Rice tillering-promoting genes and their applications

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 utilizing tiller-promoting genes.

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 of rice can promote the number of effective panicles of plants to a certain extent, thereby directly increasing the number of grains per plant. However, the dwarfing of plants helps to enhance the lodging resistance of rice in windy and rainy weather and reduce the yield loss due to lodging. Therefore, discovering 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 an important theoretical basis for the detailed analysis of the plant type structure and high-yield breeding of rice. However, these tiller genes may not necessarily be able to increase the yield per plant of rice. Only by promoting the number of effective tillers of plants 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 tillering in rice. The base sequence of the Os03g0280400 gene is shown in Sequence ID No:1.

[0005] Improvements in the application of this invention include increasing the number of tillers in rice plants and reducing their height (i.e., increasing the number of grains per plant and improving lodging resistance). This increases the yield per rice plant.

[0006] The present invention also provides a method for improving the yield per rice plant using a new functional gene, Os03g0280400, which promotes tillering in rice. The base sequence of this gene is shown in Sequence ID No:1.

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

[0008] As an improvement to 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 used to transform wild-type rice variety Nipponbare to obtain genetically modified rice.

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

[0010] The sequence shown in sequence number 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 new functional gene in rice, the number of tillers in the rice plant can be significantly increased while maintaining a moderately low plant height, resulting in an increase in the number of grains per plant and improved lodging resistance. This, in turn, increases the yield per rice plant. The technical solution of the present invention is as follows:

[0012] Using PCR technology, the coding sequence (SEQ ID NO:1) of the Os03g0280400 gene was amplified from rice cDNA and cloned into a binary expression vector to construct an Os03g0280400 gene expression vector driven by a constitutive promoter. This vector was transformed into the wild-type Japonica rice variety "Nipponbare" and the wild-type Indica rice variety "Zhefu 802" using an Agrobacterium-mediated method to obtain corresponding genetically modified plants. PCR amplification was performed on the DNA of these genetically modified plants to confirm the presence of the target sequence (SEQ ID NO:1) to obtain genetically modified plants. Next, RNA was extracted from each genetically modified plant, and plants overexpressing the Os03g0280400 gene were detected using qPCR. The expression levels of Nipponbare-OE and Zhefu 802-OE were significantly higher than those of the corresponding wild-type varieties, Nipponbare and Zhefu 802 (Figure 1). Statistical analysis comparing each wild-type control variety with the overexpressing Os03g0280400 gene at the maturation stage of rice showed that the number of effective tillers was significantly higher in plants overexpressing the Os03g0280400 gene than in wild-type control varieties (Figure 2). Rice plant height was significantly lower than in wild-type control varieties (Figure 3), and yield per plant was significantly higher than in wild-type control varieties (Figure 4).

[0013] Therefore, overexpression of the Os03g0280400 gene in genetically modified rice can effectively increase the yield per plant, improve resistance to lodging, reduce yield losses due to lodging, and effectively ensure the safety of rice production. The use of this gene has significant application value in high-yield breeding. [Brief explanation of the drawing]

[0014] Hereinafter, specific embodiments of the present invention will be described in more detail with reference to the attached drawings. [Figure 1] Expression level analysis of the Os03g0280400 gene in rice. [Figure 2] Statistics on the number of effective tillers in mature rice plants. [Figure 3] Statistics on measuring the height of rice plants at maturity. [Figure 4] Yield statistics for individual mature rice plants. In the figure, (A) represents Nipponbare and its genetically modified plants, and (B) represents Zhefu 802 and its genetically modified plants.

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

[0016] (Example 1) Step 1. Extraction of total RNA from rice leaves. At the tillering stage, leaves of the wild-type rice variety Nipponbare are collected, pulverized in liquid nitrogen, and then total RNA is extracted using the RNeasy Plant Mini Kit (QIAGEN, Germany). Specific operating procedures are described in detail in this product. Next, the extracted RNA is processed using PrimeScript. TM Reverse transcription into cDNA is performed using the 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Follow the product instructions for use.

[0017] Step 2. PCR amplification of the Os03g0280400 gene. Synthesize PCR primers with 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, use the high-fidelity enzyme PrimeSTAR (R) HS DNA Polymerase (TaKaRa, Japan). The PCR reaction system is 20 μL: PrimerSTAR HS DNA Polymerase 0.2 μL, 5×PrimerSTAR Buffer 4 μL, cDNA 0.8 μL, primers F1 and R1 (10 μM) 0.5 μL each, dNTP Mixture 1.6 μL, ddH2O 12.4 μL. The PCR amplification program consists of 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. Next, perform double digestion using the restriction endonucleases BamH I and Sal I from TaKaRa Company (Japan). The enzyme digestion reaction system consists of 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 of 20 μl, and digest at 37°C for 4 hours. Then, purify the enzyme digestion products using the AxyPrep PCR Cleaning Kit (Axygen, USA) according to the product manual. At the same time, double digest and purify the pCAMBIA1300s vector plasmid (http: / / www.kelei-biology.com / plus / view.php?aid=1019) following the same method.

