Application of SlGT2 gene in regulation and control of plant dwarfing

Abstract

The invention relates to the field of genetic engineering and molecular biology, in particular to application of an SlGT2 gene in regulation and control of plant dwarfing. The nucleotide sequence of the SlGT2 gene is a nucleotide sequence as shown in SEQ ID NO: 1. The expression level of the SlGT2 gene is regulated and controlled through an SlGT2 overexpression plant so as to research the regulation and control mechanism of the SlGT2 gene on the tomato plant height. Results show that stem cells of the SlGT2 overexpressed tomato plant become smaller, the stem node spacing is shortened, and the plant height is reduced. Gene resources are provided for cultivating new tomato dwarfing varieties, potential application value is achieved, and meanwhile a theoretical basis is laid for researching a tomato dwarfing molecular mechanism.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and molecular biology, and in particular to an application of an S1GT2 gene in regulating plant dwarfing. Background Art

[0002] Plant height is a crucial trait in crops and a key goal for crop breeders in improving them. Semi-dwarf crops fueled the first agricultural "Green Revolution" and made a significant contribution to resolving the global food crisis. Tomato (Solanum lycopersicum) is a widely cultivated vegetable crop worldwide and a key model plant for studying plant growth, development, and fruit ripening. Research on the mechanisms regulating tomato growth and development has important guiding significance for tomato breeding efforts, such as promoting plant growth and improving fruit quality and yield. However, as one of the world's most important vegetable crops, the mechanisms regulating tomato plant height remain under investigation.

[0003] Plant transcription factors play important regulatory roles in plant growth and development, morphological development, and responses to environmental changes, and hold broad application prospects in plant trait improvement and new cultivar breeding. Some transcription factors, such as MYB, bZIP, MADS-box, and WRKY, play a crucial role in regulating plant height. The Trihelix transcription factor family is one of the earliest transcription factor families discovered in plants. They are named for the three tandem helical structures (helix-loop-helix-loop-helix) in their DNA-binding domain. This domain specifically binds to the GT light-responsive element (GT) on DNA sequences, leading to the family being referred to as the GT factor family. Genes in this family not only regulate light-dependent target genes but also play crucial roles in various growth and developmental processes, including the development of flowers, stomata, epidermis, embryos, and seeds. However, the role of tomato Trihelix transcription factors in plant height regulation and their regulatory mechanisms are rarely reported. Therefore, cloning the SlGT2 gene and using transgenic technology to cultivate tomato materials with different SlGT2 expression levels has great application prospects in breeding new semi-dwarf tomato varieties. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of the SlGT2 gene in regulating plant dwarfing. The present invention constructs a SlGT2 gene overexpression plant to enhance the expression level of the SlGT2 gene, thereby regulating the cells in the plant stem to become smaller and the internode distance to shorten, resulting in semi-dwarfing of the plant.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of an SlGT2 gene in regulating plant dwarfing. The nucleotide sequence of the SlGT2 gene is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the protein encoded by the SlGT2 gene is shown as SEQ ID NO.2.

[0008] Preferably, the plant is tomato.

[0009] The present invention also provides a primer pair for amplifying the SlGT2 gene, and the nucleotide sequence of the primer pair is shown in SEQ ID NOs: 3-4.

[0010] The present invention also provides a recombinant vector comprising the S1GT2 gene.

[0011] Preferably, the recombinant vector is an expression vector having a 35S promoter.

[0012] Preferably, the expression vector having a 35S promoter is pCAMBIA2301.

[0013] The present invention also provides a recombinant bacterium comprising the recombinant vector.

[0014] The present invention also provides a cultivation method for regulating plant dwarfing, comprising the following steps:

[0015] (1) constructing a recombinant vector containing the S1GT2 gene;

[0016] (2) transforming the recombinant vector into plant tissues or plant cells;

[0017] (3) Cultivate and screen positive transgenic plants.

[0018] Preferably, the plant is tomato.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This study used genetic methods to construct tomato plants that overexpress the S1GT2 gene, enhancing its expression. The researchers then manipulated the expression of the gene to study its regulatory mechanism for tomato plant height. The results showed that the S1GT2-overexpressing plants exhibited smaller stem cells and shorter internodes, resulting in semi-dwarf growth.

