Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The test materials used in the examples described below, unless otherwise specified, are all commercially available.
The LRK20 gene has a sequence shown in SEQ ID NO.1, and ：ATGTCTTTCATGCTTCTTCTTCTTCTCCTGCTCTCCCTCATCCTCAACCTTGCATCCCCGACCACCGCCGCTTCCGGCGACGGCAACGGCGACCAGTTCATCTACTCCGGCTTCCATGGCTCCAACCTCACCGTCGACGGCGCCGCCTCCATCACACCGGACGGCCTCCTCCAGCTAACCGACGGCGCAGCCTACCTCAAAGGCCACGCGTTCCACCCATCTCCGGTGCGCCTCCGCCGCGACGTCTCCACGTCCACGACGACGACCACCGTCCGCTCCTTCTCCGTCACCTTCGTCTTCGGCATCGTCTCCGTCTACCCCGACTTCAGCGCCCATGGCATGGCCTTCGTCGTCTCCCCGACAACCAACCTCTCCTCCTCCTTGCCGGCCAAGTACCTCGGCCTCACCAACGTCCAGAACGACGGCAACGCCAGCAACCACATGCTCGCCGTCGAGCTCGACACCATCCAGAGCGTCGAGTTCAGGGACATCAACGCCAACCACGTCGGCGTCGACATCAACGGCCTCCAGTCCGTCCGCGCCTACAACGCCGGCTACTACGACGACGTCTCCGGCGAGTTCAGGAGCTTGAAGCTGATCAGCCGCCAGGCGATGCAGGTCTGGGTGGACTACCATGGCGGCGAGAAGAAGCAGCTCGACGTCACCATGGCTCCTCTCAGAATGGCCAGGCCTGTCAAGCCGCTGCTCTCGGTCACCCACGACCTCTCCACGGTGCTCGCCGACGTCGTCTACCTCGGCTTCTCGGCGGCGACCGGCCGCGTGAATTCGCGCCACTGCGTTCTTGGCTGGAGCTTGGGGATCAACGGACCAGCTCCCGCCATTGACATCGACAAGCTGCCCAAGCTTCCTCGTGCTGAACCCAAGCCAAGATCCAGGGTCCTTGAAATTGTTCTACCAATTGTCACTGCAACGATTGTGCTGGTTGTGGGTGGCGCCATTGTTATGGTGGTGAGGAGGAGATCGAGGTACGCCGAGCTACGGGAAGATTGGGAGGTTGAGTTTGGGCCGCATCGATTCTCGTACAAGGAATTGTTCCGCGCAACCGATGGATTTGCGGATAAGCATCTACTCGGTTCAGGAGGATTTGGAAAGGTGTACCGAGGAGTGCTTCCTAAATCTAAATTGGAGGTTGCCGTGAAGAAAGTGTCCCACGAATCGAGACAAGGGATGAAGGAGTTTGTTGCTGAGATAGTTAGTATTGGACGTATTCGGCACCGTAATCTTGTGCAGTTGCTCGGTTACTGTCGACGGAAGGGTGAGCTTCTTTTGGTGTATGCGTATATACCAAATGGTAGCCTCGATAAATATCTGTATAGCGAGGAGGATAAGCCTATACTGAGTTGGGCTCAAAGGTTCCGAATCATTAAAGGCATCGCATCTGGTTTGCTCTACTTGCACGAGAGGTGGGAAAAAGTGGTAGTGCACCGAGACATCAAGGCAGGCAACATACTCCTCGACAAGGACATGAACGGACAGCTAGGTGACTTTGGCTTGGCAAGACTATACGACCATGGCACGGACTCACAAACCACACATGTGGTTGGCACCATGGGATACCTAGCACCAGAGCTCATTCGCACGGGCAAAGCATCCCCTCTCACCGATGTGTTCGCGTTCGGCGTGTTCCTCCTCGAGGTCACCTGTGGGCAGAAACCTATCAAGGAGAAGAACCCTCAAGGTAGCCACATAGCGCTGGTCGACTGGGTGCTCGAGCATTGGCGCGACGGTTCACTCATGGACACGGTGGACGGGAGGCTCCACGGCGAGTACGACGCCGGTGAAGCGGCGCTGGTGCTGAAGCTGGGCCTCCTGTGCTCGCACCCGTTCGCCGCGGCGAGGCCCGGGATGGGGCAGGTCACCTGCTGCCTCGCCGGCGAGGCGCCGCTGCCTGAGCTGACGCCGGCGGACATGGGCTTCGACGTGCTGGCCATGATGCAGGATAAGGGATTCGACACGTCCGTCGTGTCGTATCCCGATAG, disease resistance is sheath blight resistance.
