Genetic engineering application of rice ethylene response transcription factor osktr2

Abstract

The application discloses a genetic engineering application of a rice ethylene response transcription factor OsKTR2. The application discloses a rice ethylene response transcription factor OsKTR2, wherein the CDS sequence of the rice ethylene response transcription factor OsKTR2 is SEQ ID NO. 1. In the application, a recombinant expression vector constructed by an OsKTR2 gene is first found, the recombinant expression vector is transfected into wild-type rice Nipponbare (Oryza sativa.ssp.cv.Japonica) through an agrobacterium-mediated rice transgenic technology, and the effective tiller number, total tiller number and seed setting rate of the rice are increased, so that the yield of the rice is improved. In addition, the expression of the OsKTR2 is induced and up-regulated under low potassium stress, and then the potassium absorption of the rice is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to genetic engineering application of rice ethylene response transcription factor OsKTR2. BACKGROUND

[0002] Potassium (K) is one of the important mineral macronutrients, which widely participates in many physiological processes of plant growth and development, including osmoregulation, enzyme activation, ion balance, stomatal movement, and resistance to biotic and abiotic stresses (Wang et al., 2013; Anschütz et al., 2014). As an important inorganic osmotic ion, K + can regulate the osmotic potential of plant cells and plays an important role in plant response to osmotic / drought stress. K can also regulate photosynthesis and carbohydrate transfer and metabolism in plants, ultimately determining the yield and quality of crops. When K supply is insufficient, plants exhibit leaf water loss and yellowing, weak plant growth, and sensitivity to abiotic stresses such as drought, which seriously affect the growth and development of plants (Munson, 1985). On the contrary, under sufficient K supply, plants can enhance the resistance to biotic and abiotic stresses.

[0003] It has been reported that low potassium stress can generate potassium deficiency signals in root epidermal cells, including Ca 2+ , ROS, plant hormones, microRNAs, and the like. These signals are further conducted and trigger the expression of downstream target genes (especially K channels and high-affinity K transporters) through transcription factors, ultimately realizing the adaptation of plants to low potassium environment (Wang and Wu, 2013).

[0004] AP2 / ERF is one of the largest plant transcription factor families, comprising 119–200 members (Du et al., 2014; Nakano et al., 2006; Rao et al., 2015; Zhuang et al., 2008). These members are involved in physiological processes such as plant ethylene signaling, stress response, metabolism, and responses to external stimuli (Han et al., 2016; Koyama et al., 2013; Lee et al., 2012; Li et al., 2007; Fits and Memelink, 2000; Trujillo et al., 2008; Zhu et al., 2014). Members of the AP2 / ERF superfamily all contain a conserved AP2-binding domain consisting of approximately 57–66 amino acids, comprised of three β-stranded DNA-binding domains and an α-helical element (Okamuro et al., 1997; Danny WK Ng et al., 2018). Based on the differences in AP2 domain copy numbers among members, AP2 / ERF transcription factors are generally divided into four subfamilies: AP2, ERF, RAV, and Soloist (Nakano et al. 2006; Licausi et al. 2010a). However, different members of this family often play specific biological roles in specific physiological processes, such as ethylene signaling, stress response, metabolism, and responses to external stimuli. Kim et al. found that RAP2.11, a member of the Arabidopsis ERF transcription factor subfamily Vb, enhances plant tolerance to low potassium environments by regulating the expression of the high-affinity K transporter AtHAK5 and other genes involved in the low potassium signaling pathway (Kim et al., 2012). The expression of other AP2 / ERF family members, such as AtERF98, MsERF8, JcERF011, and CaERFLP1, can enhance plant salt tolerance (Chen et al. 2012; Lee et al. 2004; Tang et al. 2016; Zhang et al. 2004, 2012b). Furthermore, a small number of AP2 / ERF members can simultaneously respond to two or more environmental stresses. The precise biological function of a new AP2 / ERF member is unpredictable. Summary of the Invention

[0005] The purpose of the present invention is to provide a genetic engineering application of rice ethylene response transcription factor OsKTR2, mainly to increase the number of tillers, improve the fruit setting rate and yield by overexpressing its own promoter.

