Application of CsSWEET16 gene in regulation and control of sugar content of fruits

Abstract

The invention provides an application of a CsSWEET16 gene in regulating and controlling the sugar content of fruits, the nucleotide sequence of the CsSWEET16 gene is shown as SEQ ID NO.1, the amino acid sequence of protein coded by the CsSWEET16 gene is shown as SEQ ID NO.2, and the fruits comprise citrus fruits and tomato fruits. After the gene is over-expressed in citrus callus, juice sac and tomato, the sugar content of the obtained transgenic material is obviously increased; after the gene is silenced in citrus calluses and juice sacs, the sugar content of a transgenic material is obviously reduced. The result provides a new gene resource for citrus fruit quality improvement, design and breeding, and has important scientific significance for promoting directional improvement of fruit quality and breaking through germplasm innovation bottlenecks.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology and specifically relates to CsSWEET16 Application of genes in regulating sugar content in citrus fruits. Background Art

[0002] Fruit sugar content is a key indicator of flavor quality, directly impacting market value and consumer preference. Citrus is the largest fruit crop in my country and globally, and a crucial horticultural product for a healthy diet. Understanding the regulatory mechanisms for sugar accumulation in citrus is of great scientific significance for promoting targeted improvements in fruit quality and overcoming bottlenecks in germplasm innovation. As one of the origins of citrus, China leads the world in both cultivated area and production, playing a pivotal role in rural revitalization. Therefore, cultivating high-quality, nutritious fruit is a crucial requirement for the economic development of citrus-producing regions and for the pursuit of high-quality fruit in the era of the "big food" concept. Soluble sugars form the basis for fruit sweetness, as well as the synthesis of vitamins, aromatic substances, and other metabolites, and are crucial for fruit development and quality. Therefore, understanding the regulation of sugar content in citrus fruit is a key step in improving fruit quality.

[0003] Sugar accumulation provides an energy source and important signal for fruit growth. The ripening of citrus fruit is accompanied by the accumulation of soluble sugars, which mainly come from photosynthesis in the leaves. The synthesized sucrose is loaded into the phloem through the plasmodesmata-mediated symplast pathway or the membrane protein-mediated apoplast pathway, and is unloaded into the fruit through these two pathways. It is ultimately stored in the vacuole of the fruit cells in the form of sucrose, or broken down into glucose and fructose by the action of invertase, providing an energy source for fruit development. Sugar is not only a source of energy and carbon skeleton, but also an important signaling molecule. For example, sucrose promotes cell expansion and fruit development by activating the target of rapamycin (TOR) pathway.

[0004] The SWEET (Sugars Will Eventually be Exported Transporters) family is a newly discovered class of sugar transporters reported to be involved in the transport and redistribution of photosynthetic assimilates. Citrus fruits directly accumulate soluble sugars. After photosynthetic products are transported to sink tissues such as the fruit, the vast majority are stored as soluble sugars in the vacuole. Therefore, the efficient transport and unloading of photosynthetic assimilates directly influence the soluble sugar content of citrus fruit. SWEET proteins function as bidirectional sugar transporters, facilitating the diffusion of sucrose and hexoses (glucose and fructose) across cell membranes along concentration gradients, thereby regulating sugar unloading and accumulation in plant sink organs such as fruits and seeds. However, functional research on SWEET proteins in citrus is still limited. Therefore, functional studies of citrus SWEETs will facilitate understanding of their role in fruit sugar accumulation and the molecular regulatory mechanisms, providing functional genes and theoretical foundations for improving citrus fruit quality. Summary of the Invention

