Application of ShSBT1.4 protein and coding gene thereof in regulation and control of powdery mildew resistance of plants

Abstract

The invention discloses an application of ShSBT1.4 protein and a coding gene thereof in regulating powdery mildew resistance of plants. According to the invention, ShSBT1.4 silent tomatoes and ShSBT1.4 overexpressed tomatoes are respectively constructed, and the analysis on the powdery mildew resistance of the tomatoes shows that the ShSBT1.4 in the silent tomatoes can reduce the resistance of the tomatoes to the powdery mildew, and the over-expression of the ShSBT1.4 in the tomatoes can improve the resistance of the tomatoes to the powdery mildew. The ShSBT1.4 protein or a biological material related to the ShSBT1.4 protein participates in regulating and controlling the powdery mildew resistance of the plants and can be used for culturing powdery mildew resistant plant varieties in agricultural production. The invention has important significance on the cultivation of powdery mildew resistant plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the ShSBT1.4 protein and its encoding gene in regulating plant powdery mildew resistance. Background Technology

[0002] Tomato powdery mildew is caused by obligate parasitic fungi. Oldium neolycopersici Powdery mildew is a major disease affecting tomatoes, causing severe yield reduction. It occurs on leaves, petioles, stems, and fruits. Initially, small, pale green spots appear on the leaf surface, later expanding into irregular lesions covered with a white powdery substance—mycelium, conidiophores, and conidia of the pathogen. Initially, the powdery layer is sparse, gradually thickening later. The lesions enlarge and merge, or cover the entire leaf surface, with indistinct yellow-green spots on the upper side. In later stages, the diseased leaves turn brownish-black and gradually wither and die. When petioles, stems, and fruits are infected, powdery mildew also develops on the affected areas.

[0003] Currently, most cultivated tomato varieties lack resistance, and the area affected by powdery mildew in tomatoes is gradually expanding. Vigorously cultivating and applying disease-resistant varieties is the most economical, effective, and sustainable method for controlling powdery mildew, and it is also an important goal of plant breeding today and in the future. Because traditional breeding methods are lengthy, difficult, and require significant human and material resources, molecular biology techniques have been widely applied in bio-breeding in recent years. Transgenic breeding allows for targeted plant improvement, shortening the breeding cycle. However, research on genes related to powdery mildew resistance is limited, severely hindering the development of molecular breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ShSBT1.4 protein and related biomaterials in regulating plant powdery mildew resistance.

[0005] To achieve the above objectives, the present invention first provides a novel use for the ShSBT1.4 protein.

[0006] This invention provides the use of ShSBT1.4 protein in any of the following A1)-A6): A1) Improve plant resistance to powdery mildew; A2) Prepare products that enhance plant resistance to powdery mildew; A3) Cultivate plants with improved resistance to powdery mildew; A4) Prepare products for cultivating plants with enhanced resistance to powdery mildew; A5) Plant breeding; A6) Preparation of plant breeding products; The ShSBT1.4 protein is any one of the following M1)-M4): The amino acid sequence of M1 is the protein shown in sequence 2; M2) is a fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; M3) is a protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of M1. M4) is a protein that shares more than 75% identity with M1 and is associated with plant disease resistance.

[0007] In the protein described in M2 above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0008] In the protein described in M3 above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0009] In the protein described in M4 above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained. The identity includes amino acid sequences having 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 2 of the present invention.

[0010] The proteins described in M1)-M4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] To achieve the above objectives, the present invention provides new uses for biomaterials related to the aforementioned ShSBT1.4 protein.

[0012] This invention provides the application of biomaterials related to the above-mentioned ShSBT1.4 protein in any of the following A1)-A6): A1) Improve plant resistance to powdery mildew; A2) Prepare products that enhance plant resistance to powdery mildew; A3) Cultivate plants with improved resistance to powdery mildew; A4) Prepare products for cultivating plants with enhanced resistance to powdery mildew; A5) Plant breeding; A6) Preparation of plant breeding products; In the above applications, the biomaterial is any one of the following B1)-B7): B1) The nucleic acid molecule encoding the ShSBT1.4 protein described above; B2) An expression cassette containing the nucleic acid molecules described in B1; B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0013] The aforementioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0014] In this invention, the nucleic acid molecule is the DNA molecule shown in Sequence 1 or a DNA molecule that has 75% or more identity with Sequence 1 and encodes the above-mentioned ShSBT1.4 protein.

[0015] Those skilled in the art can readily mutate the nucleotide sequence encoding the ShSBT1.4 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity with the ShSBT1.4 protein nucleotide sequence isolated according to this invention, provided they encode the ShSBT1.4 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention. The identity refers to the sequence similarity to a natural nucleic acid sequence, including nucleotide sequences that have an identity of 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0016] The expression cassette described above may include a promoter, the aforementioned nucleic acid molecule, and a terminator. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters, and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0017] The aforementioned vectors refer to vectors capable of delivering the aforementioned nucleic acid molecules into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecules with a plant expression vector. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). Nos The untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing recombinant vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer resistance to glyphosate) or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0018] The aforementioned microorganisms may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The recombinant microorganisms refer to those whose genes have been manipulated and modified to obtain recombinant microorganisms with altered functions. For example, recombinant microorganisms obtained by introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0019] To achieve the above objectives, the present invention also provides novel uses for substances that inhibit the above-mentioned ShSBT1.4 protein.

