CN118240838A - Novel rice disease-resistant gene OsBRH1 capable of efficiently utilizing resources - Google Patents

Abstract

The invention provides a novel rice blast resistance gene OsBRH1 for efficiently utilizing resources, which has positive regulation and control effects in rice blast resistance reaction. Knocking OsBRH out the rice by a genetic engineering method can reduce the resistance of the rice to the rice blast fungus; in contrast, overexpression OsBRH1 promotes rice resistance to Pyricularia oryzae. The gene responds to the immune process by promoting accumulation of hydrogen peroxide and activating expression of defense related genes OsPR10a and OsPR3, and improves the defense capacity of rice. The invention can make up the deficiency of the existing rice blast resistance gene, can improve the resistance of rice to rice blast when applied to rice, and accumulates precious gene resources and theoretical basis for breeding new rice varieties resistant to rice blast.

Description

Novel rice disease-resistant gene OsBRH1 capable of efficiently utilizing resources

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice blast resistance gene OsBRH1, a recombinant vector, a recombinant engineering bacterium and a functional identification method of a gene OsBRH for efficient resource utilization.

Background

Rice (Oryza sativa l.) is a staple food for more than half of the world population, providing a direct energy intake of about 20% of humans worldwide, and is also a scientific model for cereal crops and other individual crops. Thus, rice is one of the most important grains on earth. However, with the rapid growth of the world population, the yield of rice is difficult to ensure normal demand. Meanwhile, diseases caused by bacteria, viruses or fungi can greatly reduce the yield of rice. Thus, cultivation of rice varieties with high disease resistance is the simplest and most cost effective method.

Among the most serious and widespread cultivated rice diseases, rice blast continues to threaten worldwide rice production. When rice is infected by Pyricularia oryzae, the yield of the rice is reduced by 15% -20%, and when serious, the yield is reduced by 40% -50%, even the rice is in harvest. Rice blast diseases occur in all rice planting areas in China. Especially in the southern area, is more favorable for the propagation of rice blast fungus. At present, the agricultural production mainly comprises measures such as bactericides, agronomic measures, cultivation of resistant varieties and the like to prevent diseases caused by rice blast. The bactericide has short efficacy, high cost and serious environmental pollution due to the biochemical preparation, and is not suitable for the development requirement of the modern society. Therefore, breeding and reasonably utilizing the disease-resistant variety are the most economical and effective strategies for preventing and controlling rice blast at present, and the excavation and utilization of the disease-resistant genes are key factors of disease-resistant breeding. However, the physiological race of the rice blast fungus has high variability and rapid evolutionary property, and the rice resistant variety often loses the disease resistance advantage within 3-5 years, so that the disease resistant variety cultivated in the current production can not reach the actual requirement. Therefore, the genes for resisting the diseases are mined, the molecular mechanism of rice blast resistance is revealed, theoretical guidance is provided for breeding of new varieties of high-resistance rice, and the method has important significance for safe production of the rice.

At present, more than 30 rice blast resistance genes have been cloned, but the resistance spectrum of most resistance genes is narrow. Therefore, the novel rice blast resistance gene in rice, especially novel broad-spectrum disease resistance gene, can provide important gene resources for green prevention and control of rice blast through resistance breeding.

Disclosure of Invention

The invention aims to provide a novel rice blast resistance gene OsBRH, a recombinant vector, recombinant engineering bacteria and a functional identification method of a gene OsBRH, which are capable of efficiently utilizing resources, so as to make up for the defects of the existing rice blast resistance gene, and apply the novel rice blast resistance gene to rice breeding to improve rice disease resistance, especially rice blast resistance, and provide important gene resources for green prevention and control of rice blast through resistance breeding. In order to solve the technical problems, the invention adopts the following technical scheme:

The invention provides a rice blast resistance gene OsBRH, wherein the nucleotide sequence of OsBRH is shown as SEQ ID NO.1, and the gene can be applied to improving rice blast resistance. Furthermore, the amino acid sequence of the rice blast resistance gene OsB RH1 is shown as SEQ ID NO. 2.

