Application of HT6 gene and encoded protein thereof in improving yield of rice under high temperature stress

Abstract

The invention discloses an application of an HT6 gene and an encoding protein thereof in improving the yield of rice under high temperature stress, and belongs to the technical field of gene engineering. According to the invention, a gene of which the expression quantity is up-regulated under high-temperature induction is found, and the gene is named as HT6 and encodes casein kinase. Through construction of a knockout strain and an overexpression strain of HT6, it is found that the maturing rate of the HT6 knockout strain of the knockout HT6 gene under high temperature stress is remarkably reduced compared with that of a wild type R527; and the fruiting rate of an HT6 overexpression strain of the overexpression HT6 gene under high temperature stress is obviously improved compared with that of a wild type R498. Therefore, overexpression of the HT6 can improve the maturing rate of the rice encountering high temperature in the heading stage, so that the yield of the rice at high temperature is improved, and the HT6 has breeding utilization value. Therefore, new germplasm and genetic resources are provided for cultivation of high-temperature-resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and specifically to a HT6 Application of genes and their encoded proteins in improving rice yield under high temperature stress. Background Art

[0002] Rice, my country's most important food crop, faces severe challenges in its yield stability due to global warming. Research shows that every 1°C increase in temperature leads to a 3.2% decrease in rice yield (Li et al., 2018), and frequent extreme high temperatures could further exacerbate this decline (Heino et al., 2023). Rice is particularly sensitive to high temperatures during its reproductive development. Exposure to high temperatures during the heading and flowering stages, when high temperatures frequently occur in summer, can lead to poor seed set, directly impacting final yield.

[0003] In recent years, researchers have cloned and analyzed multiple genes that respond to high temperatures and regulate rice growth and development, including the one discovered by Academician Lin Hongxuan's team. TT1 (Li et al., 2015), TT2 (Kan et al., 2022) and TT3 (Zhang et al., 2022) series of genes, among which TT1 Regulating high temperature response through the ubiquitin-proteasome pathway, TT3 The TT3.1 / TT3.2 gene pair in the locus alleviates chloroplast heat damage through antagonism. SLG1 (Xu et al., 2020), SRL10 (Wang et al., 2023) and NRT2.3 Genes such as (Zhang et al., 2022) have also been shown to influence rice heat tolerance through pathways such as flavonoid biosynthesis, ROS scavenging, and nitrogen uptake. In general, existing studies have focused on genes involved in heat tolerance during the seedling stage. However, research reports on the heading and flowering stages, which have the greatest impact on yield (and are also the most sensitive to high temperatures), are quite limited. Breeding high-yielding rice varieties that are tolerant to heat stress is an important approach to addressing these challenges. Therefore, continued research is needed to identify proteins and genes associated with heat tolerance in rice. Summary of the Invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a HT6 The application of genes and their encoded proteins in improving the yield of rice under high temperature stress, so as to improve the yield of rice under high temperature stress.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: provide a HT6Application of genes and their encoded proteins in improving rice yield under high temperature stress.

[0006] Furthermore, rice HT6 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] further, HT6 The gene can improve the fruit set rate of rice under high temperature stress at the heading stage.

[0008] The present invention provides a method for cultivating high-yield rice under high temperature, comprising introducing a gene into the genome of rice. HT6 gene and overexpressed it in rice.

[0009] Furthermore, the introduction method is selected from a genetic transformation method, a genome editing method or a gene mutation method.

[0010] The present invention provides a HT6 Application of genes in breeding high-yield rice varieties under high temperature stress.

[0011] The present invention provides a HT6 Application of genes in screening / identifying rice varieties with high yield characteristics under high temperature stress.

[0012] The present invention provides a HT6 Application of genes in improving high-yield rice germplasm resources.

[0013] The present invention also provides a preparation for increasing rice yield, characterized in that the preparation comprises the HT6 gene or its encoded protein.