[0006] The purpose of the present invention is achieved through the following technologies:

[0007] The cDNA sequence of the rice ethylene response transcription factor OsKTR2 is shown as SEQ ID NO. 1.

[0008] A recombinant expression vector containing the rice ethylene response transcription factor OsKTR2 of claim 1.

[0009] The recombinant expression vector is pCAMBIA1305 vector.

[0010] The recombinant expression vector is further obtained by inserting the rice ethylene response transcription factor OsKTR2 of claim 1 into the SacI and PstI enzyme cutting sites of the pCAMBIA1305 vector, then removing the Ubiquitin promoter by double enzyme cutting of EcoRI and SacI and inserting the OsKTR2 gene self promoter.

[0011] The rice ethylene response transcription factor OsKTR2 is applied to any one or more of the following:

[0012] (1) increasing the total tiller number, effective tiller number and seed setting rate of crops;

[0013] (2) increasing the yield of rice;

[0014] (3) promoting the absorption of potassium by rice;

[0015] The rice ethylene response transcription factor OsKTR2 is applied to any one or more of the following:

[0016] (1) increasing the total tiller number, effective tiller number and seed setting rate of crops;

[0017] (2) increasing the yield of rice;

[0018] (3) promoting the absorption of potassium by rice;

[0019] The recombinant expression vector is applied to any one or more of the following:

[0020] (1) increasing the total tiller number, effective tiller number and seed setting rate of crops;

[0021] (2) increasing the yield of rice;

[0022] (3) promoting the absorption of potassium by rice;

[0023] The recombinant expression vector is applied to any one or more of the following:

[0024] (1) increasing the total tiller number, effective tiller number and seed setting rate of crops;

[0025] (2) increasing rice yield;

[0026] (3) promoting the absorption of potassium by rice;

[0027] Advantages of the present application:

[0028] 1. In the present application, a recombinant expression vector constructed from rice ethylene response transcription factor OsKTR2 is first discovered in the world, and is transfected into wild-type rice Nipponbare (Oryza sativa. ssp. cv. Japonica) through Agrobacterium-mediated rice transgenic technology. We found that the total tiller number, effective tiller number, seed setting rate and grain yield of OsKTR2 transgenic rice materials were significantly improved compared with wild-type rice.

[0029] 2. The OsKTR2 gene in the present application is derived from rice, and the promoter is also the sequence upstream of the OsKTR2 gene, which is not an exogenous gene, and thus has biological safety. The constructed rice ethylene response transcription factor OsKTR2 plant expression vector can be directly used for Agrobacterium-mediated plant genetic transformation to obtain new germplasm that can increase the total tiller and effective tiller of plants.

[0030] 3. The OsKTR2 gene in the present application can promote the absorption of potassium by rice after overexpression, especially under low potassium conditions. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Molecular identification and plant type screening of OsKTR2 transgenic rice materials

[0032] Figure A: Phenotype of wild-type rice Nipponbare WT and OsKTR2 self-promoter overexpression material Pro-KTR2; Figure B: Expression amount of OsKTR2 in Pro-KTR2 relative to WT; Figures C-D: Biomass of roots and shoots of WT and Pro-KTR2 materials.

[0033] Figure 2 Subcellular localization of OsKTR2 and expression in different growth periods and tissues of rice

[0034] Figure A: Expression of OsKTR2 in different growth periods and different tissue parts of wild-type rice Nipponbare; Figure B: OsKTR2 is localized in the nucleus of rice cells.

[0035] Figure 3 Up-regulation of OsKTR2 expression induced by potassium deficiency, H2O2 and ACC stress

[0036] Figure A-B: Expression of OsKTR2 gene in roots and shoots respectively at different time points after 0.05 mM K stress; Figure C-D: Expression of OsKTR2 gene in roots and shoots respectively at different time points after 10 mM H2O2 stress; Figure E-F: Expression of OsKTR2 gene in roots and shoots respectively at different time points after 100 μM ACC stress.

