Rice heat shock transcription factor OsHSFC1a and application of coding gene of rice heat shock transcription factor OsHSFC1a in rice disease-resistant breeding

Abstract

The invention discloses a rice heat shock transcription factor OsHSFC1a as well as an encoded protein and application thereof, and the gene OsHSFC1a provided by the invention is a DNA molecule as shown in the following 1), 2), 3) or 4): 1) a DNA molecule as shown in SEQ ID NO.1; 2) a DNA molecule as shown in SEQ ID NO. 2; 3) a DNA molecule which is hybridized with the DNA sequence limited by 1) or 2) under strict conditions and is used for coding the protein; and 4) a DNA molecule which has more than 90% of homology with the DNA sequence limited by 1) or 2) or 3) and is used for coding the plant heat shock transcription factor. The invention also provides a protein coded by the gene, and the protein affects the disease resistance of plants. The coding gene of the protein is knocked out from a plant with normal disease resistance, and a transgenic plant with enhanced disease resistance can be cultivated. The protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a rice disease resistance gene. OsHSFC1a Its encoded proteins and applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is the staple food for over 50% of the world's population and a crucial food crop in my country, making rice production vital to the national economy. Over-reliance on chemical pesticides not only increases costs and environmental pressures but also poses the challenge of disease mutations leading to resistance loss. Therefore, creating new varieties with broad-spectrum and long-lasting disease resistance is an essential path to achieving green and sustainable rice production. Developing disease-resistant rice varieties is crucial for ensuring national food security.

[0003] Heat shock transcription factors (HSFs) were discovered by scientists in the 1960s and 70s. When organisms encounter stresses such as high temperatures, they rapidly synthesize a group of specialized heat shock proteins to protect cells from damage. Research confirmed the existence of a conserved DNA sequence upstream of the heat shock protein gene, called a heat shock element. In the late 1980s and early 1990s, researchers first isolated and identified proteins that bind to heat shock elements (HSEs), namely heat shock transcription factors, from yeast and fruit flies. Subsequently, the first HSF gene (from yeast) was cloned in 1990. In plants, whole-genome sequencing revealed an exceptionally large HSF family, far exceeding that of animals (humans have only three). Rice contains as many as 25 OsHsf members. This indicates that plant HSFs may have highly differentiated functions, with different OsHsf members exhibiting different tissue expression patterns and showing differentiated responses to various stresses such as high temperature, drought, high salinity, low temperature, and pathogen infection, suggesting a wide range of functions. Based on their structural characteristics (such as oligomerization and transcriptional activation domains), plant HSFs are divided into three major categories: A, B, and C, with categories A and B being the core of research. The rice OsHsf family also follows this classification. However, research on category C HSFs is still relatively limited. Therefore, there is an urgent need to systematically elucidate novel disease resistance mechanisms in crops, overcome the key challenge of scarce disease resistance genetic resources, discover molecular modules and breeding pathways that can be used for disease resistance improvement, and create new germplasm with sustained disease resistance capabilities. Summary of the Invention

[0004] To address the aforementioned technical problems in the existing technology, this invention provides an application of rice heat shock transcription factor OsHSFC1a and its encoding gene in rice disease resistance breeding.

[0005] The technical solution of this invention is as follows: The first object of the present invention is to provide an OsHSFC1a gene, wherein the OsHSFC1a gene is selected from one of the DNA molecules shown in 1), 2), 3), or 4) below: 1) The DNA molecule shown in SEQ ID NO.1; 2) The DNA molecule shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 3; 4) DNA molecules that have more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encode heat shock transcription factor-related proteins.

[0006] A second objective of this invention is to provide the protein encoded by the aforementioned OsHSFC1a gene.

[0007] Furthermore, selected from either (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3; (b) Proteins derived from SEQ ID NO.3 with one or more amino acid residues substituted and / or deleted and / or added in connection with disease resistance.

[0008] A third objective of this invention is to provide a knockout vector for the aforementioned OsHSFC1a gene.

[0009] Furthermore, the knockout vector is a CRISPR-Cas9 vector, and the target sequence of the CRISPR-Cas9 vector is the DNA molecule shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] A fifth objective of this invention is to provide the application of the aforementioned OsHSFC1a gene, or the aforementioned protein, or the aforementioned knockout vector in the cultivation of disease-resistant rice.

[0011] Furthermore, the disease resistance refers to resistance to rice blast and bacterial blight.