[0014] The present invention has the following beneficial effects: Through transcriptome analysis, the present invention found that a gene expression level was upregulated under high temperature induction, and it was speculated that it might respond to high temperature, and was named HT6 ( Heat tolerance 6 ), which encodes a casein kinase. HT6 The knockout and overexpression lines found that HT6 The fruit setting rate of the knockout strain under high temperature stress was significantly lower than that of the wild type R527; HT6 The fruiting rate of the overexpression strain under high temperature stress was significantly higher than that of the wild type R498. HT6 The invention can improve the seed setting rate of rice after it encounters high temperatures during the heading stage, thereby increasing the yield of rice under high temperatures, and has breeding value. Therefore, the invention provides new germplasm and genetic resources for breeding high-temperature resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for HT6 The expression of genes induced by high temperature; Figure A is the transcriptome analysis of HT6 expression induced by high temperature in different materials; Figure B is HT6 Expression of indica rice R498 after high temperature treatment; Figure C is HT6 expression patterns; Figure 2 for HT6 Figure 1 shows the reduction of rice seed setting rate after high temperature at the panicle stage after knockout; Figure A is a schematic diagram of the mutation sites of the two knockout lines; Figure B is a schematic diagram of the wild type R498 and HT6 Phenotypes of knockout mutants treated with high temperature / untreated at the ear stage; Figure C shows the statistical results of single ear seed setting rate; Figure 3 For overexpression [[ID=XXX]]HT6 It can significantly improve the seed setting rate of rice after encountering high temperature at the panicle stage; Figure A is the expression efficiency test of two overexpression lines; Figure B is the expression efficiency test of wild type R498 and [[ID=XXX]]HT6 Phenotypes of overexpressing transgenic materials at the ear stage after high temperature treatment / untreated; Figure C shows the statistical results of single ear seed setting rate. DETAILED DESCRIPTION

[0016] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0017] Example 1 [[ID=XXX]]HT6 Gene expression is upregulated by high temperature High temperatures during the rice panicle stage reduce seed set, directly impacting yield. To identify key genes regulating seed set under high temperatures during the panicle stage, different rice varieties were subjected to high-temperature treatment at the panicle stage and transcriptome sequencing was performed to identify new genes that respond to high temperatures. The treatment conditions were as follows: ① Rice seedlings of uniform growth and at the eighth stage of panicle development were selected from the field and transplanted into plastic buckets; ② After three days of cultivation in the field, the seedlings were subjected to high-temperature treatment at the panicle stage in an artificial greenhouse (42-32°C temperature gradient, 60% humidity). Two hours after treatment, the panicles were sampled and sent to our company for transcriptome sequencing.

[0018] In the transcriptome data, it was found that the expression of a gene was upregulated by high temperature in multiple materials (see [[ID=XXX]]Figure 1 A), it is speculated that it may respond to high temperature and is named [[ID=XXX]]HT6 ( [[ID=XXX]]Heat tolerance 6), which encodes a casein kinase; its nucleic acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. [[ID=XXX]]HT6 Whether it responds to high temperature, quantitative PCR was used to analyze the different high temperature treatment times. [[ID=XXX]]HT6 First, the website (https: / / qprimerdb.biodb.org / ) was used to design [[ID=XXX]]HT6 The quantitative PCR primers for the gene were BC1871-F and BC1872-R. Wild-type R498 was treated with high temperature treatment according to the above steps at the heading stage. Rice panicles treated with high temperature for 0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h were used for subsequent experiments. Total RNA was extracted using the plant RNA extraction kit of FOREGENE and reverse transcribed into cDNA using the reverse transcription kit of Chengdu Fuji Biotechnology Co., Ltd. Quantitative PCR analysis was then performed using the kit of Novozymes. [[ID=XXX]]UBQ5 and [[ID=XXX]]FhaB As a double internal reference, the primer sequences are as follows: BC1871-F: 5'-TGGAGGGGGAGTACAATGTC-3' (SEQ ID NO. 3); BC1872-R: 5'-CGGCCTAAACCCATAAGGA-3' (SEQ ID NO. 4); UBQ5-F: 5'-ACCTTCATGGCCAACCACTT-3' (SEQ ID NO.5); UBQ5-R: 5'-CTAAGCCTGCTGGTTGTAGA-3' (SEQ ID NO. 6); FhaB-F: 5'-GAGAAGCAGAAGCAGAAGGA-3' (SEQ ID NO. 7); FhaB-R: 5'-CTTGAATAGGACCATCAGGC-3' (SEQ ID NO. 8).

[0019] Quantitative PCR results showed [[ID=XXX]]HT6 The expression level is upregulated by high temperature (see [[ID=XXX]]Figure 1 B). Then [[ID=XXX]]HT6 The expression pattern of [[ID=XXX]]HT6 Highly expressed in young panicles, it is speculated that it may regulate the seed setting rate of rice at the panicle stage under high temperature (see [[ID=XXX]]Figure 1 C).