[0020] This invention provides the use of a substance that inhibits the above-mentioned ShSBT1.4 protein in any of the following C1)-C6): C1) Reduces plant resistance to powdery mildew; C2) Prepare products that reduce plant resistance to powdery mildew; C3) Cultivate plants with reduced resistance to powdery mildew; C4) Prepare products that cultivate plants with reduced resistance to powdery mildew; C5) Plant breeding; C6) Preparation of plant breeding products; The substance that inhibits the above-mentioned ShSBT1.4 protein is any one of the following P1)-P3): P1) Substances that reduce the activity and / or content of the above-mentioned ShSBT1.4 protein; P2) Substances that inhibit or interfere with the expression of the above-mentioned ShSBT1.4 protein-coding gene; P3) The substance that knocks out the above-mentioned ShSBT1.4 protein-coding gene.

[0021] Furthermore, the substance that reduces the activity of the ShSBT1.4 protein may be a protein, polypeptide, or small molecule compound that inhibits the function of ShSBT1.4.

[0022] The substance that reduces the content of the above-mentioned ShSBT1.4 protein may be a substance that inhibits the synthesis of the above-mentioned ShSBT1.4 protein, promotes the degradation of the above-mentioned ShSBT1.4 protein, or knocks down (reduces) or removes the gene encoding the above-mentioned ShSBT1.4 protein.

[0023] Furthermore, the substance that knocks down (reduces) the expression of the ShSBT1.4 protein-coding gene can be a substance that inhibits or interferes with the expression of the ShSBT1.4 protein-coding gene.

[0024] Furthermore, the substance that inhibits or interferes with the expression of the ShSBT1.4 protein-coding gene may be a VIGS vector containing a specific fragment of the ShSBT1.4 protein-coding gene.

[0025] In some embodiments, the VIGS vector containing a ShSBT1.4 protein-coding gene-specific fragment includes a pTRV2 vector containing a ShSBT1.4 protein-coding gene-specific fragment.

[0026] In some preferred embodiments, the nucleotide sequence of the specific fragment is shown as positions 1639-2038 of Sequence 1.

[0027] To achieve the above objectives, the present invention also provides a method for improving plant resistance to powdery mildew.

[0028] The method for improving plant powdery mildew resistance provided by the present invention includes the following steps: increasing the activity and / or content of the above-mentioned ShSBT1.4 protein in the target plant to improve the powdery mildew resistance of the target plant.

[0029] To achieve the above objectives, the present invention also provides a method for cultivating transgenic plants with enhanced resistance to powdery mildew.

[0030] The method for cultivating transgenic plants with enhanced powdery mildew resistance provided by the present invention includes the following steps: increasing the activity and / or content of the above-mentioned ShSBT1.4 protein in the target plant to obtain a transgenic plant; the transgenic plant has higher powdery mildew resistance than the target plant.

[0031] The method described above for increasing the activity and / or content of the ShSBT1.4 protein in the target plant is to overexpress the ShSBT1.4 protein in the target plant.

[0032] Furthermore, the overexpression method involves introducing the gene encoding the ShSBT1.4 protein into the target plant.

[0033] Furthermore, the nucleotide sequence of the gene encoding the ShSBT1.4 protein is shown in Sequence 1.

[0034] In some embodiments, the gene encoding the ShSBT1.4 protein is transmitted via... ShSBT1.4 The overexpression vector was introduced into the target plant. ShSBT1.4 The overexpression vector is a vector obtained by inserting the DNA molecule shown in sequence 1 into the BamHI and SalI restriction sites of the pBIN vector.

[0035] To achieve the above objectives, the present invention also provides a method for reducing plant resistance to powdery mildew.

[0036] The method for reducing powdery mildew resistance in plants provided by this invention includes the following steps: reducing the activity and / or content of the above-mentioned ShSBT1.4 protein in the target plant, thereby reducing the powdery mildew resistance of the target plant.

[0037] To achieve the above objectives, the present invention also provides a method for cultivating transgenic plants with reduced resistance to powdery mildew.

[0038] The method for cultivating transgenic plants with reduced powdery mildew resistance provided by the present invention includes the following steps: reducing the activity and / or content of the above-mentioned ShSBT1.4 protein in the target plant to obtain transgenic plants; the powdery mildew resistance of the transgenic plants is lower than that of the target plants.

[0039] The method described above for reducing the content and / or activity of the ShSBT1.4 protein in the target plant is to introduce a substance that inhibits or interferes with the expression of the gene encoding the ShSBT1.4 protein into the target plant.

[0040] Furthermore, the substance that inhibits or interferes with the expression of the ShSBT1.4 protein-coding gene is a VIGS vector containing a specific fragment of the ShSBT1.4 protein-coding gene.

[0041] Furthermore, the VIGS vector containing a ShSBT1.4 protein-coding gene-specific fragment includes the pTRV2 vector containing a ShSBT1.4 protein-coding gene-specific fragment.

[0042] In some preferred embodiments, the nucleotide sequence of the specific fragment is shown as positions 1639-2038 of Sequence 1.

