Application of OsDAD1-2 gene in regulating flowering time of rice in response to temperature

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of an OsDAD1-2 gene in regulating and controlling flowering time of rice in response to temperature. According to the application of the gene OsDAD1-2 in regulation and control of rice flowers through response temperature, a gDNA sequence of the gene OsDAD1-2 is shown as SEQ ID NO.1, a CDS sequence of the gene OsDAD1-2 is shown as SEQ ID NO.2, and it is found that the gene OsDAD1-2 can respond to high temperature, so that flowering in high-temperature weather can be effectively avoided through up-regulation or down-regulation expression of the gene OsDAD1-2, and the yield of rice is increased.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the OsDAD1-2 gene in regulating rice flowering time in response to temperature. Background Technology

[0002] Flowering habits are an important factor in studying the heat tolerance of rice. The peak flowering time of Asian cultivated rice is generally between 10:00 and 12:00 in the morning, while the peak flowering time of African cultivated rice is before 9:00. The earlier opening time of the spikelets under high temperature can avoid the extreme high temperature at noon, thereby improving the rice seed setting rate. Therefore, early flowering is considered to be an advantageous characteristic of rice heat tolerance.

[0003] Rice is most sensitive to high temperatures on the day of flowering, as high temperatures affect the fertility of the florets by influencing the opening time of the spikelets. As the spikelets open, the filaments elongate, the anthers press against the top of the glumes and dehisce, releasing pollen onto the stigma for double fertilization. High temperatures during flowering will hinder anther dehiscence, resulting in insufficient pollen reaching the stigma, reduced pollen viability and germination ability, and consequently, a decrease in rice yield.

[0004] Currently, there are few reports on the temperature response during rice flowering, and the regulatory mechanisms are even less understood. Therefore, identifying genes that respond to temperature during flowering and elucidating their regulatory mechanisms is an important means to improve the high-temperature fertility of rice and is of great significance for the genetic improvement of rice to tolerate high temperatures and early flowering. Summary of the Invention

[0005] This invention overcomes the shortcomings of existing technologies by providing an application of the OsDAD1-2 gene in regulating rice flowering time in response to temperature. It has been discovered that the OsDAD1-2 gene can respond to high temperatures. Introducing temperature-sensitive DAD1-2 into rice or increasing the expression level of DAD1-2 can help rice flowering avoid midday high temperatures. Therefore, by upregulating or downregulating the expression of the OsDAD1-2 gene, flowering can be effectively avoided in hot weather, which is beneficial to increasing rice yield.

[0006] To achieve the above-mentioned objectives, the following technical solution is provided:

[0007] The invention provides an application of the gene OsDAD1-2 in regulating rice flowering time in response to temperature. The gDNA sequence of the OsDAD1-2 gene is shown in SEQ ID NO.1, and the CDS sequence of the OsDAD1-2 gene is shown in SEQ ID NO.2.

[0008] In some implementations, the response temperature is greater than 28°C.

[0009] In some embodiments, the OsDAD1-2 knockout mutant is obtained by knocking out the gene OsDAD1-2 using the following primer pair:

[0010] Forward primer OsDAD1-2-gRT1: 5'-GCGACGTACCCAATGTAGCgttttagagctagaaat-3',

[0011] Reverse primer OsDAD1-2-U6bT1: 5'-GCTACATTGGGTACGTCGCCaacacaagcggcagc-3'.

[0012] A recombinant expression vector for the aforementioned gene OsDAD1-2 is provided, including a promoter, wherein the promoter is the temperature-sensitive promoter Hap4.

[0013] In some embodiments, the carrier is pGreenII0800-mini35spro-LUC.