The amino acid sequence of the protein coded by the LRK20 gene is shown as SEQ ID NO.2 ：MSFMLLLLLLLSLILNLASPTTAASGDGNGDQFIYSGFHGSNLTVDGAASITPDGLLQLTDGAAYLKGHAFHPSPVRLRRDVSTSTTTTTVRSFSVTFVFGIVSVYPDFSAHGMAFVVSPTTNLSSSLPAKYLGLTNVQNDGNASNHMLAVELDTIQSVEFRDINANHVGVDINGLQSVRAYNAGYYDDVSGEFRSLKLISRQAMQVWVDYHGGEKKQLDVTMAPLRMARPVKPLLSVTHDLSTVLADVVYLGFSAATGRVNSRHCVLGWSLGINGPAPAIDIDKLPKLPRAEPKPRSRVLEIVLPIVTATIVLVVGGAIVMVVRRRSRYAELREDWEVEFGPHRFSYKELFRATDGFADKHLLGSGGFGKVYRGVLPKSKLEVAVKKVSHESRQGMKEFVAEIVSIGRIRHRNLVQLLGYCRRKGELLLVYAYIPNGSLDKYLYSEEDKPILSWAQRFRIIKGIASGLLYLHERWEKVVVHRDIKAGNILLDKDMNGQLGDFGLARLYDHGTDSQTTHVVGTMGYLAPELIRTGKASPLTDVFAFGVFLLEVTCGQKPIKEKNPQGSHIALVDWVLEHWRDGSLMDTVDGRLHGEYDAGEAALVLKLGLLCSHPFAAARPGMGQVTCCLAGEAPLPELTPADMGFDVLAMMQDKGFDTSVVSYPDLMTSFGTISSLSGGR.
The rice LRK20 gene is knocked out by CRISPR/Cas9, so that the sheath blight resistance is reduced, the disease susceptibility is enhanced, and the sheath blight resistance is increased and the disease susceptibility is reduced by over-expressing the rice LRK20 gene.
A method for culturing high-disease-resistance rice includes over-expressing LRK20 gene or increasing protein content in application to raise the resistance of plant to sheath blight and reduce disease susceptibility. The over-expression process includes amplifying CDS sequence of LRK20 gene, recombining and connecting to pU1301-GFP vector, colibacillus transformation and sequencing to verify, extracting plasmid and mediating into rice. The sequences of the adaptor primer pair of pU1301-GFP vector are shown as SEQ ID NO. 3: CGAACGATAGCCGGTACCATGTCTTTCATGCTTCTTCT and SEQ ID NO. 4: GTCGACTCTAGAGGATCCTCGGGATACGACACGA.
Gene knockout vector pOs-Cas9-LRK20 for regulating rice sheath blight resistance gene LRK20. The construction method comprises the steps of designing and synthesizing sgRNA 5'-TCTACCTCGGCTTCTCGGCGG-3' (SEQ ID NO. 5) for identifying target sites, adding a joint ：LRK20-Cas9-F：5'-TGTGGTCTACCTCGGCTTCTCGG-3'(SEQ ID NO.6),LRK20-Cas9-R：5'-AAACCCGAGAAGCCGAGGTAGAC-3'(SEQ ID NO.7). into 18 base sequences before CGG, annealing the base sequences through a primer to obtain a PCR product, connecting the PCR product with an enzyme-digested linear vector pOs-Cas9, and carrying out transformation and sequencing verification through escherichia coli to finally obtain a gene editing vector pOs-Cas9-LRK20.
