Plant growth and development regulation gene and application thereof

Abstract

The invention discloses a plant growth and development regulation and control gene and application thereof, and relates to the technical field of plant genetic engineering, the plant growth and development regulation and control gene is a rice Os07g0477500 gene, the nucleotide sequence of the rice Os07g0477500 gene is shown as SEQ ID NO.1, the amino acid sequence of encoded protein of the rice Os07g0477500 gene is shown as SEQ ID NO.2, and the encoded protein is located in a cell nucleus. The Os07g0477500 gene is introduced into an arabidopsis thaliana genome as a target gene, the plant height of an overexpression strain of the gene is remarkably increased compared with that of a wild type, and the growth state has remarkable advantages; the Os07g0477500 gene has a high-yield and high-quality gene expression, but a homozygous mutant plant of the gene shows that the plant height is dwarf and the growth and development process is obviously blocked, and the research result shows that the Os07g0477500 gene plays a key positive regulation role in plant growth and development regulation, provides a core gene resource for high-yield and high-quality breeding of crops, provides theoretical support and practical basis for upgrading of a plant molecular breeding technology, and has a wide application prospect. The method has remarkable innovativeness and application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a plant growth and development regulatory gene and its application. Background Technology

[0002] Rice, a vital global food crop, supports the food supply of billions of people. Its growth and development directly determine yield and quality, playing a crucial role in ensuring global food security. Plant growth and development is a complex process involving the synergistic action of multiple genes and is jointly regulated by hormones and the environment. The expression timing and intensity of key genes are central to maintaining homeostasis. Currently, some genes regulating plant growth and development have been identified, but the functions of many key genes in the systemic regulatory network of rice growth and development throughout its entire life cycle remain unresolved. Existing research shows that specific gene expression differences exist at different stages of rice growth and development. These genes may affect rice growth status through pathways such as regulating cell proliferation and differentiation, nutrient absorption and transport, or stress responses. However, the pathways of action and functional stability under different environments for specific genes with global regulatory effects remain unclear. In current rice breeding, how to coordinate the various stages of growth and development through genetic engineering to improve the stress resistance and yield of rice remains a core challenge. Since the function of the target gene in rice has not been reported, its potential value in the regulation of growth and development has not been explored. Therefore, in-depth exploration of key genes related to rice growth and development and elucidation of their regulatory mechanisms are of great theoretical and practical significance for enriching the theory of plant growth and development, promoting innovation in rice molecular breeding, and cultivating high-quality and high-yield rice varieties. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a plant growth and development regulatory gene and its application.

[0004] The present invention achieves the above objectives through the following technical solutions: This invention provides a plant growth and development regulatory gene, which is the rice Os07g0477500 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein shown in SEQ ID NO.2. The encoded protein is located in the cell nucleus.

[0005] This invention also provides an application of the rice Os07g0477500 gene in regulating plant growth and development. The Os07g0477500 gene positively regulates plant growth and development, that is, overexpression of the Os07g0477500 gene significantly enhances the plant's growth and development status.

[0006] As a further optimization of the present invention, the plant growth and development status includes plant height.

[0007] As a further optimization of the present invention, the plant is Arabidopsis thaliana or rice.

[0008] The present invention also provides a recombinant plasmid, which is obtained by transforming the Os07g0477500 gene into an overexpression vector.

[0009] As a further optimization of the present invention, the overexpression vector is pCAMBIA1301 or pCAMBIA1305.

[0010] This invention also provides a method for obtaining a growth-promoting transgenic plant variety, wherein the Os07g0477500 gene is used as the target gene and is introduced into the plant genome for expression, thereby cultivating a growth-promoting transgenic plant variety.

[0011] The present invention has the following beneficial effects: This invention introduces the Os07g0477500 gene as the target gene into the Arabidopsis thaliana genome. Overexpression of this gene significantly increased plant height compared to the wild type, exhibiting a significant growth advantage. Conversely, homozygous mutants of this gene showed dwarfed plant height and significantly inhibited growth and development. These results demonstrate that the Os07g0477500 gene plays a crucial positive regulatory role in plant growth and development, filling a research gap in existing plant growth and development-related gene resource libraries for this type of functional gene. It provides core gene resources for high-yield and high-quality crop breeding, and offers theoretical support and practical basis for upgrading plant molecular breeding technology, possessing significant innovation and application value. Attached Figure Description

[0012] Figure 1 This is a diagram illustrating the tissue expression pattern of the Os07g0477500 gene; Figure 2 This is a schematic diagram of the pCAMBIA1305-Os07g0477500-GFP recombinant vector; Figure 3 This is a subcellular localization map of the protein encoded by the Os07g0477500 gene in tobacco epidermal cells; Figure 4 This is a subcellular localization map of the protein encoded by the Os07g0477500 gene in rice protoplasts. Figure 5 This is a schematic diagram of the pCAMBIA1301-Os07g0477500 recombinant vector.

