Application of LOC_Os01g34560 gene and its coded protein in breeding high temperature resistant rice

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of LOC_Os01g34560 gene and coded protein thereof in breeding high-temperature-resistant rice; the gene and the coded protein thereof are involved in high-temperature non-biological stress regulation in the flowering stage of rice, and affect the rice yield by affecting the seed setting rate; LOC_Os01g34560 is edited by using a conventional method, so as to change the expression level of the gene, and then new rice germplasm with different expression levels of LOC_Os01g34560 gene is obtained; the rice created by the application has no significant difference in the growth and development of plants and other agronomic characters compared with the parent control, but significantly affects the seed setting rate under high-temperature non-biological stress; the application provides a useful gene resource for genetic improvement of sterile lines, and has important breeding utilization value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the LOC_Os01g34560 gene and its encoded protein in the cultivation of heat-resistant rice. Background Technology

[0002] High temperatures caused by global warming are one of the major abiotic stresses in rice production, hindering fertilization and reducing seed setting rate during the flowering stage, thus leading to reduced rice yield. In recent years, high temperatures and heat damage have frequently occurred in rice-growing areas, especially in the middle and lower reaches of the Yangtze River. For example, in 2006 and 2007, the Yangtze River basin experienced widespread high temperatures lasting more than 20 consecutive days, resulting in large-scale rice yield reductions, with seed setting rate decreasing by more than 50% in some areas. In 2013 and 2017, the middle and lower reaches of the Yangtze River again experienced high temperatures lasting more than 20 days during the rice booting and heading stages. In 2017 alone, the area affected by high temperatures and heat damage to rice in Anhui Province reached 2.67 × 10⁵ hm². Particularly noteworthy is the more than 40 days of high temperatures that began in late July 2024 in the middle and lower reaches of the Yangtze River, leading to yield reductions of over 100 kg in some areas. Studies have shown that for every 1°C increase in temperature, rice yield decreases by 3.2%. Reports also indicate that by 2030, approximately 16% of global rice production will experience at least five days of above-critical temperatures during its reproductive growth period. Therefore, the development of new heat-resistant rice varieties is urgently needed.

[0003] The heat tolerance trait in rice is an extremely complex quantitative trait, controlled by multiple genes. In recent years, with the rapid development of biotechnology such as molecular markers and genome sequencing, researchers have utilized F2 and F3 genes... 2:3Forty heat tolerance QTLs associated with the heading and flowering stage of rice were identified using BC, RIL, and natural populations. Among them, HTH5, RSF9.2, and QT12 have been cloned and (preliminary) functionally validated. HTH5 belongs to the pyridoxal phosphate-binding protein (PLPBP) family. HTH5 increases the content of heat-induced pyridoxal 5'-phosphate, thereby reducing the accumulation of reactive oxygen species under high temperatures and improving rice's heat tolerance. Overexpression of HTH5 increases the seed setting rate of rice plants under high-temperature stress during the heading stage, while inhibiting the HTH5 gene leads to increased sensitivity to heat stress in rice. One gene positively regulating rice heat tolerance, RSF9.2, was identified using GWAS. Sequence alignment, RT-qPCR analysis, overexpression, and CRISPR knockout confirmed that LOC_Os09g38500 is the target gene of RSF9.2. QT12 acts like a built-in natural "air conditioning system" in crops, regulating the heat tolerance mechanism of rice. When exposed to high temperatures, the primary-secondary "twin lock" formed by the natural variation of QT12 and the NF-Y protein complex locks the high-temperature switching system, balancing the homeostasis of storage protein and starch synthesis, and stabilizing rice quality and yield. This provides a novel molecular mechanism and green breeding strategy for achieving high quality and high yield in rice under high-temperature conditions. However, the research results on heat tolerance traits alone are not enough. It is still necessary to discover and clone some new regulatory genes for heat tolerance traits during rice flowering, and further explore their breeding application potential. This has important theoretical and practical significance for current breeding research to improve rice yield and ensure food security. Summary of the Invention

[0004] To address the problem of high-temperature heat damage encountered during rice production, this invention provides a gene LOC_Os01g34560 that regulates heat tolerance traits during rice flowering, along with its encoded protein and applications, offering a new gene resource for the genetic improvement of heat tolerance traits during rice flowering.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0006] In a first aspect, the present invention provides a gene LOC_Os01g34560 that regulates the heat tolerance trait related to rice flowering period, wherein the nucleotide sequence of the rice flowering period heat tolerance trait gene LOC_Os01g34560 is as shown in SEQ ID NO.1, or the nucleotide sequence of the rice flowering period heat tolerance trait gene LOC_Os01g34560 is at least 90% homologous to the sequence shown in SEQ ID NO.1.

