Application of rice flavanone synthase key enzyme FSIBR and its encoding gene in rice disease resistance breeding

Abstract

This invention discloses the application of FSIBR, a key enzyme in rice flavanone synthesis, and its encoding gene, in rice disease resistance breeding, relating to the field of genetic engineering technology. The amino acid sequence of the key enzyme FSIBR is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. This invention found that the expression levels of FSIBR protein and its encoding gene are closely related to the development of rice blast disease. Knocking out the FSIBR gene significantly improved the resistance level of rice to rice blast disease, indicating that FSIBR negatively regulates plant disease resistance. Its encoding gene, FSIBR, can be used as a target gene to improve plant disease resistance, showing good application prospects in breeding disease-resistant rice varieties and laying an important foundation for rice resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of FSIBR, a key enzyme in rice flavanone synthesis, and its encoding gene, in rice disease resistance breeding. Background Technology

[0002] Rice, as the world's most important staple crop, has stable and high yields that are directly related to global food security and sustainable agricultural development. However, rice blast, known as "rice cancer," is the most devastating fungal disease, causing up to 30% yield loss annually and seriously threatening my country's food security. While current control methods relying on chemical pesticides may show short-term effectiveness, long-term use easily leads to pesticide resistance in pathogens and causes environmental risks such as soil microecological imbalance. In contrast, cultivating disease-resistant varieties, as a core control strategy that combines environmental friendliness and economic benefits, not only reduces pesticide use but is also a key breakthrough in building a green control system and promoting sustainable agricultural development.

[0003] Based on this, the present invention focuses on the innovation of rice germplasm resources. By systematically mining disease-resistant genetic genes with independent intellectual property rights, it breaks through the technical bottleneck of the scarcity of existing resistance gene resources. It aims to provide new molecular breeding targets for the control of rice blast and other fungi, and can also lay a theoretical foundation and technical reserves for the cultivation of high-quality and multi-resistant new generation rice varieties. It has important practical value for ensuring the national food security strategy. Summary of the Invention

[0004] The purpose of this invention is to provide the application of FSIBR, a key enzyme in rice flavanone synthesis, and its encoding gene in rice disease resistance breeding, thereby addressing the problems existing in the prior art. This invention discovers that the expression levels of FSIBR protein and its encoding gene are closely related to the development of rice blast disease and can be applied to regulate rice blast resistance.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of FSIBR, a key enzyme in rice flavanone synthesis, in regulating rice blast resistance. The amino acid sequence of FSIBR is shown in SEQ ID NO.2.

[0007] Furthermore, if the content of the key enzyme FSIBR in rice flavanone synthesis is increased in rice plants, the resistance to rice blast will decrease; if the content of the key enzyme FSIBR in rice plants is decreased, the resistance to rice blast will increase.

[0008] The present invention also provides the application of the encoding gene of FSIBR, a key enzyme in rice flavanone synthesis, in regulating rice blast resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, upregulation of the expression level of the coding gene in rice plants reduces rice blast resistance; downregulation of the expression level of the coding gene in rice plants increases rice blast resistance.

[0010] The present invention also provides the application of a biomaterial that downregulates the expression level of the gene encoding the key enzyme FSIBR in rice flavanone synthesis in improving rice blast resistance or in breeding transgenic rice blast resistant to rice blast.

[0011] Furthermore, the biological material is a gene knockout vector that knocks out the coding gene.

[0012] Furthermore, the gene knockout vector is obtained by linking a double strand of DNA into a pTCRISPR vector;

[0013] The DNA double strand comprises a forward Oligo DNA nucleotide sequence as described in SEQ ID NO. 8 and a reverse Oligo DNA nucleotide sequence as described in SEQ ID NO. 9.

[0014] Furthermore, the biological material is a recombinant host cell; the recombinant host cell includes the gene knockout vector described above.

[0015] The present invention also provides a method for improving rice blast resistance, comprising the step of knocking out the coding gene of FSIBR, a key enzyme in rice flavanone synthesis, and constructing transgenic rice with the coding gene knocked out;

[0016] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0017] Furthermore, the coding gene was knocked out using the CRISPR / Cas9 method.