[0013] Figure 6 This is an agarose gel electrophoresis image of a homozygous mutant of the Os07g0477500 gene (SALK-039841C).

[0014] Figure 7 These are phenotypic images of wild-type Arabidopsis thaliana plants, homozygous mutant plants, and transgenic Arabidopsis thaliana lines before flowering.

[0015] Figure 8 These are phenotypic diagrams of wild-type Arabidopsis thaliana plants, homozygous mutant plants, and transgenic Arabidopsis thaliana lines during the flowering period.

[0016] Figure 9 These are phenotypic diagrams of wild-type Arabidopsis thaliana plants, homozygous mutant plants, and transgenic Arabidopsis thaliana lines at maturity. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] I. Materials Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0019] II. Methods 2.1 Analysis of the expression pattern of the Os07g0477500 gene in different tissues of rice Rice roots, stems, leaves, mature embryos, and seeds were collected and stored at -80℃ for later use. RNA was extracted from the collected samples and reverse transcribed into cDNA. Using the cDNA as a template, real-time PCR was performed using Novizan's SYBR dye and a Thermo Scientific PikoReal Cycler instrument. After the reaction, 2... –ΔΔCT The algorithm analyzes the results and calculates the relative expression level of the gene.

[0020] Experimental conclusions: such as Figure 1 As shown, tissue expression pattern analysis revealed that the Os07g0477500 gene is widely expressed in rice tissues, with relatively high expression levels in rice leaves, embryos, and seeds.

[0021] 2.2 Subcellular localization analysis To investigate the subcellular localization of the protein encoded by the Os07g0477500 gene (the nucleotide sequence of the Os07g0477500 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2), the pCAMBIA1305-Os07g0477500-GFP fusion vector was constructed using the pCAMBIA1305 vector (see SEQ ID NO. 2). Figure 2 As shown in the figure, this vector uses a 35S promoter to regulate gene expression. After obtaining the Os07g0477500 gene transcript, the stop codon in the transcript was removed, and the following primers were designed with SpeI and XbaI as restriction sites: SEQ ID NO.3: pCAMBIA1305-Os07g0477500-F: 5'-GGACAGCCCAGATCAACTAGTAATTCATTCCATCCCGCGA-3'; SEQ ID NO.4: pCAMBIA1305-Os07g0477500-R: 5'-GGTCCTCGAGACGTCTCTAGAGTTTGATGATAGGAAATGCACTCG-3'; After primer design, using cDNA as a template, the Os07g0477500 gene fragment was amplified using a high-fidelity enzyme according to the instructions provided by NCBI. After amplification, the vector was ligated and recombinated using homologous recombinase. The constructed pCAMBIA1305-Os07g0477500-GFP vector (e.g., ...) was then used. Figure 2 (As shown) The pCAMBIA1305-Os07g0477500-GFP vector was transformed into tobacco epidermal cells using Agrobacterium-mediated transformation, with an empty GFP vector as a control. In addition, to further verify the accuracy of the results, the constructed pCAMBIA1305-Os07g0477500-GFP vector and the nuclear localization vector were co-transformed into rice protoplasts. After culturing for 36 hours, the localization of the protein was observed using a laser scanning confocal microscope, with the co-transformed empty GFP vector as a control.

[0022] Experimental conclusions: such as Figure 3 As shown, observation of GFP fluorescence signals revealed that the empty GFP vector was expressed in all parts of tobacco epidermal cells, while the Os07g0477500 protein was expressed only in the nucleus of tobacco epidermal cells; Figure 4 As shown, the empty vector GFP is expressed in all parts of the protoplast, while the Os07g0477500 protein and the nucleoprotein are expressed only in the nucleus of the protoplast. This indicates that the protein encoded by the Os07g0477500 gene is a nuclear localized protein.

[0023] 2.3 Construction of the pCAMBIA1301-Os07g0477500 gene overexpression vector Using collected rice cDNA as a template, PCR amplification was performed using the following primers. The PCR amplification product was obtained, and the Os07g0477500 gene sequence was recovered via gel extraction. After removing the terminator from the cloned Os07g0477500 gene, it was ligated to the pCAMBIA1301 vector via homologous recombination to construct the pCAMBIA1301-Os07g0477500 overexpression vector (e.g., ...). Figure 5 The designed primer sequences are as follows: SEQ ID NO.5: pCAMBIA1301-Os07g0477500-F: 5'-GCGGAGGGGGAAGGGGGGAGATGAGTGGTGCACCCAAGAG-3'; SEQ ID NO. 6: pCAMBIA1301-Os07g0477500-R: 5'-AACCTGCAGGTCGACTCTAGACTAGTTTGATGATAGGAAATGCACTCG-3'.

[0024] The pCAMBIA1301-Os07g0477500 overexpression vector was then transformed into DH5α competent cells using a heat shock method. Positive colonies were screened using an anti-resistant medium (kanamycin resistance gene). A suitable number of single colonies were picked from the plate for culture, and the bacterial culture was sequenced. Those that matched successfully were positive recombinant vectors.