[0037] Figure 4 Effect of OsKTR2 expression on potassium uptake and accumulation in rice

[0038] Figure A-B: Phenotype of WT and Pro-KTR2 materials after 0.05 mM K and 1 mM K treatment respectively; Figure C-D: Biomass of WT and Pro-KTR2 materials after 0.05 mM K and 1 mM K treatment respectively; Figure E-F: K concentration of WT and Pro-KTR2 materials after 0.05 mM K and 1 mM K treatment respectively. + Figure G-H: Total K amount of WT and Pro-KTR2 materials after 0.05 mM K and 1 mM K treatment respectively.

[0039] Figure 5 Phenotype of OsKTR2 transgenic materials and wild type rice in field test

[0040] Figure A: Agronomic traits, yield and tiller number of WT and Pro-KTR2 materials in low potassium soil; Figure B: Agronomic traits, yield and tiller number of WT and Pro-KTR2 materials in normal potassium soil.

[0041] Figure 6 Map of pCAMBIA1305 expression vector.

[0042] Figure 7 Map of pCAMBIA1305.1-GFP expression vector. DETAILED DESCRIPTION

[0043] Example 1. Cloning of OsKTR2 gene

[0044] 1. Template: Extract RNA from Nipponbare (NB) wild type rice leaves of normal size of 2 weeks of hydroponic culture and reverse transcribe into cDNA as template for cloning OsKTR2-CDS sequence.

[0045] 2. PCR primer design: The gene sequence of OsKTR2 was found on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and the primer design software Primer 5.0 was used to design the primer. SacI (GAGCTC) and PstI (CTGCAG) enzyme digestion site sequences were added to the 5' and 3' ends of the primer, and then a sequence on the pCAMBIA1305 plasmid vector was added to form a 41 bp homologous recombination primer (F1 and R1).

[0046] Upstream primer F1:

[0047] 5'-ATTTACGAACGATAG GAGCTC ATGGAGCTCAACTTCCAAGT-3' (SEQ ID NO. 2);

[0048] Downstream primer R1:

[0049] 5'-GTGATTTTTGCGGAC CTGCAG CTAATGGCACAGTGGCGAGA-3' (SEQ ID NO. 3).

[0050] 3. OsKTR2 gene PCR amplification: PCR Buffer 2 μl, dNTP Mix 2 μl, 1 μl of upstream and downstream primers, 1 μl of template, KOD high-fidelity enzyme 1 μl, and 13 μl of double-distilled water. The PCR amplification program is as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 62℃ annealing for 30 s, 72℃ recombination extension for 1 min, 35 cycles, 72℃ for 10 min for full extension,

[0051] 10℃. The amplified PCR product was detected by 1% agarose gel electrophoresis, and the size of the OsKTR2 gene was 948 bp.

[0052] Example 2. Construction of plant recombinant expression vector pCAMBIA1305-OsKTR2 and obtaining of rice transgenic material

[0053] 1. Construction of OsKTR2 gene expression vector: pCAMBIA1305 plasmid vector was double digested with Sac I and Pst I, and the linearized expression vector was recovered by gel recovery, and then subjected to homologous recombination with the PCR linear product containing OsKTR2. The homologous recombination product was transformed into E. coli DH5a competent cells, and after antibiotic screening, positive single clones were picked and stored. The E. coli plasmid was extracted and sent to a company for sequencing. After correct sequencing, the pCAMBIA1305-OsKTR2 intermediate vector containing the Ubiquitin strong promoter was obtained. On this basis, the vector was double digested with EcoRI (GAATTC) and SacI (GAGCTC) to obtain a pCAMBIA1305 intermediate vector carrying the OsKTR2 gene but cutting off the Ubiquitin promoter, and the digested product was recovered by gel recovery. According to the OsKTR2 gene promoter sequence, a 1964 bp sequence was extracted, and a pair of homologous recombination primers (F2 and R2) for amplifying the OsKTR2 gene promoter were designed using the software Primer 5.0. EcoRI (GAATTC) and SacI (GAGCTC) enzyme digestion site sequences were added to the 5' and 3' ends of the primers, and a sequence of the pCAMBIA1305 plasmid vector was added. The wild type Nipponbare gDNA was used as the template to obtain the linear fragment of the target promoter by PCR technology, and after verification, the homologous recombination vector was subjected to homologous recombination with the pCAMBIA1305-OsKTR2 linear vector cutting off the Ubiquitin promoter. The homologous recombination product was transformed into E. coli DH5a competent cells, and after antibiotic screening, positive single clones were picked and stored. The E. coli plasmid was extracted and sent to a company for sequencing. After correct sequencing, the pCAMBIA1305-OsKTR2 recombinant expression vector was obtained, which was transformed into EHA105 Agrobacterium competent cells by electroporation method, and after antibiotic screening, positive clones were picked and the plasmid was extracted and returned to E. coli. After sequencing verification, the Agrobacterium liquid was added to an equal volume of 30% glycerol and stored at -70°C, and was used for subsequent experiments.

