CN116286866A - Application of rice gene OsFd4 in rice bacterial leaf blight resistance - Google Patents

Abstract

The invention provides a rice geneOsFd4Application in rice bacterial leaf blight resistance. The saidOsFd4The nucleotide sequence of the open reading frame of the gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention adopts CRISPR/Cas9 method to treat rice geneOsFd4Knocking out, and respectively carrying out bacterial leaf blight pathogen inoculation experiments on wild type plants and knock-out mutant plants. The results show that the knockout isOsFd4Obviously improves the active oxygen burst and callose deposition level of rice plants after being treated by bacterial pathogen flagellin short peptide, thereby enhancingResistance of rice to bacterial leaf blight. Meanwhile, the morphological observation of the field plants shows that,OsFd4the knockout mutant has normal growth conditions and no obvious difference compared with the wild type. The above-mentioned results show that,OsFd4is a gene for negatively regulating and controlling the immunity of rice against bacterial leaf blight. The invention provides an endogenous gene target for improving rice resistance through a gene editing technology when field bacterial leaf blight occurs.

Description

Application of rice gene OsFd4 in rice bacterial leaf blight resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a rice gene OsFd4 in rice bacterial leaf blight resistance.

Background

Rice (Oryza sativa) is a staple crop of more than half of the world population and is also a model species for functional studies of crop genomes. Rice is threatened by various diseases in the whole growth period, and the yield stability of the rice is seriously affected. For example, bacterial leaf blight caused by infection with xanthomonas oryzae (Xanthomonas oryzae pv. Oryzae) is the most damaging bacterial disease of rice worldwide, and can lead to up to 50% yield loss when the disease is severe.

The ferredoxin (Fds) family has conserved iron-sulfur [2Fe-2S]A domain, which is distributed in the matrix of the plastid, acts as the most upstream electron carrier, distributing electrons to various receptors of the downstream metabolic reaction pathway. Fds in higher plants can be classified into photosynthetic type and non-photosynthetic type according to their expression pattern. The former receives electrons from chloroplast photosystem I, while the latter uses NADPH generated in the oxidized pentose phosphate pathway as electron donor. When the main Fds in the plastid is defective, the electron transfer chain is blocked, resulting in excess electron transfer to O ₂ Or H ₂ O, forming ROS.

More and more studies have shown that Fds is involved in regulating plant response to various stresses. For example, atFd2 in Arabidopsis occupies about 90% of the total expression amount of Fd in the aerial part, and its deletion results in accumulation of ROS in chloroplasts. AtFd2 knockout mutant (Fd 2-KO) can be better adapted to long-term highlight conditions by inducing photoprotection-related gene expression. Overexpression of exogenous or endogenous Fd-encoding genes in Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) reduces endogenous H ₂ O ₂ And enhances tolerance to heat stress. Similar results were found when the sweet pepper ferredoxin-encoding gene was expressed heterologously in arabidopsis thaliana. Furthermore, the regulatory role of Fds in plant immunity has been explored, e.gThe gene encoding sweet pepper ferredoxin expressed heterologously in arabidopsis thaliana or tobacco can enhance the resistance of transgenic plants to pathogenic bacteria thereof. Further, the results of the study indicate that the Arabidopsis Fd2-KO mutant exhibits higher susceptibility to infection by (semi-) biotrophic pathogens, probably due to the inhibition of salicylic acid signal pathway-mediated resistance by the high accumulation of jasmonic acid and its derivatives in the mutant.

There are 5 typical Fd proteins in rice, designated OsFd1-OsFd5.OsFd1 is the main photosynthetic Fd in rice, and plays an important role in maintaining normal survival of rice seedlings. OsFd4 is the most predominant non-photosynthetic type Fd protein in rice, however the biological function of OsFd4 in rice is still unclear. At present, no report on the function or application of rice Fd protein in disease resistance exists. According to the research of the invention, the CRISPR/Cas9 mediated gene editing technology is adopted to ensure that the rice OsFd4 gene loses function, so that the bacterial leaf blight resistance of rice can be improved, and the normal growth of rice plants is not influenced.

Disclosure of Invention

The invention aims to provide an application of a rice gene OsFd4 in bacterial leaf blight resistance of rice, specifically, a CRISPR/Cas9 method is adopted to knock out the rice gene OsFd4 and further identify the resistance, the negative regulation function of the gene OsFd4 on bacterial leaf blight resistance of the rice is clarified, and an adjustable endogenous gene target is provided for preventing and controlling bacterial leaf blight.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a rice gene OsFd4 has an open reading frame nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the protein coded by the rice gene OsFd4 is shown as SEQ ID NO. 2.