When the gene of the present invention is constructed into a plant expression vector, any one of a general promoter, an enhanced promoter or an inducible promoter such as a cauliflower mosaic virus (CAMV) 35S promoter, a Ubiquitin promoter (Ubiquitin) of maize, which can be used alone or in combination with other plant promoters, can be added before the transcription initiation nucleotide thereof, and in addition, when the plant expression vector is constructed using the gene of the present invention, enhancers including a translation enhancer or a transcription enhancer can be used, and these enhancer regions can be ATG initiation codon or adjacent region initiation codon, etc., but must be the same as the reading frame of the coding sequence to ensure proper translation of the whole sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.
In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which can produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin markers, kanamycin markers, etc.), etc., which can be expressed in plants. The expression vector of the gene can be used for transforming plant cells or tissues by using agrobacterium mediation, ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, conductance and other conventional biological methods, and the transformed plant tissues are cultivated into plants.
In the examples, the genetic transformation material of rice is provided for the rice germplasm resource pool preservation of the sheath blight-sensing variety NIP (Nippon university).
EXAMPLE 1 identification and cloning of the LRK20 Gene
In order to screen the sheath blight resistance genes, analysis of transcriptome data (Cao et al, plant Biotechnology Journal,2021,20,335-349.) of sheath blight bacteria inoculated at the early stage lemont revealed that the expression levels of a plurality of L-type LecLRK genes in LecLRK gene families were significantly different before and after inoculation, and the LRK20 gene was significantly up-regulated by sheath blight bacteria induction. Further, the invention performs knockout and overexpression studies on the same.
A cloning method of LRK20 gene, comprising the steps of:
the genome of the sheath blight variety NIP is used as a reference sequence, and a PCR amplification primer of the LRK20 gene is designed:
LRK20-F:5’-ATGTCTTTCATGCTTCTTCT-3’(SEQ ID NO.8),
LRK20-R:5’-CTATCGGGATACGACACGA-3’(SEQ ID NO.9)。
PCR amplification is carried out by taking cDNA of a sheath blight-sensing variety NIP as a template, and the PCR amplification method refers to a Invozan Phanta Max Super-FIDELITY DNA Polymerase high-fidelity enzyme using instruction. The PCR amplified product (Tiangen ordinary agarose gel DNA recovery kit DP 209) is recovered and purified, and then the correct LRK20 gene CDS sequence is obtained by sequencing (Optimus in the field of the prior art).
The nucleotide sequence of the rice LRK20 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEO ID NO. 2.
Example 2 characterization of expression of LRK20 Gene
NIP is cultivated in a normal field environment until the end of tillering is inoculated with Rhizoctonia solani, and the strain is medium-strength pathogenic strain YN-7 provided by plant protection line of Yangzhou university. Inoculating by embedding method (Pan Xuebiao, university of Jiangsu university of agricultural school, 1997, (03): 28-33), carefully embedding wood bark (with length of 1cm and width of 2mm, which is adhered with Rhizoctonia solani mycelium, onto leaf sheath of 1cm-2cm under two inverted leaves of rice, cutting leaf sheath tissue of rice in 1cm section above and below inoculum respectively before (0 h) and after (9 h, 12h, 24h and 48 h), and freezing with liquid nitrogen. The experimental procedures of RNA extraction, reverse transcription and qRT-PCR are the same as the steps for determining the expression level of rice tissues.
