Rice heading period regulation gene DGP2 and application thereof

Abstract

The invention discloses a rice heading period regulation gene DGP2 and application thereof, the nucleotide sequence of the DGP2 gene is shown as SEQ ID No.1, and the amino acid sequence of the encoding protein of the DGP2 gene is shown as SEQ ID No.2. By increasing the expression quantity of the DGP2 gene in the rice, the heading period of the rice can be remarkably shortened, a theoretical basis and gene resources are provided for precise regulation and control of the heading period of the rice, and a foundation is laid for cultivation of eurytopic high-yield and stable-yield rice varieties.

Description

Technical Field

[0001] This invention relates to molecular genetic breeding of rice, specifically to a gene regulating the heading stage of rice. DGP2 And its applications. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important cereal crops in the world. Due to the increase in the global population, the sharp decrease in arable land, serious environmental pollution, and frequent extreme weather events, the supply and demand of food have become unbalanced.

[0003] The growth period of rice is an inherent characteristic of rice varieties, determining their regional and seasonal adaptability, as well as appropriate farming methods, and significantly impacting their yield, quality, and production efficiency. Among these traits, the heading stage is a key agronomical characteristic affecting rice's adaptation to different regions and yields. It is jointly regulated by the external environment and rice flowering genes, and is also one of the important goals of rice variety breeding. Providing genes that can regulate the rice heading stage is crucial for rice variety breeding and stabilizing rice yields. For example, in some Chinese cities, winter temperatures are low, unsuitable for rice growth. Adjusting the growth period to advance or delay it can mitigate the effects of cold weather, ensuring rice grain filling and yield.

[0004] Heading date is one of the important agronomic traits of rice. The length of the heading date (from germination to the first heading) directly reflects the length of the rice variety's growth period, and a suitable growth period plays a crucial role in maintaining high and stable rice yields. Currently, varieties have been cloned... Hd1 , Hd3a , Ghd7 , Ehd1 Key functional genes controlling the heading date of rice, such as [genes name omitted], have been identified, and a preliminary molecular regulatory network for the heading date has been established. However, in rice breeding, it is necessary to identify more regulatory sites for heading date to facilitate genetic improvement and selection in rice. Summary of the Invention

[0005] To meet the demand for more heading-stage regulatory sites, this invention provides a rice heading-stage regulatory gene. DGP2 Its application, through improving rice DGP2 The expression level of genes can significantly shorten the heading period of rice, providing a theoretical basis and gene resources for precise regulation of the heading period of rice, and laying the foundation for breeding widely adaptable, high-yielding and stable-yielding rice varieties.

[0006] To achieve the above objectives, the present invention provides a gene regulating the heading stage of rice. DGP2 (Gene number LOC_Os05g38680), whose nucleotide sequence is shown in SEQ ID No.1, or in which one or more nucleotides are substituted, inserted or deleted in the nucleotide sequence shown.

[0007] SEQ ID No.1:

[0008] The second aspect of this invention provides the above-mentioned rice heading stage regulatory gene. DGP2 The encoded protein amino acid sequence is shown in SEQ ID No. 2, or an amino acid sequence or derivative thereof generated by substituting, inserting or deleting one or more amino acids, or homologous sequences of other species in the shown amino acid sequence.

[0009] SEQ ID No. 2: MGDSSSSASYIRMVHHLIEKCICFNLNKEECMEALEKHANINPVVTSTVWKELEKENKEFFETYNKDRAERNIEAETMQRIQKMLSDAAASKGSDDDDDDE.

[0010] The third aspect of this invention provides the above-mentioned rice heading stage regulatory gene. DGP2 Its application in regulating the heading stage, through genetic engineering, enhances the growth rate of rice. DGP2 Increased gene expression or enhanced DGP2 protein activity can advance the heading stage of rice.

[0011] A fourth aspect of the present invention provides a method for shortening the heading period of rice by increasing the concentration of... DGP2 The expression level of the gene may be increased or the activity of the DGP2 protein may be enhanced.

[0012] The method is as follows: constructing a... DGP2 The gene was recombinantly expressed in a vector and transformed into rice.

[0013] Through the above technical solution, the present invention achieves the following beneficial effects: This invention reveals for the first time the rice DGP 2. Gene regulation of rice heading stage. Using genetic engineering technology, construct... DGP The overexpression vector of gene 2 was used, and Agrobacterium-mediated transformation technology was employed to... DGP The overexpression vector of gene 2 was introduced into the rice variety Yandao 8 to obtain transgenic T0 generation plants. Transgenic T0 plants were then selected from the self-pollinated progeny of T0 plants. DGP The transgenic homozygotes with significantly increased gene expression had a significantly shorter heading period than the untransformed control. Attached Figure Description

[0014] Figure 1 Wild type (WT) and DGP2 In overexpressing plants (DGP2-OE) DGP2 Gene expression levels; Figure 2 Wild type (WT) and DGP2 Phenotype of plants overexpressing DGP2-OE at the heading stage; Figure 3 Wild type (WT) and DGP2 Statistical analysis of the heading stage of overexpressing plants (DGP2-OE). Detailed Implementation

[0015] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0016] 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.