The invention also provides a recombinant vector containing the novel rice blast resistance gene OsBRH and recombinant engineering bacteria.

The invention also provides a function identification method of the novel rice blast resistance gene OsBRH1, which comprises the following steps:

s1: cloning a novel rice blast resistance gene OsBRH to a PYLCRISPR/Cas9Pubi-H vector to obtain an expression vector;

S2: infecting rice with the expression vector by using an agrobacterium transformation method to obtain a mutant plant with the gene knocked out;

S3: after the seeds of the mutant plants with the knocked-out genes are cultured to the heart of three leaves, spore suspension of the rice blast fungus is inoculated, and after 7d of culture, leaf spot areas are observed, recorded and analyzed.

Further, S1 specifically includes the following steps:

Referring to the sequenced OsBRH sequences, and predicting a knockout target by using CRISPR-P online analysis software;

Designing a target primer, performing a target joint connection reaction, and constructing an sgRNA expression cassette by two rounds of PCR (polymerase chain reaction) on the target joint and a sgRN A cloning vector subjected to BsaI-HF enzyme digestion;

Carrying out homologous recombination connection reaction on the two sgRNA expression cassettes and the BsaI-HF enzyme-digested PYLCRISPR/Cas9Pubi-H vector to form a PYLCRISPR/Cas9Pubi-H-OsBRH1 expression vector and converting escherichia coli DH 5 alpha.

Further, the sgRNA cloning vector comprises pYLgRNA-OsU a/LacZ and pYLgRNA-OsU b.

Further, the target primer comprises:

OsBRH1-T1-F, the sequence of which is shown as SEQ ID NO.3, osBRH-T1-R, the sequence of which is shown as SEQ ID NO. 4;

OsBRH1-T2-F, the sequence of which is shown in SEQ ID NO.5, osBRH-T2-R, and the sequence of which is shown in SEQ ID NO. 6.

Further, S2 also comprises molecular identification of DNA of the mutant plant with the gene knocked out;

the molecular identification method specifically comprises the following steps:

designing hygromycin primers hygF and hygR, and amplifying a section of hygromycin gene of the gene knocked-out mutant plant to obtain a 1035bp fragment which is a positive plant;

collecting seeds of positive plants, and extracting DNA from young leaves after sowing to perform mutation detection of target sites;

Designing a primer OsBRH-TC-F, osBRH1-TC-R containing a target site fragment PCR, amplifying a fragment with the size of 500bp, sequencing and analyzing, wherein the sequencing result is that a plant with a frame shift mutation is a homozygous knockout plant, and the offspring is that a homozygous Cas9 knockout mutant plant;

The sequence of hygF is shown as SEQ ID NO.7, and the sequence of hygR is shown as SEQ ID NO. 8; the sequence of OsBRH-TC-F is shown as SEQ ID NO.9, and the sequence of OsBRH-TC-R is shown as SEQ ID NO. 10.

Further, S3 is inoculated with spore suspension of rice blast fungus, after 7d of culture, leaf spot areas are observed, recorded and analyzed, and the specific steps are as follows:

1.0X10 ⁵～2.0×10⁵/mL of the spore suspension of Pyricularia oryzae was inoculated by spraying with a high pressure atomizer, cultured in an inoculation room for 7d, leaf lesions (based on the second young leaf) were observed, photographed and recorded, and the area of lesions was analyzed with imageJ software.

The invention also provides a function identification method of the novel rice blast resistance gene OsBRH1, which comprises the following steps:

s1: cloning a new rice blast resistance gene OsBRH to a PUN1301 vector to obtain an over-expression vector;

S2: infecting rice with the expression vector by using an agrobacterium transformation method to obtain a gene over-expression plant;

s3: culturing the seeds of the gene over-expression plants to three leaves and one heart, inoculating spore suspension of rice blast fungus, culturing for 7 days, and observing, recording and analyzing leaf spot areas.