[0020] Example 2 [[ID=XXX]]HT6 Knockout reduces the seed setting rate of rice after high temperature during the panicle stage To verify [[ID=XXX]]HT6 Is it directly involved in regulating high temperature tolerance in rice? [[ID=XXX]]HT6 To knock out a mutant, the specific steps are as follows: (1) Design of gene editing target sites: [[ID=XXX]]HT6 The CRISPR / Cas9 gene editing vector first uses the targetDesign function to [[ID=XXX]]HT6 The target editing site was selected on the first exon of the gene, and the target site sequence was: 5'-CTGGAATTCCTCATCTGAAG-3' (SEQ ID NO.9) ( [[ID=XXX]]http: / / skl.scau.edu.cn / ?tdsourcetag=s_ [[ID=XXX]]pcqq_aiomsg ).

[0021] (2) CRISPR / Cas9 vector construction: After the editing target site is determined, CRISPR / Cas9 vector construction is performed. The specific steps are as follows: ① Mix 5 μL of 10 μM target primers and 5 μL of primers, anneal at 99°C for 5 min using a PCR instrument, and then dilute the product 10-fold for later use.

[0022] ② Use the following system: 1 μL of 0.5 μM target adaptor (i.e., primer dimer), 20 ng of gRNA, 1 μL of Plasmid, 1 μL of 1 mM ATP; 1 μL of 20 U T4 (prepared immediately for use), 0.25 μL of 5 U BsaI-HF, 1 μL of 10X Cutsmart buffer; 4.75 μL of double-distilled water; mix the dimer and Bas1 (NEB) enzyme, incubate at 37°C for 5 min, then at 20°C for 2 min for 5 cycles; ligate the target sequence to the intermediate vector pYLsgRNA-OsU6a, and dilute the product 10-fold for later use.

[0023] ③ Combine the amplification primer Y3149-F with the BC983-R target site rear primer, and the Y3150-R with the BC982-F target site front primer. After PCR amplification, two fragments, one large and one small, will be obtained. The two fragments will then be fused and recovered by overlapping. The primer sequences are as follows: Y3149-F: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO. 10); BC983-R: 5'-aaacCGAGGAACGTACCAAGGTAT-3' (SEQ ID NO. 11); Y3150-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO. 12); BC982-F: 5'-gccgATACCTTGGTACGTTCCTCG-3' (SEQ ID NO. 13).

[0024] ④ Enzyme digestion and ligation (37°C): Combine the products obtained above using the following mixture: 1.5 μL of Cutsmart buffer (NEB), 1.5 μL of ATP T4 DNA ligase buffer (NEB), 0.5 μL of Cas9 plasmid (PuBi-H), 1 μL of the amplified product from step ③, 0.5 μL of BsaI, 0.1 μL of T4 ligase (NEB), and 9.9 μL of ultrapure water. Incubate the mixture at 37°C for 3 hours while digesting and ligating to complete the final vector ligation.

[0025] (3) E. coli transformation: ① Transformation: Remove competent E. coli DH5α (Kangti Life) from a -80°C freezer and thaw on ice for 5 minutes. Add the final ligated vector to 100 μL of the competent DH5α, mix thoroughly, and incubate on ice for 30 minutes. Heat shock the cells in a 42°C metal bath for 75 seconds, then incubate on ice for another 2 minutes. Transfer the cells to a laminar flow hood, add 1 mL of antibiotic-free LB, and incubate at 37°C on a shaker for 1 hour.

[0026] ② Plate Spreading: Centrifuge the culture at 5000 rpm for 5 minutes. Pipette 30 μL of the supernatant and resuspend the cells in a clean bench (discard the remaining supernatant). Then, spread the culture evenly onto LB culture plates (kanamycin-resistant), seal the plates, and incubate overnight at 37°C in an oven.

[0027] ③ Positive clone selection: Remove the culture plate from the culture and place a single colony on a clean bench. Expand the culture in 4 mL of LB + kanamycin resistance in a 37°C shaker. Then, use a plasmid extraction kit (Zhuangmeng Biotechnology) to extract the plasmid. The extracted plasmid is sent to Sangon Biotechnology for sequencing confirmation using universal primers AN763-F and AN764-R. The positive clone solution and plasmid that have been sequenced correctly are retained for future use. The primer sequences are as follows: AN763-F: 5'-GCGGTGTCATCTATGTTACTAG-3' (SEQ ID NO. 14); AN764-R: 5'-CCCGACATAGATGCAATAACTTCG-3' (SEQ ID NO. 15).