[0021] This study, published in the journal Nature Communications, constructs tomato plants overexpressing the S1GT2 gene and conducts functional studies. The S1GT2 gene provides a genetic resource for breeding new semi-dwarf tomato varieties, has potential applications, and lays a theoretical foundation for studying the molecular mechanisms of tomato plant height regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0023] Figure 1 This is a map of the recombinant vector of the S1GT2 gene in the embodiment of the present invention;

[0024] Figure 2 The qPCR quantitative results of tomato S1GT2 in wild-type and overexpressed plants in the examples of the present invention are from three replicates; * represents a significant difference compared with WT (P < 0.05), and ** represents a significant difference compared with WT (P < 0.01);

[0025] Figure 3 The phenotypes of the wild type and S1GT2 overexpressing plants in the examples of the present invention are shown;

[0026] Figure 4 The number of stem nodes of the wild-type and S1GT2-overexpressing plants in the examples of the present invention is statistically analyzed;

[0027] Figure 5 The stem node length statistics of the wild type and S1GT2 overexpressing plants in the embodiment of the present invention are shown in FIG.

[0028] Figure 6 These are cross-sectioned paraffin sections of the stems of the wild-type and S1GT2-overexpressing plants in the examples of the present invention;

[0029] Figure 7 The cell length statistics in the stem cross-section images of the wild-type and SlGT2-overexpressing plants in the examples of the present invention are shown; three groups of each sample were measured, and 20 cells were measured in each group; * represents a significant difference compared with WT (P < 0.05), ** represents a significant difference compared with WT (P < 0.01), and *** represents a significant difference compared with WT (P < 0.001);

[0030] Figure 8 The cell width statistics in the cross-section images of the stems of the wild-type and SlGT2-overexpressing plants in the examples of the present invention are shown; three groups of measurements were made for each sample, with 20 cells measured in each group; * represents a significant difference compared with WT (P < 0.05), ** represents a significant difference compared with WT (P < 0.01), and *** represents a significant difference compared with WT (P < 0.001);

[0031] Figure 9These are longitudinal paraffin sections of the stems of the wild-type and S1GT2-overexpressing plants in the examples of the present invention;

[0032] Figure 10 The cell height statistics in the stem longitudinal section images of the wild-type and SlGT2-overexpressing plants in the examples of the present invention; three groups were measured for each sample, and 20 cells were measured in each group; * represents a significant difference compared with WT (P < 0.05), ** represents a significant difference compared with WT (P < 0.01), and *** represents a significant difference compared with WT (P < 0.001). DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0035] Example 1: Construction of S1GT2 gene overexpression vector

[0036] The SlGT2 gene originates from tomato and is identified as Solyc11g005380 in the tomato genome database. To investigate the effects of SlGT2 overexpression on tomato plant height, the SlGT2 gene was first cloned from tomato. Based on coding region sequence analysis, specific primers SlGT2-F (SEQ ID NO: 3) and SlGT2-R (SEQ ID NO: 4) were designed. The 5' ends of the primers were then introduced with homologous sequences from both ends of the linearized vector (double-digested with XbaI and KpnI), ensuring that the 5' and 3' ends of the amplified insert carried homologous sequences (15-20 bp, excluding restriction sites) corresponding to the ends of the linearized cloning vector. High-fidelity PCR amplification was performed using Max DNA Polymerase (Takara). The reaction system (50 μL) contained 25 μL of 2× PrimeSTAR Max Premix, 1 μL of each 10 μM forward and reverse primer, and <200 ng of cDNA template. The amplification program was as follows: initial denaturation at 98°C for 1 min; 35 cycles (98°C for 10 s → 55°C for 15 s → 72°C for 5 s / kb); and final extension at 72°C for 10 min. The purified SlGT2 PCR fragment was then cleaved with the enzyme-digested vector by In- Cloning system (20 μL: 5×CE II Buffer 4 μL, linearized vector 50-200 ng, SlGT2 fragment 2-200 ng, II 2 μL). Reconstitute at 37°C for 30 min and then transform into DH5α competent cells.

[0037] After initial screening by colony PCR, the cells were sent to Anshengda Biotechnology Co., Ltd. (Tianjin) for Sanger sequencing. The results showed that the cloned SlGT2 gene nucleotide sequence (SEQ ID NO: 1) was completely consistent with the SolGenomics database (Solyc11g005380), and the encoded protein amino acid sequence (SEQ ID NO: 2) contained a complete open reading frame. Finally, the correct recombinant plasmid pCambia2301::SlGT2-GFP (see Figure 1 ) and stored at -80℃ until use.

