Application of slput3 gene in inhibiting lateral branch germination of tomato

Abstract

The application relates to the field of agricultural biotechnology, in particular to application of a PUT3 gene in inhibiting lateral branch germination of tomatoes, nucleotide and amino acid sequences of the PUT3 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively; application of the tomato PUT3 gene in regulating lateral branch germination of tomatoes, application of the lPUT3 gene in inhibiting lateral branch germination of tomatoes, and the SlPUT3 gene can effectively regulate growth of plants by regulating the SlPUT3 gene.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to the application of the PUT3 gene in inhibiting the germination of lateral branches in tomatoes. Background Technology

[0002] Tomato (Solanum lycopersicum) is the vegetable with the largest planting area under greenhouse cultivation in my country. It has a vine-like growth habit and extremely strong branching ability. The branching pattern of tomatoes is one of the determining factors for indicators such as yield and quality. An appropriate branching pattern can improve yield and economic benefits, while excessive branching can cause many negative effects, such as excessively high spatial density leading to disease; rapid branching causing plants to easily shade each other, resulting in nutrient consumption; and increased management costs such as pruning and pinching. Therefore, studying the branching mechanism of tomatoes and finding a reasonable tomato plant type has significant production value.

[0003] Polyamines (PAs) are a class of compounds containing two or more amino groups. They play a variety of roles in plant growth and development and stress responses, and have gained increasing attention as a novel type of plant growth regulator. Putrescine (Put), spermidine (Spd), and spermine (Spm) are the main PAs in plants, participating in the regulation of various physiological processes, such as flower development, embryogenesis, organogenesis, senescence, and fruit ripening, as well as responses to biotic and abiotic stresses. With the development of molecular biotechnology, increasing evidence suggests that both exogenous application and endogenous control of PA production through genetic engineering can positively influence plant growth, yield, and stress resistance.

[0004] Polyamine transport plays a crucial role in regulating intracellular PA levels in plant cells. Studies have revealed that transmembrane proteins of the type I amino acid transporter (LAT) family play a vital role in regulating PA transport. In Arabidopsis and rice, the polyamine transporters AtPUT5, OsPUT1, AtPUT2, AtPUT3, and OsPUT3 precisely regulate the dynamic balance of spermidine, thereby controlling the flowering time of plants. The Arabidopsis put2 mutant excessively accumulates polyamines and actively participates in phyA (phytochrome A)-mediated seed germination.

[0005] In Arabidopsis thaliana, the LHR1 gene encodes the polyamine transporter LHR1 / PUT3, which is essential for extracellular polyamine uptake and regulates the stability of endogenous polyamines under high temperatures, thereby participating in the mRNA of several key heat stress response genes. Studies have found that plasma membrane Na... + / H + The transporter SOS1 and protein kinase SOS2 are essential for maintaining intracellular Na+. + and K +Two important homeostatic proteins, PUT3 and SOS1, can form complexes with the protein kinase SOS2 under stress conditions, regulating each other's transport activities through protein-protein interactions and phosphorylation, thereby modulating plant salt tolerance. Although the function of Put3 in plants is gradually being understood, it is still insufficient; therefore, further in-depth research on the function of the Put3 gene is necessary. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides the application of the SlPUT3 gene in inhibiting the germination of lateral branches in tomatoes. By regulating the SlPUT3 gene, plant growth can be effectively controlled.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A tomato PUT3 gene is provided, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Screening revealed that the tomato SlPUT3 gene is related to the development of tomato lateral branches. The number of lateral branches sprouted by the tomato SlPUT3 gene mutant put3 was significantly reduced compared with the wild type Ailsa Craig (WT). This gene can be used to breed tomato varieties with fewer branches and to improve the tomato plant type.

[0010] Provided is the protein encoded by the tomato PUT3 gene described above, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] This paper provides an application of the aforementioned tomato PUT3 gene in regulating the germination of tomato lateral branches.

[0012] Since the tomato PUT3 gene has been found to regulate the number of lateral branches that sprout from tomatoes, adjusting the tomato PUT3 gene on tomato plants can effectively suppress the sprouting of lateral branches.

[0013] This invention provides an application of the aforementioned tomato PUT3 gene in the breeding of tomato plants that reduce lateral branching.

[0014] Since the expression of the tomato PUT3 gene has been found to be closely related to the number of tomato lateral branches, tomato varieties with fewer lateral branches can be screened by identifying the expression of the tomato PUT3 gene in tomato plants.

[0015] A method for improving tomato plant architecture with fewer lateral branch sprouting is provided by silencing or knocking out the tomato PUT3 gene mentioned above.

[0016] When the function of the SlPUT3 gene in tomatoes is reduced or eliminated through gene silencing or knockout, the number of lateral branches in tomatoes can be reduced.