Further, S1 specifically includes the following steps:

designing a primer with a joint consistent with the sequences at two ends of the PUN1301 after double digestion by referring to the sequence OsBRH of the sequencing;

Cloning a recombinant sequence of OsBRH1 with a linker sequence, carrying out homologous recombination connection reaction on the recombinant sequence of OsBRH1 and a PUN1301 vector after BamH1-HF and Sac1-HF double digestion to form a PUN1301-OsBRH 1 over-expression vector, and converting escherichia coli DH5 alpha;

further, the adaptor primer includes:

PUN1301-OsBRH1-F has the sequence shown in SEQ ID NO.11, and PUN 1301-OsBRH-R has the sequence shown in SEQ ID NO. 12.

Further, S2 also comprises molecular identification of the over-expressed plants;

the molecular identification method specifically comprises the following steps:

Designing hygromycin primers hygF and hygR, and amplifying a section of hygromycin gene of the plant with the gene over-expressed, wherein the amplified 1035bp fragment is a positive plant;

collecting seeds of positive plants, picking young leaves after sowing to extract RNA, reversing the extracted RNA into CDNA, and then carrying out RT-qPCR to detect the relative expression quantity of gene OsBRH 1;

The sequence of the designed primers OsBRH-Q-F and OsBRH-Q-R of RT-qPCR, osBRH-Q-F is shown as SE Q ID NO.13, osBRH-Q-R is shown as SEQ ID NO. 14.

Further, S3 is inoculated with spore suspension of rice blast fungus, after 7d of culture, leaf spot areas are observed, recorded and analyzed, and the specific steps are as follows:

1.0X105-2.0X105/mL of the spore suspension of Pyricularia oryzae was inoculated by spraying with a high pressure atomizer, cultured in an inoculation room for 7d, leaf lesions (based on the second young leaf) were observed, photographed and recorded, and the area of lesions was analyzed by imageJ software.

The invention has the beneficial effects that:

The novel gene OsBRH for resisting rice blast of the invention is the function of identifying the rice blast germ from the rice for the first time, and CDS is 723p long, and codes 240 amino acid protein. Analysis of the online web site using SMART predicts that the protein has a typical HMA domain. By comparing the rice Cas9 knockout mutant with the rice tolerance of wild type rice to Pyricularia oryzae and the rice tolerance of rice over-expression plants and wild type rice to Pyricularia oryzae, osBRH genes are found to improve the rice blast resistance of rice. The gene can effectively improve the defensive ability of rice by promoting accumulation of hydrogen peroxide and expression response of defensive related genes OsPR10a and OsPR3 in the immune process. The application of the polypeptide to the field of genetic engineering has important economic value and application prospect.

Drawings

FIG. 1 is an electrophoresis chart of the PCR product OsBRH of example 1 of the present invention on agarose gel;

FIG. 2 is a predicted view of the conserved domain of the protein encoded by OsBRH gene of example 1 of the present invention;

FIG. 3 is an electrophoretogram on PYLCRISPR/Cas9Pubi-H-OsBRH1 and detected agarose gel of example 2 of the invention;

FIG. 4 is a diagram showing the electrophoresis pattern and the relative expression level of the PUN1301-OsBRH1 vector construct agarose gel of example 3 of the present invention;

FIG. 5 is a flowchart of Agrobacterium-mediated genetic transformation of rice in example 4 of the present invention;

FIG. 6 is a diagram showing the mutation detection of the young leaf DNA target site of the T0 positive transgenic plant of example 4;

FIG. 7 is a graph showing the comparison of leaf blade and leaf spot areas of OsBRH gene mutant plants and wild type, CO39 rice, osBR H1 overexpressing plants and wild type rice blast fungus 7d inoculated thereon according to example 4 of the present invention;

FIG. 8 is a graph showing the relative expression levels of OsPR10a and OsPR3 according to example 6 of the present invention;

FIG. 9 is a graph showing the accumulation of H ₂O₂ in the OsBRH gene mutant plants and the wild type, CO39 rice plants thereof, osBR H1 overexpressing plants and the wild type rice plants inoculated with Pyricularia oryzae 48H and 7d later leaves of example 4 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The novel gene OsBRH for resisting rice blast efficiently utilizes the resources, and the nucleotide sequence of the novel gene OsBRH is shown as SEQ ID NO: 1. The CDS of the novel rice blast resistance gene OsBRH of the invention is 723bp long and codes for 240 amino acid protein. Analysis of the online web site using SMART predicts that the protein has a typical HMA domain. Through researches, the gene can improve the rice blast resistance of rice caused by rice blast fungus.