[0005] In view of this, the present invention screened and obtained CsSWEET16 This gene can be used to regulate the sugar content of fruits, which provides a feasible method for breeding plants with different sugar content in fruits.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide CsSWEET16 Application of genes in regulating sugar content in fruits, CsSWEET16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; SEQ ID NO. 1: ATGGCTAGCTTAAGCTTCTTCGTCGGTATTATTGGCAATGTAATTTCGCTATTGGTTTTTGCTTCTCCCATAAAAACATTTTGGCAAATTGTGAAGAAGAAATCTACGGAGAGTTACAAGGGGGTTCCATACATAACGACTCTAATGAGCACAAGTTTGTGGACATTCTATGGTGTTATGAAGCCAGGTGGTTTGCTTGTTGCCACAGTCAATGGGGCCGGTGCCGCTTTGCAGTTCATTTATGTTTCTCTCTATCTCATTTATGCTCCCAAGGATAAGAAGGTTAAGACGGCGAAACTTGTAGCAATATTAGATGTTGGCTTCCTCGGGGCAGTGATTGCGATAACACTTCTAGCCATGCATGGAAATTTGCGGCTTACTTTTGTTGGAATCTTATGCGCTGCATTAACCATTGGCATGTATGCATCTCCTCTAGCAGTCATGACAACGGTGATAAGGACAAAGAGTGTGAAGTACATGCCATTTTTGCTCTCATTTTTCCTATTTCTCAATGCCGGCGTATGGTCAGTTTATTCCGTGCTTGTGAAAGACATCTACATCGGAGTGCCAAATGCCGTAGGGTTCGTCTTGGGTGCAGCTCAGTTGATTCTCTACATGATTTACAAGAACAAGACACCGTTACCAACAAAATCAATGGACTCGGTTAAAGAAAGATCCGCCCACAAGGTGAAAGATGGCATCGAGATGGGTGCACGTGGCGATGATCATGATAATCAGGAAGATGATCTGGAAGAAGCCAATGGGAAAAAGAAACGTACCCTCAGGCAGGGGAAGAGCCTGCCAAAGCCAACTCTTGGCAAGCAATTCAGCATTCCCAAGATCCTGAAGAAGACGGCTTCTCTGGGTCCATATGATTTATACTCCAGCTGGTACCATCACTATGACGATTCCGACGTCGATGCATGA.

[0007] The fruits include citrus fruits, tomato fruits.

[0008] The second object of the present invention is to provide the above CsSWEET16 Application of a gene-encoded protein in regulating fruit sugar content, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2; SEQ ID NO.2: MASLSFFVGIIGNVISLLVFASPIKTFWQIVKKKSTESYKGVPYITTLMSTSLWTFYGVMKPGGLLVATVNGAGAALQFIYVSLYLIYAPKDKKVKTAKLVAILDVGFLGAVIAITLLAMHGNLRLTFVGILCAALTIGMYASPLAVMTTVI RTKSVKYMPFLLSFFLFLNAGVWSVYSVLVKDIYIGVPNAVGFVLGAAQLILYMIYKNKTPLPTKSMDSVKERSAHKVKDGIEMGARGDDHDNQEDDLEEANGKKKRTLRQGKSLPKPTLGKQFSIPKILKKTASLGPYDLYSSWYHHYDDSDVDA.

[0009] The fruits include citrus fruits and tomato fruits.

[0010] A third object of the present invention is to provide a method for regulating the sugar content of fruit, wherein: Reducing fruit sugar content is achieved by silencing CsSWEET16 Genetic realization; Increasing fruit sugar content by overexpressing CsSWEET16 Genetic realization; The fruits include citrus fruits and tomato fruits.

[0011] In some embodiments, preferably, by silencing CsSWEET16 The specific steps of obtaining low-sugar fruit by gene are as follows: constructing a silencing expression vector and infecting it with Agrobacterium to obtain gene-silenced plants, thereby obtaining low-sugar fruit; Among them, the primers for constructing the silencing vector are as follows: pTRV2- CsSWEET16 -F(BamHI):5'-GAAGGCCTCCATGGGGATCCCACAAGGTGAAAGATGGCATCG-3'; pTRV2- CsSWEET16 -R (SmaI): 5'-GTCTTCGGGACATGCCCGGGTTTTCCAGCTGATCATGCATCG-3'.

[0012] In some specific embodiments, preferably, by overexpression CsSWEET16 The specific steps for obtaining high-sugar content fruits of the gene are as follows: constructing an overexpression vector, and obtaining overexpression plants by agrobacterium infection, so that high-sugar content fruits can be obtained. The primers for constructing the overexpression vector are as follows: pK7WG2D- CsSWEET16 F: 5'-AAAGGAACCAATTCAGTCGACATGGCTAGCTTAAGCTTCTTCG-3'; pK7WG2D- CsSWEET16 R: 5'-GGAATTCGGTACCGGATCCTGCATCGACGTCGGAATCGTC-3'.