[0014] A method for increasing rice yield is provided, which regulates the expression of the gene OsDAD1-2 to control the opening time of rice spikelets. By upregulating the expression of the gene OsDAD1-2, the rice flowering time is advanced, so that the opening time of rice spikelets avoids the extreme high temperature at noon. The gDNA sequence of the OsDAD1-2 gene is shown in SEQ ID NO.1, and the CDS sequence of the OsDAD1-2 gene is shown in SEQ ID NO.2.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention provides an application of the OsDAD1-2 gene in regulating rice flowering time in response to temperature. It was discovered that high temperature can induce the expression of this gene. Given the persistent high temperatures during midday, this invention avoids high temperatures by introducing a temperature-sensitive DAD1-2 variant or increasing DAD1-2 expression to induce earlier flowering in rice. Therefore, under high temperature conditions, it promotes earlier opening of rice spikelets, thereby avoiding periods of extreme high temperatures and improving pollination success rates. After knocking out the OsDAD1-2 gene, the mutant showed no significant change in flowering time at normal temperature, but delayed spikelet opening at high temperatures, indicating that this gene plays a key regulatory role in the high-temperature response. Analysis using the RiceVarMap v2.0 database and promoter activity detection experiments revealed that the Hap4 promoter in the OsDAD1-2 promoter region is temperature-sensitive and is a superior haplotype, providing an important gene resource for improving the temperature sensitivity of rice flowering time.

[0017] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 As described in the embodiments of the present invention OsDAD1-2 gDNA agarose gel electrophoresis image.

[0021] Figure 2 As described in the embodiments of the present invention OsDAD1-2 CDS agarose gel electrophoresis image.

[0022] Figure 3 This is an agarose gel electrophoresis image of a single clone of bacteria in an embodiment of the present invention.

[0023] Figure 4 This is an agarose gel electrophoresis image for HPT identification in an embodiment of the present invention.

[0024] Figure 5 As described in the embodiments of the present invention OsDAD1-2 -KO genotype identification agarose gel electrophoresis image.

[0025] Figure 6 As described in the embodiments of the present invention OsDAD1-2 -KO genotyping and mutant phenotype observation and statistics.

[0026] Note: a, carrying CRISPR / Cas9 target OsDAD1-2 - Schematic diagram of the KO gene, with mutation sites indicated in red; b, ZH11 and OsDAD1-2 - Flowering phenotypic comparison of KO mutants, scale bar = 1 cm; c, ZH11 and OsDAD1-2 -Line graph showing the daily flowering dynamics of spikelets in the KO mutant under ambient temperature (28℃) and high temperature (36℃) treatments, n = 5 Figure 7 As described in the embodiments of the present invention OsDAD1-2 Promoter haplotype analysis.

[0027] Note: a, OsDAD1-2 Promoter haplotype analysis; b, statistical distribution frequency of four haplotypes in different rice varieties. Figure 8 As described in the embodiments of the present invention OsDAD1-2 Promoter activity detection.

[0028] Note: The bubbles on the column represent data points from multiple repeated experiments, used to reflect the degree of data dispersion. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] Example 1

[0031] 1. Sequences of the flowering temperature-responsive genes OsDAD1-2 were obtained.

[0032] Based on the results of japonica rice genome sequencing, a pair of specific primers (forward primer PF1: 5'-ATGGCGGCTTCTATCTCCGGAG-3' and reverse primer PR1: 5'-CTAATATGTGCAGGCTGCCACG-3') were designed. Rice gDNA was extracted using the TPS method, and the full-length gDNA sequence of the OsDAD1-2 gene was amplified using the above primers, yielding a full-length sequence of 1,218 bp.

[0033] The gDNA sequence of the OsDAD1-2 gene is shown in SEQ ID NO.1:

[0034]

[0035] (1) Extraction of rice gDNA using TPS method: Take tender leaves about 2 cm long and place them in a centrifuge tube. Add steel beads and freeze quickly with liquid nitrogen. Then grind the sample using a grinder. Add 800 μL of TPS and heat shock at 65℃ for 1 h, inverting and mixing every 15 min. Centrifuge at 12,000 rpm for 10 min. Take 400 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add an equal volume of pre-chilled isopropanol and place in a -20℃ freezer for 1 h. Centrifuge at 12,000 rpm for 10 min. Discard the supernatant, add 600 μL of 70% ethanol, vortex to resuspend the precipitate, centrifuge at 12,000 rpm for 5 min, discard the supernatant and repeat once. Use a pipette to remove excess supernatant, dry at 55℃ for 10 min, add 100 μL of ddH2O to dissolve the precipitate, and store the sample at 4℃.

