Application of Rice Gene OsFd4 in Resistance to Bacterial Blight of Rice

Abstract

The present invention provides a rice gene OsFd4 and its application in rice resistance to bacterial blight. The OsFd4 open reading frame nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The present invention uses the CRISPR / Cas9 method to knockout the rice gene OsFd4 . The inoculation experiments of wild-type and knockout mutant plants with Xanthomonas oryzae pv. oryzae are carried out respectively. The results show that knockout of OsFd4 significantly improves the levels of reactive oxygen species burst and callose deposition in rice plants after treatment with the flagellin short peptide of bacterial pathogens, thereby enhancing the resistance of rice to Xanthomonas oryzae pv. oryzae. At the same time, through the observation of the plant morphology in the field, it is found that OsFd4 the growth status of the knockout mutants is normal and there is no significant difference compared with the wild type. The above results indicate that OsFd4 is a gene that negatively regulates the immunity of rice to bacterial blight. The content of the present invention provides an endogenous gene target for improving the resistance of rice through gene editing technology when bacterial blight occurs in the field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of a rice gene OsFd4 in rice resistance to bacterial blight. Background Art

[0002] Rice (Oryza sativa) is the staple food crop for more than half of the world's population and is also a model species for the study of crop genome function. Rice is threatened by various diseases throughout its growth period, which seriously affects the stability of its yield. For example, bacterial blight caused by the infection of Xanthomonas oryzae pv. oryzae is the most destructive bacterial disease of rice worldwide. When the disease occurs severely, the yield can be reduced by up to 50%.

[0003] The ferredoxin (Fds) family has a conserved iron-sulfur [2Fe-2S] domain and is distributed in the stroma of plastids. As the most upstream electron carrier, it distributes electrons to various receptors in downstream metabolic reaction pathways. According to their expression patterns, Fds in higher plants can be divided into photosynthetic and non-photosynthetic types. The former receives electrons from photosystem I in chloroplasts, while the latter uses NADPH generated in the oxidative pentose phosphate pathway as an electron donor. When the main Fds in plastids are defective, the electron transport chain is blocked, resulting in the transfer of excessive electrons to O2 or H2O to form ROS.

[0004] More and more studies have shown that Fds are involved in regulating the response of plants to various stresses. For example, AtFd2 in Arabidopsis accounts for about 90% of the total Fd expression in the above-ground parts. Its deletion leads to the accumulation of ROS in chloroplasts. The AtFd2 knockout mutant (Fd2-KO) can better adapt to long-term high-light conditions by inducing the expression of genes related to photoprotection. Overexpression of exogenous or endogenous Fd-encoding genes in Chlamydomonas reinhardtii can reduce the production of endogenous H2O2 and enhance the tolerance to heat stress. Similar results were also found when the sweet pepper ferredoxin-encoding gene was heterologously expressed in Arabidopsis. In addition, the regulatory role of Fds in plant immunity has been explored. For example, the sweet pepper ferredoxin-encoding gene heterologously expressed in Arabidopsis or tobacco can enhance the resistance of transgenic plants to their pathogens. Another research result shows that the Arabidopsis Fd2-KO mutant shows higher susceptibility to the infection of (hemi) biotrophic pathogens, which may be due to the high accumulation of jasmonic acid and its derivatives in the mutant, which inhibits the resistance mediated by the salicylic acid signaling pathway.

[0005] There are 5 typical Fd proteins in rice, named OsFd1 - OsFd5. OsFd1 is the main photosynthetic Fd in rice and plays an important role in maintaining the normal survival of rice seedlings. OsFd4 is the most important non - photosynthetic type Fd protein in rice, however, the biological function of OsFd4 in rice is still unclear. So far, there is no report on the function or application of rice Fd proteins in disease resistance. The research of this invention found that using CRISPR / Cas9 - mediated gene editing technology to disrupt the function of the rice OsFd4 gene can improve the resistance of rice to bacterial blight without affecting the normal growth of rice plants. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the rice gene OsFd4 in the resistance of rice to bacterial blight. Specifically, by using the CRISPR / Cas9 method to knockout the rice OsFd4 gene and further conducting resistance identification, the negative regulatory function of the OsFd4 gene on the resistance of rice to bacterial blight is clarified, providing a regulatable endogenous gene target for preventing and controlling the occurrence of bacterial blight.