[0013] The fourth object of the present application is to provide a gene expression cassette, which comprises the above-mentioned CsSWEET16 gene.

[0014] The fifth object of the present application is to provide a recombinant expression vector, which comprises the above-mentioned gene expression cassette.

[0015] The sixth object of the present application is to provide an engineering bacteria, which comprises the above-mentioned recombinant expression vector.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application screens a gene CsSWEET16 and successfully clones it. After overexpression of the gene in citrus callus, juice sacs and tomatoes, the sugar content of the transgenic materials is significantly increased. After silencing of the gene in citrus callus and juice sacs, the sugar content of the transgenic materials is significantly reduced. This result provides new gene resources for the design of breeding of citrus fruit quality improvement and has important scientific significance for promoting the directional improvement of fruit quality and breaking through the bottleneck of germplasm innovation. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The technical scheme flowchart of the present application.

[0018] Figure 2 The gene expression amount (A) and the phylogenetic tree comparison diagram (B) of the SWEET family genes of Arabidopsis and tomato after ABA induction in Example 1 of the present application. CsSWEET16

[0019] The subcellular localization vector construction mode diagram (A) and the subcellular localization detection diagram (B) in Example 1 of the present application. Figure 3 CsSWEET16 Figure 3 B is the CsSWEET16 subcellular localization detection diagram (B) of the present application.

[0020] ​​ Figure 4 In Example 1 of the present invention CsSWEET16 Schematic diagram of sugar transport activity.

[0021] Figure 5 For Examples 2-4 of the present invention CsSWEET16 Schematic diagram of gene transgenesis and expression analysis, including: A is CsSWEET16 Relative expression level of the gene in transient transformation of citrus juice cells; B is the fructose, glucose, and sucrose contents in transient overexpression and VIGS juice cells; C is CsSWEET16 Relative expression levels of gene-stable overexpressing lines in citrus callus; D is the fructose, glucose, and sucrose contents in callus overexpression; E is CsSWEET16 Relative expression levels of gene citrus callus stable RNAi lines; F is the fructose, glucose, and sucrose content in callus RNAi; G is CsSWEET16 Semi-quantitative gel images of tomato heterologous overexpression lines; H is the fructose, glucose and sucrose content of transgenic tomato fruits at maturity. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0023] Example 1 This embodiment provides CsSWEET16 Gene screening, cloning, protein subcellular localization, and sugar transport activity analysis are as follows: 1.1 CsSWEET16 Gene screening In the early stage, the citrus fruits in the developing stage were treated with the plant hormone abscisic acid (ABA) and the control group was treated with water (CK). Transcriptome sequencing screening found Cs3g14550 The expression of this gene was induced to be upregulated after ABA treatment (Figure A). The gene was named CsSWEET16 .

[0024] 1.2 CsSWEET16 Gene cloning Citrus fruit juice cell cDNA was used as a template and amplified using high-fidelity enzyme. The amplification primer sequences were as follows: forward primer: 5'-ATGGCTAGCTTAAGCTTCTTCGT-3', reverse primer: 5'-TGCATCGACGTCGGAATCGT-3'.

[0025] The PCR system is as follows: Use 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix for cloning the target gene. The reaction system is as follows:

[0026] The PCR reaction procedure is as follows:

[0027] The amplified product was purified and recovered using the AxyPrep-96 DNA Gel Extraction Kit (Axygene, USA). The purified product was then ligated into the pEASY-Blunt vector (Quanshijin, China) using the ligation system shown in the figure. After incubation at room temperature for 5 minutes, the product was transformed into competent Escherichia coli DH5α. The plate was spread and incubated upside down at 37°C. The bacteria were picked and shaken. After PCR positive detection, the positive clones were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were used to determine the clones. CsSWEET16 The full-length gene sequence.