[0054] Upstream primer F2:

[0055] 5'-TATGACCATGATTAC GAATTC TTGTGGGATAGTAAACGAGA-3' (SEQ ID NO. 4);

[0056] Downstream primer R1:

[0057] 5'-GGATCCCCGGGTACC GAGCTC GTGTTTGTGTGTGAGCTTGT-3' (SEQ ID NO. 5).

[0058] 2. Obtaining Transgenic Rice: Rice callus was infected with Agrobacterium obtained above, carrying the pCAMBIA1305-OsKTR2 self-promoter overexpression vector. After co-cultivation (dark culture) for 2.5 days, the callus was washed and air-dried. The callus was transferred to a selective medium containing 500 mg / L carbenicillin and 50 mg / L hygromycin for the first round of selection. The culture was incubated at 28°C in the light for 2 weeks. Resistant calli were transferred to a selective medium containing 500 mg / L carbenicillin and 80 mg / L hygromycin for the second round of selection. The culture was incubated at 28°C in the light until granular, resistant callus tissue emerged. Bright yellow resistant calli from the same callus were transferred to a plastic jar containing differentiation medium for differentiation and culture. The calli were allowed to differentiate into seedlings (25-30 days). When the seedlings reached approximately 2-3 cm in length, they were placed in rooting medium for growth. The differentiated seedlings were removed from the rooting tubes, sterilized with an appropriate amount of water, and hardened for one week. The agar culture medium at the root is washed off, and the plant is transplanted into rice nutrient solution for growth and positive seedling identification is performed. The positive seedling is then transferred to the field for harvesting to obtain T1 generation transgenic seeds. The culture medium used is all prior art.

[0059] 3. Identification of Overexpression Effects of OsKTR2 Transgenic Rice Materials and Plant Type Screening: T1-generation transgenic seeds of the OsKTR2 overexpressing lines obtained above and wild-type Nipponbare rice seeds were hydrolyzed. Healthy seeds were selected and soaked in 30% sodium hypochlorite solution for 30 minutes. After rinsing five times with clean water, they were placed in a paper cup lined with a 20-mesh nylon mesh. The seeds were half-submerged in clean water and then placed in a 37°C oven for germination for 24-36 hours. Ten days after emergence, all overexpressing lines were identified by GUS staining, and positive seedlings were transferred to a transfer box for hydroponics. After culturing in full IRRI nutrient solution for two weeks, a portion of the seedlings were selected and total RNA was extracted from tissues of the OsKTR2 overexpressing lines and wild-type Nipponbare rice using Trizol reagent. cDNA was obtained by reverse transcription and used as a template to identify the expression of the OsKTR2 gene at the transcriptional level using RT-qPCR. The internal reference and OsKTR2 gene primers used for RT-qPCR are shown in Table 1. Another part of the seedlings were used to screen plant types, and the biomass of all plants was counted ( Figure 1 Among the 9 OsKTR2 overexpressing seedlings tested ( Figure 1 A), the expression levels of OsKTR2 were upregulated to varying degrees ( Figure 1 B), In addition, the root dry weight of these overexpressing seedlings ( Figure 1 C) and aboveground dry weight ( Figure 1 D) showed no significant difference from the wild type. Based on the expression level of OsKTR2 and the dry weight of the seedlings, we selected three representative lines from these nine overexpressing seedlings and named them Pro1, Pro2, and Pro3 for subsequent physiological experiments.