[0043] To achieve the above objectives, the present invention finally provides a method for plant breeding.

[0044] The plant breeding method provided by the present invention includes the following steps: using transgenic plants bred according to any of the methods described above as parents for breeding.

[0045] The transgenic plants described above include not only first-generation transgenic plants obtained by transforming the target plant with a substance that overexpresses ShSBT1.4 or inhibits or interferes with the expression of the ShSBT1.4 protein-coding gene, but also their progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plants include seeds, callus tissue, intact plants, and cells.

[0046] The powdery mildew described above is caused by powdery mildew fungi. On -lz causes powdery mildew.

[0047] The indicators for plant breeding mentioned above include powdery mildew resistance.

[0048] The purpose of any of the above-mentioned plant breeding programs includes developing plant varieties resistant to powdery mildew.

[0049] The plant mentioned above is any one of the following Q1)-Q5): Q1) Monocotyledonous or dicotyledonous plants; Q2) Plants of the order Tubularflora; Q3) Solanaceae plants Q4) Plants of the genus *Tomato*; Q5) Tomatoes (such as LA1777 or Micro-Tom).

[0050] This invention demonstrates through experiments that, compared to wild-type tomatoes, ShSBT1.4 Silent tomatoes show a significant decrease in resistance to powdery mildew. ShSBT1.4 Overexpression of ShSBT1.4 protein significantly increased resistance to powdery mildew in tomatoes. This indicates that the ShSBT1.4 protein or related biological materials can regulate plant resistance to powdery mildew and can be used to breed powdery mildew-resistant plant varieties in agricultural production. This invention is of great significance for the breeding of powdery mildew-resistant plant varieties. Attached Figure Description

[0051] Figure 1 for ShSBT1.4 Analysis of expressive features. Lowercase letters indicate... p = 0.05 significance level. 'heat' indicates high temperature stress of 40°C; 'chilling' indicates low temperature stress of 8°C; 'JA', 'SA', and 'ACC' indicate stress treatments with methyl jasmonate, salicylic acid, and Eth precursors, respectively.

[0052] Figure 2 Subcellular localization analysis of ShSBT1.4. A represents pBIN- ShSBT1.4 Distribution of cells in the lower epidermal cells of tobacco leaves. B represents pBIN- after plasmolysis. ShSBT1.4 Results of the distribution of epidermal cells in the lower epidermis of tobacco leaves.

[0053] Figure 3 Structural and functional analysis of ShSBT1.4. A represents tomato LA1777 and powdery mildew fungus. On The alignment was performed with the Fn3_6 nucleotide sequence amplified in the cDNA library plasmid, where DNA_Sly is the cDNA of tomato LA1777, and DNA- On powdery mildew OncDNA, AD are cDNA library plasmids. B is the Pfam structure analysis of ShSBT1.4 protein, where the signal peptide highlighted in red is a possible signal peptide, as the predictions for SignalIP4.0 and SignalIP5.0 are inconsistent. C is tobacco overexpression. ShSBT1.4 The gene can induce necrosis of tobacco mesophyll cells. D represents inoculation. On Subsequently, the expression levels of ShSBT1.4 in LA1777 and Moneymaker were determined.

[0054] Figure 4 for ShSBT1.4 Analysis of powdery mildew resistance in silent plants. In the figure, red represents control plants, and green represents... ShSBTI.4 Silent plant. A is... ShSBT1.4 Silentness efficiency test results. B represents the statistical results of hyphal length. C represents the statistical results of reactive oxygen species production rate. D represents the statistical results of the incidence of allergic necrosis.

[0055] Figure 5 for ShSBT1.4 Different stages of plant transformation. A: Explant preparation. B: Explant pre-culture. C: Callus induction and differentiation. D: Rooting culture.

[0056] Figure 6 for ShSBT1.4 PCR identification results of overexpressing tomato lines. M: from top to bottom 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp; lane 1 is the negative control; lane 2 is the positive control; lanes 3-14 are all positive seedlings.

[0057] Figure 7 for ShSBT1.4 Western blot analysis of overexpressing tomato lines. M is the marker, lane 1 is Micro-Tom, and lanes 2-8 are lines SBT-9, SBT-22, SBT-31, SBT-32, SBT-34, SBT-35, and SBT-36, respectively.

[0058] Figure 8 for ShSBT1.4 Analysis of powdery mildew resistance in tomato strain SBT-34 overexpressing the strain. A represents the statistical results of disease index. B represents the statistical results of mycelial length. C represents the statistical results of reactive oxygen species production rate. D represents the statistical results of hypersensitive necrosis incidence rate. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] The tomatoes in the following examples ( Solanum habrochaites LA1777 is described in the literature "Lindhout, P., H. Beek and G. Pet, 1994. Wild lycopersicon species as sources for resistance to powdery mildew ( Oidium lycopersicum ): Mapping of the resistancegene ol-1 on chromosome 6 of l. Hirsutum. International Society for Horticultural Science (ISHS), Leuven, Belgium: pp: 387-394".

[0062] The tomatoes in the following examples ( Solanum lycopersicum Moneymaker is described in the literature “Yu LX, Nasrallah J, Valenta R, Parthasarathy MV. Molecular cloning and mRNA localization of tomato pollen profilin. Plant Mol Biol. 1998 Mar;36(5):699-707”.