Taking RN A samples of different tissues (roots, stems, leaves, leaf sheaths and ears) of a rice susceptible variety NIP booting stage, and extracting total RNA of each rice sample by adopting a Trizol method. By means ofIIIRT Super Mix for qPCR (Nanjinopran Biotech Co., ltd.) reverse transcriptase synthesizes first strand cD NA, and the synthesis is performed according to the description of its reverse transcriptase. Using the above cDNA of different tissues as a template, the OsActin gene of rice as an internal reference gene, osActin gene quantitative primers qActin-F5'-CTAA GCCAAGAGGAGCTGTTAT-3' (SEQ ID NO. 10) and qActin-R5'-ATAAC AGATAGGCCGGTTGAAA-3' (SEQ ID NO. 11), and LRK20 gene-specific quantitative primers qLRK-F5'-CTCGCTTTCTGGGAAACAACAGC-3' (SEQ ID NO. 12) and qLRK-R5'-CAATGTGCCGCTGCTTGAAAGG-3' (SE Q ID NO. 13), real-time quantitative PCR was performed to detect the expression level of the LRK20 gene in different tissues of rice. The qRT-PCR reaction system is as follows: QPCR MASTER Mix (Nanjinouzan Biotechnology Co., ltd.) 5. Mu.L, 0.5. Mu.L (10. Mu. Mol/L) each of the front and rear primers, 5. Mu.L (100 ng/. Mu.L) of the cDN A template. qRT-PCR was performed using a Bio-Rad CFX96 TouchTM fluorescent quantitative PCR apparatus under conditions of 95℃pre-denaturation for 3min, 95℃denaturation for 15s,60℃annealing extension for 30s,40 cycles. The expression value data are subjected to conversion analysis by a 2-delta ct method. The reaction conditions were 95℃30sec,95℃10sec,60℃10sec, 40 cycles of reaction, and 95℃15sec,60℃95℃15sec, dissolution curves were read.
As shown in FIG. 1A, the LRK20 gene starts to be up-regulated 12 hours after infection with Rhizoctonia solani, reaches the highest value for 24 hours, and then starts to fall back for 48 hours. As shown in FIG. 1B, the LRK20 gene is expressed predominantly in roots, stems, leaves and leaf sheaths, and relatively low in spikes.
Example 3 construction of vector, genetic transformation and detection
1. The construction method of the CRISPR/Cas9 editing vector for the rice LRK20 gene comprises the following steps:
(1) The target site is knocked out with high selection efficiency, and the sgRNA for identifying the target site is designed and synthesized, and the sequence is sgRNA 5'-TCTACCTCGGCTTCTCGGCGG-3' (SEQ ID NO. 5). The linker was added to the first 18 base sequences of CGG as follows ,LRK20-Cas9-F：5'-TGTGGTCTACCTCGGCTTCTCGG-3'(SEQ ID NO.6),LRK20-Cas9-R：5'-AAACCCGAGAAGCCGAGGTAGAC-3'(SEQ ID NO.7).
(2) Intermediate products are obtained by primer annealing to introduce a knockout target of LRK20, wherein the reaction system and conditions are that the front primer (10 mu M) is 1 mu L, the rear primer (10 mu M) is 1 mu L, the Anneal Buffer (1xTE buffer,50mM NaCl) is 8 mu L, the temperature is 95 ℃ for 10min, and the temperature is 0.1 ℃ per sec and is reduced to 20 ℃.
(3) The specific cleavage site was linearized by cleavage with the restriction enzyme BsaI (Takara) on pOs-Cas9 vector under the conditions of 1. Mu.g of plasmid, 5. Mu.L of 10 Xbuffer, 1. Mu.L of BsaI, and 50. Mu.L of ddH2 O were filled up to 37℃for 2-3 hours and recovered according to the DNA purification kit (Tiangen ordinary agarose gel DNA recovery kit DP 209).
(4) And connecting the annealed intermediate product with the pOs-Cas9 linear carrier after enzyme digestion, wherein the connecting reaction system and the conditions are that the annealed product is 2 mu L, the linear carrier is 2 mu L,10x T4 DNALigase Buffer 0.5 mu L, T4 DNALIGASE is 0.5 mu L and the room temperature is 15min.