[0028] (4) Agrobacterium transformation: Take out the competent Agrobacterium EHA105 (Qingke Biotechnology) from the -80℃ freezer and melt it on ice. Add 2 μL of the positive clone plasmid, mix well and let it stand on ice for 5 min. Then, freeze it instantly in liquid nitrogen for 5 min, place it in a 37℃ water bath for 5 min, and then let it stand on ice for 5 min. Then, add 1 mL of antibiotic-free LB to the product, culture it on a shaker at 28℃ for 2-3 h, centrifuge it and plate it (kanamycin + rifampicin dual antibodies). The plate coating steps are the same as above.

[0029] (5) Rice genetic transformation: ① Callus induction: Approximately 500 seeds of the indica rice variety Shuhui 527 (R527) were collected and hulled for later use. Callus induction was then performed using the following steps: disinfection with 70% alcohol for 1 minute and 50% sodium hypochlorite for 30 minutes, respectively. The seeds were then rinsed 15 times with sterile ultrapure water. After rinsing, the surface moisture of the seeds was completely absorbed with sterile filter paper. Seeds in good condition were then cultured on NMB medium.

[0030] ② Agrobacterium infection: First, expand the transformed Agrobacterium-positive clones to 50 mL in a shaker at 28°C. Collect the cells after centrifugation at room temperature. Resuspend the cells in AAM liquid medium supplemented with acetosyringone and set aside. Select well-growing callus and incubate it in AAM medium in a sterile environment for 30 minutes. Blot the callus medium with sterile filter paper and return the callus to the culture room for at least 2 days.

[0031] ③ Screening: After culturing for 2 days, the callus tissue is washed with sterile water and sterile water with cephalosporin respectively. After washing, the culture medium is returned to the culture room for more than three weeks, and then transferred to the differentiation medium for rooting. The culture is continued until the plants can be used for seedling hardening and transplanting.

[0032] (6) Transgenic seedling detection: After obtaining transgenic seedlings, the transgenic plants were first amplified using the hygromycin detection primers BC2499-F and BC2500-R to detect the vector transformation status. Then, the strains with successful vector transformation were selected and amplified and sequenced using the cross-target site amplification primers BC1461-F and BC1462-R. The primer sequences are as follows: BC2499-F: 5'-CAAAGCAAGTGGATTGATGTGATA-3' (SEQ ID NO. 16); BC2500-R: 5'-GAAGAAGATGTTGGCGACCTCGTA-3' (SEQ ID NO. 17); BC1461-F: 5'-GCACTGATTACTTGTCCT-3' (SEQ ID NO. 18); BC1462-R: 5'-CATCAACCACACAGAGCA-3' (SEQ ID NO. 19).

[0033] A total of two homozygous strains with different mutation patterns were obtained and named [[ID=XXX]]HT6-KO1 and [[ID=XXX]]HT6-KO2 .in [[ID=XXX]]HT6- [[ID=XXX]]KO1 Inserting an A base into the target site [[ID=XXX]]HT6-KO2 Inserting a C base at the target site, both mutations lead to premature termination of protein translation ( [[ID=XXX]]Figure 2 A).

[0034] (7) For two [[ID=XXX]]HT6 The knockout lines were subjected to high-temperature treatment at the heading stage, and their seed set rate was measured under high temperature. The treatment conditions were as follows: rice seedlings of uniform growth and at the eighth stage of panicle development were selected from the field and transplanted into plastic buckets. After incubation in the field for three days, panicles that had flowered before treatment and those awaiting treatment were marked. The high-temperature treatment was then applied for three days in an artificial greenhouse (42-32°C temperature gradient, 60% humidity) at the heading stage. The plants were then transplanted to the field, and after grain maturity, the marked panicles were classified and counted.

[0035] [[ID=XXX]]HT6 The results of high temperature treatment of knockout mutants showed that wild type R498 and [[ID=XXX]]HT6 The fruit setting rate of the knockout strains after high temperature treatment was significantly lower than that of the plants without high temperature treatment. [[ID=XXX]]HT6 The knockout lines showed an increased decrease in seed setting rate compared to the wild type R527; R527 decreased by 18%, and the two [[ID=XXX]]HT6 The seed setting rates of the knockout lines decreased by 39% and 38%, respectively (see [[ID=XXX]]Figure 2 BC). The above results show that [[ID=XXX]]HT6 It is necessary for rice to withstand high temperatures. [[ID=XXX]]HT6 After the function is lost, rice's tolerance to high temperature at the heading stage is reduced.