[0007] The amino acid sequence encoded by LOC_Os01g34560 is as shown in SEQ ID NO.2, or the amino acid sequence encoded by LOC_Os01g34560 is at least 90% homologous to the sequence described in SEQ ID NO.2.

[0008] Secondly, this invention provides the application of the gene LOC_Os01g34560, which regulates the heat tolerance related traits of rice flowering period, in improving the heat tolerance of rice under high temperature abiotic stress during flowering period, thereby ensuring stable rice yield.

[0009] The application method is as follows: the high-temperature resistance-related trait gene LOC_Os01g34560 in rice flowering period is edited and knocked out, thereby changing the expression level of the LOC_Os01g34560 gene in the target rice variety, and thus obtaining rice plants with different phenotypes.

[0010] Thirdly, the present invention also provides a gene-specific marker M1 for LOC_Os01g34560, the nucleotide sequence of which is shown in SEQ ID NO.3, and can be used for marker-assisted selection of materials with different heat resistance.

[0011] The present invention has the following beneficial effects:

[0012] This invention reveals that disrupting the biological function of the protein encoded by the LOC_Os01g34560 gene can significantly reduce the relative seed setting rate of rice, indicating that this gene plays an important role in improving heat tolerance during the flowering period of rice. This invention provides useful genetic resources and technical routes for the genetic improvement of heat tolerance-related traits during the flowering period of rice. Attached Figure Description

[0013] Figure 1 This study analyzes the expression pattern of the LOC_Os01g34560 gene in rice in Example 1 of the present invention.

[0014] Figure 2 This is a schematic diagram of the LOC_Os01g34560 gene editing target site and mutation type in Embodiment 2 of the present invention;

[0015] Figure 3 This is an analysis of the relative seed setting rate of rice lines with the LOC_Os01g34560 gene knockout in Example 3 of the present invention; "**" indicates extremely significant differences. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0017] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.

[0018] All primers used are indicated when they first appear, and subsequent use of the same primers will use the same indication as the first indication.

[0019] Unless otherwise specified, the methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0020] Example 1: Expression pattern analysis of the LOC_Os01g34560 gene in rice

[0021] 1. Obtaining the LOC_Os01g34560 gene sequence

[0022] This study focuses on how to solve the problem of high-temperature heat damage encountered in the production of rice. A gene, LOC_Os01g34560, controlling heat tolerance during the flowering period of rice, was identified. Its encoded protein consists of 478 amino acids (SEQ ID NO. 2), and the corresponding gene contains 1948 nucleotides (SEQ ID NO. 1). Both the gene and amino acid sequences are derived from the genome of the rice variety Yuedao 12 (http: / / rice.plantbiology.msu.edu).

[0023] SEQ ID NO.1

[0024]

[0025] SEQ ID NO.2

[0026] MLLLKAVYRVVVNNFLALAAAAVAAVLLRRPDELAARLRAVSSPLHAATAAVLAAGVARLVRARRARGVYLVEYGCFRPRPCYRAPFATCLEHAHLMPYLVDEESVAFAIRLLERSGLG DETCVPDAYHYMPPDRSLRASRDESELVIFSAVDDVFARSALTPADIDVLIVNCSIFTPTPVFADMVVNRYKLRAEVQNVNLSGMGCSAGLVSVGLAKNLLQVSPPGTNVLIVSTEILSS QYYVGTERAMLLPNCLFRMGAAAMILSNSPDHARFRLGRVVRTVTAARDSDYRCVFQEEDEQGNTGIRLSKDLATTAGHALKSNIAAFGPLVLPASEQLLVAISFLKRKLKQLSGHAGK VRLYRPDFRTAFEHFCIHAGGRGVIDEVQHGLGLSDDDVEASRMTLHRRFGNTSSSSVLYELAYLEAKGRMKRGDRVWMISFGAGFDCNSVAWECVKPAPDADGPWVDCIHRYPVQLPEIA

[0027] 2. Validation of LOC_Os01g34560 gene expression pattern

[0028] To verify the expression characteristics of the LOC_Os01g34560 gene, a pair of cross-exon quantitative analysis primers were designed in the exon region of the LOC_Os01g34560 gene using the online software QuantPrime (https: / / quantprime.mpimp-golm.mpg.de / ). The primer sequences are shown in Table 1.

