Rice tiller promotion gene and its use

Abstract

The present invention belongs to the field of crop genetic breeding, and particularly relates to a method for increasing the yield per plant of rice by utilizing the tiller-promoting gene. The present invention discloses the application of the Os03g0280400 gene in improving rice tillering promotion, and the nucleotide sequence of the Os03g0280400 gene is shown in SEQ ID NO:1. The present invention also provides a method for increasing the yield per plant of rice by utilizing the novel tiller-promoting rice functional gene Os03g0280400.

Description

Technical Field

[0001] The present invention belongs to the field of crop genetic breeding, and particularly relates to a method for increasing the yield per plant of rice by using a tiller-promoting gene.

Background Art

[0002] Rice is the most important food crop in the world. In the first "Green Revolution" that occurred in the mid-20th century to solve the food problems in developing countries, new semi-dwarf varieties and multi-tiller varieties were bred using semi-dwarf genes such as sd1 (semi-dwarf 1) and Rht-1 (reduced height-1), effectively increasing the yields of rice, wheat, and other food crops. The multi-tiller trait in rice can promote the number of effective panicles of the plant to a certain extent, thereby directly increasing the number of grains per plant. However, the dwarfing of the plant helps to enhance the lodging resistance of rice in windy and rainy weather and reduce the yield loss due to lodging. Therefore, discovering the dwarf multi-tiller genes in rice is very valuable for increasing the yield per plant of rice. Currently, it has been reported that genes such as MOC1, TAD1, D53, HTD1, and others are involved in the coordinated regulation of tillering and plant height in rice, which provides important theoretical basis for the detailed analysis of the plant type structure and high-yield breeding of rice. However, these tiller genes do not necessarily increase the yield per plant of rice. Only by promoting the number of effective tillers of the plant can the yield per plant of rice be increased. Therefore, discovering new genes that increase the number of effective tillers from rice is very important for food crop production.

Summary of the Invention

Problems to be Solved by the Invention

[0003] An object of the present invention is to provide a method for effectively increasing the yield per plant of rice.

Means for Solving the Problems

[0004] To solve the above technical problems, the present invention provides the application of the Os03g0280400 gene in improving tiller promotion of rice. The nucleotide sequence of the Os03g0280400 gene is shown in SEQ ID NO:1.

[0005] The improvements in the application of the present invention include increasing the number of tillers of rice and reducing the plant height (i.e., increasing the number of grains per plant and improving lodging resistance), thereby increasing the yield per plant of rice.

[0006] The present invention also provides a method for improving the yield per plant of rice by utilizing the novel functional gene Os03g0280400 for promoting tillering of rice. The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] As an improvement of the method of the present invention, overexpression of the functional gene Os03g0280400 in rice can increase the number of tillers of rice and reduce the plant height.

[0008] As an improvement of the method of the present invention, an Os03g0280400 gene overexpression vector (the Os03g0280400 gene overexpression vector contains the nucleotide sequence shown in SEQ ID NO:1) is prepared. This overexpression vector is transformed into the wild-type rice variety Nipponbare to obtain genetically modified rice.

[0009] In summary, the present invention provides a method for improving the yield per plant of rice by utilizing a novel functional gene for promoting tillering of rice. The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0010] The sequence shown in SEQ ID NO:1 is publicly available in the rice database (https: / / rapdb.dna.affrc.go.jp / ), but its function is unknown.

[0011] By overexpressing this novel functional gene in rice, the tiller number of rice can be significantly increased while moderately reducing the plant height, increasing the number of grains per plant, and improving lodging resistance. As a result, the yield per plant of rice increases. The technical solution of the present invention is as follows.

[0012] Using the PCR technique, the coding sequence of the Os03g0280400 gene (SEQ ID NO:1) was amplified from rice cDNA, cloned into a binary expression vector, and an Os03g0280400 gene expression vector driven by a constitutive promoter was constructed. By the method via Agrobacterium, this vector was transformed into the wild-type japonica rice variety "Nipponbare" and the wild-type indica rice variety "Zhefu 802" to obtain the corresponding transgenic plants. PCR amplification was performed on the DNA of these transgenic plants to confirm whether they contained the target sequence (SEQ ID NO:1), and transgenic positive plants were obtained. Next, RNA was extracted from the transgenic positive plants respectively, and qPCR was used to detect the plants overexpressing the Os03g0280400 gene. The expression levels of Nipponbare-OE and Zhefu 802-OE are much higher than those of the corresponding wild-type varieties Nipponbare and Zhefu 802 (Figure 1). At the mature stage of rice, the results of statistical analysis compared with each wild-type control variety showed that the effective tiller number of the strains overexpressing the Os03g0280400 gene was significantly higher than that of the wild-type control variety (Figure 2). The plant height of rice was significantly lower than that of the wild-type control variety (Figure 3), and the yield per plant was significantly higher than that of the wild-type control variety (Figure 4).

[0013] Therefore, overexpression of the Os03g0280400 gene in transgenic rice can effectively increase the yield per plant of rice, improve the ability to resist lodging, reduce the yield loss caused by lodging, and effectively ensure the production safety of rice. The use of this gene has important application value in high-yield breeding.

