Sgsup gene of momordica grosvenori, protein, gene editing target, recombinant expression vector, application and breeding method

Abstract

The present application discloses a momordica grosvenori SgSUP gene and its protein, gene editing target point, recombinant expression vector, application and breeding method, belongs to the field of plant genetic engineering and genetic breeding technology, the nucleotide sequence of the gene is shown as SEQ ID NO:1.The present application clones the momordica grosvenori plant type, flowering, inflorescence structure and flower fertility key gene SgSUP for the first time, reveals that it affects plant height, flowering, inflorescence branching, small flower number and flower fertility by regulating meristem cell division and flower organ development.Through gene editing or transgenic technology, new varieties of momordica grosvenori with dwarf, stringing and self-pollination can be cultivated directionally, and the planting yield and breeding efficiency are improved significantly.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and genetic breeding technology, specifically a method for the SgSUP gene and its protein, gene editing target, recombinant expression vector, application and breeding of Luo Han Guo (Siraitia grosvenorii). Background Technology

[0002] Monk fruit (Siraitia grosvenorii) is a dioecious plant, with significant differences between female and male plants in terms of inflorescence branching, number of florets, flower morphology, and fertility.

[0003] Existing female varieties have long flowering times and require artificial pollination to produce fruit. Their tall stature necessitates trellises, leading to low planting density, difficulty in large-scale production, and high costs. Furthermore, their inability to self-pollinate makes it difficult to cultivate superior inbred lines, severely restricting the efficiency of breeding superior varieties. These challenges have resulted in high raw material costs for monk fruit processing, a persistent bottleneck restricting the industry's growth. While existing SUP gene regulation focuses primarily on dicotyledonous model plants, it does not address the unique needs of monk fruit regarding flower sex differentiation and plant type improvement.

[0004] This invention is the first to clone the SgSUP gene (SEQ ID NO:1) of *Monk Fruit*, discovering its unique SNP sites in the zinc finger domain and LZ-like transcriptional repression domain, and confirming that this gene regulates plant height, flowering period, inflorescence branching, floret number, and flower sex development. By targeting the SgSUP gene using gene editing technology, new varieties with short stems, clustered fruit, and self-pollination can be created, significantly improving planting density, yield, and breeding efficiency of superior varieties of *Monk Fruit*, effectively overcoming the bottlenecks restricting the development of the *Monk Fruit* industry. Summary of the Invention

[0005] To address the challenges of planting and breeding of monk fruit due to its dioecious nature, this invention clones and verifies the function of the SgSUP gene in monk fruit for the first time. It was found that the gene regulates plant height, flowering period, inflorescence branching, number of florets, and flower sex differentiation, providing a molecular target for creating new monk fruit varieties that are short-stemmed, produce clusters of fruit, and are self-fertile.

[0006] To achieve these objectives of the present invention, the present invention provides a SgSUP gene from Luo Han Guo, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] Furthermore, the SgSUP gene has a G / A SNP missense mutation (R / K) at position 521 of the C-terminal LZ-like transcriptional repression region, which is used to regulate the plant height, flowering period, inflorescence branching, number of florets, or flower fertility of monk fruit.

[0008] The present invention provides a protein encoded by the SgSUP gene of monk fruit, wherein the amino acid sequence of the protein is the SgSUP protein sequence shown in SEQ ID NO:2.

[0009] This invention provides a gene editing target that targets at least one of the following regions:

[0010] a) The zinc finger domain or LZ-like transcriptional repression domain of the SgSUP gene described above;

[0011] b) The promoter region of the SgSUP gene, which contains the regulatory region from 2000 bp upstream of the SgSUP gene to the transcription start site;

[0012] It is used to regulate the plant height, flowering period, inflorescence branching, number of florets, or fertility of monk fruit.

[0013] The present invention provides a recombinant expression vector comprising the SgSUP gene, wherein the gene is operatively linked to a heterologous promoter.