[0063] The carriers pTRV1 and pTRV2 used in the following examples are described in the literature Sun, G., C. Feng, J. Guo, A. Zhang, Y. Xu, Y. Wang, B. Day and Q. Ma, 2019. The tomato arp2 / 3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici .Plant Cell Environ, 42(9): 2664-2680. DOI 10.1111 / pce.13569.

[0064] The vector PGR106 in the following examples is described in the literature “Uhde-Holzem K, Fischer R,Commandeur U. Genetic stability of recombinant potato virus X virus vectors presenting foreign epitopes. Arch Virol. 2007;152(4):805-11. DOI: 10.1007 / s00705-006-0892-y.”.

[0065] The vector pBIN in the following examples is described in the literature “Lian Q, He X, Zhang B, Wang Y, Ma Q. Identification and Characterization of WRKY41, a Gene Conferring Resistance to Powdery Mildew in Wild Tomato (Solanum habrochaites) LA1777. Int J MolSci. 2022 Jan 23;23(3):1267. DOI: 10.3390 / ijms23031267.”.

[0066] Powdery mildew in the following examples On The -lz strain is documented in the literature "Sun, G., C. Feng, J. Guo, A.Zhang, Y. Xu, Y. Wang, B. Day and Q. Ma, 2019. The tomato arp2 / 3 complex is required for resistance to the powdery mildew fungus O idium neolycopersici .Plant Cell Environ, 42(9): 2664-2680. DOI 10.1111 / pce.13569".

[0067] Example 1: Obtaining the ShSBT1.4 protein and its encoding gene 1. Total RNA was extracted from wild-type tomato LA1777, and the total RNA was reverse transcribed to obtain cDNA.

[0068] 2. Using the obtained cDNA as a template, PCR amplification was performed with 5'-ATGGCGGAAATCTCCGTTCTTTC-3' as the forward primer and 5'-TCACATGGACACAGCAGATTGG-3' as the reverse primer to obtain the PCR amplification product.

[0069] 3. After detecting the PCR amplification products by agarose gel electrophoresis, the PCR amplification products were separated, purified, and sequenced.

[0070] Sequencing results showed that the nucleotide sequences of the PCR amplification products were as shown in Sequence 1, where Sequence 1 was... ShSBT1.4 The coding region sequence of a gene, ShSBT1.4 The amino acid sequence of the gene-encoded ShSBT1.4 protein is shown in Sequence 2.

[0071] Example 2 ShSBT1.4 Expression characteristic analysis 1-Aminocyclopropanecarboxylic acid (ACC), a direct precursor of Eth biosynthesis, was sprayed onto 5-leaf stage LA1777 plants at a concentration of 0.5 mM, with water treatment serving as a control. Samples were collected at 0 h, 1 h, 3 h, 6 h, and 10 h after treatment, and the relative expression levels of the gene were analyzed by RT-qPCR. Simultaneously, the effects of 40℃ high-temperature stress, 8℃ low-temperature stress, and 1 mM SA (salicylic acid) and 100 μM MeJA (methyl jasmonate) stress on the expression of the gene in tomato LA1777 were analyzed. ShSBT1.4 Gene expression levels.

[0072] The results are as follows Figure 1 As shown. The results show that after high-temperature treatment at 40℃, ShSBT1.4 The expression level decreased, and except for 1.18 at 6 hpi, the expression level was significantly lower than 0 hpi at all other time points; under low temperature treatment at 8℃, ShSBT1.4 The expression of JA was significantly decreased, but increased to 1.36 at 10 hpi; hormone treatment results indicated that treatment with JA, SA, and the Eth precursor ACC induced ShSBT1.4 Gene expression was significantly high, especially after JA treatment. ShSBT1.4 The expression level increased sharply, reaching a maximum of 486.64 and a minimum of 92.99; while after SA and ACC treatment, ShSBT1.4 The expression patterns are basically the same, with the expression level gradually increasing over time after processing.

[0073] Example 3: Subcellular localization analysis of ShSBT1.4 protein 1. ShSBT1.4The CDS sequence of the gene is inserted into pBIN- EGFP The vector, as detected and analyzed by Sanger sequencing, was found to be... ShSBT1.4 The start codon ATG and pBIN- EGFP The 24 bp nucleotide interval between the last codon AAG of the GFP protein in the vector indicates that the recombinant vector pBIN- ShSBT1.4 No frameshift mutations were found in the nucleotide sequence encoding the SBT1.4 protein.

[0074] 2. pBIN- is expressed in the lower epidermal cells of tobacco leaves. ShSBT1.4 It was found that GFP signaling is mainly located in the cell nucleus and cell membrane. Figure 2 A).

[0075] 3. To verify whether there was a GFP signal in the cell wall, plasmolysis was performed on tobacco lower epidermal cells. The results showed that some GFP fluorescent signals were present in the cell wall. Figure 2 B).

[0076] The above results indicate that the ShSBT1.4 protein is mainly distributed in the cell nucleus, cell membrane, and cell wall.