(5) E.coli DH5 alpha is transformed, after DH5 alpha is melted, the connection product of the previous step is added, the mixture is subjected to ice bath for 30min, heat shock is carried out at 42 ℃ for 90sec, the mixture is placed on ice for 2min, 700 mu L of LB liquid medium without antibiotics is added, the mixture is cultured for 1h at 37 ℃ and 200rpm, the mixture is centrifuged for 1min at 5000rpm, most of the supernatant is discarded, 100 mu L of liquid is reserved, the mixture is sucked and evenly mixed, the mixture is coated on an LB solid medium plate containing kanamycin, and the mixture is cultured for 12h at 37 ℃.
(6) Selecting a monoclonal, placing the monoclonal into a liquid LB liquid culture medium containing kanamycin, culturing for 6 hours at 37 ℃, and carrying out bacterial liquid PCR reaction according to a conventional PCR amplification instruction of Novain 2X TAQ MASTER Mix (Dye Plus), wherein the primer sequences of the PCR reaction are as follows:
M13F:5’-TGTAAAACGACGGCCAGT-3’(SEQ ID NO.14),
LRK20-Cas9-R:5'-AAACCCGAGAAGCCGAGGTAGAC-3'(SEQ ID NO.15),
amplifying the positive monoclonal bacteria liquid, extracting plasmid and carrying out sequencing verification, wherein the plasmid extraction kit is (NovainPLASMID MINI KIT), sequencing Co-Optima Prime biotechnology Co Ltd.
(7) The recombinant positive plasmid is transferred into agrobacterium EHA105, and the reaction system and conditions are 50 mu LEHA105 competent cells, 1 mu L recombinant plasmid, ice bath for 5min, liquid nitrogen quick freezing for 5min,37 ℃ water bath for 5min, and ice bath for 5min. 700. Mu.L of LB liquid medium without antibiotics was added, cultured at 28℃for 2h at 200rpm, centrifuged at 5000rpm for 1min, most of the supernatant was discarded, 100. Mu.L of liquid was left to be sucked and mixed uniformly, and spread on LB solid medium plates containing kanamycin and rifampicin antibiotics, and cultured at 28℃for two days.
(8) Selecting a monoclonal, placing the monoclonal into a liquid LB culture medium containing kanamycin and rifampicin, culturing for two days at a temperature of 28 ℃, and performing bacterial liquid PCR reaction, wherein the PCR reaction is the same as that of the step (6). The positive monoclonal bacterial liquid can be used for the subsequent rice genetic transformation experiment.
2. The construction steps of the super-expression vector are as follows:
(1) The CDS sequence of the LRK20 gene is taken as a template, primers with pU1301-GFP carrier joints are designed and synthesized, the primer sequences are LRK20_OE-F CGAACGATAGCCGGTACCATGTCTTTCATGCTTCTTCT (SEQ ID NO. 3) and LRK20_OE-R GTCGACTCTAGAGGATCCTCGGGATACGACACGA (SEQ ID NO. 4), and the LRK20 gene CDS sequence is amplified by using high-fidelity enzyme (Novain Phanta Max Super-FIDELITY DNA Polymerase high-fidelity enzyme).
(2) The overexpression vector pU1301-GFP was digested with restriction enzymes KpnI and BamHI (Takara), linearized, and the digestion reaction system and conditions were the same as in step 1 (3).
(3) By homologous recombination enzymes (NorvainIIOne Step Cloning Kit) connecting the LRK20 gene CDs with linearization vectors pU1301-GFP to obtain recombinant plasmids pU1301-LRK20-GFP.
3. And (3) sending the constructed knocked-out and overexpressed vector agrobacterium tumefaciens liquid to a non-rice biotechnology company to carry out genetic transformation on rice, wherein the acceptor rice variety is Nippon.