[0018] The present invention discloses the following technical effects:

[0019] This invention identified a key enzyme in flavanone synthesis induced by rice blast fungus, named FSIBR. Experimental results show that the expression levels of the FSIBR protein and its encoding gene, FSIBR, are closely related to the development of rice blast. This invention constructed overexpression and knockout vectors for the rice FSIBR gene, transferred these vectors into the Zhonghua 11 (ZH11) recipient material, and obtained FSIBR overexpressing and knockout plants. Disease resistance analysis of the obtained transgenic plants showed that knocking out the FSIBR gene significantly improved rice resistance to rice blast, indicating that FSIBR negatively regulates plant disease resistance. Its encoding gene, FSIBR, can be used as a target gene to improve plant disease resistance, showing good application prospects in breeding disease-resistant rice varieties and laying an important foundation for rice resistance breeding. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The figure shows the expression statistics of FSIBR in rice leaves before and after infection with rice blast fungus; the figure shows the expression changes of FSIBR in the leaves of three-week-old Zhonghua 11 (ZH11) plants after inoculation with rice blast fungus spores (physiological race zhong10-8-14) and Mock (0.1% Tween-20) at 0h, 6h, 12h, 24h, 36h, 48h, 60h, and 72h.

[0022] Figure 2 The figures show the phenotypes and statistical results of puncture-inoculated plants in control, FSIBR knockout, and FSIBR overexpressing plants; where A is a representative image of lesions on leaves of three-week-old plants 5 days after inoculation with rice blast fungus (physiological race zhong10-8-14), with a scale bar of 1 cm; B is a statistical graph of the lesion length on leaves of puncture-inoculated plants. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The rice variety Zhonghua 11 (ZH11) used in this invention was provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0029] The rice variety Nipponbare (O. sativa L.spp. japonica, var. Nipponbare, AA genome, Nip) belongs to the japonica subspecies and is described in the following literature: Rice Variety “Nipponbare”, Agricultural Science and Technology Communications, 1973(02):31; provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0030] The FSIBR knockout and overexpression transgenic rice was developed by Boyuan Biotechnology Co., Ltd.

[0031] The plant binary expression vector pTCRISPR was provided by Associate Professor Tang Yongyan of the State Key Laboratory of Sichuan Agricultural University.

[0032] The plant binary expression vector pCAMBIA-35s-3×HA was provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0033] Total RNA Extraction Kit: Purchased from Invitrogen's TRIzol (catalog number 15596026).

[0034] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper) purchased from Vazyme (China), catalog number R323-01.

[0035] Homologous recombination kit: ClonExpress II One Step Cloning Kit, catalog number C112-01, purchased from Vazyme in China.

[0036] In the study of rice immune responses, this invention identified a key enzyme in flavanone synthesis induced by rice blast fungus, which was named FSIBR. FSIBR plays an important role in plant growth, development, and the synthesis of secondary metabolites; therefore, previous research on FSIBR has focused on its regulation of flower color formation and antioxidant defense. This invention shows that FSIBR knockout plants exhibit enhanced resistance to rice blast, while FSIBR overexpression plants are more susceptible to rice blast. This indicates that FSIBR negatively regulates rice resistance to rice blast and can be used as a target gene for molecular breeding to improve plant disease resistance. Specific research is as follows:

[0037] Example 1: FSIBR, a key enzyme in rice flavanone synthesis, and its cloning.

[0038] A key enzyme for flavanone synthesis, FSIBR, was screened in rice. Primers were designed based on the rice Nip genome reference gene sequence.

[0039] FSIBR-F: ATGGTCGGTGGCTCGA (SEQ ID NO. 3);

[0040] FSIBR-R: TCACACTCCCAGCTTCAAGCTCT (SEQ ID NO. 4).

[0041] Using total cDNA from rice ZH11 as a template, a DNA band of approximately 1.1 kb was amplified using the primers described above. Sequencing confirmed that it belongs to the FSIBR gene, a key enzyme in the flavanone metabolic pathway.

[0042] The nucleotide sequence of the FSIBR gene obtained from total cDNA amplification of rice ZH11 is shown in SEQ ID NO.1, and the amino acid sequence of the FSIBR protein is shown in SEQ ID NO.2.

