ( Figure 3 B). These results suggest that VvSPX-MFS3 plays an important role in fruit ripening.

[0075] At the physiological level, the total phosphorus content, soluble solids and hardness of the transgenic fruits were further determined, and it was found that the phosphorus content in the VvSPX-MFS3 overexpressing fruits was higher than that in the control fruits, while the phosphorus content in the RNAi fruits was lower ( Figure 3 C). The soluble solid content in VvSPX-MFS3 OE fruits was significantly higher than that in the control, while that in VvSPX-MFS3 RNAi fruits was significantly lower than that in the control ( Figure 3 D). In terms of fruit hardness, the RNAi fruit hardness was significantly greater than the control, while the overexpression fruit hardness was significantly less than the control ( Figure 3 .11D), indicating that VvSPX-MFS3 promotes the softening process during fruit ripening.

[0076] The above experiments show that VvSPX-MFS3 positively regulates the ripening and sugar accumulation of grape fruits.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A VvSPX-MFS3 protein, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

1.

4. A biomaterial, characterized in that: The biological material is a recombinant vector or a recombinant microorganism, and comprises the nucleic acid molecule according to claim 2.

5. Use of the protein according to claim 1, the nucleic acid molecules according to claims 2-3, or the biological material according to claim 4 in regulating the development of grape fruit.

6. The use according to claim 5, characterized in that The fruit development is one or more of fruit coloring, fruit soluble solids content, fruit phosphorus content, fruit ripening, or fruit sugar accumulation.

7. The use according to claim 5 or 6, characterized in that The regulation is positive regulation.

8. The use according to claim 5, characterized in that The fruit develops into fruit firmness.

9. The use according to claim 8, characterized in that The regulation is negative regulation.

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