[0036] (2) PCR amplification system and procedure: Gene sequences were found at the National Rice Data Center (https: / / www.ricedata.cn / gene / ). Primers were designed using Primer5 and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer names and sequences are listed in the appendix. The DNA obtained above was used as a template for PCR amplification. The PCR reaction system used for amplifying the target fragment sequence in this experiment is shown in Table 1, and the PCR amplification procedure is shown in Table 2. The reaction system in Table 1 uses the KOD high-fidelity enzyme kit (KFX-101, TOYOBO).

[0037] Table 1 PCR reaction system

[0038]

[0039] Table 2 PCR amplification program

[0040]

[0041]

[0042] (3) Electrophoresis detection and sequencing: The size of the PCR amplification product bands was observed by 1% agarose gel electrophoresis. PCR amplification products with the correct band size were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Figure 1 ).

[0043] 2. Obtaining the OsDAD1-2 cDNA sequence

[0044] Based on the japonica rice genome sequencing results, a pair of specific primers (forward primer PF2: 5'-ATGGCGGCTTCTATCTCCGGAG-3' and reverse primer PR2: 5'-CTAATATGTGCAGGCTGCCACG-3') were designed to extract total RNA from the leaves of ZH11 seedlings and utilize the technology of Shanghai Yisheng Biotechnology Co., Ltd. The 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) reverse transcription kit (catalog number 11141ES60) was used to synthesize cDNA via reverse transcription. The CDS sequence of the OsDAD1-2 gene was amplified from the cDNA obtained by reverse transcription using the primers PF2 and PR2, with a full length of 1,218 bp.

[0045] The CDS sequence of the OsDAD1-2 gene is shown in SEQ ID NO.2:

[0046]

[0047] (1) RNA extraction using the TRIZOL method: Take 2 cm of young leaf tissue into a 1.5 mL RNase-free centrifuge tube, add several clean small steel balls, freeze quickly in liquid nitrogen, crush the sample with a sampler, add 1 mL of TRIZOL extraction solution, and vortex vigorously to mix. Let stand on ice for 5 min. Add 200 μL of chloroform, gently invert to mix, and let stand on ice for 5 min. Centrifuge at 12,000 rpm for 15 min using a centrifuge pre-cooled to 4 °C. Transfer 400 μL of the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of pre-cooled isopropanol, invert to mix, and let stand at -20 °C for 30 min. Centrifuge at 12,000 rpm for 12 min using a centrifuge pre-cooled to 4 °C, discard the supernatant, add 600 μL of 70% ethanol (prepared with DEPC water) to resuspend the precipitate, and centrifuge at 12,000 rpm for 5 min (wash twice). Remove the supernatant and keep the precipitate. Place the precipitate on a clean bench and blow it dry. Add 50-100 μL of DEPC water to dissolve it and store it at -80℃ for later use.

[0048] (2) Obtaining cDNA: Using the total RNA from (1) as a template, cDNA was obtained from Shanghai Yisheng Biotechnology Co., Ltd. The 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) reverse transcription kit (catalog number 11141ES60) was used to reverse transcribe and synthesize cDNA. The reaction conditions were performed according to the kit instructions.

[0049] (3) Obtaining the target fragment: Using the cDNA in (2) as a template, the PCR reaction system is shown in Table 3. The reaction system uses the KOD high-fidelity enzyme kit (KFX-101, TOYOBO) and the PCR amplification program (Table 4) is used for PCR amplification.