[0007] To achieve the above - mentioned purpose, this invention adopts the following technical solutions:

[0008] A rice gene OsFd4, whose open reading frame nucleotide sequence is as shown in SEQ ID NO.1.

[0009] A protein encoded by the above - mentioned rice gene OsFd4, whose amino acid sequence is as shown in SEQ ID NO.2.

[0010] The application of the above - mentioned rice gene OsFd4 in the resistance of rice to bacterial blight, the specific operation includes: selecting two target sites in the open reading frame coding region of the rice OsFd4 gene, constructing an OsFd4 knockout vector, then transforming the OsFd4 knockout vector into Agrobacterium tumefaciens, and obtaining an OsFd4 knockout mutant by means of Agrobacterium - mediated transformation of rice mature embryos; using the plant pathogenic bacterial flagellin - conserved polypeptide flg22 to treat the leaves of wild - type and OsFd4 knockout mutant plants, and detecting the ROS burst and callose deposition to further verify the resistance of the osfd4 mutant to bacterial blight pathogens.

[0011] The nucleotide sequences of the above - mentioned two target sites are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0012] The application of the rice gene OsFd4 in the resistance of rice to bacterial blight, knocking out the OsFd4 gene significantly improves the resistance of rice to bacterial blight; and the levels of reactive oxygen species burst and the number of callose depositions in the OsFd4 knockout mutant are higher than those of the wild - type after responding to flg22 treatment.

[0013] The present invention has the following beneficial effects:

[0014] After knocking out the rice gene OsFd4 of the present invention, the ability to resist bacterial blight is significantly improved compared with wild-type rice, and this gene provides a regulatable endogenous gene target for preventing and controlling the occurrence of bacterial blight. Brief Description of the Drawings

[0015] Figure 1 : Structure diagram of OsU3:gRNA in the intermediate vector SK-gRNA.

[0016] Figure 2 : Structure diagram of 2×35S:Cas9 in the binary vector pC1300-Cas9.

[0017] Figure 3 : Identification of OsFd4 knockout mutants. Reference: reference gene sequence; osfd4-1, osfd4-2 and osfd4-3 are homozygous mutant lines of the OsFd4 gene.

[0018] Figure 4 : Identification of the field growth phenotype of OsFd4 knockout mutants. ZH11: wild-type rice variety Zhonghua 11; osfd4-1, osfd4-2 and osfd4-3: homozygous mutant plants of the OsFd4 gene; scale bar length: 10 cm.

[0019] Figure 5 : Investigation results of inoculating the bacterial blight pathogen strain Pxo86 on OsFd4 knockout mutants. ZH11: wild-type rice variety Zhonghua 11; osfd4-1, osfd4-2 and osfd4-3: homozygous mutant plants of the OsFd4 gene. a, diseased leaves 15 days after inoculating the bacterial blight pathogen (scale bar length: 3 cm); b, statistics of lesion length; c, statistics of the number of bacterial blight bacteria in diseased leaves of the same length.

[0020] Figure 6 : Detection results of the production of reactive oxygen species levels induced by flg22 in OsFd4 knockout mutants. flg22: conserved polypeptide of flagellin of phytopathogenic bacteria; H2O: water control treatment; ZH11: wild-type rice variety Zhonghua 11; osfd4-1: homozygous mutant plant of the OsFd4 gene.

[0021] Figure 7 : Detection results of callose induced by flg22 in OsFd4 knockout mutants. ZH11: wild-type rice variety Zhonghua 11; osfd4-1: homozygous mutant plant of the OsFd4 gene; scale bar length: 30 μm. Detailed Embodiments

[0022] The present invention will be further described in detail below in conjunction with the specific embodiments.