[0043] SEQ ID NO.1:

[0044]

[0045] SEQ ID NO.2:

[0046] MVGGFDKLKVEFIDQDESVQVVADTIRSSGEIPERYARPEMEADPVIIDTDGYNLPVIDMSRLINPEFSEEEIAKLGSACEDWGFFQLVNHGVDGELLQRIKDDITEFFRLPLQEKMSVAIPPNGLQGFGHHFVFSKEQKLDWVDLLFLTTRPVEDRTTEFWPTKPPTFRDSLDKYSLEIA NVSAKLFKFMAINLGVDEEALLAAFKPEQPQSVRINHYPPCSQANKVLGLSPHTDGVGMTLLLQVNDVQGLQIRKDGRWFAVKNLPGALVVNVGDVLEILTNGKYKSIEHRAVINPDKERITLAAFQSVPLSSTVGPLQELLMKGEARYKTVDGAEFTKGYFAAKLEGRRYLESLKLGV*.

[0047] Based on the nucleotide sequence of FSIBR, the following real-time quantitative PCR primers were designed:

[0048] F: CTGGACTGGGTCGATTTGCT (SEQ ID NO.5);

[0049] R: CCCTGAATGTGGGAGGCTTT (SEQ ID NO. 6).

[0050] Material cultivation and rice blast fungus infection

[0051] (1) The experimental rice material was ZH11, and ZH11 material was planted in the field until the three-leaf stage.

[0052] (2) The tested rice blast fungus was Zhong10-8-14 rice blast fungus spores. Spray inoculation was performed at a concentration of 5 × 10⁻⁶. 5 Rice blast fungus spores at a concentration of 1 / mL were inoculated onto ZH11 rice leaves at the tillering stage. Leaves were collected at 0h, 6h, 12h, 24h, 36h, 48h, 60h, and 72h after inoculation, flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA was extracted from the rice using Trizol reagent and reverse transcribed to obtain cDNA for real-time quantitative PCR detection. 0.1% Tween-20 served as a control (designated as the Mock group). Detection results are shown below. Figure 1 As shown, the expression of the FSIBR gene in ZH11 was significantly upregulated after infection with rice blast fungus, indicating that this gene plays an important role in the development of rice blast disease.

[0053] Example 2: Construction of FSIBR plant knockout vector

[0054] FSIBR knockout vector pTCRISPR-sgRNA FSIBR Construction:

[0055] The knockout target sequence was designed as: TCTGCAGGAGAAGATGTCCG (SEQ ID NO.7). Forward OligoDNA (F) and reverse OligoDNA (R) were synthesized.

[0056] Forward Oligo DNA (F): TGTGTCTGCAGGAGAAGATGTCCGG (SEQ ID NO.8);

[0057] Reverse Oligo DNA(R):AAAAC CGGACATCTTCTCCTGCAGA (SEQ ID NO.9).

[0058] After denaturation at 95°C, annealing was performed to form a DNA double strand. The pTCRISPR vector was digested with BsaI, and the linearized vector was recovered. The DNA double strand and linearized vector were mixed in the following system (2 μL DNA double strand fragment, 2 μL linearized vector, 2 μL T4 ligase, 4 μL ddH2O, total volume 10 μL), and the T4 DNA ligase ligation reaction was performed. The mixture was incubated at room temperature for 30 minutes to obtain pTCRISPR-sgRNA. FSIBR The vector was knocked out. The cells were transformed into DH5α competent cells, and after kanamycin selection, single colonies were picked for colony PCR identification. Plasmids were extracted from positive colonies and sequenced to confirm the pTCRISPR-sgRNA. FSIBR Yes, genetic transformation can be performed.

[0059] Gene knockout was performed using ZH11 as the base material to obtain FSIBR knockout transgenic rice (fsibr-ko#1 and fsibr-ko#2), which was completed by Boyuan Biotechnology Co., Ltd.

[0060] Example 3: Construction of FSIBR plant overexpression vector

[0061] Construction of the FSIBR overexpression vector pCAMBIA-35s-FSIBR-3×HA:

[0062] Using cDNA obtained by reverse transcription of total RNA from rice ZH11 as a template, the cloning primers were:

[0063] OE-F: ATTTGGAGAGAACACGGGGGACGAGCTCATGGTCGGTGGCTTCGA (SEQ ID NO. 10);

[0064] OE-R: TATGGGTACATGGATCCGGTACCCACTCCCAGCTTCAAGCTCT (SEQ ID NO. 11).