[0017] In some embodiments, the silencing or knockout step includes: designing a gene editing target sequence based on the tomato PUT3 gene target, transferring the gene editing target sequence into a host cell, and obtaining a recombinant CRISPR vector;

[0018] The CRISPR vector was transferred into tomatoes via Agrobacterium-mediated transformation to obtain gene-edited tomato plants with improved plant type.

[0019] Gene editing target sequences were designed targeting the tomato PUT3 gene. These sequences were then loaded into host cells, which acted as vectors to introduce the gene editing target sequences into tomato plants to edit the tomato PUT3 gene, thereby silencing or knocking out the SlPUT3 gene.

[0020] In some embodiments, the sequence of the tomato PUT3 gene target site is as follows:

[0021] GTGTGGGTTTCATCTGCTTT; and ATGGGGGACTACAATGGTGC.

[0022] In some embodiments, the host cell is Agrobacterium GV3101.

[0023] In some embodiments, the Agrobacterium-mediated transformation includes: infecting tomato cotyledons with a CRISPR vector containing Agrobacterium tumefaciens solution, re-obtaining seedlings through tissue culture, and screening for mutant plants with the PUT3 gene deletion.

[0024] This invention helps to reveal the molecular mechanism of tomato lateral branch development, laying the foundation for the creation of new tomato germplasm with few branches and the breeding of new varieties, thereby improving the efficiency of simplified tomato cultivation and reducing production costs. Attached Figure Description

[0025] Figure 1 This is a graph showing the detection results of SlPUT3 gene knockout plants in a specific embodiment.

[0026] Figure 2 The images show the tomato mutant Slput3 with few lateral branches and the wild-type Ailsa Craig plant, representing specific embodiments.

[0027] Figure 3 This is a comparison diagram of lateral bud abortion in wild-type control (WT) and tomato SlPUT3 gene knockout plants in a specific embodiment.

[0028] Figure 4 This is a comparison chart of the plant height of the mutant Slput3 in a specific embodiment and the plant height of WT.

[0029] Figure 5This is a comparison chart of the relative expression levels of the SlPUT3 gene in the mutant Slput3 of a specific embodiment and the relative expression levels of the SlPUT3 gene in WT. Detailed Implementation

[0030] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0031] Example 1

[0032] Cloning of the tomato SlPUT3 gene and construction of a tomato SlPUT3 gene knockout vector

[0033] 1. Total RNA extraction from tomatoes

[0034] Total RNA was extracted from young tomato leaves using the Plant Total RNA Extraction Kit (TIANGEN). The steps were as follows:

[0035] (1) Take 0.1g of tomato leaves, grind them in liquid nitrogen, add 1mL of lysis buffer, and then treat them with a homogenizer;

[0036] (2) Place the homogenized sample at 15-30℃ for 5 min to allow the nucleic acid-protein complex to be completely separated;

[0037] (3) Centrifuge at 4℃ and 12,000 rpm for 5 min, remove the supernatant and transfer it to a new RNase-free centrifuge tube.

[0038] (4) Add 200 μL of chloroform, cap the tube, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0039] (5) Centrifuge at 4℃ and 12,000 rpm for 10 min. The sample will separate into three layers: a yellow organic phase, an intermediate layer, and a colorless aqueous phase. RNA is mainly in the aqueous phase, which has a volume of approximately 50% of the RZ lysis buffer used. Transfer the aqueous phase to a new tube for the next step.

[0040] (6) Slowly add 0.5 times the volume of anhydrous ethanol and mix well (precipitation may occur at this time). Transfer the resulting solution and precipitate together into the adsorption column CR3, centrifuge at 12,000 rpm for 30 s at 4℃, and discard the waste liquid in the collection tube;

[0041] (7) Add 500 μL of protein removal solution RD to the adsorption column CR3, centrifuge at 12,000 rpm for 30 s at 4 °C, and discard the waste liquid.

[0042] (8) Add 600 μL of washing solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s at 4℃, and discard the waste liquid.

[0043] (9) Repeat step (8);

[0044] (10) Place the adsorption column into a 2mL collection tube, centrifuge at 4℃ and 12,000rpm for 2min to remove residual waste liquid;

[0045] (11) Transfer the adsorption column CR3 into a new centrifuge tube, add 50 μL LNase-Free ddH2O, place at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min at 4℃.

[0046] (12) The content and purity of RNA samples were tested by measuring OD260 / OD280 using a UV spectrophotometer. The concentration was 835 ng / μL and OD260 / OD280 = 2.12.

[0047] 2. Gene cloning and construction of the CRISPR / Cas9-PUT3 knockout vector for the tomato PUT3 gene.

[0048] 1 μg of tomato total RNA was reverse transcribed into cDNA using the ReverTraAce qPCR RT Kit (Toyobo).