[0017] Example 1 DGP Construction of 2 gene overexpression vectors according to DGP PCR primers designed from the CDS sequences of two genes: DGP 2-OE-F:5'-ggtacccggggatcctctagaATGGGAGACTCATCATCCTCAGC-3', DGP 2-OE-R: 5'-atccttgtaatctccgtcgacGCTCTCGTCGTCGTCGTCG-3'.

[0018] Using rice leaf cDNA as a template, PCR amplification was performed using the high-fidelity enzyme Phanta Max Master Mix. The reaction system was as follows: PCR reaction system

[0019] PCR program: 95 ℃ pre-denaturation for 5 min; 98 ℃ denaturation for 5 s; 55 ℃ annealing for 5 s, 72 ℃ extension for 1 min 30 s; 72 ℃ extension for 10 min; 30 cycles in total.

[0020] The target fragment was cloned into the pC1300-3FLAG expression vector (with a strong 35S promoter) to obtain the overexpression vector pC1300-3FLAG- DGP2 .

[0021] Example 2 DGP Obtaining rice with 2 gene overexpression Plasmid pC1300-3FLAG- DGP2Agrobacterium EHA105 was transformed by electroporation. The steps are as follows: (1) Take 1 µL of plasmid and add it to 50 µL of competent Agrobacterium cells and mix gently. Carefully add the mixture to an electroporation cup and electroporate once. (2) Add 1 mL of liquid LB to the electroporation cup, mix by pipetting, and transfer to a 1.5 mL centrifuge tube. Place it in a shaker at 28°C and activate for 2 h. Place the kanamycin-resistant solid LB medium on a clean bench and dry it. (3) Spread the activated Agrobacterium onto the prepared solid medium and invert it in a 28°C oven for two days. (4) Add kanamycin-resistant liquid medium to a sterile 10 mL centrifuge tube, pick a single colony with a sterile pipette tip, place it in a centrifuge tube, and incubate it in a shaker at 28°C for 12 h. Pick out the centrifuge tubes that have been successfully propagated and perform colony PCR identification. Store the single clones that are identified as positive for later use.

[0022] Recombinant Agrobacterium was used to infect callus tissue of wild-type Salt Rice 8. T0 generation transformed seedlings were obtained through a series of processes including resistance selection, differentiation, and rooting.

[0023] Design hygromycin resistance selection marker gene ( HPT Specific primers are used for amplification. HPT The gene and primer sequences are as follows: HPT-F:5'-CTGCCCGCTGTTCTACAACCGG-3'; HPT-R:5'-GGAGCATATACGCCCGGAGTC-3'.

[0024] The obtained T0 generation transformed seedlings were used HPT Specific primers were used to amplify and obtain positive transformants containing the target gene. The T0 generation transgenic rice containing the target gene was self-pollinated to obtain T1 and T2 generation transgenic rice, which were then used... HPT Specific primer identification was used to obtain homozygous rice of the target gene from the T2 generation. DGP2 -OE). For homozygous transgenic rice... DGP 2-gene qRT-PCR analysis showed that the transgenic DGP 2. Gene expression levels were significantly increased ( Figure 1 ).

[0025] Example 3 DGP Phenotypic observation and statistical analysis of heading date of plants with 2 overexpressed genes Under the natural growing conditions in Yangzhou DGP Rice plants overexpressing the two genes headed earlier than the control variety, Salt Rice No. 8. Figure 2 Statistical analysis shows that wild-type seedlings typically head after 83.75 days of sowing. DGP Rice with overexpression of the two genes takes an average of 78.2 days to head after sowing. DGPThe heading period of rice with overexpression of the two genes was significantly shortened compared with that of wild type. Figure 3 ).

[0026] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0027] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0028] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A gene regulating the heading stage of rice. DGP2 Its characteristics are, The nucleotide sequence is as shown in SEQ ID No. 1, or one or more nucleotides are substituted, inserted, or deleted in the shown nucleotide sequence.

2. Genes regulating rice heading stage DGP2 The encoded protein is characterized by, The amino acid sequence is as shown in SEQ ID No. 2, or one or more amino acids are substituted, inserted, or deleted in the shown amino acid sequence.

3. The rice heading stage regulating gene as described in claim 1 DGP2 Its application in regulating the heading stage is characterized by... Through genetic engineering, improve the quality of rice DGP2 Increased gene expression or enhanced DGP2 protein activity can advance the heading stage of rice.

4. A method for shortening the heading period of rice, characterized in that, Improving the rice according to claim 1 DGP2 The expression level of the gene may be increased or the activity of the DGP2 protein may be enhanced.

5. The method according to claim 4, characterized in that, The method is as follows: constructing a... DGP2 The gene was recombinantly expressed in a vector and transformed into rice.

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