Brief Description of the Drawings

[0014] Hereinafter, with reference to the accompanying drawings, specific embodiments of the present invention will be described in more detail.

Figure 1

Figure 2

Figure 3

Figure 4

[0015] Nipponbare and Zhefu 802 are wild-type control varieties of rice. Nipponbare-OE-#1 and Nipponbare-OE-#2 are genetically modified plants of two different lines of Nipponbare that overexpress the Os03g0280400 gene. Zhefu 802-OE-#1 and Zhefu 802-OE-#2 are two different lines of Zhefu 802 plants that overexpress the Os03g0280400 gene. * indicates a significant (P<0.05) difference in the t-test. ** indicates a very significant (P<0.01) difference in the t-test.

Mode for Carrying Out the Invention

[0016] (Example 1) Step 1. Extraction of total RNA from rice leaves At the tillering stage, collect the leaves of the wild-type rice variety Nipponbare, grind them into powder in liquid nitrogen, and then extract total RNA using the RNeasy Plant Mini Kit (QIAGEN, Germany). The specific operation procedure is described in detail in this product. Next, reverse transcribe the extracted RNA into cDNA using the PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Follow the instructions of the product for the usage method.

[0017] Step 2. PCR amplification of the Os03g0280400 gene Synthesize PCR primers having the following sequences. F1: 5′-cgcggatccATGGCTCTGGGATGACTC-3′ R1: 5′-acgcgtcgacTCAGGAGCTGCTCACGCCTC-3′. The lowercase sequences are the sequences used for restriction endonucleases BamH I and Sal I.

[0018] For PCR amplification, the high-fidelity enzyme PrimeSTAR (R) HS DNA Polymerase (TaKaRa, Japan) is used. The PCR reaction system is 20 μL: 0.2 μL of PrimerSTAR HS DNA Polymerase, 4 μL of 5× PrimerSTAR Buffer, 0.8 μL of cDNA, 0.5 μL each of primers F1 and R1 (10 μM), 1.6 μL of dNTP Mixture, and 12.4 μL of ddH2O. The PCR amplification program is 30 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 5 minutes. The sequence of the Os03g0280400 gene, i.e., SEQ ID NO:1, is obtained.

[0019] Step 3. Enzymatic digestion of the Os03g0280400 gene PCR product and the vector First, purify the PCR products using the AxyPrep PCR Cleaning Kit (Axygen, USA) according to the product manual, and then perform double digestion using the restriction endonucleases BamH I and Sal I from TaKaRa Company (Japan). The enzyme digestion reaction system is prepared by adding 1 μl each of BamH I and Sal I, 3 μl of Buffer T (TaKaRa), 10 μl of PCR products, and ddH2O to make a total volume of 20 μl, and digest at 37°C for 4 hours. Next, the enzyme digestion products are purified using the AxyPrep PCR Cleaning Kit (Axygen, USA) according to the product manual. At the same time, the pCAMBIA1300s vector plasmid (http: / / www.kelei-biology.com / plus / view.php?aid=1019) is double digested and purified following the same method.

[0020] Step 4. Ligation of the Os03g0280400 gene PCR product and the vector The Os03g0280400 gene PCR product obtained after the enzyme digestion and purification in Step 3 is ligated to the vector using T4 ligase (Promega, USA). The reaction system consists of 1 μl of vector plasmid, 2 μl of PCR product, 0.5 μl of T4 ligase, 1 μl of Buffer, and ddH2O added to 10 μl, and incubate at 4°C for 12 hours. Next, take 10 μl of the ligation product, incubate it with JM109 competent cells on ice for 30 minutes, apply a heat shock at 42°C for 90 seconds, and immediately cool it in an ice bath for 10 minutes. Add 800 μl of liquid LB medium without antibiotics, incubate with constant shaking at 37°C for 1 hour, then evenly spread the bacterial solution on a solid plate medium containing Kan (25 mg / L) resistance, and culture at 37°C for 12 hours.

[0021] Step 5. PCR and sequencing of the Os03g0280400 gene overexpression vector Add 4 l of LB liquid medium containing Kan (25 mg / L) resistance to a 15-ml test tube, pick up the single clone colonies grown on the solid plate obtained in Step 4, and transfer them to the test tube. Incubate in the liquid medium on a shaker at 200 rpm and 37 °C for 12 hours. Perform PCR verification of the bacterial solution using 2× Taq PCR premix reagent (Tiangen, Beijing). Reaction system: Add 1 μl of the bacterial solution, 10 μl of 2× Taq PCR MasterMixII, 1 μl of F1+R1 primer (10 μM), and ddH2O to make up to 20 μl. The PCR amplification program was 35 cycles of pre-denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. Then, extend at 72 °C for 5 minutes. Collect 5 μl of the PCR product and detect it by 1% agarose gel electrophoresis.