[0023] Example 1 Obtaining the OsFd4 Gene

[0024] Using the protein sequence of Arabidopsis thaliana ferredoxin AtFd2 in Arabidopsis thaliana for sequence alignment in the NCBI database, we identified the homologous protein of AtFd2 in rice with the gene number LOC_Os03g61960 (OsFd4), and its similarity was 68%. The protein encoded by the LOC_Os03g61960 gene is 153 amino acids in length, and its N-terminus has a chloroplast localization signal peptide. According to the predicted cDNA sequence (XM_015774279.1) of this gene in the Nipponbare rice genome on the NCBI website, we successfully cloned the open reading frame of OsFd4 from the total cDNA of Zhonghua 11 leaves. The full length is 462 bp, and sequencing found it to be consistent with the predicted sequence. The nucleotide sequences of the primers used to clone the open reading frame of OsFd4 are as follows:

[0025] OsFd4ORF-F (SEQ ID NO.5): 5’-ATGGCGACGTGCACACTTG-3’,

[0026] OsFd4ORF-R (SEQ ID NO.6): 5’-TTAGTAAAGGTCTCCTTCCT-3’

[0027] The nucleotide sequence of the open reading frame of the OsFd4 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0028] Example 2 Construction of the OsFd4 Knockout Vector

[0029] Two target sites (the sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively) were selected in the open reading frame coding region of the OsFd4 gene to construct a gRNA expression cassette, and then the two target gRNA expression cassettes were ligated into the knockout vector pC1300-Cas9 to obtain the OsFd4 knockout vector. The detailed vector construction method refers to the patent "Construction and Application of Plant Multigene Knockout Vectors, ZL201510485573.2", specifically as follows:

[0030] (1) Selection of target sequences and primer design

[0031] Search for target sequences containing the 5’-(N) X -NGG-3’ structure in the open reading frame coding region of the OsFd4 gene, where N represents any one of A, T, C, and G, and X is 19. According to the above principle, two target sequences, namely OsFd4-T1 and OsFd4-T2, were designed, and the sequences are as follows:

[0032] OsFd4-T1 (SEQ ID NO.3): 5’-ATGCAGTTTCCAAGCCTGAAGG-3’

[0033] OsFd4-T2 (SEQ ID NO.4): 5’-GGGCAAGAGCACGAGTTCGAGG-3’

[0034] Primer pairs designed for constructing gRNA: Add GGCA in front of the forward sequences of OsFd4-T1 and OsFd4-T2 to obtain primers OsFd4-T1F and OsFd4-T2F; add AAAC in front of the reverse complementary sequences of OsFd4-T1 and OsFd4-T2 to obtain primers OsFd4-T1R and OsFd4-T2R. The specific sequences are as follows:

[0035] OsFd4-T1F (SEQ ID NO.7): 5’-ggcaATGCAGTTTCCAAGCCTGA-3’,

[0036] OsFd4-T1R (SEQ ID NO.8): 5’-aaacTCAGGCTTGGAAACTGCAT-3’;

[0037] OsFd4-T2F (SEQ ID NO.9): 5’-ggcaGGGCAAGAGCACGAGTTCG-3’,

[0038] OsFd4-T2R (SEQ ID NO.10): 5’-aaacCGAACTCGTGCTCTTGCCC-3’

[0039] (2) Construction of a single gRNA expression cassette:

[0040] SK-gRNA( Figure 1 ) is digested with AarI enzyme (purchased from Ferment company) to form a vector with sticky ends. The enzyme digestion reaction system is as follows:

[0041]

[0042] Digest at 37°C for 3 hours, and purify it using the Biomed Gel Extraction Kit (Biomed, DR0103) according to the product manual; obtain the linear vector SK-gRNA / AarI.

[0043] Mix 20 μL each of 100 μM primers OsFd4-T1F and OsFd4-T1R, place at 100°C for 5 minutes and then at room temperature, and gradually cool to denature and anneal to form a fragment with sticky ends. Ligate the linear vector and the fragment with T4 enzyme (purchased from NEB company). The reaction is as follows:

[0044]

[0045] React at room temperature for 1 hour. Take 5 μL of the ligation product to transform Escherichia coli competent cells DH5α to obtain the ligation plasmid. Use the primer T7 (SEQ ID NO.11) on SK: 5’-TAATACGACTCACTATAGG-3’ to sequence and determine that the clone construction is correct, and obtain the gRNA expression cassette SK-gRNA-1. Similarly, use the annealing product of the primer pair OsFd4-T2F / T2R to ligate with the linear vector SK-gRNA / AarI to obtain SK-gRNA-2.

