Use of melon callose arabinogalactan protein in controlling sugar content of melon fruit

Abstract

The application belongs to the field of plant genetic engineering, and relates to application of a melon fasciculate arabinogalactan protein in controlling sugar content of melon fruits, wherein the protein has an amino acid sequence as shown in SEQ ID NO. 2 in the sequence table. CmFLA1 The gene is found to have a negative correlation with sucrose accumulation in melon fruits. Meanwhile, it is found that CmFLA1 and vacuole membrane sugar transporter CmTST2 interact and inhibit the transport of sucrose to the vacuole. By transiently knocking out the gene in melon fruits, the sucrose content of the fruits is significantly increased; meanwhile, the melon fruits are treated with an arabinogalactan protein inhibitor beta-Yariv, which significantly inhibits CmFLA1 expression of the gene and increases the sucrose content. It is shown that the cloned CmFLA1 gene in the application has a function of regulating the sucrose content of melon fruits. The gene discovery provides a new gene resource for melon fruit quality improvement and molecular design breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering. Specifically, it relates to the application of melon bundle-like arabinogalactan protein in controlling the sugar content of melon fruit. The applicant isolated and cloned a key gene CmFLA1 for regulating sucrose accumulation in melon fruit from melon (Cucumis melo L.). Knocking out or reducing the expression of this gene in melon fruit significantly increases the sucrose content in the fruit. This gene can be used to improve the quality of melon fruit. Background Technology

[0002] Fasciclin-like arabinogalactan proteins (FLAs) belong to a subfamily of the arabinogalactan protein (AGP) family. AGPs are highly glycosylated proteins found in the plasma membrane, cell wall, extracellular matrix, and secretions (such as stigma surfaces and wound flow) (Elliset et al., 2010). Unlike other AGP subfamilies, FLAs, in addition to possessing 1-2 AGP motifs, also have 1-2 unique fasciclin (FAS) domains (Huber and Sumper, 1994; Kim et al., 2002; Johnson et al., 2003; Faik et al., 2006; Showalter et al., 2010). Structural analysis of Arabidopsis thaliana AtFLA4 revealed that the FAS1 domain is used to maintain plasma membrane localization; while the glycosylphosphatidylinositol (GPI) modification signal is responsible for anchoring proteins to the membrane after they are transported from the endoplasmic reticulum; N-glycosylation is used for protein transport from the endoplasmic reticulum, while O-glycosylation affects the destination of proteins after secretion (Xue et al., 2017).

[0003] FLAs are involved in plant growth and development, such as fiber development (Huang et al., 2013; Liu et al., 2013; Wang et al., 2015; Guerriero et al., 2017), reproductive growth (Li et al., 2010; Tan et al., 2012; Miao et al., 2021; Deng et al., 2022), cell wall regeneration (Showalter et al., 2016), and responses to the environment (Ma and Zhao, 2010; Takahashi et al., 2016; Cagnola et al., 2018; Meng et al., 2020). There are numerous studies on FLA genes in Arabidopsis thaliana. AtFLA1 plays a role in shoot regeneration and lateral root development (Johnson et al., 2011); AtFLA18 regulates root elongation (Allelign Ashagre et al., 2021). AtFLA11, 12, and 16 are involved in stem development and differentiation (Ito et al., 2005; Macmillan et al., 2010; Liu et al., 2020). AtFLA3 and AtFLA14 are specifically expressed in pollen grains and play a crucial role in pollen development (Li et al., 2010; Miao et al., 2021). Some FLA genes respond to stress: for example, FLA4 responds to salt stress and affects intercellular adhesion (Shi et al., 2003); while drought induces FLA9 expression and increases seed abortion rates in maize and Arabidopsis (Cagnola et al., 2018). However, the role of FLA genes in fruit sugar accumulation has not yet been reported.