The application of the rice gene OsFd4 in bacterial leaf blight resistance of rice comprises the following specific operations: selecting two target sites in an open reading frame coding region of a rice OsFd4 gene, constructing an OsFd4 knockout vector, then converting the OsFd4 knockout vector into agrobacterium tumefaciens, and obtaining an OsFd4 knockout mutant by means of agrobacterium-mediated rice mature embryo conversion technology; wild-type and OsFd4 knockout mutant plant leaves are treated by using plant pathogenic bacteria flagellin conserved polypeptide flg22, ROS outbreak and callose deposition are detected, and the resistance of the osFd4 mutant to bacterial blight pathogenic bacteria is further verified.

The nucleotide sequences of the two target sites are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

The application of the rice gene OsFd4 in resisting bacterial leaf blight of rice, and the knockout of the gene OsFd4 obviously improves the resistance of rice to bacterial leaf blight; and the OsFd4 knockout mutant responds to the flg22 treatment, and the active oxygen burst level and the callose deposition quantity are higher than those of the wild type.

The invention has the following beneficial effects:

after the rice gene OsFd4 is knocked out, compared with wild rice, the capability of resisting bacterial leaf blight is obviously improved, and the gene provides an adjustable endogenous gene target for preventing and controlling bacterial leaf blight.

Drawings

Fig. 1: construction of OsU3:gRNA in the intermediate vector SK-gRNA.

Fig. 2: 2X 35S in binary vector pC1300-Cas 9: structure of Cas 9.

Fig. 3: identification of OsFd4 knockout mutants. Reference: is a reference gene sequence; the OsFd4-1, osFd4-2 and OsFd4-3 are homozygous mutant lines of the OsFd4 gene.

Fig. 4: and (5) performing field growth phenotype identification on the OsFd4 knockout mutant. ZH11: flower 11 in wild rice variety; osfd4-1, osfd4-2 and osfd4-3: homozygous mutant plants of OsFd4 gene; length of scale: 10cm.

Fig. 5: the OsFd4 knockout mutant was inoculated with bacterial leaf blight pathogen strain Pxo86 findings. ZH11: flower 11 in wild rice variety; osfd4-1, osfd4-2 and osfd4-3: homozygous mutant plants of OsFd4 gene. a, inoculating bacterial leaf blight pathogen, and then, curing disease leaves (length of scale: 3 cm) after 15 days; b, counting the length of the lesion; and c, counting the number of the white leaf mold in the diseased leaves with the same length.

Fig. 6: flg22 induced OsFd4 knockout mutants to produce a test result of reactive oxygen species levels. flg22: plant pathogenic bacteria flagellin conserved polypeptides; h ₂ O: water control treatmentThe method comprises the steps of carrying out a first treatment on the surface of the ZH11: flower 11 in wild rice variety; osfd4-1: homozygous mutant plants of OsFd4 gene.

Fig. 7: flg22 induced OsFd4 knockout mutant callose detection results. ZH11: flower 11 in wild rice variety; osfd4-1: homozygous mutant plants of OsFd4 gene; length of scale: 30 μm.

The specific embodiment is as follows:

the present invention will be described in further detail with reference to the following embodiments.

Example 1 obtaining of OsFd4 Gene

By using the protein sequence of the Arabidopsis thaliana ferredoxin AtFd2 in Arabidopsis thaliana to carry out sequence alignment in NCBI database, we determine that the homologous protein of AtFd2 in rice with the gene number of LOC_Os03g61960 (OsFd 4) has the similarity of 68 percent. The LOC_Os03g61960 gene encodes a protein 153 amino acids long and has a chloroplast localization signal peptide at its N-terminus. Based on the predicted cDNA sequence (XM_ 015774279.1) of the gene in the genome of Nippon Temminck variety on NCBI website, we succeeded in cloning the open reading frame of OsFd4 from the total cDNA of Chinese flower 11 leaves, and found that the full length is 462bp, and the sequence is consistent with the predicted sequence. The nucleotide sequence of the primers used to clone the OsFd4 open reading frame was as follows:

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

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

the nucleotide sequence of the open reading frame of the OsFd4 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

EXAMPLE 2 construction of OsFd4 knockout vector

Two target sites (the sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4) are selected from an open reading frame coding region of an OsFd4 gene to construct a gRNA expression cassette, and then the two target gRNA expression cassettes are connected into a knockout vector pC1300-Cas9 to obtain the OsFd4 knockout vector. The detailed vector construction method refers to the construction and application of a plant polygene knockout vector, ZL201510485573.2, and specifically comprises the following steps:

(1) Selection of target sequences and primer design

On OsFd4 groupThe coding region of the open reading frame contains 5' - (N) _X -a target sequence of NGG-3' structure, wherein N represents any one of A, T, C and G and X is 19. Two target sequences, namely OsFd4-T1 and OsFd4-T2, are designed according to the principle, and the sequences are as follows:

