Protein HTT7 capable of simultaneously improving high temperature tolerance of rice at seedling and heading stages, encoding gene and use thereof

Abstract

The present invention discloses a protein HTT7 that improves the high temperature tolerance of rice at both the seedling and heading stages, a coding gene and a use, and belongs to the field of genetic engineering technology. The amino acid sequence of the protein HTT7 is shown in SEQ ID NO.1, and the nucleic acid sequence encoding the protein HTT7 is shown in SEQ ID NO.2. The present invention creates a loss-of-function mutant of the protein through gene editing technology, and can quickly obtain genetic materials with enhanced high temperature tolerance at the seedling and heading stages, which provides new gene resources for the study of the mechanism of high temperature resistance of rice and the cultivation of new high temperature resistant varieties, and has important breeding application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a protein HTT7 capable of improving high temperature tolerance of rice at both seedling and heading stages, a coding gene and uses thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops, and more than half of the world's population relies on rice as their staple food. Rice is a plant that is sensitive to high temperature stress. Under high temperature stress, seedlings show slow growth, yellowing, stunted development, wilting, drying, and eventually death (Xu et al., 2021). High temperatures during the reproductive growth period of rice directly lead to a decrease in rice yield and quality. Studies have shown that for every 1°C increase in global surface temperature, rice yield will decrease by 3.2% (Zhao et al., 2017). Therefore, exploring heat-resistant-related genes, exploring the molecular mechanisms that regulate heat resistance in rice, and cultivating heat-resistant rice varieties will help to meet the challenges brought about by global climate change.

[0003] So far, many high temperature-related QTLs have been identified, but only a few of them have been cloned, including OsTT1 (Li et al., 2015), OsTT2 (Kan et al., 2021), HTH5 (Cao et al., 2022), OsTT3.1 and OsTT3.2 (Zhang et al., 2022), etc. In addition, researchers have also cloned some other high temperature-related genes through different genetic materials, including HTG3 (Wu et al., 2022), SLG1 (Xu et al., 2020), OsEDS1 (Liao et al., 2023), OsbZIP58 (Xu et al., 2020), OsbZIP14 (Qiu et al., 2023), OsCNGC14 and OsCNGC16 (Cui et al., 2020), etc. Although many high temperature-related genes have been reported and cloned, there are few reports of genes that can simultaneously improve the high temperature tolerance of rice at both the seedling and heading stages. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a protein HTT7, a coding gene and uses thereof, which can simultaneously improve the high temperature tolerance of rice at both the seedling and heading stages. By using gene editing technology to create a loss-of-function mutant of the protein, genetic materials with enhanced high temperature tolerance at both the seedling and heading stages can be quickly obtained, which provides new gene resources for the study of the mechanism of high temperature resistance of rice and the breeding of new high temperature resistant varieties, and has important breeding application value.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] A protein HTT7 that improves high temperature tolerance of rice at both seedling and heading stages, the amino acid sequence of which is shown in SEQ ID NO.1;

[0007] Or a protein sequence that has more than 80% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same structure and function.

[0008] A nucleic acid encoding the protein HTT7 according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0009] An expression vector, which can knock out the gene or nucleic acid encoding the above protein.

[0010] A preparation for improving high temperature tolerance of rice at seedling and heading stages, comprising the above expression vector.

[0011] Use of the above protein HTT7, nucleic acid or expression vector in improving high temperature tolerance of rice.

[0012] The use of the above protein HTT7, nucleic acid or expression vector in breeding rice varieties with high temperature tolerance or improving germplasm resources.

[0013] The use of the above protein HTT7, nucleic acid or expression vector in improving rice yield under high temperature.

[0014] The use of the above nucleic acid in screening rice varieties with high temperature tolerance.

[0015] Beneficial effects of the present invention:

[0016] The present invention discovered that OsHTT7 negatively regulates high temperature tolerance of rice at both the seedling and heading stages. By creating loss-of-function mutants through gene editing, genetic materials that simultaneously enhance high temperature tolerance of rice at both the seedling and heading stages can be quickly obtained, providing a theoretical and genetic material basis for breeding new high temperature-resistant rice varieties, and having important breeding utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the detection of the expression of OsHTT7 gene under high temperature induction; (A) Transcriptome sequencing was used to analyze the expression of OsHTT7 in different materials before and after high temperature treatment at the seedling stage (3 biological replicates). The P value was calculated using the t-test, and ** indicates a significant difference at the 0.01 level; (B) Quantitative PCR was used to analyze the expression of OsHTT7 induced by high temperature.