[0004] Muskmelon (Cucumis melo L.) is an important horticultural crop in the Cucurbitaceae family, with its fruit being its product organ. This invention cloned a muskmelon bundle-like arabinogalactan protein gene, named CmFLA1, and found that the expression of this gene is negatively correlated with the sucrose content of muskmelon fruit. Experiments demonstrated that CmFLA1 is a gene that negatively regulates the sucrose content of muskmelon fruit. A search of domestic and international patent literature, journals, and other publicly published literature (such as the internet) revealed no reports of this invention domestically or internationally, and we have not published any articles relating to this invention; therefore, this invention is unknown to the public both domestically and internationally. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the coding gene of melon bundle arabinogalactan protein or melon bundle arabinogalactan protein in controlling the sugar content of melon fruit, wherein the melon bundle arabinogalactan protein is shown in SEQ ID NO.2.

[0006] Another objective of this invention is to provide the application of the coding gene of melon bundle arabinogalactanin or melon bundle arabinogalactanin in improving the quality of melon fruit. By knocking out or reducing the expression of the coding gene of melon bundle arabinogalactanin in melon fruit, the sucrose content in the fruit can be significantly increased.

[0007] The final objective of this invention is to provide the application of a melon bundle arabinogalactan protein activity inhibitor in improving the sweetness of melon fruit.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The applicant cloned a CmFLA1 gene from melon using plant gene cloning technology. Its nucleotide sequence is shown in SEQ ID NO.1, and its corresponding amino acid sequence is shown in SEQ ID NO.2. Open reading frame (ORF) prediction revealed that the gene contains one ORF, 1245 bp in length, encoding a 414-amino acid protein with a predicted molecular weight of 44.15 kDa and an isoelectric point of 5.88. The applicant designed a pair of primers and, using melon fruit cDNA as a template, cloned the full-length coding sequence of the CmFLA1 gene using PCR technology. The primers are as follows:

[0010] CmFLA1-F: 5'-ATGCTGCTGCAACTCTGCCGCGGCGCCGCCGCCGGGT-3'

[0011] CmFLA1-R: 5'-TTACACTAGAAGAAGCGCTGCCAAACCTGAAG-3'.

[0012] The scope of protection of this invention includes:

[0013] The application of the gene encoding melon bundle arabinogalactanin or melon bundle arabinogalactanin in controlling the sugar content of melon fruit, wherein the melon bundle arabinogalactanin is shown in SEQ ID NO.2;

[0014] The applications described above are specifically as follows:

[0015] Inhibiting or reducing the expression of fascicular arabinogalactan protein in melon can increase the sugar content of melon fruit;

[0016] Knockout, suppression, or silence of the gene encoding bundle arabinogalactan protein in melon to increase the sugar content of melon fruit;

[0017] Increasing the expression level of bundled arabinogalactan protein in melon can reduce the sugar content of melon fruit;

[0018] Increasing the expression level of the gene encoding bundle arabinogalactan protein in melon can reduce the sugar content of melon fruit.

[0019] In the above-described applications, a preferred method is to introduce a substance that inhibits or reduces the expression level of arabinogalactan protein in melon into the melon; the substance is a nucleic acid molecule, its expression cassette, recombinant vector, or recombinant microorganism that inhibits or reduces the expression level of arabinogalactan protein in melon.

[0020] In the above-described applications, preferably, the knockout is performed using the CRISPR / Cas9 system, where the target site of the gRNA is: ATCTTGCCGTCTCAAGTGG.

[0021] In the applications described above, the preferred melons with increased sugar content contain the gene shown in SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

[0022] The above applications also apply to improving the quality of melon fruits.