[0077] Example 4: Structural and Functional Analysis of ShSBT1.4 I. Structural Analysis of ShSBT1.4 Nucleotide searches revealed that the Fn3_6 domain is widely present in plants and fungi. Using 5'-ACCCATACGACGTACCAGATTA-3' as the forward primer and 5'-AGCATAGTTCCTCAGTCACAAC-3' as the reverse primer, samples were obtained from tomatoes and powdery mildew. On The Fn3_6 domain was amplified from the interaction library, and the product was subjected to Sanger sequencing. Based on the amino acid sequence of ShSBT1.4, its conserved domains were analyzed online using Pfam.

[0078] The results are as follows Figure 3 A and Figure 3 As shown in B. The results showed that the Fn3_6 domain selected by the interaction screening originated from tomato, not powdery mildew. Amino acid sequence structure analysis revealed that amino acids 1 to 24 at the N-terminus were likely a signal peptide (the signal peptide cleavage site was between 24 and 25 aa). Pfam conserved domain analysis revealed that amino acids 31-110 aa were the Peptidase inhibitor I9 domain, and amino acids 133-604 aa were the Barwin Peptidase_S8 domain. Figure 3 B), 661-765 aa is the Fn3_6 structural domain, and 371-458 is the PA structural domain.

[0079] II. Functional Analysis of ShSBT1.4 1. ShSBT1.4 The CDS sequence of the gene was ligated into the vector pGR106, resulting in the recombinant vector pGR106- ShSBT1.4 .

[0080] 2. Transient expression was performed in *Nicotiana benthamiana* using an Agrobacterium-mediated method. The specific steps are as follows: pGR106- ShSBT1.4 Single colonies of Agrobacterium GV3101 were cultured in LB broth containing antibiotics at 28°C with shaking until OD200. 600nm =0.8~1.0, collect bacterial cells by centrifugation, and resuspend in osmotic buffer (10 mM MES pH5.6, 10 mM MgCl2, 200 μMAS) to OD. 600nm =0.5, after activating the toxicity system by standing at room temperature in the dark for 3 hours, the toxicity was injected into healthy 4-6 week old tobacco leaves with negative pressure using a sterile syringe or needleless syringe, and cultured in the dark for 24 hours. The HR response phenotype was observed and recorded after 72 hours.

[0081] The results showed that heterologous overexpression in tobacco ShSBT1.4 Not only does it not inhibit the Bax-induced HR response, but it can also induce the tobacco-induced HR response. Figure 3 C), Explanation ShSBT1.4 It may be directly involved in the process of LA1777's resistance to powdery mildew. Therefore, RT-qPCR was used to analyze the inoculation with powdery mildew ( On -lz strain) after LA1777 and Moneymaker ShSBT1.4 The relative expression levels of [a substance] were analyzed, and the results showed that at 36 and 72 h after inoculation with powdery mildew, [a substance] in Moneymaker [was present]. ShSBT1.4 The expression level of [a specific substance] was significantly higher than that of LA1777, while at 120 h, the expression level of [a specific substance] in LA1777 was significantly higher. ShSBT1.4 The expression level reached 7.16, significantly higher than 1.06 in Moneymaker ( ). Figure 3 D), Explanation ShSBT1.4 It participated in the defense response of LA1777 against powdery mildew.

[0082] Example 5 ShSBT1.4 Preparation of Silent Tomatoes and Analysis of Their Powdery Mildew Resistance I. ShSBT1.4 Preparation of Silent Tomatoes 1. Virus-induced ShSBT1.4 Construction of silent carriers Based on SGN-VIGS (https: / / vigs.solgenomics.net / ) Online tool At the same time Solanum lycopersicumITAG v2.4 and Solanum lycopersicum ITAG v3.2 was used to screen for gene-specific silencing fragments, and the final selected silencing fragment was located at positions 1639-2038 of sequence 1. The silencing fragment was then ligated into the pTRV2 vector via homologous recombination. BamH At the I restriction site, the vector pTRV2- is obtained. ShSBT1.4 .

[0083] 2. ShSBT1.4 Analysis of the acquisition and silence efficiency of silent tomatoes The vectors pTRV1, pTRV2, and pTRV2- were respectively carried out. PDS and the vector pTRV2- ShSBT1.4 Agrobacterium GV3101 was inoculated into LB liquid medium containing secondary antibodies (rifampin and kanamycin) and incubated at 28°C and 180 rpm until OD200 was reached. 600nm =1.0. Centrifuge at 5000 rpm for 5 min, collect the bacterial cells, and administer an equal volume of 10 mmol / L. . L -1 After washing with MgCl2 solution, the cells were centrifuged again at 5000 rpm for 5 min to collect the cells. The cells were then injected with 0.5 volume of MES (pH 5.5, 10 mmol / L). . L -1 The bacterial suspensions were resuspended in MgCl2 to obtain recombinant bacterial solutions carrying each vector.