[0012] Furthermore, knocking out the OsHSFC1a gene in rice, or reducing the expression level of the aforementioned protein in rice, or transferring the aforementioned knockout vector into rice can improve the disease resistance of rice. Beneficial effects

[0013] This invention is the first to discover heat-shock transcription factors. OsHSFC1a The related protein influences the plant's disease resistance process. Knocking out this gene can enhance the plant's disease resistance, thereby enabling the breeding of edited plants with enhanced resistance to rice blast and bacterial blight. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0014] Figure 1 For wild-type Ningjing 7 and HSFC1a Phenotypic diagram of rice blast disease after knockout of the inoculated strain.

[0015] Figure 2 For wild-type Ningjing 7 and HSFC1a Statistics on the length of lesions after inoculation of knockout lines with rice blast.

[0016] Figure 3 For wild-type Ningjing 7 and HSFC1a The knockout system is based on the statistical analysis of lesion length after inoculation with bacterial blight.

[0017] Figure 4 The expression level of OsHSFC1a induced by the pathogen after inoculation with rice blast fungus.

[0018] Figure 5 for HSFC1a The knockout mutation site. Detailed Implementation

[0019] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental 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 stores.

[0020] Example 1: Obtaining and Identifying Transgenic Plants I. Knockout Vector Construction 1. Knockout Primer Design (1) On the CRISPR-P website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), select Oryza sativa (RAP-DB) in the targetgenome option; (2) Enter the gene accession number in the Locus Tag: genbankOs01g0625300; (3) Click submit to run the program and wait for the results to appear; (4) Select primers located on the CDS that are close to the ATG start site and have a high score to replicate the first 20 bp; perform blast analysis and select primers with high specificity; (5) Primer synthesis: The back primer needs to be reverse complementary to the 20 bp sequence before adding AAAC at the 5' end, while the front primer directly adds GGCA at the 5' end of the 20 bp sequence; After primer design and synthesis, the knockout vector was constructed. CRISPR-OsHSFC1A .

[0021] The knockout primer sequences are as follows: Primer1: 5' GGAGTACGGCGGACCGGCGC3' (SEQ ID NO. 4); Primer2: 5' TGTGGTACGTGCAGGGATTC3' (SEQ ID NO. 5); 2. Knockout vector construction (1) Add 1 mL each of Primer 1 and 2 stock solutions (100 mM) to a 200 mL PCR tube, and then add 8 mL of ddH2O; incubate at 95℃ for 5 min, and then allow to cool to room temperature naturally; (2) Preparation of reaction system: ATP 1 mL, pCAMBIA1305.1 carrier 1 mL, buffer 1 mL, oligo 0.2 mL, AarⅠ 0.2 mL, T4ase 0.2 mL, ddH2O 5.5 mL, (1) mixture 1 mL; (3) Reaction program: 37℃ for 5 min; 20℃ for 5 min; 4℃ for 30 min (1×10 cycles).

[0022] Transformation of competent *E. coli* cells: Add 10 mL of ligation vector, gently pipette to mix, and incubate on ice for 30 min; incubate at 42°C for 45 s, then immediately incubate on ice for 2 min; add 700 mL of LB liquid medium (antibiotic-free), and incubate at 37°C and 220 rpm for 45 min; spread the bacterial culture evenly on LB medium (containing appropriate antibiotics), place in a clean bench for about 30 min, and after the culture medium is completely dried, invert it in a 37°C incubator for overnight incubation; after obtaining colonies, colony PCR detection is performed. The primers used are Ubipro-R: gctcattatctctagagaggggca (SEQ ID NO.6) paired with Primer 2. Select 1-2 positive colonies for sequencing. After successful sequencing alignment, plasmid extraction is performed to obtain the OsHSFC1a gene knockout vector, named... CRISPR-OsHSFC1a. HSFC1a Knockout mutation sites such as Figure 5 As shown.

[0023] II. Obtaining Recombinant Agrobacterium Agrobacterium EHA105 strain was transformed using the freeze-thaw transformation method. Commercial Agrobacterium competent (EHA105) samples were retrieved, thawed on ice, and then 2-3 μL of transformation plasmid were added. CRISPR-OsHSFC1aMix thoroughly by pipetting, then flash-freeze in liquid nitrogen for 5 minutes; immediately heat-shock in a 37°C water bath for 5 minutes; remove and place on ice, add 1 mL of antibiotic-free LB broth, and incubate at 28°C on a shaker for 3-4 hours; evenly spread the bacterial culture onto LB broth (containing the corresponding antibiotic), air-dry naturally in a clean bench, and then incubate upside down in a 28°C incubator for 2-3 days to obtain... CRISPR-OsHSFC1a strains.