[0028] Sequencing results showed that the gene contained an open reading frame (ORF) of 924 bp, encoding 308 amino acids, and molecular weight prediction showed that the protein had a molecular weight of 33.83 kDa. The gene was named CsSWEET16 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0029] 1.3 CsSWEET16 Analysis of subcellular localization and sugar transport activity of the encoded protein Amplification CsSWEET16 The ORF region (excluding the stop codon) of CsSWEET16 The ORF region of the PCR product was cloned as in step 1.2 of Example 1. The following primers were used: pRI101YFP- CsSWEET16 -F: 5'-ATGGGATCTACTAGTGAATTCATGGCTAGCTTAAGCTTCTTCGT-3'; pRI101YFP- CsSWEET16 -R: 5'-GGGGGTACCGTCGACGGATCCTGCATCGACGTCGGAATCGT-3'.

[0030] The target gene was constructed into the pRI101-YFP vector, with the YFP protein located at the 3' end of the gene and its expression driven by the CaMV35S promoter ( Figure 5 A). The experimental group is 35S: CsSWEET16 -YFP CsSWEET16 Fusion of green fluorescent protein, the control group is 35S:YFP. The membrane marker protein fused with red fluorescent protein 35S:CBL1n-OFP-mCherry was mixed with the experimental group and the control group Agrobacterium, respectively, and transiently injected into the epidermal cells of Nicotiana benthamiana leaves. Laser confocal fluorescence observation showed that the fluorescence of the control group filled the entire epidermal cells, including the cytoplasm and nucleus. It was found that the transformed 35S: CsSWEET16 -YFP fluorescence is only concentrated in the cell membrane, indicating that CsSWEET16 is a cell membrane localized protein ( Figure 5 B).

[0031] CsSWEET16 The results of gene subcellular localization showed that it was located on the cell membrane. In order to analyze the sugar transport activity of CsSWEET16, CsSWEET16 The CDS sequence of the recombinant protein was cloned into the yeast expression vector pDR196, pDR196-EV was used as the negative control group, and the plasmid was transformed into the yeast mutant CSY.VW4000 that was defective in hexose uptake. CsSWEET16 -CSY.VW4000 yeast mutant culture was diluted to 1, 10 -1 , 10 -2 and 10 -3 After three concentrations, the yeast colonies were spotted on SC / -Ura solid medium containing different sugar sources, cultured in the dark at 30°C for 3 days, and the growth of the colonies was observed. The yeast colonies were able to grow on SC / -Ura solid medium supplemented with glucose and fructose, indicating that CsSWEET16 has glucose and fructose transport activity ( Figure 5 ).

[0032] Example 2 This example provides vector construction, transient transformation of citrus juice cells, and sugar content determination, as follows: 2.1 Build 2.1.1 VIGS vector construction Using citrus fruit juice cell cDNA as template, specific primers were designed to amplify CsSWEET16 The primer sequences for the 257 bp fragment of the non-conserved region in the gene CDS (shown in SEQ ID NO. 3) are as follows: pTRV2- CsSWEET16-F(BamHI):5'-GAAGGCCTCCATGGGGATCCCACAAGGTGAAAGATGGCATCG-3'; pTRV2- CsSWEET16 -R(SmaI): 5'-GTCTTCGGGACATGCCCGGGTTTTCCAGCTGATCATGCATCG-3'.

[0033] Among them, amplification CsSWEET16 The non-conserved regions in the gene CDS are as follows: SEQ ID NO.3: CACAAGGTGAAAGATGGCATCGAGATGGGTGCACGTGGCGATGATCATGATAATCAGGAAGATGATCTGGAAGAAGCCAATGGGAAAAAGAAACGTACCCTCAGGCAGGGGAAGAGCCTGCCAAAG CCAACTCTTGGCAAGCAATTCAGCATTCCCAAGATCCTGAAGAAGACGGCTTCTCTGGGTCCATATGATTTATACTCCAGCTGGTACCATCACTATGACGATTCCGACGTCGATGCATGATCAGCTGGAAA.

[0034] The PCR reaction system is as follows:

[0035] The PCR reaction procedure is as follows:

[0036] The ClonExpress II One Step Cloning Kit (Novozymes, China) was used to insert the gene between the BamHI and SmaI restriction sites on the pTRV2 vector in one step. The constructed vector was then transformed into GV3101 Agrobacterium competent cells for transient gene silencing.