[0084] The recombinant bacterial culture carrying vector pTRV1 and the recombinant bacterial culture carrying vector pTRV2 were mixed at a 1:1 volume ratio to obtain the recombinant bacterial culture TRV1+TRV2; the recombinant bacterial culture carrying vector pTRV1 and the recombinant bacterial culture carrying vector pTRV2- were mixed at a 1:1 volume ratio to obtain the recombinant bacterial culture TRV1+TRV2. PDS The recombinant bacterial cultures were mixed at a 1:1 volume ratio to obtain the recombinant bacterial culture TRV1+TRV2- PDS ; The recombinant bacterial culture carrying vector pTRV1 and the recombinant bacterial culture carrying vector pTRV2- ShSBTU.4 The recombinant bacterial cultures were mixed at a 1:1 volume ratio to obtain the recombinant bacterial culture TRV1+TRV2- ShSBT1.4 .

[0085] The recombinant bacterial cultures TRV1+TRV2 and TRV1+TRV2- were respectively... PDS and recombinant bacterial culture TRV1+TRV2- ShSBT1.4 After being placed at 25℃ for 3 hours, the inoculated bacterial solution was used for injection. The specific injection method is as follows: Using a 1 mL sterile syringe without a needle, each recombinant bacterial solution was taken and inoculated onto the underside of tomato LA1777 leaves using the compression method. The inoculated tomatoes were cultured in the dark at 22℃ for 2 days, and then placed in an artificial climate chamber with a light / dark cycle of 16 h / 8 h, 30% humidity, and 20℃ to observe the effects of the inoculated recombinant bacterial solution TRV1+TRV2-. PDSDo the tomato leaves show any phenotypic changes?

[0086] After inoculation with recombinant bacterial culture TRV1+TRV2- PDS One month later, large areas of the leaves were bleached. Recombinant bacterial suspension TRV1+TRV2- was then inoculated at the same locations. ShSBT1.4 Tomato leaves were sampled, with tomato leaves injected with recombinant bacterial suspension TRV1+TRV2 serving as a control. The control plants and tomato leaves were then tested. ShSBT1.4 The Silent Tomato ShSBT1.4 By comparing relative expression levels and calculating silencing efficiency, the results showed that... ​ The silencing efficiency was 68% ( ​ A). Selection ​ Silent tomatoes were used for powdery mildew resistance analysis.

[0087] two, ​ Analysis of powdery mildew resistance in silent tomatoes The fresh spores were sprayed using a spore suspension method. ​ -lz conidia at 10 5 Inoculate 5-leaf stage tomatoes at a concentration of spores / mL. ​ The growth conditions were as follows: temperature 23 ± 2 ℃, humidity 75 ± 3%, 16 h light / 8 h dark. Samples were collected at 12 h, 36 h and 72 h post-inoculation for histological observation, including... ​ Conidial germination, H2O2 production, and cell necrosis were observed. Disease severity was assessed at 7 and 14 days following the method described in the literature "Correll, James C. 'Powdery Mildew of Tomato: The Effect of Planting Date and Triadimefon on Disease Onset, Progress, Incidence, and Severity.' Phytopathology 78 (1988):512." H2O2 production was observed using DAB staining for ease of observation. ​ Spores and hyphae were stained with DAB followed by counterstaining with 5% Coomassie brilliant blue for 5 minutes. Cell necrosis was observed using trypan blue staining.

[0088] The results of the observation of disease severity indicate that: ​ Silent plant inoculation ​ On day 14, a small number of lesions appeared on the leaves of the control plants, indicating a disease severity level of 1. ​ The plant exhibits numerous white powdery spots on its leaves, covering approximately 20.06% of the leaf area, indicating a disease severity level of 3.

[0089] Histological observations indicate that: ​ After silence ​ Conidial infection rate was faster than the control group LA1777, and hyphal length data indicated that... ​ Inoculation on silent plants ​ Twelve hours after conidia, the average hyphal length was 45.67 μm, significantly higher than the 3.68 μm of the control LA1777 plants. Thirty-six hours after inoculation, the average hyphal length of silent plants was 217.59 μm, while that of the control plants was only 82.61 μm, significantly lower than the hyphal length on silent plants. After 72 hours of inoculation, the control plants... ​ The hyphal length was 135.92 μm, significantly lower than the 478.38 μm on silent plants. ​ B).

[0090] The staining observation results of reactive oxygen species showed that: ​ The level of H2O2 production in silent plants was lower than that in control plants. ​ C). After 12 hpi inoculation, the reactive oxygen species production rate in the control plants was 2.67%, significantly higher than that in the control plants. ​ The percentage of silent plants was 1.21%; after 36 h of inoculation, the reactive oxygen species production rate in the control plants reached 11.83%, while... ​ Only 6.82% of plants were silent; 72 hours after inoculation, ​ The production rate of reactive oxygen species in the silent plants was 36.29%, significantly lower than the 55.16% in the control plants. Simultaneously, it was found that the silent plants... ​ Afterwards, vaccination ​ The incidence of allergic necrosis in the plants was significantly lower than that in the control plants ( ​ D), Silent plants after inoculation ​ At 12, 36, and 72 hpi, the rates of allergic necrosis were 9.28%, 32.16%, and 41.52%, respectively, while those in the control plants were 13.96%, 46.89%, and 63.15%, respectively.

[0091] VIGS Silent ​ Experiments show that, ​ Silencing reduces the resistance of LA1777 to powdery mildew by decreasing the production of reactive oxygen species and the occurrence of allergic necrosis.