[0018] Figure 2 Schematic diagram of OsHTT7-CRISPR / Cas9 vector;

[0019] Figure 3 Schematic diagram of the OsHTT7 gene knockout target site and knockout plant mutation method;

[0020] Figure 4 The wild-type R498 and OsHTT7 knockout materials were treated with high temperature during the seedling stage; (A) The phenotypes of wild-type R498 and OsHTT7 knockout materials before treatment; (B) The phenotypes of wild-type R498 and OsHTT7 knockout materials after 7 days of recovery from high temperature treatment at 42°C; (C) Comparison of the survival rates of wild-type R498 and OsHTT7 knockout materials after high temperature treatment. The P value was calculated using the t-test, and ** indicates a significant difference at the 0.01 level; (D) Growth conditions before and after high temperature treatment;

[0021] Figure 5 High temperature treatment of wild-type R498 and OsHTT7 knockout materials at the heading stage; (A) fruiting phenotypes of wild-type R498 and OsHTT7 knockout materials at normal and high temperatures; (B) normal and high temperature treatment conditions; (C) comparison of fruiting rate data of wild-type R498 and OsHTT7 knockout materials at normal and high temperatures; t-test was used to calculate P value, ** indicates significant difference at 0.01 level. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0023] Example 1: Identification of the high temperature response gene OsHTT7

[0024] From the 33 fine genome sequencing materials completed by our team (Qin et al., 2021, Cell), 7 representative resources were selected for high temperature treatment at the seedling stage (14-day-old seedlings, sampled after 9 hours of treatment at 45℃), and transcriptome sequencing was performed. By screening the genes induced by high temperature in different materials, a gene that was significantly upregulated by high temperature in different materials was identified. This gene may be involved in high temperature response and was named OsHTT7 ( H igh t emperature t olerance 7)( Figure 1 A).

[0025] To further confirm whether OsHTT7 expression is induced by high temperature, quantitative primers Y840-F (5′-ACCAGGACAACAGGTGAGGA-3′) and Y841-R (5′-AGTTGCTATGATGGTCTCCC-3′) were designed on the adjacent exons of the OsHTT7 gene using the website (https: / / biodb.swu.edu.cn / qprimerdb / download-primers).

[0026] After the wild-type Shuhui 498 (R498) was grown in an incubator for 14 days, it was transferred to a 45°C light box for high temperature treatment. During this period, the aboveground parts were sampled at different time points, namely 0h, 1.5h, 3h, 9h and 12h. Three independent strains were selected at each time point for subsequent experiments. After sampling, the plant RNA extraction kit of FOREGENE was used to extract the total RNA of the three independent strains at different time points according to the operating steps of the instruction manual. The total RNA was reverse transcribed to form cDNA using the reverse transcription kit of Chengdu Fuji Biotechnology Co., Ltd. The quantitative kit of Novozymes was used in qTOWER 3 Quantitative PCR was performed on the GReal-Time PCR thermocycler system, using UBQ5 and FhaB as double internal references. The sequences are as follows:

[0027] UBQ5-F:5'-ACCTTCATGGCCAACCACTT-3'

[0028] UBQ5-R: 5'-CTAAGCCTGCTGGTTGTAGA-3'

[0029] FhaB-F: 5'-GAGAAGCAGAAGCAGAAGGA-3'

[0030] FhaB-R: 5'-CTTGAATAGGACCATCAGGC-3'

[0031] The results are as follows Figure 1 As shown in B, the expression level of OsHTT7 was significantly upregulated 1.5 h after high temperature induction, which was consistent with the transcriptome data, proving that OsHTT7 is involved in the high temperature stress response.

[0032] Example 2: Construction and genetic transformation of OsHTT7 gene editing vector

[0033] 1. Design of OsHTT7 gene editing target site

[0034] In order to investigate whether OsHTT7 is involved in regulating high temperature tolerance in rice, we used the website http: / / skl.scau.edu.cn / ?tdsourcetag=s_pcqq_aiomsgThe targetDesign tool was used to design a knockout target in exon 1 of OsHTT7. The target site sequence is as follows:

[0035] Target: 5'-CACCACTCCGCCGGGCGGTACGG-3'

[0036] 2. CRISPR / Cas9 vector construction

[0037] Primer dimer acquisition: Take 5 μL of 10 μM target site front and rear primers respectively, mix them evenly, and anneal them at 99°C for 5 min to obtain primer dimers. Dilute the product 10 times for later use.