[0037] 2.1.2 Overexpression vector construction Citrus fruit juice cell cDNA was used as template to amplify CsSWEET16 The ORF region (excluding the stop codon) of CsSWEET16 The ORF region of the PCR product was cloned as in step 1.2 of Example 1. The following primers were used: pK7WG2D- CsSWEET16 -F: 5'-AAAGGAACCAATTCAGTCGACATGGCTAGCTTAAGCTTCTTCG-3'; pK7WG2D- CsSWEET16 -R: 5'-GGAATTCGGTACCGGATCCTGCATCGACGTCGGAATCGTC-3'.

[0038] The ClonExpress II One Step Cloning Kit (Novozymes, China) was used to insert the target gene between the SalI and KpnI restriction sites on the pENTR1A vector. After the target gene was constructed into the pENTR1A vector, LR Clonase TM enzyme (ThermoFisher, 11791043, USA) was inserted into the pK7WG2D vector, and the obtained pK7WG2D-CsSWEET16 recombinant plasmid was transformed into EHA105 Agrobacterium competent cells for gene overexpression.

[0039] 2.2 Transient infection of citrus juice spores 1) Will be used for VIGS (TRV1, TRV2-EV, TRV2- CsSWEET16 ) and overexpression (pK7WG2D-EV, pK7WG2D- CsSWEET16 ) were streaked onto LB (50 mg / L Rif + 50 mg / L Kana) solid medium and cultured in an inverted manner at 28°C for 2-3 days to obtain single colonies.

[0040] 2) Pick one single colony from each group and place it in 3 mL of LB liquid medium containing the same antibiotics. Incubate at 28°C, 220 rpm, and shake gently for 24 hours to fully activate the cells.

[0041] 3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a ratio of 1:10, cultured at 28°C, 220 rpm / min, and expanded for about 6 hours. Centrifuged at 6000 rpm / min for 5 minutes, collected the bacteria, and suspended in MES buffer (10 mmol / L MES + 10 mmol / L MgCl2 + 50 μmol / L AS, pH = 5.8). The OD 600 Adjust to 0.5.

[0042] 4) Mix TRV1 and TRV2-EV, TRV1 and TRV2- CsSWEET16 The two combined resuspensions were mixed and incubated in the dark at 28°C incubator for 2-3 h to prepare the infection solution.

[0043] 5) Harvest fresh citrus fruits from the orchard, disinfect the peel surface with 75% alcohol, remove intact, plump, and unpeeled juice sacs, and completely immerse them in MES buffer. Mix the removed juice sacs with the bacterial solution, shake incubate at 120 rpm / min for 5 min, vacuum pump for 30 min, and let it stand for 15 min. After discarding the bacterial solution, rinse the juice sacs three times with sterile water, dry the surface moisture with filter paper, and place the juice sacs in culture medium for incubation in the dark at room temperature for 4 days before sampling.

[0044] 2.3 Determination of relative expression of target genes Total RNA from juice cell samples was extracted using the EasySpin Plus RNA extraction kit (Aidlab Biotech, Beijing, China), and reverse transcription was performed using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix EasyScript (Transgen, Beijing, China) to obtain cDNA. Relative expression levels were determined using an RT-qPCR instrument (Applied Biosystems, Foster City, CA, USA) using the SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). CsActin is the internal reference gene, CsSWEET16 For the target gene, use 2 −ΔΔCt Methods To calculate relative expression, the primer sequences are as follows: CsActin -F:5'-CCGACCGTATGAGCAAGGAAA-3'; CsActin -R: 5'-TTCCTGTGGACAATGGATGGA-3'; TRV- CsSWEET16 -F:5'-GCACGTGGGCGATGATCATGATAA-3'; TRV- CsSWEET16 -R: 5'-CAGAGAAGCCGTCTTCTTCAGG-3'.

[0045] The qRT-PCR detection reaction system is as follows:

[0046] The reaction conditions are as follows:

[0047] The results are as follows Figure 5A: Compared with the control group (grey column), CsSWEET16 The expression level of was upregulated by about 2.3 times in SW16-OE (orange column) and downregulated to 0.3 times in SW16-TRV (blue column), indicating that the results of transient infection experiment were effective. CsSWEET16 Gene overexpression or silencing.