[0036] Example 3 Overexpression [[ID=XXX]]HT6 Can significantly increase the seed setting rate of rice after encountering high temperature during the panicle stage To determine [[ID=XXX]]HT6 Whether it has breeding value, the indica rice Shuhui 498 (R498) background was constructed. [[ID=XXX]]HT6 Overexpression of transgenic materials ( [[ID=XXX]]HT6 - [[ID=XXX]]OE ), the specific steps are as follows: (1) Overexpression vector construction: Using R498 cDNA as a template, amplify with primers BC1723-F and BC1724-R [[ID=XXX]]HT6 The CDS sequence (excluding the stop codon) of the pCAMBIA2300 expression vector was obtained by ligating the amplified product to the KpnI / BamHI multiple cloning site of the pCAMBIA2300 expression vector using the single-fragment recombination method. The C-terminal fused eGFP was obtained. 35S :: [[ID=XXX]]HT6-eGFP Vector. The primer sequences are as follows: BC1723-F: 5'-ggtacccggggatcctctagaATGGAGCATGTGATCGGGG-3' (SEQ IDNO. 20); BC1724-R: 5'-cttgctcaccatggtactagtTTTCCTTCTGTCAGCACTGATTGA-3' (SEQ ID NO. 21).

[0037] (2) Rice genetic transformation steps Example 2 Knockout strain construction process, according to the quantitative PCR steps above, the overexpression efficiency of the positive strains was detected, and two high expression efficiency strains were obtained (see [[ID=XXX]]Figure 3 A).

[0038] The two overexpression lines were subjected to high temperature treatment at the heading stage to detect the fruit setting rate under high temperature. The treatment conditions were the same as those in Example 2. [[ID=XXX]]HT6 The results of high temperature treatment of overexpression materials showed that wild type R498 and [[ID=XXX]]HT6 The fruit setting rate of plants overexpressing high temperature treatment was significantly lower than that of plants without high temperature treatment. [[ID=XXX]]HT6 Compared with the wild type R527, the overexpression lines showed a lower decline in fruit set rate; R498 decreased by 90%, and the two [[ID=XXX]]HT6 The seed setting rates of the overexpression lines decreased by 70% and 64%, respectively (see [[ID=XXX]]Figure 3 BC). The above results indicate that overexpression [[ID=XXX]]HT6 It can increase the fruit setting rate of rice after encountering high temperature at the heading stage, thereby increasing the yield of rice under high temperature, and has breeding utilization value.

[0039] In the present invention [[ID=XXX]]HT6 The coding region sequence of the gene and the amino acid sequence of its encoded protein are shown below: (1) Coding region sequence: (2) Amino acid sequence: MEHVIGGKFKLGRKIGSGSFGELYLGVNIQSSEEVAIKLESVKSRHPQLHYESKLYMLLQGGTGIPHLKWFGVEGEYNVMVIDLLGPSLEDLFNYCNRKFSLKTVLMLADQMINRV EYMHTRGFLHRDIKPDNFLMGLGRKASQVYVIDYGLAKKYRDLQTHKHIPYRENKNLTGTARYASVNTHLGVEQSRRDDLESLGYVLMYFLRGSLPWQGLKAGTKKQKYDKISEKKM LTPVEVLCKSYPTEFISYFHYCRSLRFEDKPDYSYLKRLFRDLLFIREGYQLDYIFDWTKQGSESNRLRSSGTSGLVGPSAERTERAAARQDVPDRFSGTVDPFARRRTGSGSGHYGE HTKHRNILDSLLAPKTAVDLDKRRPTSSSRNGSTSRKALLSSSRPSSGDPIDPNRSNLIPTSGSSRPSTMQRLHQSTGLETRSSLTKTARNVHDDPTLRTFERLSISADRRK (SEQ ID NO.2).

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A HT6 Application of genes and their encoded proteins in improving rice yield under high temperature stress.

2. The use according to claim 1, characterized in that The rice HT6 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that described HT6 The gene can improve the fruit set rate of rice under high temperature stress at the heading stage.

4. A method for cultivating high-yield rice under high temperature, characterized in that: Including the introduction of HT6 gene and overexpressed it in rice.

5. The method according to claim 4, characterized in that The introduction method is selected from a genetic transformation method, a genome editing method or a gene mutation method.

6. The method according to claim 1 HT6 Application of genes in breeding high-yield rice varieties under high temperature stress.

7. The method according to claim 1 HT6 Application of genes in screening / identifying rice varieties with high yield characteristics under high temperature stress.

8. The method according to claim 1 HT6 Application of genes in improving high-yield rice germplasm resources.

9. A preparation for increasing rice yield, characterized in that: The preparation comprises the HT6 gene or its encoded protein.

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