[0023] The scope of protection of this invention also includes:

[0024] Application of melon bundle arabinogalactan protein activity inhibitor in improving melon fruit sweetness;

[0025] In the above-described applications, preferably, the inhibitor is β-glucosyl-Yariv.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The discovery of bundled arabinogalactan protein has enabled the regulation of sucrose accumulation, enriching the understanding of the role of this gene family in the regulation of plant fruit quality. This provides new gene resources for improving melon fruit quality and molecular design breeding, and offers new ideas for achieving precise regulation of melon production. Attached Figure Description

[0028] Figure 1 This is an agarose gel electrophoresis image of the CmFLA1 gene after amplification.

[0029] The blue arrows indicate the target fragment. The red arrows indicate the corresponding DNA marker fragments.

[0030] Figure 2 The correlation between the expression level of the CmFLA1 gene and sucrose content.

[0031] Figure A shows the relationship between sucrose content and CmFLA1 gene expression levels in different melon varieties.

[0032] Figure B shows the relationship between sucrose content and CmFLA1 gene expression in fruits of a single high-sugar melon variety at different developmental stages.

[0033] Figure 3 Analysis of heterologous functions of CmFLA1 in yeast

[0034] Figure A shows that the yeast two-hybrid assay indicates that CmFLA1 can interact with the melon saccharide transporter CmTST2.

[0035] Figure B shows that when CmFLA1 and CmTST2 were simultaneously transferred into the yeast vacuolar sugar accumulation deletion mutant W303, the original function of CmTST2 in transporting sucrose to vacuoles was lost.

[0036] Figure 4 Functional analysis of CmFLA1 in melon fruit

[0037] Figure A shows GFP expression in melon fruit after transient transformation with a CRISPR / Cas editing vector carrying a GFP fluorescent tag. Figure B shows the editing of the FLA1 gene in transiently transformed melon pulp cells.

[0038] Figure C shows the sucrose content in melon pulp cells undergoing transient transformation.

[0039] Figure 5 Treating melon fruit with the FLA protein inhibitor β-Yariv

[0040] Figure A shows the changes in CmFLA1 gene expression in melon fruits after β-Yariv treatment.

[0041] Figure B shows the change in sucrose content in melon pulp after β-Yariv treatment. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0043] Example 1: Cloning of the full length of the CmFLA1 translation region

[0044] Seeds of the melon variety "Wheat Crisp" were sown in a seedling substrate. Once the plants reached four leaves and a central bud, they were transplanted into a plastic greenhouse and managed routinely. After flowering and fruiting, total RNA was extracted from melon fruits 30 days post-pollination (DAA) and reverse transcribed into cDNA. Using this cDNA as a template, PCR primers were designed based on the CmFLA1 reference sequence obtained from melon genome sequence alignment.

[0045] CmFLA1-F: 5'-ATGCTGCTGCAACTCTGCCGCGGCGCCGCCGCCGGGT-3'

[0046] CmFLA1-R: 5'-TTACACTAGAAGAAGCGCTGCCAAACCTGAAG-3'.

[0047] The full-length coding region of the CmFLA1 gene was cloned using PCR. The reaction mixture is as follows:

[0048]

[0049]

[0050] PCR reaction conditions: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0051] 50 μL of PCR product was subjected to agarose gel electrophoresis. The target fragment was excised and recovered from the gel. The purified product was ligated into the pEasy-Blunt vector, and the ligation product was transformed into competent E. coli DH5α cells. After PCR identification, positive bacterial cultures were sent to the company for sequencing. Based on the sequencing results, the full-length coding region of the CmFLA1 gene was obtained.

[0052] Sequencing results revealed that the coding region of this gene is 1245 bp in length, encoding a protein of 414 amino acids. The predicted molecular weight of this protein is 44.15 kDa, and its isoelectric point is 5.88. This gene was named CmFLA1, and its amino acid sequence is shown in SEQ ID NO. 2. The polynucleotide encoding this protein is shown in SEQ ID NO. 1.