[0050] Table 3 PCR reaction system

[0051]

[0052]

[0053] Table 4 PCR Amplification Program

[0054]

[0055] (4) Electrophoresis detection: The size of the PCR amplification product bands was observed using 1% agarose gel electrophoresis. PCR amplification products with the correct band size were sent to the company for sequencing. Figure 2 )

[0056] 3. Obtaining the OsDAD1-2 knockout mutant

[0057] (1) Target design: First, based on the gDNA sequence of OsDAD1-2, the target sequence was designed, and the pYLgRNA-U6b promoter adapter sequence was added to the target sequence, which became the primers for constructing the expression cassette (OsDAD1-2-gRT1: 5'-GCGACGTACCCAATGTAGCgttttagagctagaaat-3' and the reverse primer OsDAD1-2-U6bT1: 5'-GCTACATTGGGTACGTCGCCaacacaagcggcagc-3'). The primers were synthesized by Sangon Biotech.

[0058] (2) Construction of sgRNA expression cassette: The sgRNA expression cassette was constructed using the overlapping PCR method. The target sequence was inserted downstream of the promoter and upstream of the sgRNA. The PCR was performed in two steps. The PCR reaction system for the first step is shown in Table 5. The reaction program was: 95℃ for 15s, 55℃ for 15s, 72℃ for 10s, for 20-22 PCR cycles (no electrophoresis check is required).

[0059] (3) Colony identification: For the colonies obtained above, several single-clone colonies were selected and PCR identification was performed according to the system prepared in Table 5 and the procedure in Table 6. The Taq enzyme in Table 5 was T5Super PCR Mix (TSINGKE TSE005) provided by Beijing Qingke Biotechnology Co., Ltd. Afterwards, the correct band size was detected by electrophoresis, and single-clone colonies with the correct band size were selected. Figure 3 The bacteria were shaken overnight and sent to Sangon Biotech for sequencing. The sequencing results were analyzed, and single clones with correct sequencing results were selected for plasmid extraction.

[0060] Table 5 PCR reaction system

[0061]

[0062]

[0063] Table 6 PCR Amplification Procedure

[0064]

[0065] (3) Obtaining knockout mutant plants: The plasmid constructed above was sent to Wuhan Aidijing Company for genetic transformation via Agrobacterium-mediated transformation (Saba-Mayoral A, Bassie L, Christou P, Capell T. Development of a facile genetic transformation system for the Spanish eliterice paella genotype Bomba. Transgenic Res. 2022 Jun; 31(3):325-340. doi:10.1007 / s11248-022-00303-z.), and the obtained positive plants were tested.

[0066] Identification of positive plants: Using leaves of transgenic T0 generation rice as gDNA templates, and the hygromycin resistance genes Hyg-F (5'-ACGGTTGTCCATCACAGTTTGCC-3') and Hyg-R (5'-TCCGACCTGATGCAGCTCTCGGAG-3') were used as primers to detect whether transgenic rice contained hygromycin resistance. Figure 4 In addition, a forward primer OsDAD1-2-KO-F (5'-CGTACGCCAGCTTCGACTTC-3') and a reverse primer OsDAD1-2-KO-R (5'-GGTGAAGAGCCTCCAGAATCC-3') were designed upstream and downstream of the target site, respectively, for PCR amplification. The samples were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing and sequence alignment. Figure 5 ).

[0067] (4) Mutant phenotypic observation: When the main panicle of rice begins to develop boots, plants with similar developmental stages and growth vigor are selected and transplanted into plastic buckets, with 4 plants per bucket and 6 pots per variety. Each variety is then moved into an artificial climate chamber for temperature treatment. After the rice adapts to the artificial climate chamber conditions for 2-3 days and flowering stabilizes, the flowering time is investigated using the flower counting method for 3-4 days. After the temperature treatment is completed, the rice is moved outdoors to grow naturally until the grains mature. The experimental conditions are divided into two temperature treatments (28℃ and 36℃), with light hours basically consistent with natural conditions and humidity controlled at 75%. The specific temperature settings are shown in Table 7, with the temperature set at 21℃ and humidity at 75% from 17:00 to 7:00.