[0025] 2.4 Genetic Transformation of Os07g0477500 Transgenic Arabidopsis The pCAMBIA1301-Os07g0477500 positive plasmid was transformed into Agrobacterium competent cells using a heat shock method to obtain positive Agrobacterium. The Agrobacterium was then tested using the same detection method to ensure that the Agrobacterium used for transformation contained the target gene. The positive Agrobacterium was then transformed into Arabidopsis thaliana, and the T0 generation seeds were tested. Since the constructed vector contained both the vector and the target gene, PCR amplification was performed using specific primers (SEQ ID NO. 5 and 6) to screen for positive plants containing the target gene. Three positive lines were selected from the detected positive plants, and then these three lines were further propagated to the T3 generation. The positive plants that no longer exhibited phenotypic segregation were selected as homozygous Arabidopsis lines, which were then used as transgenic plants for subsequent experiments.

[0026] 2.5 Identification of homozygous mutants Information about Arabidopsis thaliana with the Os07g0477500 gene mutation was obtained through the Tair website. A homozygous mutant (SALK-039841C) was purchased from Fuzhou Airosha Biotechnology Co., Ltd. After receiving the mutant seeds, they were planted. After the Arabidopsis thaliana had grown for one month, DNA was extracted from the leaves of the mutant to be tested. At the same time, primers for verifying the mutant were designed. The specific primer sequences are as follows: SEQ ID NO.7: LP: 5'-CTCTGGCCTCTCCAGCTTTAG-3'; SEQ ID NO.8: RP: 5'-TTAATTGATGTTTTCCCGCTGG-3'; SEQ ID NO.9: BP: 5'-ATTTTGCCGATTTCGGAAC-3', Using the extracted DNA as a template, PCR verification was performed with two primer pairs, LP+RP and BP+RP, respectively. Plants with correct verification results were identified as homozygous Arabidopsis mutants (e.g., Figure 6 (As shown in the image), the seeds were harvested and then planted on a larger scale for further research.

[0027] 2.5 Observation of plant growth phenotype (1) Put appropriate amounts of wild-type, homozygous mutant (SALK-039841C) and transgenic Arabidopsis seeds into 2.0 mL centrifuge tubes respectively, add 1.5 mL of 12% sodium hypochlorite solution, shake vigorously for 10 min, use a micropipette to remove the sodium hypochlorite solution, minimize the remaining sodium hypochlorite solution, and then wash with sterile water 5-10 times to remove the sodium hypochlorite solution; (2) Spread the cleaned Arabidopsis seeds evenly on a square petri dish containing 1 / 2 MS medium, seal it with 3M breathable tape, and place it in an artificial climate chamber for cultivation (parameter settings: light intensity of 20 Klux, temperature of 22℃, 16 h light / 8 h dark, humidity of 70%). (3) After 10 days, transplant the Arabidopsis thaliana into the substrate (sterilized black soil and vermiculite mixed evenly in a ratio of 1:3). During the transplanting process, avoid damage to the Arabidopsis thaliana. Place the Arabidopsis thaliana in an artificial climate chamber for continued cultivation and ensure water supply during the growth period. (4) Take photos at appropriate times before, during, and after the flowering of Arabidopsis thaliana.

[0028] Experimental conclusions: such as Figure 7-9As shown, by introducing the Os07g0477500 gene as the target gene into Arabidopsis thaliana, it was found that the transgenic plants were superior to the wild type. However, after the gene was mutated, the growth and development status of the transgenic plants was significantly lower than that of the wild type and the transgenic lines. These results indicate that the Os07g0477500 gene is involved in plant growth and development and is a gene that regulates plant height.

[0029] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A plant growth and development regulatory gene, characterized in that, The plant growth and development regulatory gene is the rice Os07g0477500 gene, whose nucleotide sequence is shown in SEQ ID NO.1 and whose encoded protein amino acid sequence is shown in SEQ ID NO.

2.

2. An application of the rice Os07g0477500 gene as described in claim 1 in regulating plant growth and development, characterized in that, The Os07g0477500 gene positively regulates plant growth and development; that is, overexpression of the Os07g0477500 gene significantly enhances plant growth and development.

3. The application according to claim 2, characterized in that, The plant growth and development includes plant height.

4. The application according to claim 3, characterized in that, The plant in question is either Arabidopsis thaliana or rice.

5. A recombinant plasmid, characterized in that, The recombinant plasmid was obtained by transforming the Os07g0477500 gene as described in claim 1 into an overexpression vector.

6. The recombinant plasmid according to claim 5, characterized in that, The overexpression vector is pCAMBIA1301 or pCAMBIA1305.

7. A method for obtaining a growth-promoting transgenic plant variety, characterized in that, The Os07g0477500 gene as described in claim 1 was used as the target gene and introduced into the plant genome for expression, thereby cultivating a growth-promoting transgenic plant variety.

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