[0053] Example 2:

[0054] Analysis of sucrose content and CmFLA1 expression levels in different melon varieties and at different developmental stages:

[0055] Fruits of the following melon varieties were sampled: 'Baicai Gua' (BCG), 'Huapi Cai Gua' (HPC), 'Baling Cui' (BLC), 'Mai Mai Su' (XM), 'Yangjiao Su' (YJ), 'Longtian No. 4' (LT4), 'Gaoshi Cui Gua' (GS), and 'M43' melons at 30 DAA (i.e., 30 days after flowering), as well as melons 'Elizabeth' at 15, 20, 28, 32, and 38 DAA.

[0056] The expression level of CmFLA1 in the above samples and the sugar content in the fruits were determined;

[0057] The expression pattern of the CmFLA1 gene was analyzed using quantitative real-time PCR (qRT-PCR). The quantitative reagents were as follows: Tip Green qPCR SuperMix (Truly Gold, Beijing), the method is as per the manufacturer's instructions, and the reaction system is shown in the table below:

[0058]

[0059] The quantitative fluorescence primers designed using the NCBI website are: CmFLA-qRTf: 5'-TACTGGCTCTGCTCCTGG-3';

[0060] CmFLA-qRTr: 5'-ATCGCATCAATGTCGTCACTATC-3', using the muskmelon Tublin as the internal reference gene, with primers for the internal reference gene: CmTub-qRTf: 5'-AACTCCACCAGTGTAGCCG-3'; CmTub-qRT-r: 5'-CACCCACCTCTTCATAATCC-3'. The quantitative reaction is described in the instructions. After the reaction, 2... -ΔΔCt The algorithm calculates gene expression levels.

[0061] The method for determining the sugar content in fruit is as follows:

[0062] Weigh approximately 0.5 g of sample into a glass bottle, add 5.0 mL of 80% ethanol, and extract by sonication at room temperature for 30 min (500 W, 40 kHz). Centrifuge the supernatant at 14000 g for 10 min, and inject the supernatant into the sample. Accurately weigh 2.08 mg of sucrose into a 2.0 mL volumetric flask, add 2.0 mL of 50% acetonitrile to dissolve it, and dilute successively to prepare standard solutions of 0.1, 0.2, 0.4, 0.6, and 0.8 times. Take 200 μL of each standard solution and mix them to prepare a mixed standard solution.

[0063] HPLC analysis: Samples were separated and determined using an Ultimate 3000 high-performance liquid chromatograph with an Alltech ELSD6000 evaporative light scattering detector. The chromatographic column was a Welch Ultimate HILIC Amide (4.6 × 250 mm, 5 μm), the mobile phase was 0.02% ammonia-acetonitrile (25:75) with isocratic elution, the flow rate was 1.0 mL / min, the column temperature was 40 °C, the drift tube temperature was 75 °C, and the carrier gas (air) flow rate was 2.5 mL / min. Data were processed and analyzed.

[0064] Experimental results are as follows Figure 2 As shown in Figure A, the sucrose content in the thin-skinned melons BCG, HPC, and BLC with 30DAA was low, while that in XM, YJ, LT4, GS, and M43 was high. In the thin-skinned melons with 30DAA, the expression level of CmFLA1 in the low-sucrose varieties BCG, HPC, and BLC was significantly higher than that in the high-sucrose varieties XM, YJ, LT4, GS, and M43.

[0065] from Figure 2 As can be seen in the B-type sample, in the thick-skinned melon Elizabeth 15–38DAA fruit, the sucrose content gradually increases as the fruit matures, while the expression of CmFLA1 gradually decreases.

[0066] All of the above results indicate that CmFLA1 is negatively correlated with the sucrose content of melon fruit.