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

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

primer pairs were designed for construction of gRNA: GGCA is added before forward sequences of OsFd4-T1 and OsFd4-T2 to obtain primers OsFd4-T1F and OsFd4-T2F; AAAC is added before the reverse complementary sequences of the OsFd4-T1 and the OsFd4-T2 to obtain primers OsFd4-T1R and OsFd4-T2R. The specific sequence is as follows:

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

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

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

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

(2) Construction of a single gRNA expression cassette:

SK-gRNA (FIG. 1) was digested with AarI (available from Ferment Inc.) to form a vector with a sticky end, and the cleavage reaction was performed as follows:

digestion at 37℃for 3 hours, purification was performed using a Biomed gel recovery kit (Biomed, DR 0103) according to the product specifications; obtaining the linear vector SK-gRNA/AarI.

100. Mu.M of the primer OsFd4-T1F was mixed with 20. Mu.L of each of OsFd4-T1R, left at 100℃for 5 minutes, and then left at room temperature, gradually cooled, and denatured and annealed to form a fragment having a cohesive end. The linear vector and fragment were subjected to T4 enzyme ligation (purchased from NEB company) as follows:

the reaction was carried out at room temperature for 1 hour. Ligation product 5. Mu.L of transformed E.coli competent cells DH 5. Alpha. Were used to obtain a ligation plasmid. Primer T7 on SK (SEQ ID NO. 11): 5'-TAATACGACTCACTATAGG-3', sequencing to determine that the cloning construction is correct, and obtaining the gRNA expression cassette SK-gRNA-1. The annealed product of the primer pair OsFd4-T2F/T2R is connected with a linear carrier SK-gRNA/AarI to obtain SK-gRNA-2.

(3) Polymerization of SK-gRNA-1 and SK-gRNA-2

Polymerization of two SK-gRNA intermediate vectors was performed using BamHI and BglII as isotail enzymes. The SK-gRNA-1 plasmid was digested with BamHI and KpnI, and purified using a Biomed gel recovery kit (Biomed, DR 0103) according to the product specifications to give the linear vector SK-gRNA-1/BamHI+KpnI. The SK-gRNA-2 plasmid is digested with BglII and KpnI, and the SK-gRNA-2/BglII+KpnI fragment with the size of about 0.56kb is recovered by gel cutting; the gRNA-2 fragment was ligated between BamHI and KpnI recognition sites of the SK-gRNA-1 vector to obtain a SK-gRNA-1-gRNA-2 plasmid.

The vector and fragment were subjected to the T4 enzyme (purchased from NEB company) ligation reaction as follows:

the reaction was carried out at room temperature for 1 hour. Ligation product 5. Mu.L of transformed E.coli competent cells DH 5. Alpha. Were used to obtain a ligation plasmid. Cloning was tested for success using the universal primers T7 (SEQ ID NO. 11): 5'-TAATACGACTCACTATAGG-3' and T3 (SEQ ID NO. 12): 5'-ATTAACCCTCACTAAAGGGA-3' on SK. When the amplification gave a band of about 1.1kb, the cloning was judged to be successful; otherwise, the cloning was unsuccessful.

(4) Ligation of target gRNA expression cassette and knockout vector pC1300-Cas9

The SK-gRNA-1-gRNA-2 plasmid is digested with Bgl II and Kpn I, the band with the size of about 1.1kb is recovered by cutting gel, and the band is connected between Kpn I and BamH I recognition sites of pC1300-Cas9 (figure 2) binary vector, so as to obtain the binary expression vector pC1300-Cas9-SK-gRNA-1-gRNA-2 with the final OsFd4 knocked out.

The ligation reaction was as follows:

the reaction was carried out at room temperature for 1 hour. Ligation product 5. Mu.L of transformed E.coli competent cells DH 5. Alpha. Were used to obtain a ligation plasmid.

Primer pC1300-F (SEQ ID NO. 13) was used: 5'-ACACTTTATGCTTCCGGCTC-3', osFd4-T2R sequencing confirmed that the clone was constructed correctly. When the sequencing result is aligned with the design sequence, judging that the construction is correct; otherwise, the construction is incorrect.

Example 3 obtaining of OsFd4 knockout mutant

The binary expression vector pC1300-Cas9-SK-gRNA-1-gRNA-2 with correct sequencing is transformed into agrobacterium tumefaciens EHA105 by an electric shock method, and after the transformant is verified to be correct, the transformation of the flower 11 callus in the japonica rice variety is carried out to obtain the OsFd4 gene knockout mutant rice plant. OsFd4-1, osFd4-2 and OsFd4-3 are transgenic homozygous mutant plants obtained by knocking out the OsFd4 gene in medium flower 11 rice.