[0029] Table 1 Primer sequence information for Primer l-2

[0030] sequence name sequence Serial Number Primer 5' CGTGTACCTGGTGGAGTA 3' SEQ ID NO.3 Primer 2 5' GCATGTAGTGGTACGCGTC 3' SEQ ID NO.4

[0031] Using SSSL10 as material, different tissues (roots, stems, leaves, leaf sheaths, and young panicles) and young panicles at different developmental stages (stages 4-8) of rice were collected. Total RNA was extracted using a plant rapid RNA extraction kit (Novizan, FastPure Universal PlantTotal RNA Isolation Kit), and first-strand cDNA was synthesized using a reverse transcription kit (Novizan, HiScript IIIRTSuperMix for qPCR). The expression level of LOC_Os01g34560 in different tissues was detected using a quantitative PCR kit (Novizan, ChamQUniversal SYBR qPCR Master Mix) and the aforementioned quantitative PCR primers (SEQ ID NO.3 and SEQ ID NO.4). The detection results are as follows: Figure 1 As shown, this gene is expressed in different tissues, but the expression level is highest in the young spikelet at stage 8, indicating that this gene may have very important biological significance in the development of the young spikelet.

[0032] Example 2: Construction of LOC_Os01g34560 gene knockout vector in rice and transgenic detection

[0033] 1. Design of gene editing sites

[0034] This study is based on existing CRISPR / Cas9 related experimental methods.

[0035] Select a knockout target site on the LOC_Os01g34560 exon (e.g.) Figure 2 As shown, primers Primer 3 and Primer 4 (SEQ ID No. 5 and SEQ ID No. 6) were designed using the online tool targetDesign software (http: / / skl.scau.edu.cn / targetdesign / ) for gene editing vector construction.

[0036] The CRISPR primer sequences are shown in Table 2:

[0037] Table 2 Primer sequence information for Primer 3-4

[0038] sequence name sequence Serial Number Primer 3 5' CCGCGTGGTGGTGAACAACTTCC 3' SEQ ID NO.5 Primer 4 5' AGGACGAGCAGGGCAACACGGGG 3' SEQ ID NO.6

[0039] 2. CRISPR / Cas9 vector construction and genetic transformation methods

[0040] The CRISPR / Csa9 vector system used in this study contains the intermediate vector SK-gRNA and the final vector pC1300-Cas9, whose DNA backbones are derived from the pBlueScript (SK+) vector and the pCAMBLA1300 vector, respectively.

[0041] Specific steps: Primers 3 and 4 were diluted to 100 μM, and 10 μL of each primer were mixed and denatured at 100°C for 5 min, followed by natural cooling to obtain double-stranded sequences containing the knockout target site; 7 μL of annealed primers were mixed with 100 ng of linear intermediate vector SK-gRNA digested with Aar I restriction endonuclease and ligated using T4 DNA ligase; E. coli DH5α competent cells (Nanjing Novizan) were removed from -80°C and thawed on ice. After cell lysis, the ligation product from the previous step was quickly added, gently mixed with a pipette, and incubated on ice for 30 min, followed by heat shock at 42°C for 30 s, and then incubated on ice again for 2 min. Next, 10 volumes of LB antibiotic-free culture medium were added to the transformation product, and the mixture was incubated at 37°C and 200 rpm for 50 min; the activated bacterial culture was then removed and 4000... After centrifugation at rpm, most of the supernatant was removed. The remaining liquid (about 100 μL) after resuscitation was plated (LB + ampicillin resistant) and incubated overnight at 37°C. The next day, a single colony was picked and cultured in 3 mL (LB + ampicillin resistant). Dedicated sequencing primers were constructed using the vector to sequence and verify the target site sequence. Positive clones were selected and plasmids were extracted for later use.