[0049] The genome sequence of tomato PUT3 was obtained from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). Using CRISPR-GE (http: / / skl.scau.edu.cn / targetdesign / ), candidate target sequences for the SlPUT3 gene sgRNA (small guide RNA) GGTGGGGTTTCATCTGCTTT and ATGGGGGACTACAATGGTGC were designed. These two target sequences were inserted into a single guide RNA (sgRNA) expression cassette using overlap extension PCR, and then cloned into the pYLCRISPR / Cas9Pubi-H vector using the GoldenGate ligation method. The correctly sequenced pFGC1008-Put2-3HA and pYLCRISPR / Cas9Pubi-H-put2 binary vectors were transformed into Agrobacterium tumefaciens GV3101 strain via point transfection.

[0050] (1) Construction of sgRNA expression cassettes using overlapping PCR method

[0051] In the first round of PCR, the target sequence was introduced downstream of the U3 / U6 promoter and upstream of the sgRNA sequence.

[0052] The PCR reaction system is as follows:

[0053]

[0054]

[0055] PCR reaction conditions settings:

[0056]

[0057] In the second round of PCR, the promoter, target site, and sgRNA were constructed into a complete expression cassette.

[0058] The reaction mixture of reaction 1 + reaction 2 dilution buffer = 1 μL of reaction 1 product + 1 μL of reaction 2 product + ddH2O 8 μL is as follows:

[0059]

[0060] PCR reaction conditions settings:

[0061]

[0062] (2) Cloning the sgRNA expression cassette into the pYLCRISPR / Cas9 vector

[0063] The PCR reaction system is as follows:

[0064]

[0065] PCR reaction conditions settings:

[0066]

[0067] Example 2

[0068] Constructing transgenic plants with SlPUT3 gene knockout in tomatoes

[0069] 1. Tomato Ailsa Craig seeds were soaked in tap water (or shaken at 28°C and 200 rpm) for 6-8 hours, then sterilized with 75% alcohol for 30 seconds, followed by sterilization in 10% NaClO for 15 minutes (shaking at 28°C and 200 rpm). The seeds were rinsed three times with sterile distilled water and transferred to sterile containers, inoculated onto 1 / 2 MS medium. Germination was achieved at 25°C in the dark, followed by transfer to a light-controlled culture chamber. Seedling growth conditions were 25°C, 16 hours of light / 8 hours of darkness, and a light intensity of 1800 lx.

[0070] 2. Preparation of explants and culture of Agrobacterium

[0071] Six days after seed germination, cut off the cotyledons of sterile seedlings with a knife, leaving a small section of the petiole attached. Pre-culture these cotyledons in KCMS culture medium for 1 day (avoid light, overnight is sufficient; prolonged culture may lead to over-infection). Pick a single Agrobacterium colony from an LB agar plate containing antibiotics and inoculate it into 20 mL of LB agar containing antibiotics. Incubate overnight at 28°C and 200 rpm until mid-log phase (OD600≈1.0, approximately 16-24 h). Shake the culture first, then cut off the cotyledons (inoculate between 12-20 h).

[0072] 3. Conversion and Regeneration

[0073] 3.1 Infection

[0074] Transfer the cultured Agrobacterium tumefaciens strain A containing the tomato SlPUT3 gene knockout vector to a 10 mL centrifuge tube (sealed with sealing film), and centrifuge at 4000 rpm for 10 min at 4°C. Discard the culture medium, add MS0.2 suspension medium, and mix well. Transfer 3-4 dishes of cotyledonary explants to sterile culture dishes containing MS0.2, and add the resuspended bacterial solution until OD600 ≈ 0.2-0.3 (7-8 mL MS0.2, with 2-3 mL poured into the culture dish). Inoculate in the dark for 4-5 min, gently agitating the culture dish. Transfer the explants to sterile filter paper, blot off any remaining bacterial solution, and re-inoculate onto KCMS. Co-culture at 22°C for 2 days (in the dark). Place the tubes with the reverse side facing up.

[0075] 3.2 Selection, Cultivation, and Regeneration

[0076] After co-culturing, the explants were carefully transferred to MRS1 (2Z+) and then transferred to MRS2 (0.2Z+) for 2-3 weeks. During the culture process, contaminated materials should be cleaned up in time until regenerated shoots grow.

[0077] 4. Regenerating buds, rooting, and transplanting

[0078] When the regenerated shoots grow to about 1cm, cut them off (optional, to avoid damaging the rooting area) and place them in rooting medium to root. Two weeks later, harden off the well-rooted seedlings that have grown to about 5cm, transplant them into disposable plastic cups, and after they have survived, transplant them into flowerpots to obtain tomato SlPUT3 gene knockout plants. The test results of the SlPUT3 gene knockout plants are as follows. Figure 1 Tomato mutant Slput3 with few lateral branches and wild-type Ailsa Craig plants, such as Figure 2 .