[0046] (3) Polymerization of SK-gRNA-1 and SK-gRNA-2

[0047] Utilize the property that Bam HI and BglII are isocaudamers to perform the polymerization of the two SK-gRNA intermediate vectors. The SK-gRNA-1 plasmid is digested with BamH I and KpnI, and purified using the Biomed Gel Extraction Kit (Biomed, DR0103) according to the product instructions to obtain the linear vector SK-gRNA-1 / BamH I+KpnI. The SK-gRNA-2 plasmid is digested with BglII and KpnI, and a fragment of about 0.56 kb in size, SK-gRNA-2 / Bgl II+KpnI, is recovered by gel extraction; the gRNA-2 fragment is ligated into the recognition sites between BamH I and KpnI of the SK-gRNA-1 vector to obtain the SK-gRNA-1-gRNA-2 plasmid.

[0048] The vector and the fragment are subjected to a T4 ligase (purchased from NEB) ligation reaction as follows:

[0049]

[0050] React at room temperature for 1 hour. Take 5 μL of the ligation product to transform Escherichia coli competent cells DH5α to obtain the ligation plasmid. Use the universal primers T7 (SEQ ID NO.11): 5’-TAATACGACTCACTATAGG-3’ and T3 (SEQ ID NO.12): 5’-ATTAACCCTCACTAAAGGGA-3’ on SK for colony PCR to detect whether the cloning is successful. When an amplified band of about 1.1 kb is obtained, it is determined that the cloning is successful; otherwise, the cloning is unsuccessful.

[0051] (4) Ligation of the target gRNA expression cassette and the knockout vector pC1300-Cas9

[0052] The SK-gRNA-1-gRNA-2 plasmid was digested with Bgl II and Kpn I, and a band of about 1.1 kb was recovered by gel cutting. This fragment was ligated into the recognition sites between Kpn I and BamH I of the binary vector pC1300-Cas9( Figure 2 ) to obtain the final binary expression vector pC1300-Cas9-SK-gRNA-1-gRNA-2 for knocking out OsFd4.

[0053] The ligation reaction was as follows:

[0054]

[0055] React at room temperature for 1 hour. Take 5 μL of the ligation product and transform it into Escherichia coli competent cells DH5α to obtain the ligation plasmid.

[0056] Use the primer pC1300-F (SEQ ID NO.13): 5’-ACACTTTATGCTTCCGGCTC-3’ and OsFd4-T2R for sequencing to determine that the clone construction is correct. When the sequencing result is consistent with the designed sequence, it is determined that the construction is correct; otherwise, it is incorrect.

[0057] Example 3 Obtaining of OsFd4 knockout mutants

[0058] The correct binary expression vector pC1300-Cas9-SK-gRNA-1-gRNA-2 was transformed into Agrobacterium tumefaciens EHA105 by electroporation. After the transformants were verified to be correct, callus transformation of the japonica rice variety Zhonghua 11 was carried out to obtain OsFd4 gene knockout mutant rice plants. osfd4-1, osfd4-2, and osfd4-3 are transgenic homozygous mutant plants obtained by knocking out the OsFd4 gene in Zhonghua 11 rice.

[0059] Example 4 Verification of OsFd4 knockout mutants

[0060] Primers were designed near the front and back ends of the OsFd4 gene target site. The primer sequences were

[0061] OsFd4-F (SEQ ID NO.14): 5’-CTCTGTTCAAGTTAGAGCA-3’ and OsFd4-R (SEQ ID NO.15): 5’-GCCGACGACATTGAGAGTT-3’. Using the genomic DNA of the T0 generation plants of the OsFd4 knockout mutants and their offspring segregating rice single plants as templates, PCR amplification was carried out. The PCR products were compared by sequencing, and the results are shown in Figure 3 as follows.