[0092] Example 6 ​ Preparation of overexpressed tomatoes and analysis of their powdery mildew resistance one, ​ Preparation of overexpressed tomatoes 1. ​ Construction of overexpression vectors Total RNA was extracted from wild-type tomato LA1777, and the total RNA was reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed with a forward primer of 5'-ccccggggtcgacGGATCCATGGCGGAAATCTCCGTTCTTTC-3' and a reverse primer of 5'-ctctagttcatctagaGGATCCTCACATGGACACAGCAGATTGG-3', yielding a 2328 bp cDNA. ​ CDS sequence, and then homologous recombination method ​ The CDS sequence was ligated between the BamHI and SalI restriction sites of the pBIN vector to obtain... ​ Overexpression vector.

[0093] 2. Construction of recombinant bacteria The construction in step one ​ The overexpression vector was introduced into Agrobacterium GV3101 (Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant bacteria.

[0094] 3. Genetic transformation Using Agrobacterium-mediated genetic transformation to ​ The expression vector was introduced into tomato Micro-Tom, and the following was obtained. ​ Tomato processing. The specific steps are as follows: 1) Obtaining sterile vaccines First, soak mature tomato seeds in sterile water for 30 minutes, then disinfect them with 75% alcohol for 30 seconds, then disinfect them with sodium hypochlorite solution (available chlorine of 2%) for 15 minutes, and finally rinse them with sterile water 4 times. Then, dry the seeds with sterile filter paper and inoculate them onto 1 / 2 MS solid medium (pH 5.8).

[0095] 2) Preparation of explants After culturing on 1 / 2 MS solid medium for 6-7 days, take the newly unfolded cotyledons of sterile seedlings, remove both ends, cut them from the middle, and place them in pre-culture medium (MS medium containing 1 mg / L KT, pH 5.8) for 24 hours to obtain pre-cultured explants.

[0096] 3) Agrobacterium propagation The recombinant bacteria were streaked onto LB solid medium containing 50 mg / L kanamycin, 50 mg / L gentamicin, and 50 mg / L rifampin. After incubation at 28°C for two days, single colonies were picked and inoculated into 3 mL of LB liquid medium containing 50 mg / L kanamycin, 50 mg / L gentamicin, and 50 mg / L rifampin. The medium was then incubated overnight at 28°C with shaking at 220 rpm until OD reached. 600nmWhen the OD value is approximately 0.6, centrifuge at 5000 rpm for 10 min, collect the bacterial cells, and resuspend the collected bacterial cells in MS liquid medium to OD. 600nm =0.4, thus obtaining OD 600nm =0.4% Agrobacterium bacterial suspension.

[0097] 4) Agrobacterium infection and co-culture Pre-cultured explants were placed in OD 600nm Add the Agrobacterium tumefaciens to a 0.4 g / L culture medium, gently shake for 5 minutes, blot dry the bacterial solution on the surface of the explant with sterile filter paper, and then place it back on the pre-culture medium and incubate in the dark for 2-3 days.

[0098] 5) Bud differentiation induction After co-culture, the explants were transferred to shoot induction differentiation medium (MS medium containing 2 mg / L ZT, 300 mg / L Timentin, and 50 mg / L Kan, pH 5.8) to induce shoot growth, and the medium was changed every two weeks.

[0099] 6) Screening of rooted and resistant plants When the resistant shoots reach 2 cm in length, the shoots are cut and transferred to rooting medium (MS medium containing 1 mg / L IAA, 300 mg / L Timentin, and 50 mg / L Kan, pH 5.8). Once a complete plantlet has formed, some leaves are cut off, and genomic DNA is extracted using the TransDirect Plant Tissue PCR Kit. PCR amplification is performed using 5'-AACACATACTGCTTCAAC-3' and 5'-GAACACCATCATCAACAG-3'. Plantlets with a size of 600 bp are considered positive.

[0100] 7) ​ Expression level detection The expression of transgenic plants was detected at the RNA and protein levels using qRT-PCR and Western blot, respectively. ​Expression levels. qRT-PCR quantification was performed using a kit from Yeasen (Shanghai Yisheng Biotechnology Co., Ltd.). First-strand reverse transcription was performed using the Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) first-strand synthesis kit (11141es60). Quantitative PCR was performed using the Hieff® qPCR SYBRGreen Master Mix (Low Rox Plus) kit, and expression levels were analyzed using the 2-ΔΔCt method. Western blot assays used the following primary antibody: anti-GFP-tagged mouse monoclonal antibody (Beijing TransGen Biotechnology); secondary antibody: HRP-labeled goat anti-mouse IgG (H+L) antibody (Beijing TransGen Biotechnology); PVDF membrane: Millipore (Millipore, Germany); skim milk powder: Yili Group; protein marker: BakerKade Biotechnology; ECL Plus chemiluminescence kit: Tiangen (Tiangen, China); and all other reagents were domestically produced analytical grade commercial reagents.

[0101] qRT-PCR expression analysis showed that compared with wild-type tomato, the expression levels of transgenic lines SBT-9, SBT-22, SBT-31, SBT-32, SBT-34, and SBT-36 were all increased, with expression levels of 1.02, 7.06, 1.12, 1.52, 3.67, and 1.53, respectively. Western blot analysis showed that ShSBT1.4 expression was high in all transgenic lines SBT-9, SBT-22, SBT-31, SBT-32, SBT-34, and SBT-36. Based on these results, transgenic line SBT-34 was selected as the overexpression material for subsequent functional validation.