[0079] The results showed that, compared with the wild-type control (WT), the lateral buds of tomato SlPUT3 gene knockout plants were aborted. Figure 3 The mutant Slput3 was significantly taller than the WT mutant. Figure 4 Furthermore, the relative expression level of the SlPUT3 gene in the mutant Slput3 was significantly lower than that in the WT mutant. Figure 5 ).

[0080] The nucleotide sequence of tomato SlPUT3 is shown in SEQ ID NO.1.

[0081] The nucleotide sequence is SEQ ID NO.1:

[0082]

[0083] The amino acid sequence of the protein encoded by tomato SlPUT3 is shown in SEQ ID NO.2.

[0084] Amino acid sequence SEQ ID NO.2:

[0085] MVPTTTTTSSETLQNSSISVADKKTSQQNGDNNNQVLRGVNVGTFKIELHFFMKLRVSAQREAAIPMGDYNGAEYIGINEVPSPRANNSNKVSLLPLIFLIFYEVSGGPFGVEDTVQAAGPLFALLGFLIFP LIWSVPEALITAEMGTMFPENGGYVVWVSSALGPYWGFQQGWMKWLSGVIDNALYPVMFLDYLKSAIPALGGGLPRIVAVLALTVVLTYMNYRGLTIVGWVAVSLGILSMLPFVVMGLISIPKIRPERWLVADV HSIDWNLYLNTLFWNLNYWDSISTLAGEVRNPKKTLPKALFYAVLLVVLSYLFPLLIGTGAVPLERELWTDGYFSDIAKILGGVWLRFWLQGAAAVSNMGMFVAEMSSDSFQLLGMAERGMLPEFFAKRSRHG TPILGIIFSASGVLLLSWLSFQEIVAAENFLYCFGMILEFIAFVWLRIKYPNAPRPFKIPGGIIGAILLCVPPAILIGVVLAFSTIKIMIVSLAAVAIGMVLQPCIKLIERKRWLKFSTSSDLPDITAHGPLIR

[0086] This invention cloned the SlPUT3 gene from tomato. Transgenic experiments demonstrated that the mutant slput3, resulting from a mutation in the SlPUT3 gene, produced fewer lateral branches than the wild-type 'Ailsa Craig'. This gene can be used to screen for tomato varieties with fewer branches, thus improving tomato plant architecture. This research helps elucidate the molecular mechanisms of tomato lateral branch development, laying the foundation for the creation of new tomato germplasm and the breeding of new varieties with fewer branches, thereby improving the efficiency of simplified tomato cultivation and reducing production costs.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An application of the tomato PUT3 gene in regulating tomato lateral branch germination, characterized in that, The nucleotide sequence of the tomato PUT3 gene is shown in SEQ ID NO.1; The amino acid sequence of the protein encoded by the tomato PUT3 gene is shown in SEQ ID NO.2; The regulation of tomato lateral branch germination is achieved by silencing or knocking out the tomato PUT3 gene to reduce the number of tomato lateral branches.

2. The application of the tomato PUT3 gene in breeding tomato plants that reduce lateral branching, characterized in that, The nucleotide sequence of the tomato PUT3 gene is shown in SEQ ID NO.1; The amino acid sequence of the protein encoded by the tomato PUT3 gene is shown in SEQ ID NO.2; Tomato plant breeding that reduces lateral branching is achieved by silencing or knocking out the tomato PUT3 gene.

3. A method for improving the tomato plant type with fewer lateral branches, characterized in that, Silencing or knocking out the tomato PUT3 gene as described in claim 1.

4. The method for improving tomato plant type with fewer lateral branch sprouts according to claim 3, characterized in that, The silencing or knockout is performed according to the following steps: designing a gene editing target sequence based on the tomato PUT3 gene target, transferring the gene editing target sequence into host cells to obtain a recombinant CRISPR vector; transferring the CRISPR vector into tomatoes via Agrobacterium-mediated transformation to obtain gene-edited tomato plants with improved plant type.

5. The method for improving tomato plant type with fewer lateral branches according to claim 4, characterized in that, The sequence of the tomato PUT3 gene target site is as follows: GTGTGGGTTTCATCTGCTTT; and ATGGGGGACTACAATGGTGC.

6. The method for improving tomato plant type with fewer lateral branches according to claim 4, characterized in that, The host cell was Agrobacterium GV3101.

7. The method for improving tomato plant type with fewer lateral branch sprouts according to claim 4, characterized in that, The Agrobacterium-mediated transformation follows these steps: CRISPR vectors containing Agrobacterium tumefaciens are used to inoculate cotyledons of common wild-type tomatoes, seedlings are obtained again through tissue culture, and mutant plants with the PUT3 gene deletion are screened for.

Images