[0038] Intermediate vector connection: Use BsaI (NEB) restriction enzyme system: 0.5μM target adaptor 1μL; 20ng gRNA Plasmid 1μL; 1mM ATP 1μL; 20U T4 (freshly prepared for use) 1μL; 5U BsaI-HF 0.25μL; 10XCutsmart buffer 1μL; ddH2O 4.75μL, and perform restriction enzyme connection at 37℃ to connect the target site sequence with the intermediate vector pYLsgRNA-OsU6a.

[0039] PCR amplification: Primers Y3149-F (5'-CTCCGTTTTACCTGTGGAATCG-3') and Y2635-R (5'-aaacCACCTGTTGTCCTGGTTGAT-3') were used in combination, and Y3150-R (5'-CG GAGGAAAATTCCATCCAC-3') and Y2634-F (5'-gccgATCAACCAGGACAACAG GTG-3') were used in combination. The product obtained in the previous step was amplified using MAX enzyme (Novozymes) according to the instruction manual amplification system to obtain two fragments, one large and one small, which were then fused and recovered using overlapping.

[0040] Enzyme digestion and ligation (37°C): according to the following system: 10X Cutsmart buffer 1.5μL; 1mM ATP NEBT4 DNA ligase buffer 1.5μL; 60-100ng Cas9 plasmid (PuBi-H) 0.5μl (60ng / mL); 100ng sgRNA (gel recovery product) 1μL (based on the amount of 100ng after purification); 10UBsaI-HF 0.5μL; 40U T4 ligase 0.1μL; ddH2O is added to make up to 15μL, and the gel recovery product and the final vector are connected while digesting at 37°C. Figure 2 shown.

[0041] 3. CRISPR / Cas9 vector E. coli transformation

[0042] (1) Conversion

[0043] 10 minutes before the completion of the above connection process, take out a competent E. coli DH5α (Kangti Life) from the -80℃ refrigerator and put it on ice to melt. After the connection is completed, add the connection product to the competent E. coli, blow it to mix it, and place it on ice for 30 minutes. Then, heat shock the product in a 42℃ metal bath for 1 minute, and then place it on ice for 2 minutes. Pipette 1mL of non-antibody LB on the clean bench and add it to the mixed product, place it on a 37℃ shaker, and culture it at 200rpm for 1 hour.

[0044] (2) Coated board

[0045] After the above 1h of culture, take out the bacterial solution and centrifuge it at 5000rpm for 5min at room temperature. Then transfer it to the clean bench for subsequent operations. First prepare the plate with LB+kanamycin and mark it. Then use a 100μL pipette to absorb 30μL of supernatant from the centrifuged bacterial solution, and discard the rest of the supernatant. Then resuspend the bacteria with the 30μL supernatant, evenly spread it on the prepared plate, and after sealing, culture it at 37℃.

[0046] (3) Identification of positive clones

[0047] Take out the overnight culture plate, pick up the monoclonal colony in 5mL LB+kanamycin liquid culture medium on the clean bench, and culture it overnight at 37℃200rpm. The next day, take out the cultured bacterial solution and extract the plasmid using a plasmid extraction kit (Zhuangmeng International Biogene). Then, use the CRISPR / Cas9 vector to construct a dedicated sequencing primer Y3160-R (5'-GCGGTGTCATCTATGTTACTA-3') and sequence it at Sangon Biotech Co., Ltd. to confirm it. The positive plasmid and bacterial solution with correct sequencing are saved for future use.

[0048] 4. CRISPR / Cas9 vector Agrobacterium transformation

[0049] Take out the competent Agrobacterium EHA105 (Qingke Bio) from -80℃ and put it on ice to melt, add 3μL of the above positive clone plasmid, mix well and let it stand on ice for 5min, then freeze it in liquid nitrogen for 5min. Then put it in a 37℃ metal bath for 5min, and then put it on ice for 5min. Then add 1mL of antibiotic-free LB to the clean bench, and then culture it in a shaker at 200rpm and 28℃ for 2-3h. Then take out the bacterial solution and centrifuge it at 7000rpm for 5min, and the subsequent plating (LB+Rifampicin+Kanamycin) steps are as described above. Then pick a single clone for bacterial solution PCR detection, and save the bacterial solution of the detected positive clone for later use.

[0050] 5. Genetic transformation of rice using CRISPR / Cas9 vector

[0051] (1) Induced healing

[0052] Take about 500 seeds of indica rice Shuhui 498 (R498), remove the seed shells and set aside. Then treat with 70% alcohol for 1 minute, disinfect with 50% sodium hypochlorite for 30 minutes, then wash repeatedly with sterilized ultrapure water 10-15 times and dry with filter paper for later use. Then, evenly spread the treated seeds on NMB medium.