[0042] The SK-gRNA-D560 plasmid was digested with restriction endonucleases Kpn I and Bgl II. The 300 bp fragment was recovered and mixed with the pC1300-Cas9 vector, which had been double-digested with Kpn I and BamHI. The mixture was then ligated using T4 DNA ligase. The mixture was transformed into *E. coli* using the same method as in the previous step, and clones were selected for sequencing. The plasmid with correct sequencing was named pC1300-Cas9-D560. Agrobacterium EHA105 competent cells (Qingke Biotechnology Co., Ltd.) were taken out from -80°C and thawed on ice. 1 μL of the prepared positive clone plasmid was added, and the mixture was gently mixed and placed on ice for 30 min. Then, the cells were frozen in liquid nitrogen for 2 min and quickly removed and placed in a 37°C water bath to lyse the cells for 2 min. Next, 10 volumes of LB antibiotic-free culture medium were added to the transformation product and cultured at 28°C and 250 rpm for 2-3 h. The activated bacterial culture was taken out, centrifuged at 5000 rpm, and most of the supernatant was removed. The bacterial cells were resuspended in the remaining liquid (about 100 μL) and plated (LB + kanamycin). The cells were then incubated at 28°C overnight for 36-48 h. Single colonies were picked and sequenced to obtain a positive strain named Cas9-D560.

[0043] The above-mentioned positive Agrobacterium Cas9-D560 strain was used to transform rice Yuedao 12 callus tissue using the Agrobacterium-mediated transformation of mature rice embryos (Liu Qiaoquan et al., Acta Physiologica Sinica, 1998). Successfully transformed callus cells were obtained through hygromycin resistance screening, and after redifferentiation, positive transgenic rice seedlings were formed. When the seedlings reached approximately 10 cm in height, they were tested and identified before transplanting to obtain T0 generation rice plants.

[0044] Example 3: Phenotypic Analysis of LOC_Os01g34560 Gene Knockout Lines

[0045] 1. Detection of genetically modified seedlings

[0046] Twenty-two seedlings were obtained from Agrobacterium infection and transformation. Positive seedlings were first screened using hygromycin detection primers. Then, sequencing primers primers 5 and 6 (SEQ ID NO. 7 and SEQ ID NO. 8) were designed upstream and downstream of the target site genomic sequence to detect mutations near the target site. After sequence amplification and sequencing analysis based on the target site, two gene mutation types, CR-1 and CR-2 (e.g., ...), were obtained. Figure 2 (As shown).

[0047] The sequencing primer sequences are shown in Table 3:

[0048] Table 3 Primer sequence information for Primer 5-6

[0049] sequence name sequence Serial Number Primer 5 5' GCTACGTACGAACGTACGT 3' SEQ ID NO.7 Primer 6 5' GATGCAGAAGTGCTCGAAC 3' SEQ ID NO.8

[0050] Phenotypic analysis of LOC_Os01g34560 gene knockout lines

[0051] In T0 and T1 generation plantings, agronomic traits of different lines were investigated. It was found that compared to wild-type Yuedao12, the knockout mutant line had a significantly lower relative seed setting rate (relative seed setting rate = the ratio of seed setting rate under high temperature to seed setting rate under normal conditions), while panicle length, grain length, grain width, grain thickness, and thousand-grain weight showed no significant differences. Subsequently, stable mutant lines were obtained in the T2 generation. Three replicates were planted in the field, with two rows planted in each replicate, to further investigate the relative seed setting rate trait of rice. The results showed that compared to wild-type Yuedao 12, the relative seed setting rates of the knockout mutants CR-1 and CR-2 were significantly lower. This indicates that LOC_Os01g34560 is a rice heat tolerance regulatory gene (e.g., ...). Figure 3 (As shown).

[0052] Example 4: Development of LOC_Os01g34560 gene-specific markers

[0053] Based on the deletion of 2 bases in the coding region sequences of LOC_Os01g34560 in Yanhui 559 and Yuedao 12, 1 Indel marker M1 (F: AGAGATCCTGTCGTCGCAGT, as shown in SEQ ID NO: 9; R: CTCAACTGCTTGAGCTTCCG, as shown in SEQ ID NO: 10) was designed. Using this marker M1 to genotype 100 germplasm resources, it was found that the materials showing no bands were of the same type as Yanhui 559, that is, the heat-intolerant type; the materials showing a 357bp band were of the same type as Yuedao 12, that is, the heat-tolerant type. Therefore, M1, as a specific marker for the LOC_Os01g34560 gene, is used for molecular marker-assisted selection of materials with different heat tolerance levels.