[0067] Example 3:

[0068] Verification of protein interactions between CmFLA1 and CmTST2

[0069] Yeast two-hybrid screening revealed that CmFLA1 and CmTST2 (accession number: XP_008448165.1; CmTST2 is a sugar transport protein located on the vacuolar membrane, capable of transporting sugars from the cytoplasm into the vacuoles for storage) interact. CmFLA1 was constructed into the yeast expression vector pPR3-N, and CmTST2 into the yeast expression vector pBT-SUC. Both recombinant plasmids were transformed into yeast strain NMY51 for co-expression, along with positive and negative control plasmids. Yeast growth was observed on media with two-deficient, four-deficient, and four-deficient plus 3-AT conditions. The yeast two-hybrid results showed that CmFLA1 and CmTST2 could grow on four-deficient plus 3-AT medium, while the negative control could not, indicating their interaction (see [link to article]). Figure 3 (A)

[0070] Example 4:

[0071] sucrose transport analysis after co-expression of CmFLA1 and CmTST2 in yeast

[0072] Yeast strain W303 mutated all sugar transporters on the vacuolar membrane, allowing for the detection of sugar transport capabilities of vacuolar membrane-localized sugar transporters. AtSUC2, a previously reported plasma membrane-localized sucrose transporter, can transport sucrose into the cytoplasm of yeast strain W303. CmTST2 was constructed into the yeast expression vector pDR196. The CmTST2-pDR196 expression vector was then used.

[0073] The three plasmids / CmFLA1-pPR3-N / AtSUC2 were co-transformed into yeast strain W303, with CmTST2-pDR196 / AtSUC2 serving as a control.

[0074] Esculin is a fluorescent analog of sucrose, and it can be used to reflect sucrose uptake. Yeast W303 transformed with the plasmid was shaken to OD... 600 =0.6-0.8, after washing with 50mM phosphate buffer, the bacterial culture was resuspended to OD. 600 =2.0. Add esculin to a final concentration of 1 mM, and incubate at 30°C in the dark for 4-6 hours. After washing with 50 mM phosphate buffer, observe and photograph under a fluorescence microscope at a wavelength of 494-551 nm.

[0075] In CmTST2+AtSUC2 yeast, esculin was observed to be concentrated in the vacuoles, indicating that CmTST2 has the function of transferring sucrose from the cytoplasm into the vacuoles. In CmTST2+CmFLA1+AtSUC2 yeast, esculin was concentrated in the cytoplasm, indicating that the interaction between CmFLA1 and CmTST2 inhibits its ability to transfer sucrose into the vacuoles (see...). Figure 3 (B)

[0076] Example 5:

[0077] Application of knocking out the gene encoding bundle arabinogalactan in melon in increasing the sugar content of melon fruit

[0078] The 19nt target sequence (ATCTTGCCGTCTCAAGTGG) on the CDS of the CmFLA1 gene was designed and the target sgRNA primers were synthesized by the company. The target sequence was constructed into the CRISPR / Cas9 knockout vector pKSE402 with a GFP fluorescent tag, and the FLA1-pKSE402 positive gene editing plasmid was obtained and then transformed into Agrobacterium tumefaciens strain EH105 for transient transformation of melon fruits.

[0079] The Agrobacterium obtained above was shaken to OD 600=0.6-0.8, after centrifuging to collect the bacterial culture, dilute to OD with infection solution (containing 50 mM MES, pH 5.6; 0.1 mM AS; 10 mM MgCl2) to 0.6-0.8. 600 =0.3-0.4, and then injected into thin-skinned melon fruits that were 10 days old. At the same time, an empty control was injected. Each treatment was injected with at least 6 melons of the same size.

[0080] Observe GFP fluorescence after 4-5 days of cultivation; the presence of fluorescence indicates successful vector transformation and expression. Experimental results show that both the knockout vector and the empty vector were successfully transformed into melon fruits (see...). Figure 4 (Central A, only one fruit from each is shown as a representative). Fluorescent pulp was removed and sequenced to determine gene editing status. Sequencing results showed that the CmFLA1 gene was not edited in the pulp transiently transformed with the empty vector control, while the pulp transformed with the FLA1-pKSE402 editing vector showed three types of editing (see...). Figure 4 The three editing types (B and sequences 3, 4, 5) resulted in the deletion of 1, 2, and 4 bases, respectively, which led to reverse translocation of the gene coding sequence and loss of gene function, indicating that the editing was effective.