[0053] (2) Agrobacterium infection

[0054] The selected positive clones were expanded to 50 mL, centrifuged at 5000 rpm to collect the cells, and then resuspended in AAM liquid medium (with acetosyringone). The good callus was immersed in the AAM medium containing the cells in a sterile environment for about 30 minutes, and then the medium was removed with sterile filter paper, and the callus was placed back on the culture rack for 2 days of incubation.

[0055] (3) Screening

[0056] The cultured callus tissue is washed with sterile water and sterile water with cephalosporin respectively and then placed back on the culture rack for further culture for about three weeks. The callus tissue is then induced to take root using differentiation medium until the seedlings are about 10 cm tall. They can then be hardened indoors and can be used for transplanting and testing.

[0057] Example 3: Phenotypic analysis of OsHTT7 knockout strains treated with high temperature at seedling stage

[0058] 1. Knockout transgenic seedling detection

[0059] After obtaining the transgenic seedlings, the hygromycin detection primers Y214-F (5'-TACACAGCCATCGGT CCAGA-3') and Y215-R (5'-TAGGAGGGCGTGGATATGTC-3') were used to detect the transformation of the vector. Then, the cross-target site amplification primers Y3833-F (5'-CACAAAAGCCAGCAAAAAAA-3') and Y3834-R (5'-CGAGCATAACAAGCGAAGAT-3') were used to amplify the transformed positive strains and sent them to Sangon Biotech for sequencing. The results are as follows Figure 3 As shown, two knockout transgenic plants, OsHTT7-KO1 and OsHTT7-KO2, were obtained, each with a base insertion in the OsHTT7 coding region, both of which resulted in premature termination of OsHTT7 protein translation.

[0060] 2. High temperature treatment at seedling stage

[0061] The seedlings of wild-type R498, knockout mutants OsHTT7-KO1 and OsHTT7-KO2 were hydroponically cultivated in the laboratory. After 14 days of growth, they were placed in a 42°C light incubator for 72 hours of high temperature treatment, and then placed in a 28°C light incubator to recover growth for 7 days, and the survival of each material was counted. The results showed that the survival rate of knockout mutants OsHTT7-KO1 and OsHTT7-KO2 after high temperature treatment in the seedling stage was significantly higher than that of wild-type R498, indicating that the loss of OsHTT7 function can enhance the high temperature tolerance of rice seedlings ( Figure 4 ).

[0062] Example 4: Phenotypic analysis of OsHTT7 knockout lines treated with high temperature at the heading stage

[0063] To further investigate whether OsHTT7 is involved in regulating high temperature tolerance at the heading stage of rice, the ears of R498, knockout mutants OsHTT7-KO1 and OsHTT7-KO2 that were marked in the field and flowering at the same time were moved back to the artificial climate chamber and heated to 40 ℃ for 24 h. Figure 5 The conditions shown in B were cycled for 3 days. Afterwards, the plants were moved back to the field and grown to maturity, and the fruit setting rate was counted. Compared with the plants grown in the normal artificial climate chamber, the fruit setting rate of wild-type R498 decreased by 76.73% after high temperature treatment, and the fruit setting rate of knockout mutants OsHTT7-KO1 and OsHTT7-KO2 decreased less significantly after high temperature treatment, only 62.04% and 61.57% respectively ( Figure 5 C), indicating that the loss of OsHTT7 function can enhance the high temperature tolerance of rice at the heading stage, which is of great significance for the stable yield of rice under high temperature.

[0064] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. The use of knocking out the gene encoding protein HTT7 in improving the high temperature tolerance of rice at both the seedling and heading stages, wherein the amino acid sequence of the protein HTT7 is shown in SEQ ID NO.1; the nucleotide sequence encoding the protein HTT7 is shown in SEQ ID NO.

2.

2. Use of knocking out the gene encoding the protein HTT7 as claimed in claim 1 in improving the high temperature tolerance of rice.

3. Use of knocking out the gene encoding the protein HTT7 in claim 1 in breeding rice varieties with high temperature tolerance or improving germplasm resources.

4. Use of knocking out the gene encoding the protein HTT7 in claim 1 to increase rice yield under high temperature.

5. Use of knocking out the gene encoding the protein HTT7 in claim 1 in screening rice varieties with high temperature tolerance.

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