The invention also provides a functional identification method of the novel rice blast resistance gene OsBRH1, which comprises the following steps:

S1: cloning a new rice blast resistance gene OsBRH1 of the rice with high-efficiency utilization of resources into a PYLCRISPR/Cas9Pubi-H vector to obtain an expression vector; the construction and transformation of the expression vector comprise the following specific steps: referring to the sequence of OsBRH of sequencing, predicting a knocked-out target by using CRISPR-P online analysis software, designing a target primer, then carrying out a connection reaction of a target joint, and constructing an sgRNA expression cassette by using the target joint and an sgRNA cloning vector subjected to BsaI-HF enzyme digestion through two rounds of PCR, wherein the sgRNA cloning vector comprises pYLgRNA-OsU a/LacZ and pYLgRNA-OsU b; and finally, carrying out homologous recombination connection reaction on the two sgRNA expression cassettes and the BsaI-HF enzyme digestion and then carrying out PYLCRISPR/Cas9Pubi-H vector, constructing a PYLCRISPR/Cas9Pubi-H-OsBRH1 expression vector and converting escherichia coli DH5 alpha.

The target primer sequences include:

OsBRH1-T1-F(SEQ ID NO.3)：GCCGAACCGAGGAGACGGCCTTCA；

OsBRH1-T1-R(SEQ ID NO.4)：AAACTGAAGGCCGTCTCCTCGGTT；

OsBRH1-T2-F(SEQ ID NO.5)：GTTGATCGAAATGGCGTCGATTCC；

OsBRH1-T2-R(SEQ ID NO.6)：AAACGGAATCGACGCCATTTCGAT。

S2: infecting rice with the expression vector by using an agrobacterium transformation method to obtain a mutant plant with a knocked-out gene (preferably, agrobacterium adopts agrobacterium EHA 105); then carrying out molecular identification on the DNA of the mutant plant with the knocked-out gene; wherein, the molecular identification method comprises the following steps:

Hygromycin primer hygF (SEQ ID No. 7) was designed: CCGGAAGTGCTTGACATTGG and hygR (SEQ ID No. 8): GCCGAATTAATTCGGGG, amplifying a section of hygromycin gene of the mutant plant with the knocked-out gene, and amplifying a section with 1035bp size as a positive plant; collecting seeds of the positive plants, picking young leaves to extract DNA after sowing to carry out mutation detection of target sites, and designing a primer OsBRH-TC-F (SEQ ID NO. 9) containing target site fragment PCR: ATGTCGAAGAAAATTGTGGT, OSBRH1-TC-R (SEQ ID NO. 10): TCAGCAAATGGCGCACGAGT, amplifying a 489bp fragment for sequencing analysis, wherein the sequencing result is that the frame shift mutant plant is a homozygous knockout plant, and the offspring is a homozygous Cas9 knockout mutant plant.

S3: taking a proper amount of seeds of the mutant plants with the knocked-out genes, culturing until the seeds are three leaves and one heart, inoculating spore suspension of rice blast fungus (Guy 11), culturing for 7 days, and observing, recording and analyzing leaf spot areas.

Further, the spore suspension inoculated with the rice blast fungus is cultured for 7d, and the specific steps of observing, recording and analyzing the leaf spot area are as follows: 1.0X10 ⁵～2.0×10⁵/mL of the spore suspension of Pyricularia oryzae was inoculated by spraying with a high pressure atomizer, cultured in an inoculation room for 7d, leaf lesions (based on the second young leaf) were observed, photographed and recorded, and the area of lesions was analyzed with imageJ software.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a cloning method of a novel gene OsBRH for resisting rice blast by using resources efficiently, which comprises the following steps:

(1) Preparation of materials

Rice variety material and E.coli DH 5. Alpha (Rhizoctonia cerealis TSC-C14). Wherein, the rice variety is used for RNA extraction and is provided by a key laboratory of the co-construction education department of the Fujian university crop genetic breeding and comprehensive utilization department.