[0062] Example 5 Identification of the resistance of OsFd4 knockout mutants to Xanthomonas oryzae pv. oryzae

[0063] The growth phenotypes of ZH11 (wild type), osfd4-1, osfd4-2, and osfd4-3 rice plants grown in the field until maturity were observed. The results are as Figure 4 shown. There were no significant differences in growth and development between the OsFd4 knockout mutants and the wild type. At the peak tillering stage in the field, the above-mentioned rice plants were inoculated with the Xanthomonas oryzae pv. oryzae strain Pxo86 using the leaf tip clipping method. The concentration of the bacterial suspension used for inoculation was OD 600 of 1.0. Fifteen days after the onset of the disease, the resistance differences between the wild type and the OsFd4 knockout mutants were preliminarily evaluated by measuring the lesion lengths. In addition, the resistance differences between the wild type and the mutants were further compared by calculating the number of Xanthomonas oryzae pv. oryzae in the diseased leaves of the same length. The results are as Figure 5 shown. The lesion lengths of the homozygous OsFd4 knockout mutants were significantly shorter than those of the wild type ZH11 ( Figure 5 a and Figure 5 b), and at the same time, the number of pathogenic bacteria in the leaves was also significantly less than that of the wild type ( Figure 5 c).

[0064] Example 6 Identification of the level of reactive oxygen species production induced by flg22 in OsFd4 knockout mutants

[0065] Expand the leaves were cut from 15-day-old ZH11 and osfd4-1 seedlings, and circular leaf tissues with a diameter of 2.5 mm were obtained by punching holes in the same part of the leaves. Then they were placed in a 96-well plate filled with ddH2O overnight. Before the measurement the next day, the reaction solution was prepared: 10 μL of 20 mM Luminal, 5 μL of 5 mg / mL HRP, and made up to 10 mL with ddH2O; after pouring out the ddH2O in the 96-well plate from the previous day, 100 μL of the reaction solution was added to the control group wells, and 100 μL of the reaction solution containing 100 nM flg22 was first added to the experimental group wells; then immediately placed in a Berthold Mithras luminometer for measurement, measured once per minute for 1 hour continuously. The results are as Figure 6 shown. The level of ROS burst in the homozygous OsFd4 knockout mutant osfd4-1 was significantly higher than that of the wild type.

[0066] Example 7 Identification of the level of callose deposition induced by flg22 in OsFd4 knockout mutants In order to observe the effect of OsFd4 gene knockout on the level of callose deposition in rice in response to flg22 treatment, the present invention took the leaves of 7-day-old ZH11 and osfd4-1 rice seedlings and treated them with a final concentration of 100 nM flg22. The specific method is as follows:

[0067] After immersing the leaves of rice seedlings in an aqueous solution of 100 nM flg22 for 30 minutes, they were fixed with a fixative (ethanol: glacial acetic acid = 3:1) for 5 hours (the fixative was changed 3 times during this period). After being treated with 70% and 50% ethanol for 2 hours respectively, they were placed in water overnight. The next day, they were washed 3 times with ddH2O, then treated with 10% NaOH solution for 1 hour to make the tissue transparent, and then washed 4 times with ddH2O. Then, after staining with 0.01% aniline blue solution (dissolved in 150 mM dipotassium hydrogen phosphate, pH 9.5) for 4 hours, callose deposition on the leaves was observed under ultraviolet light, and the number of callose depositions was analyzed and measured by ImageJ (v1.49) software. The results are as Figure 7 shown. The number of callose depositions in the OsFd4 homozygous knockout mutant osfd4-1 was significantly higher than that in the wild type.

[0068] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. Application of knocking out OsFd4 gene in enhancing rice resistance to bacterial blight, characterized in that: The OsFd4 open reading frame nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded thereby is shown in SEQ ID NO.2; The application includes the following steps: in rice OsFd4 Select two target sites in the open reading frame coding region of the gene, and construct OsFd4 a knockout vector, and then OsFd4 transform the knockout vector into Agrobacterium tumefaciens, and obtain OsFd4 knockout mutants by means of Agrobacterium-mediated transformation technology of mature rice embryos.

2. The application according to claim 1, wherein: The sequences of the two target sites are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

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