[0048] 2.4 Sugar content determination The samples were ground into powder in liquid nitrogen. 0.2 g of each sample was extracted with 1.4 mL of 75% (v / v) methanol and chloroform. Ribitol (0.12 mg) was added as an internal reference during the extraction process. The samples were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed using a high-performance gas chromatograph (GC9720Plus, Fuli Instruments, Zhejiang, China). Quantitative analysis was performed using the internal standard method (FL97Plus. lnk). Each sample was determined independently three times.

[0049] The results are as follows Figure 5 As shown in B: Compared with the control group (gray columns), the fructose, glucose and sucrose contents after SW16-OE were significantly increased. On the contrary, the sugar contents in SW16-TRV were decreased, indicating that CsSWEET16 can increase the sugar content in citrus fruit juice cells.

[0050] Example 3 This example provides genetic transformation and sugar content determination of citrus callus, as follows: 3.1 Vector construction 3.1.1 RNAi vector construction Using citrus fruit juice cell cDNA as template, specific primers were designed to amplify CsSWEET16 The primer sequences for the 147 bp fragment of the non-conserved region in the gene CDS (shown in SEQ ID NO. 4) are as follows: 221- CsSWEET16 -F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCTCCCAAGGATAAGAAGGTTAA-3'; 221- CsSWEET16 -R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTGGTTAATGCAGCGCATAAGATTC-3'.

[0051] Among them, amplification CsSWEET16 The 147 bp fragment of the non-conserved region in the gene CDS is as follows: SEQ ID NO. 4: GCTCCCAAGGATAAGAAGGTTAAGACGGCGAAACTTGTAGCAATATTAGATGTTGGCTTCCTCGGGGCAGTGATTGCGATAACACTTCTAGCCATGCATGGAAATTTGCGGCTTACTTTTGTTGGAATCTTATGCGCTGCATTAACC.

[0052] The reaction system is as follows:

[0053] The PCR reaction procedure is as follows: First pass BP Clonase TM The fragment was inserted into the pDONOR221 vector by LR Clonase TM enzyme (Thermo Fisher, 11791043, USA) and the fragment was inserted into the pK7GWIWG2D (II) vector to obtain pK7GWIWG2D (II)- CsSWEET16 The recombinant plasmid was transformed into EHA105 competent Agrobacterium for callus genetic transformation.

[0054] 3.1.2 Overexpression vector construction The overexpression vector of Example 2 was used.

[0055] 3.2 Genetic transformation of citrus callus 1) The RNAi vectors (pK7GWIWG2D(II)-EV, pK7GWIWG2D(II)- CsSWEET16 ) and overexpression (pK7WG2D-EV, pK7WG2D- CsSWEET16 ) were streaked onto LB (50 mg / L Rif + 50 mg / L Kana) solid medium and cultured inverted at 28°C for 2-3 days to obtain single colonies.

[0056] 2) Pick one single colony from each group and place it in 3 mL of LB liquid medium containing the same antibiotics. Incubate at 28°C, 220 rpm, and shake gently for 24 hours to fully activate the cells.

[0057] 3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a ratio of 1:10, and cultured at 28°C, 220 rpm / min for about 6 hours. Centrifuged at 6000 rpm / min for 5 minutes, the bacteria were collected, and MT suspension (MT + 40 g / L sucrose + 0.5 g / L maltose extract + 1.5 g / L glutamine + 50 μmol / L AS, pH = 5.8) was added to suspend the bacteria. The OD 600 Adjust to 0.5.

[0058] 4) Mix the callus with the bacterial solution, shake and culture at 120 rpm / min for 5 minutes, vacuum-extract for 10 minutes, and let it stand for 5 minutes. Discard the bacterial solution and use filter paper to absorb the moisture on the surface of the callus. Place the callus in the culture medium and culture it in the dark at room temperature for 2 days before sampling.