[0081] The sugar content in the pulp of the above samples was measured, and the results showed that the sucrose content in the pulp of melons with the CmFLA1 gene knocked out was significantly higher than that in the control (see...). Figure 4 The results indicate that inhibiting CmFLA1 can promote sucrose accumulation in melon fruits.

[0082] Example 6:

[0083] Application of arabinogalactan protein inhibitors in increasing sugar content in melon fruits:

[0084] In this embodiment, the inhibitor used is β-glucosyl-Yariv, which can inhibit the expression of the fascicular arabinogalactan protein family genes (including CmFLA1). Other fascicular arabinogalactan protein inhibitors can also produce the same effect.

[0085] The experiment used the low-sugar melon variety 'Baicai Gua'. After pollination and fruit set, melons with consistent node positions and similar fruit size (22 DAA after pollination) from different plants were selected. A β-glucosyl-Yariv solution (200 mg / L, dissolved in 0.15 M NaCl solution) was sprayed on the fruit surface three times daily from 8:00 to 9:00 AM, ensuring even distribution. A water treatment served as a control. Three replicates were used, and each melon was labeled.

[0086] Samples were taken after 5 days of treatment, and the contents of sucrose, glucose and fructose in the samples were measured. At the same time, RNA was extracted, and cDNA was obtained by reverse transcription. The expression level of CmFLA1 gene was analyzed by fluorescence quantitative analysis.

[0087] Experimental results showed that β-Yariv treatment significantly reduced CmFLA1 gene expression, indicating that β-glucosyl-Yariv effectively inhibited CmFLA1 expression (see Figure 5A). Simultaneously, sugar content analysis showed that inhibiting CmFLA1 expression significantly increased sucrose content (see Figure 5A). Figure 5 (B) indicates that the fascicular arabinogalactan protein inhibitor has the effect of increasing the sugar content of melon fruit. It achieves this by inhibiting CmFLA1 protein and regulating CmTST2 protein, thereby increasing the accumulation of sucrose in melon fruit.

Claims

1. Application of the coding gene of melon bundle arabinogalactan in controlling the sucrose content of melon fruit, wherein the sucrose content of melon fruit is increased by knocking out, inhibiting or silencing the coding gene of melon bundle arabinogalactan, wherein the melon bundle arabinogalactan is shown in SEQ ID NO.

2.

2. The application according to claim 1, which increases the sucrose content of melon fruit by inhibiting or reducing the expression of fascicular arabinogalactan protein in melon.

3. The application according to claim 2, wherein a substance that inhibits or reduces the expression level of arabinogalactan protein in melon is introduced into melon; wherein the substance is a nucleic acid molecule, or its expression cassette, recombinant vector, or recombinant microorganism that inhibits or reduces the expression level of arabinogalactan protein in melon.

4. The application according to claim 1, wherein the knockout is performed using the CRISPR / Cas9 system, and the target site of the gRNA in the system is: ATCTTGCCGTCTCAAGTGG.

5. The application according to claim 4, wherein the melon with increased sucrose content obtained after editing by the CRISPR / Cas9 system contains the gene shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

6. Application of the coding gene of melon bundle arabinogalactan protein in improving the quality of melon fruit: The sucrose content of melon fruit is increased by knocking out, inhibiting or silencing the coding gene of melon bundle arabinogalactan protein, wherein the melon bundle arabinogalactan protein is shown in SEQ ID NO.

2.

7. Application of a melon bundled arabinogalactan protein activity inhibitor in increasing the sucrose content of melon fruit, wherein the melon bundled arabinogalactan protein is shown in SEQ ID NO.2, and the inhibitor is... β -glucosyl- Yariv .

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