[0060] Table 1 Primer sequences of OsActin and OsKTR2 for RT-qPCR

[0061]

[0062] Example 3. Identification of the temporal and spatial expression pattern of OsKTR2 and its response to environmental stresses by RT-qPCR

[0063] To explore the temporal and spatial expression pattern of OsKTR2, we used the primers of OsKTR2 and the internal control in Example 2 to perform RT-qPCR analysis on samples of different tissues and different growth stages of Nipponbare wild-type rice. The specific steps of RT-qPCR were as follows: according to the instructions of TaKaRa kit, a 10 μL reaction system was used: SYBR Premix Ex Taq 5 μL, 0.2 μL of upstream and downstream primers, 50x ROX Reference Dye 0.2 μL, ddH2O 2.4 μL, and 2 μL of diluted cDNA template. The reaction conditions were as follows: 95°C for 5 min, 95°C for 5 s, 60°C for 30 s, 40 cycles; 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. The quantitative results were calculated according to the C T (Threshold Cycle) values of the internal control gene and the gene to be detected using the formula 2 -ΔCt . The results are shown in Figure 2 Figure 3A, which shows that OsKTR2 is expressed in different tissues of rice at different growth stages, indicating that OsKTR2 may be widely involved in the regulation of the expression of downstream genes.

[0064] To explore the response of OsKTR2 to environmental stresses, we performed different environmental stresses on 10-day-old Nipponbare wild-type rice, and took samples of the roots and aboveground parts to analyze the expression pattern of OsKTR2 by RT-qPCR, with samples without any treatment as the control group. The results showed that, in both the roots and the aboveground parts, the transcription level of OsKTR2 was induced by low potassium (0.05 mM K + ) stress by about 4-10 times Figure 3 (A-B), induced by H2O2 (10 mM) by about 3 times Figure 3 (C-D), and induced by ACC (0.1 mM) by about 2 times Figure 3 (E-F).

[0065] Example 4. Subcellular localization analysis of OsKTR2

[0066] According to the CDS sequence of OsKTR2, a double enzyme digestion site containing BamHI and XbaI and a single enzyme digestion site containing BglII were designed, and a homologous recombination primer containing an eGFP empty sequence was also designed. Then, using the total cDNA sequence of rice as a template, two pairs of specific homologous recombination primers (Table 2) were used for PCR amplification reaction to amplify the open reading frame (ORF) of the OsKTR2 gene. Then, the PCR amplification product was subjected to homologous recombination with the linearized vector pCAMBIA1305.1-GFP Figure 7 ) that had been subjected to double enzyme digestion and single enzyme digestion, respectively, to obtain a homologous recombination product. After sequencing, the positive strain was stored in a -70°C refrigerator.

[0067] To verify whether OsKTR2 has the nuclear localization characteristics of a transcription factor, we constructed 2x 35S::OsKTR2::GFP fusion protein and 2x 35S::GFP::OsKTR2 fusion protein recombinant plasmids with 2x 35S cauliflower mosaic virus (CaMV) as the promoter. At the same time, we used 2x 35S::GFP fusion protein with 2x 35S cauliflower mosaic virus (CaMV) as the promoter as a positive control. Then, the three groups of recombinant plasmids were transiently expressed in rice protoplasts, and the subcellular localization of the target protein in rice cells was observed under a laser scanning confocal fluorescence microscope. The results showed that the green fluorescence emitted by both 2x 35S::OsKTR2::GFP and 2x 35S::GFP::OsKTR2 fusion proteins could completely overlap with the blue light emitted by the nuclear dye DAPI Figure 2 B), while the green fluorescence emitted by the positive control 2x 35S::GFP fusion protein filled the entire cell and could not completely overlap with the blue light emitted by the nuclear dye DAPI. This indicates that OsKTR2 is localized in the nucleus and has the characteristics of a transcription factor nuclear localization.