[0038] The CDS sequence of the S1GT2 gene (shown in SEQ ID NO: 1) is:

[0039]

[0040] The amino acid sequence of the S1GT2 protein (as shown in SEQ ID NO: 2) is:

[0041] MLESSVLLENTAAGGAVTGADGEASELKNEGGGGGGSVGGGSEEEDKNFSGGNRWPHEETLALLKIRSEMDVAFRDSNLKSPLWDEISRKMAELGYNRNAKKCREKFENIYKYHKRTKDGRSGR QTGKNYRFFEQLELLDSQSLFSSPPLNHSQINRMETMPVPMPMPMTMIKPAASGCQDFGMDHSRVRGFNPGFMSTSTSTTSSSGKESDGSVKKKRKLASYFERLMKEVLDKQEDLQNKFLEAME KCEKDRIARDEAWKMQEIARLKKEQEALAHERAISAAKDAAVIAFLQKVSDQTIQLQLPTDLPHRHTEERESESMKTIGNQENVVMQQDNDKENIDKQEIDSAGENSNSFQTNSSSRWPKAEVE ALIKLRTNVDLQYQDNGSSKGPLWEDISCGMKKLGYDRNAKRCKEKWENINKYYRRVKESQKKRPEDSKTCPYFHQLDSIYQNKSKKQLPIMETPGSNMKAGEILMQIINQQQQQQALERTEC.

[0042] Specific primers SlGT2-F and SlGT2-R (as shown in SEQ ID NOs: 3-4);

[0043] SlGT2-F:GAGAACACGGGGGACTCTAGAATGCTAGAAAGTTCTGTTTTGT

[0044] SlGT2-R:GCCCTTGCTCACCATGGTACCACATTCTGTTCTTTCTAATGCT

[0045] Example 2: Acquisition of S1GT2 transgenic plants

[0046] The plant overexpression vector pCambia2301::SlGT2-GFP was chemically transformed into Agrobacterium GV3101 and then infected with wild-type (MicroTom) tomato cotyledons. T0 generation tissue culture seedlings were obtained through callus induction, resistance induction differentiation, and rooting culture. Genomic DNA from T0 generation tomato leaves was extracted and positive seedlings were identified by PCR. The results are as follows: Figure 2 The expression levels of the SlGT2 gene in tomato were significantly different between wild-type and overexpressing plants. T0-generation plants that tested positive were harvested individually to obtain T1-generation plants. The T1-generation overexpressing seeds were tested for kanamycin resistance, and lines with a ratio of 3:1 positive plants were selected, indicating single-copy positive lines. Three-quarters of these positive plants were harvested individually, and T2-generation plants that were completely positive (SlGT2 overexpressing lines L13 and L14) were selected for subsequent experimental observation.

[0047] Example 3 Phenotypic Identification of S1GT2 Transgenic Plants

[0048] The tomato varieties used in this experiment were the wild-type MicroTom and the SlGT2-overexpressing strains L13 and L14 obtained in Example 2. Seeds were sown in plastic pots filled with a 3:1 peat-vermiculite composite culture medium. After emergence, the medium was watered to maintain moisture, and Hoagland's nutrient solution was used throughout the cultivation process. Plant height, internode length, and number of internodes were measured at different times after sowing, and paraffin sections of tomato stems were analyzed.

[0049] Paraffin section analysis was performed as follows: paraffin sections were prepared from the third section of wild-type and SlGT2-overexpressing tomato plants, with transverse and longitudinal sections. Sections from the third section of wild-type and SlGT2-overexpressing tomato plants were fixed in FAA fixative, dehydrated with ethanol, and then cleared, wax-impregnated, and embedded. Finally, sections were sectioned, stained, and observed under a microscope. Statistical analysis was performed using ImageJ software.

[0050] The results show that ( Figures 3 to 10 ), compared with the wild type, SlGT2-overexpressing plants showed a semi-dwarf phenotype, with the number of stem nodes unchanged but the internode distance shortened. Cytological observation results showed that the stem cell length, width and height of SlGT2-overexpressing plants were significantly reduced compared with the wild type.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of the S1GT2 gene in regulating plant dwarfing, characterized in that: The nucleotide sequence of the S1GT2 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the SlGT2 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The plant is tomato.

4. A primer pair for amplifying the S1GT2 gene according to claim 1, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs: 3-4.

5. A recombinant vector comprising the S1GT2 gene according to claim 1.

6. The recombinant vector according to claim 5, characterized in that The type of the recombinant vector is an expression vector with a 35S promoter.

7. The recombinant vector according to claim 6, characterized in that The expression vector with 35S promoter is pCAMBIA2301.

8. A recombinant bacterium comprising the recombinant vector according to any one of claims 5 to 7.

9. A method for regulating plant dwarfing, characterized in that: The following steps are involved: (1) constructing a recombinant vector containing the S1GT2 gene according to claim 1; (2) transforming the recombinant vector into plant tissues or plant cells; (3) Cultivate and screen positive transgenic plants.

10. The cultivation method according to claim 9, characterized in that The plant is tomato.

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