[0014] This invention provides the application of the SgSUP gene in regulating plant height, flowering period, inflorescence branching, number of florets, or flower fertility, wherein the plant includes monk fruit.

[0015] This invention provides a method for breeding monk fruit, comprising the following steps:

[0016] Transform the aforementioned gene or recombinant expression vector into monk fruit cells, and after cultivation and screening, obtain monk fruit plants that are short-stemmed, produce clusters of fruit, or are self-fertile.

[0017] Ideally, high-yield cultivation can be achieved by combining monk fruit plants with dense planting.

[0018] Preferably, one or more of the following plant phenotypes are obtained through overexpression or knockout of the SgSUP gene:

[0019] a) Plant height decreased;

[0020] b) Increased branching of the inflorescence;

[0021] c) The number of small flowers increases;

[0022] d) Self-fertile.

[0023] The present invention has at least the following beneficial effects:

[0024] This invention is the first to clone the key gene SgSUP, which influences the plant type, flowering period, inflorescence structure, and fertility of *Siraitia grosvenorii* (monk fruit). It reveals that SgSUP affects plant height, flowering period, inflorescence branching, floret number, and flower fertility by regulating meristematic cell division and floral organ development. Through gene editing or transgenic technology, new *Siraitia grosvenorii* varieties that are short-stemmed, produce clusters of fruit, and are self-fertile can be selectively bred. Compared to existing tall varieties that require artificial pollination to bear fruit, this invention can significantly improve planting density, yield, and breeding efficiency.

[0025] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0026] Figure 1 This is a conserved domain of the SgSUP gene;

[0027] Figure 2 It is a Zinc-finger structure field;

[0028] Figure 3 It is an LZ-like transcriptional repressor domain;

[0029] Figure 4 The evolutionary relationship of SUP genes between monk fruit and other plants;

[0030] Figure 5 Comparison of vigor, plant height, inflorescence, and flowering period of wild-type and SgSUP gene-transformed Arabidopsis thaliana seedlings planted at the same time;

[0031] Figure 6 Comparison of fertility variation between wild-type and SgSUP gene-transformed Arabidopsis thaliana planted at the same time;

[0032] Figure 7 This is a picture of the wild-type plant in bloom; in the picture, the pistil is higher than the stamen.

[0033] Figure 8 The first image shows the flower of a plant transgenic SUP plant in bloom; in the image, the pistil is noticeably lower than the stamen when the flower is open.

[0034] Figure 9 This is the second image of a flower from a plant transgenic SUP; in this image, the flower lacks a pistil, with only the stamens developing normally. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0036] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0037] Example 1

[0038] Isolation of the SgSUP gene from monk fruit

[0039] 1. Primer sequence

[0040] Cloning primer SgSUP-F: 5'-ATGGAGAGGAACTGTTTACGC-3';

[0041] Cloning primer SgSUP-R: 5'-CTAAGAGTACCCTAATCGAAGCTC-3'.

[0042] The cloning primers were designed based on the Luo Han Guo transcriptome data, targeting the full-length CDS region of the SgSUP gene. SgSUP-F corresponds to the start codon ATG, and SgSUP-R contains the stop codon TAG. The amplification products are expected to cover the complete open reading frame (ORF) of SgSUP.

[0043] 2. Experimental Methods

[0044] The reagents include:

[0045] RNase-free ddH2O: Double-distilled water (ddH2O) that is treated to ensure it is free of ribonuclease (RNase).

[0046] 5×gDNA wiper mix: A 5x concentrated genomic DNA removal reagent containing DNase I enzyme. Used to degrade residual genomic DNA (gDNA) in RNA samples, ensuring specificity for subsequent RT-PCR or cDNA synthesis.

[0047] 10×RT Mix: A 10-fold concentrated premix for reverse transcription reaction.

[0048] HiScript III 1st Strand cDNA Synthesis Kit: A kit for synthesizing first-strand cDNA.