[0059] 3.3 Determination of relative expression of target genes The method is the same as Example 2, and the primer sequences are as follows: CsActin -F:5'-CCGACCGTATGAGCAAGGAAA-3' CsActin -R:5'-TTCCTGTGGACAATGGATGGA-3' q- CsSWEET16 -F:5'-GGCATGTATGCATCTCCTCTAG-3' q- CsSWEET16 -R:5'-CGGAATAAACTGACCATACGCC-3' The qRT-PCR detection reaction system is as follows:

[0060] The reaction conditions are as follows:

[0061] The results are as follows Figure 5 C and E show: compared with the control group (grey bars), CsSWEET16 The expression level of SW16OE-1 / 7 / 8 was upregulated by 44-125 times ( Figure 5 C), was downregulated to 0.05-0.5 times in the three silenced strains SW16Ri-1 / 2 / 4 ( Figure 5 E), indicating that the lines obtained by stably genetically transforming citrus calli CsSWEET16 Gene overexpression or silencing is successful.

[0062] 3.4 Sugar content determination The method is the same as in Example 2, and the results are as follows Figure 5As shown in D and F: Compared with the control group (gray columns), the fructose, glucose and sucrose contents after SW16-OE were significantly increased, while the sugar contents in SW16-Ri decreased, indicating that CsSWEET16 can increase the sugar content in citrus callus.

[0063] Example 4 This example provides genetic transformation of tomato with heterologous overexpression and determination of sugar content, as follows: 4.1 Vector Construction The overexpression vector of Example 2 was used.

[0064] 4.2 Tomato genetic transformation 1) Seed preparation: Disinfect seeds with 75% ethanol for 20 seconds and 40% sodium hypochlorite solution for 10-14 minutes. Rinse the disinfected seeds 3-5 times with sterile water, place them on sterile filter paper to absorb moisture, sow them in seed culture medium, and incubate them at 26°C with a 16 / 8 h light / dark cycle for 5-7 days.

[0065] 2) Explant preparation: When the cotyledons are extended but true leaves have not yet grown, cut off the tips and ends of the cotyledons to form rectangular explants. Soak the explants in MSO suspension (MS + 25 g / L sucrose, pH = 5.8) for 0.5 h. Transfer them to pre-culture medium and incubate them in the dark at 26°C for 2 days.

[0066] 3) Preparation of bacterial liquid: Agrobacterium was streaked onto LB (50 mg / L Rif + 50 mg / L Kana) solid medium and inverted at 28°C for 2-3 days to obtain a single colony. A single colony was picked and placed in 3 mL of LB liquid medium containing the same antibiotics. The culture was shaken at 28°C, 220 rpm, and inoculated into the same LB liquid medium at a ratio of 1:10. The culture was expanded at 28°C, 220 rpm, and incubated for about 6 hours. The cells were centrifuged at 6000 rpm for 5 minutes, and the cells were suspended in MSO (MS + 25 g / L sucrose + 50 μmol / L LAS, pH = 5.8). The OD 600 Adjust to 0.5; 4) Explant infection: Centrifuge the prepared Agrobacterium at 6000 rpm for 5 min at 28°C. Discard the supernatant and fully suspend the bacteria in an equal volume of MSO solution. Co-incubate the explants with the bacterial solution for 15 min. After absorbing the excess bacterial solution with filter paper, place the explants back into the pre-incubation medium. Place sterile filter paper on the medium with the back side facing up and incubate in the dark at 26°C for 2 days.

[0067] 5) Change the culture medium: Transfer the dark-cultured explants from the pre-culture medium to the screening medium and culture them at 28°C with a 16 / 8 h light / dark cycle for 7 days. Then transfer them to the budding medium and culture them at 28°C with a 16 / 8 h light / dark cycle for 14 days. When regenerated buds grow from the cut ends of the explants, cut them off and transfer them to the budding medium and culture them at 28°C with a 16 / 8 h light / dark cycle for 10-15 days. Continue to remove the callus tissue around the regenerated buds until the regenerated buds reach 3 cm in length.

[0068] 6) Transfer regenerated shoots that have grown to 3 cm from the budding medium to the rooting medium and grow until they root. When the rooted shoots reach the top of the bottle, transplant them into soil to harvest the seeds. Use T2 fruit samples for semi-quantitative identification and sugar content determination.