[0068] This study used CRISPR / Cas9 technology to knock out the OsDAD1-2 gene in a ZH11 background, screening for two homozygous mutant lines, OsDAD1-2#1 and OsDAD1-2#2. The OsDAD1-2#1 genotype has an insertion of one A base, and the OsDAD1-2#2 genotype has an insertion of one T base. Figure 6 a). ZH11, OsDAD1-2#1, and OsDAD1-2#2 were treated with ambient and high temperatures, respectively, and their flowering phenotypes were examined. At ambient temperature (28℃), there was no significant difference in flowering time between the wild-type ZH11 and the OsDAD1-2 mutants. Figure 6 b left and Figure 6 c left), Figure 6 The ordinate of c represents the number of open flowers. Under a high-temperature treatment of 36℃, wild-type ZH11 reached full bloom at 12:00 noon, while the flowering time of OsDAD1-2#1 and OsDAD1-2#2 was delayed by 0.5 hours. Figure 6 b right and Figure 6 c (right).

[0069] Table 7 Temperature Setting Procedure for Artificial Climate Chamber

[0070]

[0071] Example 2

[0072] 4. OsDAD1-2 Haplotype Analysis

[0073] The promoter sequences and coding regions of Zhonghua 11 and 9311 were amplified, and the PCR products were sent to Sanger sequencing at Sangon Biotech. Sequence alignment using Snapgene software revealed six SNPs in the promoter region, located 121bp, 914bp, 999bp, 1000bp, 1192bp, 1339bp, and 1344bp upstream of the ATG. To identify whether haplotype variation existed in the OsDAD1-2 promoter region, haplotype analysis was performed using the RiceVarMap v2.0 database (https: / / ricevarmap.ncpgr.cn / hap_net / ). This revealed four haplotypes of OsDAD1-2. Figure 7 ).

[0074] 5. Temperature sensitivity of OsDAD1-2 haplotypes

[0075] (1) Vector construction: Promoters of different haplotypes of OsDAD1-2 were amplified by PCR. The reaction system and reaction program were the same as in Table 3 and Table 4, respectively. After the amplification program was completed, the target band was detected by 1% agarose gel. Bands of the expected size were excised and recovered (Magen, D2120). The recovered products were stored at -20℃ for later use. The pGreenII0800-mini35spro-LUC vector was digested with NEB restriction endonuclease (HindIII). The reaction system was the same as in Table 8. The reaction was carried out at 37℃ for 1-2 h. After the digestion reaction, the digested vector was detected by 1% agarose gel and excised. The recovered digested vector was stored at -20℃. The target fragment was ligated with the pGreenII0800-mini35spro-LUC digested vector using the ClonExpress Ultra One Step Cloning Kit (Vazyme, C112-01). The reaction was carried out at 50℃ for 30 min. The ligated product was added to 50 μL. In DH5α competent E. coli cells, gently swirl to mix, incubate on ice for 30 min, heat shock at 42℃ for 1 min, incubate on ice for 2 min, add 500 μL of antibiotic-free LB, and incubate at 37℃ and 200 rpm for 45-60 min; centrifuge at 6,000 rpm for 1 min, remove the supernatant, plate the bacterial culture, and incubate overnight at 37℃; select single clones as templates, and perform PCR amplification using primers 0800-F: TGTAAAACGACGGCCAGT; 0800-R: CTTATGCAGTTGCTCTCCAG. The PCR reaction system is shown in Table 5, and the reaction procedure is shown in Table 6. The PCR products are detected by agarose gel, and single clones with the correct band are sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The plasmid is extracted using the plasmid extraction kit (Tiangen, DP117) for later use.