[0049] Rapid Taq Master Mix: A premixed PCR reaction solution containing Taq DNA polymerase, dNTPs, buffer, etc.

[0050] pEASY-T1 Cloning Vector: A TA cloning vector used for the rapid ligation of PCR products via TA cloning (adding an A tail to the 3' end of the PCR product using Taq enzyme).

[0051] DH5α competent cells: Commonly used Escherichia coli (E. coli) competent cells.

[0052] Oligo(dT) 20 VN: Anchor primer, the sequence is Oligo(dT) plus variable nucleotide (VN).

[0053] TRIzol reagent: used for rapid extraction of total RNA (including RNA, DNA, and protein separation functions).

[0054] LB medium: contains tryptone, yeast extract, and NaCl.

[0055] The steps include:

[0056] Total RNA was extracted from Siraitia grosvenorii using a plant RNA extraction kit and used as a template. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers SgSUP-F and SgSUP-R were designed, and the target gene was amplified by RT-PCR. The amplified product was then recovered from gel ligation and sequenced to obtain the target gene sequence.

[0057] Specifically, 0.1 g of female flower buds (0.5 mm in diameter) from *Siraitia grosvenorii* were ground in liquid nitrogen and total RNA was extracted using TRIzol reagent (Invitrogen). cDNA was then synthesized via reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme).

[0058] RNA template denaturation: 5 μg of total RNA was added to 8 μL with RNase-free ddH2O, heated at 65℃ for 5 min, rapidly cooled on ice, and left to stand on ice for 2 min to obtain denatured RNA;

[0059] Genomic DNA removal: Add 2 μL of 5 × gDNA wiper Mix (5-fold concentrated genomic DNA removal reagent) to the denatured RNA from the previous step, gently mix with a pipette, and react at 42℃ for 2 min to obtain denatured RNA mixture;

[0060] Reverse transcription to synthesize cDNA:

[0061] 10 μL of denatured RNA mixture;

[0062] 10 × RT Mix 2 μL;

[0063] HiScript III Enzyme Mix 2 μL;

[0064] Oligo (dT) 20 VN 1 μL;

[0065] RNase-free ddH2O 5 μL;

[0066] React at 37°C for 45 min, then at 85°C for 5 sec. The product can be used immediately for PCR reactions or stored at -20°C.

[0067] PCR reaction system (50 μL):

[0068] 2 × Rapid Taq Master Mix (Vazyme) 25 μL;

[0069] 2 μL each of 10 μM SgSUP-F / SgSUP-R primers;

[0070] 1 μL of cDNA template;

[0071] Add ddH2O to a final volume of 50 μL.

[0072] Amplification procedure:

[0073] Pre-denaturation: 95℃ for 3 min;

[0074] Cycle (35 times): 95℃ 15 sec → 58℃ 15 sec → 72℃ 20 sec;

[0075] Final extension: 72℃ for 5 min;

[0076] The PCR product was obtained, and the target band size was 612 bp.

[0077] The PCR product was recovered and ligated into the pEASY-T1 Cloning Vector (Full Gold), transformed into DH5α competent cells, and the SgSUP gene was obtained by sequencing verification. Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0078] SEQ ID NO:1