[0069] 4.3 Semi-quantitative identification of positive plants Total RNA from tomato fruit samples was extracted using the EASYspin Plus RNA extraction kit (Aidlab Biotech, Beijing, China). Reverse transcription was performed using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix EasyScript (Transgen, Beijing, China) to obtain cDNA. PCR amplification was performed using the cDNA as a template using 2× Rapid Taq Master Mix. Bands were detected by DNA gel electrophoresis. The primer sequences are as follows: SlSAND -F: 5'-TGACATGACTCCTTTGCTTGG-3'; SlSAND -R: 5'-ATGCAAGATGGCACTAGCAGC-3'; q- CsSWEET16 -F:5'-GGCATGTATGCATCTCCTCTAG-3'; q- CsSWEET16 -R:5'-CGGAATAAACTGACCATACGCC-3'.

[0070] Wherein, the reaction system is as follows:

[0071] The PCR reaction procedure is as follows:

[0072] The results are as follows Figure 5 G shows: SlSANDThe target gene was not present in WT. The amplifications using wild-type (WT) and transgenic tomato (L11 and L12) cDNA as templates showed bands of the same size, indicating that the cDNA extraction was successful and the concentration was relatively consistent. CsSWEET16 The bands were not found in L11 and L12, indicating that the gene was overexpressed in tomato. CsSWEET16 success.

[0073] 4.4 Sugar content determination The method is the same as in Example 2, and the results are as follows Figure 5 As shown in H: Compared with WT (gray columns), the fructose and glucose contents in tomato fruits heterologously overexpressing CsSWEET16 were significantly increased, indicating that CsSWEET16 can increase the sugar content of the fruit.

[0074] After the above series of studies, we know that: CsSWEET16 After the gene was overexpressed in citrus callus, juice cells and tomatoes, the sugar content of the transgenic materials was significantly increased; after the gene was silenced in citrus callus and juice cells, the sugar content of the transgenic materials was significantly reduced.

[0075] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0076] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, 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. CsSWEET16 The application of the gene in regulating the sugar content of fruit is characterized in that: described CsSWEET16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The fruits include citrus fruits and tomato fruits.

2. The method according to claim 1 CsSWEET16 The application of the gene-encoded protein in regulating the sugar content of fruit is characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.2; The fruits include citrus fruits and tomato fruits.

3. A method for regulating the sugar content of fruit, characterized in that: In the method: Reducing fruit sugar content is achieved by silencing CsSWEET16 Genetic realization; Increasing fruit sugar content by overexpression CsSWEET16 Genetic realization; The fruits include citrus fruits and tomato fruits.

4. The method according to claim 3, characterized in that Through silence CsSWEET16 The specific steps of obtaining low-sugar fruit by gene are as follows: constructing a silencing expression vector and infecting it with Agrobacterium to obtain gene-silenced plants, thereby obtaining low-sugar fruit; Among them, the primers for constructing the silencing vector are as follows: pTRV2- CsSWEET16 -F(BamHI):5’-GAAGGCCTCCATGGGGATCCCACAAGGTGAAAGATGGCATCG-3’; pTRV2- CsSWEET16 -R(SmaI):5’-GTCTTCGGGACATGCCCGGG TTTCCAGCTGATCATGCATCG-3’。 5. The method according to claim 3, characterized in that Through overexpression CsSWEET16 The specific steps of obtaining high-sugar-content fruits by gene are as follows: constructing an overexpression vector and infecting it with Agrobacterium to obtain overexpression plants, thereby obtaining high-sugar-content fruits; Among them, the primers for constructing the overexpression vector are as follows: pK7WG2D- CsSWEET16 -F:5’-AAAGGAACCAATTCAGTCGACATGGCTAGCTTAAGCTTCTTCG-3’; pK7WG2D- CsSWEET16 -R:5’-GGAATTCGGTACCGGATCCTGCATCGACGTCGGAATCGTC-3’。 6. A gene expression cassette, characterized in that The gene expression cassette comprises the gene expression cassette according to claim 1 CsSWEET16 Gene.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene expression cassette according to claim 6.

8. An engineered bacterium, characterized in that: The engineered bacteria includes the recombinant expression vector described in claim 7.

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