[0076] Table 8 Enzyme digestion reaction system

[0077]

[0078] (2) Protoplast transformation: Sterilized seeds were planted on 1 / 2 MS medium and cultured in the dark at 30℃ for 7-10 days; the leaf sheaths of the yellow flower seedlings obtained from the above culture were cut into 0.5 mm segments, and the cut leaf sheaths were placed in the enzymatic hydrolysate and cultured at 28℃ and 70 rpm for 4-6 h; the enzymatic hydrolysate was filtered through a 300-400 mesh sieve, and the protoplasts were collected; the protoplasts were resuspended in W5 solution, 200 g for 4 min, and the supernatant was removed; the precipitate was resuspended in MMG solution, and the protoplasts were examined under a hemocytometer; 10 μL of plasmid was added to a 2.0 mL RNase-free centrifuge tube containing 200 μL of protoplasts, and gently mixed, then 220 μL of 40% PEG solution was added, gently mixed, and incubated in the dark for 25 min, then 880 μL of W5 solution was added, gently inverted and mixed, 200 g for 4 min, the supernatant was removed, and 1 mL of the solution was used to extract the precipitate. The protoplasts were resuspended in W5 solution, and the protoplasts of the transformed plasmids were transferred to cell culture plates and incubated overnight at 28°C and 36°C, respectively.

[0079] (3) Promoter activity assay: Protoplasts of the transformed plasmid were placed on cell culture plates and incubated overnight at 28°C and 36°C, respectively. The next day, they were transferred to a fresh 2.0 mL cell culture plate. Centrifuge at 200g for 2 min in RNase-free centrifuge tubes and remove supernatant. Transfer 1 μL of protoplasts to a glass slide and observe their condition using an upright microscope (Nikon, DS-Ri2). Protoplasts in good condition can be used for subsequent dual-luciferase activity assays. Using a dual-luciferase reporter gene assay kit (Yisheng, 11402ES60), first add 50 μL of cell lysis buffer to the protoplasts, vortex for 10 s, and incubate in the dark for 5 min. Centrifuge at 12,000 rpm for 5 min, collect 10 μL of supernatant, add 25 μL of firefly luciferase reaction solution, and detect firefly luciferase activity using GloMax 20 / 20 luminescence detection solution (Promega). Then add 25 μL of Renida luciferase reaction solution to detect Renida luciferase activity.

[0080] In this study, promoters of different haplotypes were ligated into the pGreenII0800-mini35spro-LUC vector and transiently transformed into rice protoplasts. After incubation at 28℃ and 36℃, the enzyme activity of the dual-luciferase LUC / REN was measured to determine the activity of different haplotypes. The results showed that the promoter activities of Hap1, Hap2, and Hap3 were lower than those of Hap4. Figure 8 This indicates that Hap4 type promoters may be more sensitive to temperature.

[0081] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. The application of the gene OsDAD1-2 in regulating rice flowering time in response to temperature, wherein the gDNA sequence of the OsDAD1-2 gene is shown in SEQ ID NO.1 and the CDS sequence of the OsDAD1-2 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The response temperature is greater than 28°C.

3. The application according to claim 1, characterized in that, The OsDAD1-2 knockout mutant was obtained by knocking out the OsDAD1-2 gene using the following primer pair: Forward primer sDAD1-2-gRT1: 5'-GCGACGTACCCAATGTAGCgttttagagctagaaat-3', Reverse primer OsDAD1-2-U6bT1: 5'-GCTACATTGGGTACGTCGCCaacacaagcggcagc-3'.

4. The recombinant expression vector of gene OsDAD1-2 according to any one of claims 1 to 3, characterized in that, It includes a promoter, wherein the promoter is the temperature-sensitive promoter Hap4.

5. The recombinant expression vector for gene OsDAD1-2 according to claim 4, characterized in that, The carrier is pGreenII0800-mini35pro-LUC.

6. A method for increasing rice yield, characterized in that, By regulating the expression of the gene OsDAD1-2 to control the opening time of rice spikelets, the rice flowering time is advanced by upregulating the expression of the gene OsDAD1-2, thus avoiding the extreme high temperature at noon. The gDNA sequence of the OsDAD1-2 gene is shown in SEQ ID NO.1, and the CDS sequence of the OsDAD1-2 gene is shown in SEQ ID NO.2.

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