[0079] atggagagga actgtttacg caccagcttc agataccaga gagttaacac gagcgcgatc 60

[0080] ggcagaggtg gaggagg agaatccaac gccaccatga aaagccagag aaacaagctg

[0081] ggcagggcgg aggaggattt cataaatggg atttcatggc ctcccagaag ttacacgtgc

[0082] aacttctgca aaagggaat tagatcggct caagctctgg gcggccatat gaatgttcat

[0083] cggagagata gggccatgct cagacagtac tctcccccag ctgatggtga tcagttcacc 300

[0084] 360. aatcttaacc ttaacctcca acacaaccct aaccctaatc tctcttcttc acgttctgct

[0085] cctgaaacta acaaatgggc caccgcccgc gacgacgacg tcgcattcgg tctccggttc 420

[0086] aaccggaag tttccgattt gaaaacgtcg accacggaga ttgtgaacc taatctgttt

[0087] gagcgaatcc atggttgtgg agattggac aaggcggaga gaatcattgg gttggacatt

[0088] gaaagcagta ctactatact tgctgactcc aagaaggaca ttgatttgga gcttcgatta

[0089] gggtactctt 612

[0090] SEQ ID NO:2

[0091] MERNCLRTSF RYQRVNTSAI GRGGGGGESN ATMKSQRNKL GRAEEDFING ISWPPRSYTC 60

[0093] NFCKREFRSA QALGGHMNVH RRDRAMLRQY SPPADGDQFT NLNLNLQHNP NPNLSSSRSA 120

[0095] PETNKWATAR DDDVAFGLRF NRKVSDLKTS TTEIVKPNLF ERIHGCGDWN KAERIIGLDI 180

[0097] ESSTTILADS KKDIDLELRL GYS 203

[0099] The SgSUP gene is 612 bp in length, encoding 203 amino acids; it has a start codon (ATG) and a stop codon (TAG), and its conserved gene domains include... Figure 1 As shown.

[0100] Analysis using the NCBI CDD database revealed that the SgSUP protein contains a typical C2H2 type zinc finger motif (C-X2-CX). 12 -H-X3-H), located at positions 58-82 of the amino acid sequence, the Zinc-finger domain is as follows Figure 2 As shown.

[0101] The C-terminal amino acids 173-203 of SgSUP belong to the LZ-like transcriptional repression domain, among which the DLELRL motif (amino acid positions 195-200) is an essential domain for transcriptional repression. Figure 3 As shown.

[0102] The evolutionary relationship between the SgSUP gene of monk fruit and the SUP genes of the following plants, in descending order of genetic relationship, is as follows: cucumber CsSUP; alfalfa MtSUP; tomato SlySUP; chrysanthemum CmSUP; white bottlebrush SlSUP; and Arabidopsis thaliana AtSUP.

[0103] Phylogenetic analysis showed that *Siraitia grosvenorii* SgSUP was closest in genetic distance to *Cucumber* CsSUP, and showed the highest degree of divergence from *Arabidopsis thaliana* AtSUP. The results are as follows: Figure 4 As shown.

[0104] Example 2

[0105] Functional identification of Arabidopsis thaliana transformed with SgSUP gene

[0106] 1. Carrier Construction

[0107] The recombinant plasmid PHB-35S-SgSUP (containing the SgSUP gene driven by the CaMV 35S promoter) was constructed as follows: the CDS region of the SgSUP gene (SEQ ID NO:1) and the PHB vector were digested with NcoI and XhoI enzymes. After recovering the target fragment, it was ligated overnight at 16°C using T4 ligase. The plasmid was transformed into DH5α Escherichia coli, kanamycin-resistant colonies were screened, and the insertion direction and sequence correctness were verified by sequencing.

[0108] 2. Agrobacterium-mediated transformation

[0109] The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. The cells were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin and cultured at 28°C with shaking until OD600=1.0. The cells were collected by centrifugation and resuspended in osmotic buffer until OD600=1.0, which is the transformation solution.

[0110] 3. Arabidopsis genetic transformation and screening

[0111] Wild-type Arabidopsis thaliana was selected at the early flowering stage (main inflorescence height about 5-10 cm). The inflorescence was immersed in the transformation solution for 30 seconds, covered with plastic wrap and kept moist in the dark for 24 hours. The inoculation was repeated twice, with an interval of 3 days, and then cultured normally (22℃, 16 hours light / 8 hours dark).

[0112] T0 generation seeds were harvested, disinfected with 70% ethanol for 1 minute and sodium hypochlorite for 10 minutes, and sown on 1 / 2 MS solid medium containing 50 μg / mL hygromycin to obtain resistant plants;

[0113] Resistant plants were transplanted into a nutrient soil with a peat:vermiculite:perlite mass ratio of 2:7:1 and the plants were screened.