[0068] Table 3 Primer sequences for subcellular localization of OsKTR2

[0069]

[0070] Example 5. Exploring the effect of OsKTR2 expression on potassium absorption and accumulation in rice

[0071] To investigate whether OsKTR2 affects potassium uptake in rice, we grew 10-day-old Nipponbare wild type and OsKTR2 overexpression materials in low potassium (0.05 mM K) and normal potassium (1 mM K) IRRI nutrient solution for 4 weeks. The pH of the nutrient solution was adjusted to 5.1-5.8 during the experiment, and 6 replicates were set for each material. After the treatment, plant samples were collected from the root and shoot parts, weighed, and the potassium content was measured. The results showed that, under both low potassium and normal potassium conditions, the biomass of the OsKTR2 overexpression material was not significantly different from that of the wild type ( Figure 4 A-D), but the potassium content and total potassium content were significantly higher than those of the wild type ( Figure 4 E-H). Under low potassium conditions, the potassium content of the root and shoot of the OsKTR2 overexpression material was about 23-33% higher than that of the wild type ( Figure 4 E); the total potassium content was about 14-18% higher than that of the wild type ( Figure 4 G). Under normal potassium conditions, the potassium content of the root of the OsKTR2 overexpression material was about 18% higher than that of the wild type, and there was no significant difference in the shoot ( Figure 4 F); the total potassium content was about 23-30% higher than that of the wild type ( Figure 4 H). The above results show that the expression of OsKTR2 is induced by low potassium stress, which promotes the uptake and accumulation of potassium in rice.

[0072] Example 6. Bucket cultivation and field phenotypes and major agronomic trait indicators of wild type and transgenic rice

[0073] To study the effects of OsKTR2 on the field phenotypes and yield of rice at maturity, we conducted field trials on wild type and transgenic rice materials. The field trials were conducted at the Banliao Test Base of Nanjing Agricultural University. The soil at the test base was acidic yellow-brown soil with a pH of about 5.20, and the soil available potassium concentration was 120 mg / kg (normal K content, and the rest of the nutrients were normal) as determined by the ammonium acetate extraction method. The field trials were planted in small plots, with 7 x 7 replicates for each strain, a total of 49 replicates. During the experiment, water, fertilizer, and pesticide were applied regularly until the rice was fully mature. By comparing the differences in plant height, panicle length, total tiller, effective tiller, grain number per panicle, seed setting rate, thousand-grain weight, and yield per plant of the wild type and transgenic rice materials, we found that the OsKTR2 overexpression material had a higher yield per plant than the wild type ( Figure 5As shown in Table 3-4, we found that overexpression of OsKTR2 promoter (Pro) increased rice yield (21-50%) under both normal and low potassium conditions; total tiller number and effective tiller number were significantly increased, with an average of 3-6 effective tillers per plant; seed setting rate was increased by about 25%. In addition, under normal potassium conditions, the number of grains per spike of OsKTR2 promoter (Pro) overexpression lines was increased by 11-17% compared with wild type, while under low potassium supply conditions, there was no significant difference. Plant height, main spike length and 1000-grain weight had no significant difference compared with wild type, and the yield per plant was significantly increased compared with wild type. Therefore, we found that the tillering (total tiller and effective tiller) ability of OsKTR2 promoter (Pro) overexpression material was stronger than that of wild type under both normal and low potassium conditions, thereby achieving the effect of increasing rice yield.

[0074] Table 3 Statistics of agronomic traits of wild type and transgenic rice field test (normal soil)

[0075] Genotype WT (NB) KTR2PRO1 KTR2PRO2 KTR2PRO3 Plant Height (cm) 90.0±2.07a 90.0±3.92a 85.7±5.05a 88.2±3.43a Total tiller number per plant 16.8±2.12b 21.2±3.77a 20.8±0.45a 20.2±1.94a Effective tiller number per plant 12.3±1.39b 14.6±1.95a 15.7±2.50a 14.3±1.03a Panicle Length (cm) 21.4±1.23a 22.5±1.49a 20.1±0.91a 21.9±1.27a Grain number per panicle 101.5±5.90b 118.8±8.14a 117.8±5.40a 112.5±7.97b 1000-grain weight (g) 23.4±0.50a 23.0±1.26a 23.0±1.99a 24.0±2.31a Seed setting rate (%) 73.2±6.87b 82.8±4.00a 82.5±4.36a 83.4±5.26a Yield (g / plant) 14.7±3.24b 21.4±2.81a 19.1±1.20a 21.4±2.91a