[0102] two, ​ Analysis of powdery mildew resistance in tomatoes with overexpression According to the powdery mildew resistance analysis method in Example 4, ​ Powdery mildew resistance was analyzed using the overexpression of tomato line SBT-34.

[0103] Resistance analysis results show that ( ​ Compared to wild-type tomato Micro-Tom, the overexpression line SBT-34 showed significantly enhanced resistance to powdery mildew, 14 days after inoculation. ​ The disease index of the overexpressing SBT-34 strain was only 10.24, while the disease index of the wild-type tomato was as high as 31.42. Simultaneously, histological observation data at 12 h, 36 h, and 72 h post-inoculation showed that... ​Overexpression of the SBT-34 strain inhibited the mycelial expansion of powdery mildew by exacerbating the production of reactive oxygen species and the occurrence of hypersensitive necrosis (HR) response. These results indicate that... ​ Overexpression can effectively enhance the resistance of Micro-Tom in powdery mildew-susceptible tomatoes to powdery mildew infection.

[0104] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of a ShSBT1.4 protein in any one of A1) to A6) below: A1) increasing the resistance of a plant to powdery mildew; A2) producing a product for increasing the resistance of a plant to powdery mildew; A3) breeding a plant with increased resistance to powdery mildew; A4) producing a product for breeding a plant with increased resistance to powdery mildew; A5) plant breeding; A6) producing a product for plant breeding; said ShSBT1.4 protein being any one of M1) to M4) below: M1) a protein having the amino acid sequence shown in SEQ ID NO: 2; M2) a fusion protein related to plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; M3) a protein related to plant disease resistance obtained by substitution and / or deletion and / or addition of one or several amino acid residues in M1); M4) a protein having more than 75% identity to M1) and being related to plant disease resistance.

2. Use of a biological material related to the ShSBT1.4 protein of claim 1 in any one of A1) to A6) below: A1) increasing the resistance of a plant to powdery mildew; A2) producing a product for increasing the resistance of a plant to powdery mildew; A3) breeding a plant with increased resistance to powdery mildew; A4) producing a product for breeding a plant with increased resistance to powdery mildew; A5) plant breeding; A6) producing a product for plant breeding; 3. Use according to claim 2, characterized in that: said biological material being any one of B1) to B7) below: B1) a nucleic acid molecule encoding the ShSBT1.4 protein of claim 1; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1), or an expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3); B5) a transgenic plant cell line comprising the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3); B6) a transgenic plant tissue comprising the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3); B7) a transgenic plant organ comprising the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3).

4. Use of a substance inhibiting the ShSBT1.4 protein of claim 1 in any one of C1) to C6) below: C1) decreasing the resistance of a plant to powdery mildew; C2) producing a product for decreasing the resistance of a plant to powdery mildew; C3) breeding a plant with decreased resistance to powdery mildew; C4) producing a product for breeding a plant with decreased resistance to powdery mildew; C5) plant breeding; C6) producing a product for plant breeding; said substance inhibiting the ShSBT1.4 protein of claim 1 being any one of P1) to P3) below: P1) a substance decreasing the activity and / or content of the ShSBT1.4 protein of claim 1; P2) a substance that inhibits or interferes with the expression of the ShSBT1.4 protein-encoding gene as defined in claim 1; P3) a substance that knocks out the ShSBT1.4 protein-encoding gene as defined in claim 1.

5. A method for increasing the powdery mildew resistance of a plant, comprising the step of increasing the activity and / or amount of the ShSBT1.4 protein as defined in claim 1 in a plant of interest, thereby increasing the powdery mildew resistance of the plant of interest.

6. A method for breeding a transgenic plant with increased powdery mildew resistance, comprising the step of increasing the activity and / or amount of the ShSBT1.4 protein as defined in claim 1 in a plant of interest, thereby obtaining a transgenic plant; wherein the powdery mildew resistance of the transgenic plant is higher than that of the plant of interest.

7. A method for decreasing the powdery mildew resistance of a plant, comprising the step of decreasing the activity and / or amount of the ShSBT1.4 protein as defined in claim 1 in a plant of interest, thereby decreasing the powdery mildew resistance of the plant of interest.

8. A method for breeding a transgenic plant with decreased powdery mildew resistance, comprising the step of decreasing the activity and / or amount of the ShSBT1.4 protein as defined in claim 1 in a plant of interest, thereby obtaining a transgenic plant; wherein the powdery mildew resistance of the transgenic plant is lower than that of the plant of interest.

9. A method for breeding plants, comprising the step of breeding from a transgenic plant obtained by the method according to claim 6 or 8.

10. Use according to any one of claims 1 to 4 or method according to any one of claims 5 to 9, characterized in that: The plant is any one of the following Q1) to Q5): Q1) a monocotyledonous plant or a dicotyledonous plant; Q2) a plant of the order Tubiflorae; Q3) a plant of the Solanaceae family Q4) a plant of the genus Lycopersicon; Q5) a tomato.

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