[0114] 4. Inflorescence phenotypic detection

[0115] Number of inflorescence branches: When the main inflorescence reaches about 10 cm in length, count the number of lateral branches. Transgenic lines have 0-1 branches, while wild-type lines have an average of 6-8 branches. Figure 5 );

[0116] Number of florets: The number of florets in the main inflorescence of a single plant was counted. The average number of florets in the transgenic lines was 12±3, while that in the wild type was 28±5.

[0117] Floral organ morphology: In wild-type flowers, the pistil is significantly longer than the stamen when open. Figure 7 In SgSUP overexpression lines, pistil development was severely restricted, with the number of pistils in the flower significantly lower than that of the stamens. Figure 8 ), and some flowers even completely lack pistils, retaining only normal stamens ( Figure 9 This indicates that SgSUP directly regulates floral sex differentiation by affecting pistil elongation.

[0118] 5. Plant height and fertility testing

[0119] Plant height measurement: Plant height was measured at the end of flowering (45 days after sowing). The average height of transgenic lines was 4-13 cm, and that of wild-type lines was 28±3 cm. Figure 6 );

[0120] Fruit set rate: The number of pods per plant was counted, with transgenic lines averaging 5±2 pods and wild-type lines averaging 25±4 pods. Pod length was shortened by 40%. Results are as follows: Figure 6 As shown.

[0121] This invention, through the construction of the PHB-35S-SgSUP vector and its transformation into Arabidopsis thaliana, demonstrates that overexpression of the SgSUP gene can reduce plant height, delay flowering, decrease in inflorescence branching and the number of florets, and simultaneously reduce fertility. Reduced fertility refers to a decrease in the reproductive capacity of plants through gene regulation, specifically manifested as weakened pollen viability, inhibited ovule development, reduced pollination success rate, or reduced seed yield. Editing or knocking out the SgSUP gene can promote the development of stamens in female flowers (or pistils in male flowers), forming hermaphroditic flowers and achieving self-pollination.

[0122] The above results indicate that SgSUP has the function of regulating plant height, flowering period, inflorescence branching, floret number, and flower fertility. Therefore, it can be used to regulate plant height, flowering period, inflorescence branching, floret number, or flower fertility in *Siraitia grosvenorii* plants. In the breeding process, the SgSUP gene or its recombinant expression vector can be transformed into *Siraitia grosvenorii* cells, and after cultivation, *Siraitia grosvenorii* plants with short stems, fruit clusters, or (hermaphroditic) self-pollinating characteristics can be obtained. The SgSUP gene can serve as a key target for regulating plant type, inflorescence structure, and flower fertility. Specifically, this gene editing target targets at least one of the following regions: a) the zinc finger domain or LZ-like transcriptional repression domain of the SgSUP gene; b) the promoter region of the SgSUP gene, which contains the regulatory region from 2000 bp upstream of the SgSUP gene to the transcription start site; used to regulate plant height, flowering period, inflorescence branching, floret number, or fertility in *Siraitia grosvenorii*.

[0123] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A SgSUP gene from monk fruit, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A protein encoded by the SgSUP gene of *Monk Fruit* as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, It contains the SgSUP gene as described in claim 1.

4. The application of overexpression of the SgSUP gene as described in claim 1 in reducing plant height, wherein the plant is monk fruit.

5. A method for breeding monk fruit, characterized in that, The procedure includes the following steps: genetically transforming the monk fruit with the gene described in claim 1 or the recombinant expression vector described in claim 3, and then cultivating and screening to obtain dwarf monk fruit plants.

6. The method for breeding monk fruit as described in claim 5, characterized in that, High-yield cultivation can be achieved by combining dense planting with high-density planting.

7. The method for breeding monk fruit as described in claim 5, characterized in that, The following plant phenotypes were obtained by overexpressing the SgSUP gene: a) Reduced plant height.

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