[0020] Step 4. Ligation of the Os03g0280400 gene PCR product and the vector Ligate the Os03g0280400 gene PCR product obtained after the enzyme digestion and purification in Step 3 with the vector using T4 ligase (Promega, USA). The reaction system consists of adding 1 μl of vector plasmid, 2 μl of PCR product, 0.5 μl of T4 ligase, 1 μl of Buffer, and ddH₂O 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, give 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, perform constant temperature shaking culture 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 liters of LB liquid culture containing Kan (25 mg / L) resistance to a 15 ml test tube. Collect single clonal colonies grown on the solid plate obtained in step 4 and transfer them to a test tube. Incubate in liquid medium on a shaker at 200 rpm and 37°C for 12 hours. Perform PCR validation of the bacterial suspension using 2× Taq PCR premix reagent (Tiangen, Beijing). Reaction system: Add 1 μl of bacterial suspension, 10 μl of 2× Taq PCR Master Mix II, 1 μl F1+R1 primer (10 μM), and ddH2O up to 20 μl. The PCR amplification program consisted of 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 PCR product and detect by 1% agarose gel electrophoresis.

[0022] Positive clones identified by PCR were sent to a biotechnology company for sequencing using the universal primers P1: 5′-CCAGGCTTTACACTTTATGC-3′ and P2: 5′-GCGATTAAGTTGGGTAACGC-3′ of pCAMBIA1300s, and all inserted gene sequences were measured and confirmed. In this way, the 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 using the Os03g0280400 gene overexpression vector. The overexpression vector constructed above was used to transform 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 levels in genetically modified rice. For the genetically modified plants obtained in Step 6, total RNA is extracted from the leaves according to the method in Step 1, and cDNA is synthesized. QPCR analysis is performed using the specific primers for the Os03g0280400 gene: F2: 5′-CCCTGCCTCTTACATCCACA-3′, R2: 5′-CCTTTCTCCAGCTCCTTCCAT-3′.

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

[0026] Note: The rice actin gene is used as the internal reference. In other words, it is the same except that the F3+R3 primer is used instead of the F2+R2 primer. Repeat each sample three times. -ΔΔCT Using the method (Livak et al., 2001), the cDNA amount of each sample was normalized and calibrated using the expression level of the internal reference gene actin, and the relative expression level of the Os03g0280400 gene was 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. Statistics on the number of effective tillers in genetically modified rice. Seeds of two lines (Nipponbare-OE-#1 and Nipponbare-OE-#2) overexpressing the Os03g0280400 gene, along with their wild-type control, Nipponbare, were sown in a rice experimental field. Sowing took place in early May, and transplanting in early June followed the conventional rice planting procedure. At the rice maturation stage (October of the same year), 10 plants each of Nipponbare-OE-#1, Nipponbare-OE-#2, and 10 control wild plants were randomly selected. The number of tillers per plant was counted, plant height was measured, and seed yield per plant was determined. A t-test was used to analyze any significant difference between the genetically modified plants and the wild-type control.

[0028] As a result, compared to the wild-type control variety Nipponbare, plants overexpressing the Os03g0280400 gene showed a significant increase in tillering (Figure 2-A), a substantial decrease in plant height (Figure 3-A), and a remarkable increase in yield per plant (Figure 4-A).

[0029] (Example 2) In Example 1, the wild-type rice variety was changed from Nipponbare (Japonica rice, suitable for planting in northern regions) to another variety with a broader genetic background, Zhefu 802 (Indica rice, suitable for planting in southern regions). The genetic background is significantly different from Nipponbare, and this change was made to demonstrate that the function of the Os03g0280400 gene can be generally applied to various rice varieties. Otherwise, the results were the same as in Example 1.

[0030] The results showed that, compared to the wild-type control variety Zhefu 802, plants overexpressing the Os03g0280400 gene had a significantly increased tillering rate (Figure 2-B), a significantly reduced plant height (Figure 3-B), and a significantly increased yield per plant (Figure 4-B).

[0031] Finally, it should be noted that the above enumeration represents only some specific embodiments of the present invention. The present invention is not limited to these embodiments, and various modifications are possible. All modifications that a person skilled in the art can directly derive or associate with the disclosure of the present invention should be considered to fall within the scope of protection of the present invention.

Claims

[Claim 1] A method for increasing the number of tillers in a Nipponbare variety of rice, reducing the height of the plant, and thereby increasing the yield per plant of the rice by overexpressing the Os03g0280400 gene, the nucleotide sequence of which is shown in Sequence ID No. 1, A method characterized by preparing an overexpression vector for the Os03g0280400 gene by ligating the Os03g0280400 gene and the pCAMBIA1300s vector plasmid (trademark) using T4 ligase, and using the overexpression vector to transform the Nipponbare variety of rice by an Agrobacterium-mediated method to obtain genetically modified rice.

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