[0024] III. Obtaining Transgenic Plants Will CRISPR-OsHSFC1a The specific method for transforming the strain into wild-type Ningjing No. 7 is as follows: (1) Incubate at 28℃ CRISPR-OsHSFC1a (Or, for the control strain with empty vector) After 16 hours, collect the bacterial cells and dilute them in N6 liquid medium (Sigma, C1416) to a concentration of OD600 ≈ 0.5 to obtain the bacterial culture. (2) Mix the mature embryonic callus of rice Ningjing 7, which has been cultured for one month, with the bacterial solution in step (1) and infect for 30 min. After the bacterial solution is dried with filter paper, transfer it to co-culture medium (N6 solid co-culture medium, Sigma) and co-culture at 24℃ for 3 days. (3) The callus from step (2) was inoculated on N6 solid selection medium containing 100 mg / L hygromycin for the first screening (16 days). (4) Select healthy callus and transfer it to N6 solid selection medium containing 100 mg / L hygromycin for a second selection. Subculture every 15 days. (5) Select healthy callus and transfer it to N6 solid selection medium containing 50 mg / L hygromycin for the third selection, and subculture every 15 days; (6) Select resistant callus and transfer it to differentiation medium for differentiation; obtain CRISPR-OsHSFC1a (HSFC1a) T0 generation positive plants that differentiated into seedlings.

[0025] IV. Identification of Transgenic Plants PCR amplification was performed to identify the transgenic plants. The PCR primer sequences are as follows: Primer3: 5'CTTCTCCAGCTTCGTTCGC 3' (SEQ ID NO. 7).

[0026] Primer4: 5'AACGACTCGTGCGCGAACT 3' (SEQ ID NO. 8).

[0027] 1. PCR molecular identification The result obtained in step four CRISPR-OsHSFC1A ( HSFC1aGenomic DNA was extracted from T0 generation plants. Primer 3 and Primer 4 were used for amplification. HSFC1a Genomic DNA extracted from T0 generation plants was amplified using Primer7 and Primer8 as primers. pGluC-OsHSFC1A Genomic DNA extracted from T0 generation plants.

[0028] PCR reaction system: DNA (20 ng / μL) 2 μL, Primer3 (10 pmol / μL) and Primer4 (10 pmol / μL) 2 μL each or Primer7 (10 pmol / μL) and Primer8 (10 pmol / μL) 2 μL, 10xBuffer (MgCl2 free) 2 μL, dNTP (10 mM) 0.4 μL, MgCl2 (25 mM) 1.2 μL, rTaq (5 U / μL) 0.4 μL, ddH2O 10 μL, total volume 20 μL.

[0029] The amplification reaction was performed on a PTC-200 (MJ Research Inc.) PCR instrument: 94℃ for 3 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0030] 2. Identification of disease resistance phenotype T2 will be converted respectively CRISPR-OsHSFC1a Positive plants and Ningjing 7 were planted in a transgenic field at the Tuqiao Rice Breeding Base of Nanjing Agricultural University. Resistance to rice bacterial blight was assessed during the peak tillering stage. After seed maturity, seeds from each material were collected for resistance assessment. The assessment methods are as follows: Methods for identifying and investigating bacterial blight: Material cultivation: The materials used were cultivated in Tuqiao Community, Chunhua Street, Jiangning District, Nanjing City, Jiangsu Province, and field management was carried out in accordance with the field production method.

[0031] Preparation of pathogens: The bacterial blight pathogen strain used in this experiment was PXO99 stored in the laboratory at -80℃. A Three days before inoculation, 20-30 μL of the culture was spread onto NA medium and incubated at 28°C for 2 days. The resulting bacterial block was then scraped off and placed in 500 ml of liquid NA medium. The culture was incubated on a shaker at 28°C for 12-16 hours. Oddi concentration (OD) was measured the following morning. 600 The OD was determined by using dd water. 600 Prepare for inoculation when adjusted to version 1.0.

[0032] Inoculation and investigation methods for rice bacterial leaf blight: The inoculation method for bacterial blight is the leaf-cutting method. Inoculation is performed during the peak tillering stage of rice. When inoculating, dip scissors in the bacterial solution and cut off approximately 2 cm from the tip of the unfolded leaf at the top of the plant. At least 3 leaves should be inoculated per plant, and at least 3 plants should be inoculated per number. The length of bacterial blight lesions should be surveyed 14 days after inoculation. During the survey, the length of lesions should be measured on at least 10 diseased leaves for each number, and statistical analysis should be performed.

[0033] Methods for identifying and investigating rice blast: Material cultivation: After soaking and germinating the seeds in the laboratory, they were sown in small black squares and disease resistance was assessed when they reached the three-leaf-one-heart stage.