4. And (5) detecting transgenic rice plants.
(1) For the knocked-out plants, primers are designed and synthesized on two sides of the knocked-out target point, the obtained T0 transgenic plants are sequenced, and the sequencing primer sequences are as follows:
LRK20-CXF:ATGCAGGTCTGGGTGGACTACCAT(SEQ ID NO.16),LRK20-CXR:GCTCGGCGTACCTCGATCTCCT(SEQ ID NO.17)。
As shown in FIG. 2A, there are 3 types of variation in the transgenic knockout line in which lrk20-ko1 is inserted with base G, lrk20-ko2 is inserted with base C, and lrk20-ko3 is deleted with base C. All three mutation types resulted in frame shift mutations of the LRK20 protein (fig. 2B).
(2) Aiming at the over-expression plants, western Blot detection is firstly carried out on all T0 single plants by using GFP antibodies (figure 2C), then RNA levels of two single plants with higher protein level expression of LRK20-OE#1 and LRK20-OE#3 are further detected, as shown in figure 2D, compared with a wild type, the expression quantity of LRK20 genes of two over-expression lines is obviously improved. Wherein the primer sequences of qRT-PCR are qLRK20-F:5'-CTCGCTTTCTGGGAAACAACAGC-3' (SEQ ID NO. 12), and qLRK-20-R: 5'-CAATGTGCCGCTGCTTGAAAGG-3' (SEQ ID NO. 13).
Example 4 identification of sheath blight resistance of transgenic Rice plants
The wild type (WT, NIP), knockout line (LRK 20-ko1, LRK20-ko2, LRK20-ko 3) and over expression line (LRK 20-OE #1, LRK20-OE # 3) of the gene were identified for banded sclerotial blight resistance by using an artificial climate chamber in vitro resistance identification method.
The method for identifying the in vitro resistance of the artificial climate chamber comprises the following steps:
Selecting a main stem with consistent growth vigor and inverted two leaf sheaths in the booting stage, cutting the stem from the base, pruning the stem, reserving the inverted one leaf for 3cm, removing the rest leaves, reserving the length of the stem to the position 1cm below the inverted two leaf sheaths, immersing the stem in water, and placing the stem into a climatic chamber for pre-culture for 24 hours. A cuboid framework with the length of 55cm and the length of 75cm is built in a climatic chamber, a bottom basin with the length of 60cm and the length of 80cm is placed at the bottom of the cuboid framework, water is stored in the basin, a timing humidifier is installed, the cuboid framework covers a polyester film, a moisturizing space is formed, and the humidity of the moisturizing space is maintained at 75% -90%. The cultivated bark is placed at a position 1cm below the leaf sheath of the inverted leaf, the stem is inserted into the flower mud, and the flower mud is placed in a cultivation room of a manual climate removing room for cultivation for 7d. Not less than 10 stems were inoculated per material. The lighting of the greenhouse is set to be 14h lighting and 10h darkness day-night alternation, the temperature is set to be 30 ℃ in the daytime and 24 ℃ in the evening, and a spray humidifier is arranged in the greenhouse, so that the whole greenhouse can be controlled to be moderately between 75% and 90%. The culture medium in the flower mud is changed every two days during the culture period. The difference of the sheath blight disease spots among different materials can be seen 7d after inoculation, and the length of the disease spots of the materials is investigated.
As shown in FIG. 3A, the average lesion length of each of the 3 transgenic knockout lines LRK20-ko1, LRK20-ko2 and LRK20-ko3 was significantly higher than that of the wild-type NIP, while the average lesion length of each of the 2 transgenic overexpressing lines LRK20-OE #1 and LRK20-OE #3 was significantly lower than that of the wild-type NIP (FIG. 3B). The result shows that the LRK20 gene positively regulates the rice sheath blight resistance, and the sheath blight resistance of rice can be obviously improved by over-expressing the gene.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.