[0022] Send the positive clones identified by PCR to a biotechnology company, determine the sequences using the universal primers P1: 5′-CCAGGCTTTACACTTTATGC-3′ and P2: 5′-GCGATTAAGTTGGGTAACGC-3′ of pCAMBIA1300s, and measure and confirm all the inserted gene sequences. In this way, an Os03g0280400 gene overexpression vector was obtained. That is, the Os03g0280400 gene overexpression vector contains the nucleotide sequence shown in SEQ ID NO:1.

[0023] Step 6. Genetic transformation of rice with the Os03g0280400 gene overexpression vector Transform the overexpression vector constructed above into the wild-type rice variety Nipponbare using the method of Nishimura et al (Nishimura et al, Nat Protoc, 2006) to obtain genetically modified rice plants.

[0024] Step 7. Analysis of Os03g0280400 gene expression level in genetically modified rice For the genetically modified plants obtained in Step 6, total RNA is extracted from the leaves and cDNA is synthesized according to the method of Step 1. qPCR analysis is performed using the specific primers F2: 5′-CCCTGCCTCTTACATCCACA-3′ and R2: 5′-CCTTTCTCCAGCTCCTTCCAT-3′ for the Os03g0280400 gene.

[0025] SYBR of TaKaRa Company (R) Premix Ex Taq TM II kit is used. The reaction system is as follows: 10 μl of SYBR (R) Premix Ex Taq (R) II, 2 μl of cDNA template, 1 μl of 10 μM F2+R2 primer, 0.4 μl of ROX Reference Dye, and ddH2O is added to make up to 20 μl. The rice actin Actin gene is used as an internal reference, and the PCR primers are F3: 5′-TGGCATCTCTCAGCACATTCC-3′ and R3: 5′-TGCACAATGGATGGGTCAGA-3′. The PCR program is 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds.

[0026] Note: The rice actin gene is used as an internal reference. That is, it is the same except that the F3+R3 primer is used instead of the F2+R2 primer. Each sample is repeated 3 times, and 2 -ΔΔCT methods (Livak et al., 2001) are used to normalize and calibrate the cDNA amount of each sample using the expression level of the internal reference gene actin, and the relative expression level of the Os03g0280400 gene is calculated. In two different genetically modified lines (Nipponbare-OE-#1 and Nipponbare-OE-#2), the expression level of the Os03g0280400 gene was significantly higher than that of the control Nipponbare (Figure 1-A).

[0027] Step 8. Counting the effective tiller numbers of genetically modified rice Seeds of two lines overexpressing the Os03g0280400 gene (Nipponbare - OE - #1, Nipponbare - OE - #2) and their wild - type control Nipponbare were sown in a rice experimental field, sown in early May, transplanted in early June, and conventional rice transplanting was carried out. At the maturity stage of rice (October of the same year), 10 plants of the Nipponbare - OE - #1 line, 10 plants of the Nipponbare - OE - #2 line, and 10 plants of the control wild - type strain were randomly selected. The number of tillers per plant was counted, the plant height was measured, the seed yield per plant was determined, and the t - test method was used to analyze the significant differences between the genetically modified plants and the wild - type control.

[0028] As a result, compared with the wild - type control variety Nipponbare, the number of tillers of the plants overexpressing the Os03g0280400 gene increased significantly (Figure 2 - A), the plant height decreased significantly (Figure 3 - A), and the yield per plant increased significantly (Figure 4 - A).

[0029] (Example 2) The wild - type rice variety in Example 1 was changed from Nipponbare (japonica rice, suitable for planting in the north) to another variety Zhefu 802 (indica rice, suitable for planting in the south) with a large genetic background. This is to prove that the function of the Os03g0280400 gene can be generally applied to different varieties of rice. Other procedures were the same as in Example 1.

[0030] The results obtained were that, compared with the wild - type control variety Zhefu 802, the number of tillers of the plants overexpressing the Os03g0280400 gene increased significantly (Figure 2 - B), the plant height decreased significantly (Figure 3 - B), and the yield per plant increased significantly (Figure 4 - B).

[0031] Finally, it should also be noted that the above enumeration is only some specific embodiments of the present invention. It goes without saying that the present invention is not limited to the above - described embodiments, and various modifications are possible. All changes that can be directly derived or associated by those skilled in the art from the disclosure of the present invention should be considered to be within the protection scope of the present invention.

Claims

**Claim 1** Application of the Os03g0280400 gene in improving tillering of rice, characterized in that the nucleotide sequence of the Os03g0280400 gene is shown in SEQ ID NO:

1. **Claim 2** The application according to claim 1, characterized in that it increases the tiller number of rice, reduces the plant height, and thereby increases the yield per plant of rice. **Claim 3** A method for increasing the yield per plant of rice, characterized in that a novel functional gene Os03g0280400 that promotes tillering of rice is used, and the nucleotide sequence of the gene is shown in SEQ ID NO:

1. **Claim 4** The method according to claim 3, characterized in that overexpression of the functional gene Os03g0280400 in rice increases the tiller number of rice and reduces the plant height. **Claim 5** The method according to claim 3, characterized in that an Os03g0280400 gene overexpression vector is prepared, and the overexpression vector is transformed into the wild-type rice variety Nipponbare to obtain transgenic rice.

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