[0034] Preparation of the pathogen: The strain used in this experiment was 24-586, provided by the research group of Liu Yongfeng at the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. All strains were grown in PDA medium and incubated at 28℃ in the dark for approximately 5-7 days. Afterward, the mycelial blocks from the edge of the culture dish were removed and placed in the sporulation medium for sporulation. When the mycelial blocks had completely covered the sporulation medium (approximately 5-7 days), the medium was rinsed with sterile water to collect the spores. The spores were then filtered through a special filter cloth, and the spore concentration was measured and adjusted to 1×10⁻⁶. 5 spores / ml.

[0035] Inoculation and investigation methods for rice blast: Take fully expanded leaves at the 3-leaf stage, cut 5-8 cm sections, place them in a suitable concentration of 6-BA solution, puncture the leaf surface with a yellow nozzle, and drop the spore solution onto the leaves. Incubate at 26℃ in the dark for 24 h, followed by 12 h light / 12 h dark. Observe the phenotype after 5-10 days of incubation.

[0036] V. Results Disease resistance experiments showed that inoculation with Cr- blast fungus... HSFC1a The lesion length of the plant was only half that of the wild type, and statistically, it was significantly shorter than that of the wild type. Figure 1 ,2), while cr- inoculated with bacterial blight HSFC1a The length of lesions on the plants was also significantly shorter than that of the wild type. Figure 3 The rice exhibits significantly enhanced disease resistance. Following inoculation with *O. blast fungus*, it displays a unique and sustained pattern of transcriptional upregulation; specifically, significant upregulation begins 6 hours post-inoculation and maintains high expression levels for at least 72 hours. This expression characteristic suggests that the *O. blast fungus* gene differs from early transient response genes and likely plays a central role in the sustained defensive response or maintenance of disease resistance in rice against *O. blast fungus*. Figure 4 ).

[0037] The above results indicate that knocking out OsHSFC1a can enhance the resistance of rice to rice blast and bacterial blight.

[0038] SEQ ID NO.1 Rice heat shock transcription factorOsHSFC1a gene sequence SEQ ID NO.2 Rice heat shock transcription factor OsHSFC1A CDS sequence SEQ ID NO.3 Rice heat shock transcription factor OsHSFC1A amino acid sequence MDGLHTELALGLIGCCGGDGQQQTAPFVAKTYQMVCDPRTDALVRWGRDNNSFVVVDPAAFSQLLLPCFFKHGNFSSFVRQLNTYGFRKVHPDRWEFAHESFLRGQTHLLPRIVRRKKRGEGGGGGGGASCSFGGGAGEHQVAAAAASVGMSGEEEDAAEDVLAKEAALF EEVQRLRHEQTAIGEELARMSQRLQATERRPDQLMSFLAKLADDPNAVTGHLLEQAAERKRRRQHLPSHEPTVCPLPPAPPPQPPQPLLALAGAAAMDGTYWWTTEHHHHHHHQMKPMTVLPSLEPPTASCGVHQVPELGGGGVMGLTTDGEAKVEPPFPFCLLGQAFF* The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The OsHSFC1a gene, characterized by: The OsHSFC1a gene is selected from one of the DNA molecules shown in 1), 2), 3), or 4) below: 1) The DNA molecule shown in SEQ ID NO.1; 2) The DNA molecule shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 3; 4) DNA molecules that have more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encode heat shock transcription factor-related proteins.

2. The protein encoded by the OsHSFC1a gene as described in claim 1.

3. The protein according to claim 2, characterized in that, Choose from either (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3; (b) Proteins derived from SEQ ID NO.3 with one or more amino acid residues substituted and / or deleted and / or added in connection with disease resistance.

4. The OsHSFC1a gene knockout vector as described in claim 1.

5. The knockout carrier according to claim 4, characterized in that, The knockout vector is a CRISPR-Cas9 vector, and the target sequence of the CRISPR-Cas9 vector is the DNA molecule shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. A recombinant expression vector, expression cassette, or recombinant bacteria containing the OsHSFC1a gene as described in claim 1.

7. The application of the OsHSFC1a gene of claim 1, or the protein of claim 2 or 3, or the knockout vector of claim 4 or 5, or the recombinant expression vector, expression cassette, or recombinant bacteria of claim 6 in the cultivation of disease-resistant rice.

8. The application according to claim 7, characterized in that, The disease resistance refers to resistance to rice blast and bacterial blight.

9. The application according to claim 7, characterized in that, Knocking out the OsHSFC1a gene in rice, or reducing the expression level of the protein described in claim 2 or 3 in rice, or transferring the knockout vector described in claim 4 or 5 into rice can improve the disease resistance of rice.

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