[0054] Example 5: Application of Rice LOC_Os01g34560 Gene in the Creation of New Heat-Tolerant Materials and Breeding

[0055] 1. Creation of New Heat-Tolerant Materials

[0056] In winter 2021, at the Lingshui Base of the Rice Research Institute of Anhui Academy of Agricultural Sciences, the backbone restorer lines R900, R17, and 19 Xiang with weak heat tolerance in production were used as female parents, and the germplasm material Yuedao 12 containing the LOC_Os01g34560 gene and strong heat tolerance was used as male parent for hybridization to obtain F1 seeds of the combinations R900 / Yuedao 12, R17 / Yuedao 12, and 19 Xiang / Yuedao 12. In the main season of 2022, the seeds of the 3 F1 combinations and the parents were planted at the Lujiang Base of Anhui Academy of Agricultural Sciences. At the flowering stage, the F1 seeds of the 3 combinations were used as female parents and were backcrossed with the corresponding parents respectively to obtain seeds of R900 / Yuedao 12 / / R900 BC1F1, R17 / Yuedao 12 / / R17 BC1F1, and 19 Xiang / Yuedao 12 / / 19 Xiang BC1F1. In winter 2022, the BC1F1 seeds of the 3 combinations were planted at the Lingshui Base, and molecular marker-assisted selection of the heat-tolerant gene LOC_Os01g34560 was carried out using the M1 marker. Single plants containing this gene and with plant types close to the recurrent parents (R900, R17, and 19 Xiang) were selected for seed retention, and BC1F2 seeds of the 3 combinations were obtained. Repeat this process until stable lines of BC1F5 containing the LOC_Os01g34560 heat-tolerant gene and with plant types close to R900, R17, and 19 Xiang were obtained in the main season of 2024, and were named HR900, HR17, and H19 Xiang respectively. Multiplication of seeds was carried out at Lingshui in winter 2024.

[0057] 2. Identification of Heat Tolerance

[0058] In 2025, the heat resistance of HR900, HR17, and H19 Xiang rice varieties was assessed according to the Anhui Provincial Local Standard DB34 / T 3484-2019 "Technical Regulations for Identification of Heat Resistance of Single-Season Indica Rice Varieties". R900, R17, and H19 Xiang were used as controls. The seed setting rate of the six materials under different treatments is shown in Table 4.

[0059] Table 4 Comparison of the settling rate of 6 materials under different treatments

[0060] As shown in Table 4, the seed setting rates of R900 in greenhouses and fields were 24.7% and 63.6%, respectively, with a relative seed setting rate of 38.8%; the seed setting rates of R17 in greenhouses and fields were 28.9% and 68.5%, respectively, with a relative seed setting rate of 42.2%; and the seed setting rates of H19 Xiang in greenhouses and fields were 39.1% and 74.3%, respectively, with a relative seed setting rate of 52.6%. After introducing the heat-resistant gene LOC_Os01g34560, the seed setting rates of HR900 in greenhouses and fields increased to 67.4% and 84.1%, respectively, with a relative seed setting rate of 80.1%; the seed setting rates of HR17 in greenhouses and fields were 62.4% and 80.3%, respectively, with a relative seed setting rate of 77.7%; and the seed setting rates of H19 Xiang in greenhouses and fields increased to 76.8% and 90.4%, respectively, with a relative seed setting rate of 85.0%. The T-test showed that the improved material had significantly higher seed setting rate and relative seed setting rate than the original material in both field and greenhouse conditions. These results indicate that introducing the heat-resistant gene LOC_Os01g34560 can improve the seed setting rate and enhance the heat resistance of rice.

Claims

1. The application of the LOC_Os01g34560 gene in improving the heat stress tolerance of rice, characterized in that, The nucleotide sequence of the LOC_Os01g34560 gene is shown in SEQ ID NO.

1.

2. The application of the LOC_Os01g34560 protein encoded by the LOC_Os01g34560 gene in improving the heat stress tolerance of rice, characterized in that... The nucleotide sequence of the LOC_Os01g34560 gene is shown in SEQ ID NO.1; the amino acid sequence of the LOC_Os01g34560 protein is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The high-temperature stress mentioned refers to high-temperature stress during the flowering period.

4. A method for breeding rice varieties with improved heat resistance, characterized in that, The LOC_Os01g34560 gene was transferred into rice for expression.

5. A molecular marker for identifying the heat resistance of rice, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3.

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