Example 4 verification of OsFd4 knockout mutant

Primers are designed near the front end and the rear end of the OsFd4 gene target site, and the sequences of the primers are respectively as follows

OsFd4-F (SEQ ID NO. 14): 5'-CTCTGTTCAAGTTAGAGCA-3' and OsFd4-R (SEQ ID NO. 15): 5'-GCCGACGACATTGAGAGTT-3' the genomic DNA of the T0 generation plant of the OsFd4 knockout mutant and the rice single plant isolated from the progeny thereof is used as a template for PCR amplification, and the result of the PCR product is shown in figure 3 after sequencing comparison.

Example 5 identification of bacterial leaf blight pathogen inoculated with OsFd4 knockout mutant

The observation of growth phenotypes was performed on ZH11 (wild type), osFd4-1, osFd4-2 and OsFd4-3 rice plants grown in the field to maturity, and as shown in FIG. 4, the OsFd4 knockout mutant was not significantly different in growth and development from the wild type. In the field tillering stage, the rice plant is inoculated with bacterial strain Pxo86 by blade tip shearing method, and the bacterial liquid concentration used in inoculation is OD ₆₀₀ 1.0. 15 days after onset, wild type and Os were treated by measuring lesion lengthThe differences in resistance of Fd4 knockout mutants were initially assessed. Furthermore, the difference in resistance between wild type and mutant was further compared by counting the number of bacterial wilt bacteria contained in diseased leaves of the same length. As a result, as shown in FIG. 5, the length of the lesion of the homozygous knockout mutant of OsFd4 was significantly shorter than that of the wild type ZH11 (FIGS. 5a and 5 b), and the number of pathogenic bacteria contained in the leaf was significantly smaller than that of the wild type (FIG. 5 c).

Example 6 identification of the level of active oxygen production by flg 22-induced OsFd4 knockout mutants

Cutting the stretched leaf from 15-day-old ZH11 and osfd4-1 seedling, punching the same part of the leaf to obtain round leaf tissue with diameter of 2.5mm, and placing into ddH ₂ O in 96-well plates overnight; the reaction solution was prepared before the next measurement: 10. Mu.L of 20mM Luminal, 5. Mu.L of 5mg/mL HRP, with ddH ₂ O constant volume to 10mL; ddH was cleared from the previous day in 96-well plates ₂ After O, 100. Mu.L of reaction solution was added to the control wells, and 100. Mu.L of reaction solution containing 100nM flg22 was added to the experimental wells; the measurement was then carried out immediately in a Berthold Mithras luminometer instrument, 1 time per minute, for 1 hour. As a result, as shown in FIG. 6, the level of ROSFd 4 burst in the homozygous knockout mutant OsFd4-1 was significantly higher than that in the wild type.

Example 7 flg22 Induction of OsFd4 knockout mutant leaf callus deposition levels identification to observe the effect of OsFd4 gene knockout on callus deposition levels generated in response to flg22 treatment of rice, the present invention resorts to stripping 7-day-old ZH11 from leaves of OsFd4-1 rice seedlings and treating with 100nM flg22 final concentration. The specific method comprises the following steps:

after immersing rice seedling leaves in 100nM flg22 in water for 30 minutes, they were fixed with a fixing solution (ethanol: glacial acetic acid=3:1) for 5 hours (3 fixing solutions were replaced). After 2 hours of treatment with 70% and 50% ethanol, respectively, the mixture was left in water overnight. The next day, use ddH ₂ O-washing 3 times, treating with 10% NaOH solution for 1 hr to make the tissue transparent, and then using ddH ₂ O was washed 4 times. Then, after 4 hours of staining with 0.01% aniline blue solution (in 150mM dipotassium hydrogen phosphate, pH 9.5), callose deposition on leaves was observed under UV light and analyzed by imageJ (v 1.49) softwareThe number of callose deposits was determined. As shown in FIG. 7, the amount of callose deposited in the OsFd4 homozygous knockout mutant OsFd4-1 was significantly higher than that in the wild type.

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (3)

1. Rice plantOsFd4The application of the gene in enhancing the resistance of rice to the white leaf blight is characterized in that: the saidOsFd4The nucleotide sequence of the open reading frame of the gene is shown as SEQ ID NO. 1; the coded amino acid sequence is shown as SEQ ID NO. 2.

2. The use according to claim 1, characterized in that: the method comprises the following steps: in riceOsFd4Two target sites are selected from the open reading frame coding region of the gene to constructOsFd4Knocking out the vector, and thenOsFd4The knocked-out vector is transformed into agrobacterium tumefaciens, and is obtained by means of agrobacterium-mediated rice mature embryo transformation technologyOsFd4The mutant is knocked out.

3. The use according to claim 2, characterized in that: the sequences of the two target sites are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

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