[0065] PCR reactions were performed on a PE9600 PCR instrument. The program was as follows: 94℃ pre-denaturation for 2 min; then 94℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 2 min, for a total of 30-32 cycles; followed by 72℃ extension for 10 min; and storage at 4℃. Full-length FSIBR cDNA was obtained by PCR amplification. The pCAMBIA-35s-3×HA vector was double-digested with SacI and KpnI to recover the linearized vector. The recovered PCR product, full-length FSIBR cDNA, and the linearized vector were mixed in the following system (4 μL DNA fragment, 4 μL linearized vector, 4 μL 5×CE II Buffer, 2 μL Exnase II, and 6 μL ddH2O, total volume 20 μL) and incubated at 37℃ for 30 min for homologous recombination to obtain the pCAMBIA-35s-FSIBR-3×HA recombinant vector.

[0066] After transformation into DH5α competent cells and selection with kanamycin, single clones were selected for colony PCR identification. Plasmids were extracted from positive colonies and sequenced to confirm that pCAMBIA-35s-FSIBR-3×HA was correct and could be genetically transformed.

[0067] Gene overexpression was performed using ZH11 as the base material to obtain FSIBR overexpressing transgenic rice (FSIBR-OE#1 and FSIBR-OE#2), which was completed by Boyuan Biotechnology Co., Ltd.

[0068] Example 4: FSIBR participates in regulating plant disease resistance

[0069] In plants, research on FSIBR has primarily focused on flower color formation and antioxidant defense. This invention reveals that FSIBR plays a crucial role in regulating plant disease resistance responses. Therefore, the effect of the FSIBR gene on disease resistance in rice was tested.

[0070] Seedling puncture and inoculation treatment: The test material was Zhonghua 11 (ZH11), the genetically stable FSIBR knockout lines fsibr-ko#1 and fsibr-ko#2 with ZH11 as the background, and the genetically stable FSIBR overexpression lines FSIBR-OE#1 and FSIBR-OE#2 with ZH11 as the background. Plump seeds were selected and placed in conical flasks filled with tap water. These flasks were then placed in a 37℃ dark incubator for germination, with the water changed daily. After 2 days, seeds showing signs of sprouting were selected and placed in 96-well seedling trays. The trays were then placed on floats and grown in Hoagland nutrient solution. After 21 days, the second-to-last rice leaf of uniform growth and size was selected, punctured, and inoculated with 5 μL of a 3×10⁻⁶ concentration. 5 Five days after inoculation with *Blastomyces oryzae* spores (Zhong10-8-14), the length of lesions was observed and counted. The results showed that, compared to ZH11, lesion length was significantly reduced and disease resistance was enhanced in fsibr-ko#1 and fsibr-ko#2 plants under the ZH11 background following puncture inoculation. Figure 2 Compared to ZH11, under the ZH11 background, the lesion length of FSIBR-OE#1 and FSIBR-OE#2 plants was significantly increased, and the plant's disease resistance was significantly reduced. Figure 2 This indicates that FSIBR negatively regulates plant disease resistance, and its encoding gene FSIBR can be used as a target gene for molecular breeding to improve plant disease resistance.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a biomaterial that downregulates the expression level of the gene encoding FSIBR, a key enzyme in rice flavanone synthesis, in improving rice blast resistance or breeding transgenic rice resistant to rice blast, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The biological material is a gene knockout vector that knocks out the encoding gene; The gene knockout vector is obtained by linking double-stranded DNA into a pTCRISPR vector.

2. The application according to claim 1, characterized in that, The DNA double strand comprises forward Oligo DNA as described in SEQ ID NO. 8 and reverse Oligo DNA as described in SEQ ID NO.

9.

3. A method for improving rice blast resistance, characterized in that, The method includes the step of knocking out the coding gene of FSIBR, a key enzyme in rice flavanone synthesis, and constructing transgenic rice with the coding gene knocked out. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, The coding gene was knocked out using the CRISPR / Cas9 method.

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