[0076] Table 4 Statistics of agronomic traits of wild type and transgenic rice field test (low potassium soil)

[0077] Genotype WT (NB) KTR2PRO1 KTR2PRO2 KTR2PRO3 Plant Height (cm) 80.1±3.40a 78.0±3.39a 78.3±3.20a 80.5±3.70a Total tiller number per plant 12.9±2.59b 17.8±3.70a 17.7±2.94a 16.8±2.05a Effective tiller number per plant 9.0±1.60b 14.1±1.14a 12.9±3.23a 13.0±1.0a Panicle Length (cm) 20.9±0.92a 21.0±1.38a 20.2±0.80a 20.4±0.88a Grain number per panicle 90.9±5.96a 90.2±9.01a 88.33±9.58a 90.2±9.65a 1000-grain weight (g) 22.92±1.10a 23.75±1.56a 22.40±1.61a 23.0±2.47a Seed setting rate (%) 66.9±5.24b 73.6±3.53a 78.6±6.32a 73.7±3.41a Yield (g / plant) 10.7±1.84b 15.8±3.62a 12.9±1.22a 15.3±2.29a

[0078] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, alternatives and variations can be made to these embodiments without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Rice ethylene response transcription factor having a cDNA sequence as shown in SEQ ID NO. 1 OsKTR2 for use, characterized in that, selected from any one or more of: (1) increasing total tiller number, effective tiller number, and seed setting rate of rice; (2) increasing yield of rice; (3) promoting potassium absorption of rice.

2. Rice ethylene response transcription factor having a cDNA sequence as shown in SEQ ID NO. 1 OsKTR2 Use in the construction of transgenic rice, characterized in that, The transgenic rice has any one or more of the following properties: (1) increasing total tiller number, effective tiller number, and seed setting rate of rice; (2) increasing yield of rice; (3) promoting potassium absorption of rice.

3. Use of a recombinant expression vector comprising the ethylene response transcription factor of claim 1 in rice. OsKTR2 selected from any one or more of: ​ (1) increasing total tiller number, effective tiller number, and seed setting rate of rice; (2) increasing yield of rice; (3) promoting potassium absorption of rice.

4. Use according to claim 3, characterized in that The starting vector is pCAMBIA1305 vector.

5. Use according to claim 4, characterized in that The recombinant expression vector is a rice ethylene response transcription factor OsKTR2 The self promoter replaces the self promoter of the pCAMBIA1305 vector Ubiquitin Promoter.

6. Use according to claim 5, characterized in that The recombinant expression vector is a watermelon ethylene response transcription factor OsKTR2 inserted into the original vector pCAMBIA1305 Sac I and Pst I enzyme sites and inserted into EcoR I and Sac I double enzyme digestion Ubiquitin promoter and inserted into OsKTR2 the gene itself promoter.

7. A method comprising the rice ethylene-responsive transcription factor according to claim 1 OsKTR2 The use of a recombinant expression vector in constructing transgenic rice is characterized in that: The transgenic rice has any one or more of the following properties: (1) increasing total tiller number, effective tiller number, and seed setting rate of rice; (2) increasing yield of rice; (3) promoting potassium absorption of rice.

8. Use according to claim 7, characterized in that The starting vector is pCAMBIA1305 vector.

9. Use according to claim 8, characterized in that The recombinant expression vector is a rice ethylene response transcription factor OsKTR2 The self promoter replaces the self promoter of the pCAMBIA1305 vector Ubiquitin Promoter.

10. Use according to claim 9, characterized in that The recombinant expression vector is a watermelon ethylene response transcription factor OsKTR2 inserted into the pCAMBIA1305 vector Sac I and Pst I enzyme sites and inserted into EcoR I and Sac I double-digested Ubiquitin promoter and inserted into OsKTR2 the gene itself

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