(2) Cloning and transformation of novel Rice blast resistance Gene OsBRH A novel Rice blast resistance Gene OsBRH1 was designed according to the annotation information of OsBRH1 (NCBI ReferenceSequence: XM_ 015762849.2), whose CDS was 1389bp in length, encoding 462 amino acids, using NCBI on-line website to design a pair of specific primers OsBRH1-F (ATGTCGAAGAAAATTGTGGTGAAGC) and OsBRH1-R (TCAGCAAATGGCGCACGAGTTGGGG).

And (3) taking trefoil-stage rice seedlings, extracting total RNA by using a plant RNA mass extraction kit (OMEGAbia-tek), and reversely transcribing the total RNA into cDNA as a PCR reaction template. The PCR reaction system for amplifying OsBRH gene is shown in Table 1, and the total volume is 50. Mu.L. The PCR reaction procedure was:

Carrying out at 94 ℃ for 2min; denaturation: annealing at 94℃for 30 s: extension was performed at 55℃for 30 s: 1mi n,30 cycles at 72 ℃; finally, extending for 5min at 72 ℃; preserving at 4 ℃.

The PCR product was detected and purified by 1% agarose gel electrophoresis (as shown in FIG. 4 a), then treated with DNA A-TAILING KIT (Takara 6109) and TA cloned, the vector was pMD18-T, and 2 positive clones were selected for sequencing.

CDS of the novel rice blast resistance gene OsBRH is 723bp long, the protein of 240 amino acids is encoded, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The structure of OsBRH protein was analyzed using SMART online tools and its conserved domain prediction diagram is shown in figure 2. As can be seen from fig. 2, the protein comprises an HMA domain.

TABLE 1 PCR reaction System for amplifying OsBRH Gene

Example 2

The embodiment provides a PYLCRISPR/Cas9Pubi-H-OsBRH1 vector construction method, which comprises the following steps:

(1) Material preparation: osBRH1 mutant material and wild rice variety thereof, CO39 (used as pathogenic control inoculated with rice blast fungus), cas9 knockout vector: PYLCRISPR/Cas9Pubi-H, sgRNA

(2) Cloning vector: pYLgRNA-OsU a/LacZ and pYLgRNA-OsU b, agrobacterium EHA105, all of which are offered by the university of agricultural in North China, liu Yaoguang, and Agrobacterium EHA105 are available from Shanghai, biotechnology Inc.

(3) Referring to the sequenced OsBRH1 sequence, a CRISPR-P (http:// cbi. Hzau. Edu. Cn/cgi-bin/CRISPR) online analysis software is utilized to predict a knockout target, and target primers are designed:

OsBRH1-T1-F(GCCGAACCGAGGAGACGGCCTTCA)；

OsBRH1-T1-R(AAACTGAAGGCCGTCTCCTCGGTT)；

OsBRH1-T2-F(GTTGATCGAAATGGCGTCGATTCC)；

OsBRH1-T2-R(AAACGGAATCGACGCCATTTCGAT)；

followed by ligation of the target linker. Wherein, the target joint connection reaction condition is 95 ℃,3min, naturally cooling to room temperature, and the connection reaction system is shown in table 2:

Positive and negative primer ligation systems for target spots of Table 2

(4) Cutting by using sgRNA cloning vector: the reaction was carried out using BsaI-HF (NEB Co.) endoenzyme for pYLgRNA-OsU a and pYLgRNA-OsU b at 37℃for 20min. The reaction is completed and then the next step is directly carried out, and the reaction can be stored for-20 days without recovery and used for a period of time. The reaction system is shown in Table 3:

TABLE 3pYLgRNA-U cleavage System

(5) And (3) connecting the target point joint with the corresponding cut sgRNA: the target linker was ligated to the digested cloning vector pYLgRNA-U using T4 ligase (TAKARA Co.), the target 1 linker fragment was ligated to digested pYLgRNA-OsU a, the target 2 linker fragment was ligated to digested pYLgRNA-OsU6b, the ligation conditions were 37℃for 18min, and the reaction system was as shown in Table 4:

TABLE 4 target fragment ligation pYLgRNA-U cleavage plasmid reaction System

(6) Construction of sgRNA expression cassette: using the ligation reaction as a template, the sgRNA expression cassette was cloned by two rounds of PCR using KOD high-fidelity enzyme (TOYO BO Co.). The first round of PCR reaction conditions were:

Pre-denaturation: carrying out at 94 ℃ for 30s; denaturation: annealing at 94℃for 10 s: extension was performed at 60℃for 15 s: 20s at 68℃for 20 cycles; finally, extending at 68 ℃ for 5min; preserving at 4 ℃. Each target corresponds to two first rounds of PCR reactions, and the first round of PCR reaction systems are shown in Table 5. The two targets, i.e., 4 reactions, were reacted with primer pairs U-F/T-R and T-F/gR-R, respectively, and the primers were paired as shown in Table 6 below.

TABLE 5 first round PCR cloning of sgRNA expression cassette reaction System

TABLE 6 first round PCR cloning of sgRNA expression cassette primer template pairing Table

Four reaction products are obtained from the first round of PCR products, 1 mu L of each of the reaction 1 and the reaction 2 is mixed with 18 mu L of sterile water, and the mixture is named as a target 1 template and used as a template of a second round of PCR target 1. Likewise, reactions 3 and 4 were mixed as templates for target 2. After the second round of PCR, two fragments of target 1 and target 2 are connected into PYL CRISPR/Cas9Pubi-H expression vector, and an in-fusion homologous recombination cloning connection method is adopted, so that primers including an fusion-F and an fusion-R with a linker with 17bp homologous sequences at two ends after the vector is digested, and the two fragments are connected through in-fusion, and the primers including the homologous sequences include the fusion-F1 and the fusion-R1. The second round PCR primer template pairs are shown in Table 7.

TABLE 7 primer template pairing Table for second round PCR cloning of sgRNA expression cassette

The second round of PCR reaction conditions were: pre-denaturation: carrying out at 94 ℃ for 30s; denaturation: annealing at 94℃for 10 s: extension was performed at 58℃for 15 s: 20s at 68℃for 30 cycles; finally, extending at 68 ℃ for 5min; preserving at 4 ℃.2% agarose gel electrophoresis, recovering two fragments of target 1 and target 2 with the linker. The second round PCR reaction system is shown in Table 8:

TABLE 8 reaction System for cloning sgRNA expression cassette by second round PCR

(7) Cleavage PYLCRISPR/Cas9Pubi-H expression vector: the reaction was performed using BsaI-HF (NEB Co.) endonuclease at 37℃for 30min. About 50ng of the concentration of the cleavage recovery was required, and the cleavage system was as shown in Table 9:

table 9PYLCRISPR/Cas9 cleavage System

(8) And connecting the two sgRNA expression cassettes obtained by cloning with the expression vector fragments (fusion) after enzyme digestion to construct PYLCRISPR/Cas9Pubi-H-OsBRH1. The ligation reaction conditions were 50℃for 18min. The reaction system of the sgRNA expression cassette linked to the cleaved expression vector fragment is shown in table 10:

table 10PYLCRISPR/Cas9Pubi-H-OsBRH1 ligation System

(9) And (3) converting the ligation reaction connection solution into escherichia coli DH5 alpha competent cells, and selecting a bacterial solution for identification. Both sgRNA expression cassettes were identified as successfully linked to PYLCRISPR/Cas9Pubi-H vectors. Plasmid extraction kit (SanPrep column type plasmid extraction kit) of Shanghai Biotechnology company is used for extracting plasmid, extracting plasmid of positive bacterial liquid, and performing enzyme digestion identification of AscI. After identification and digestion, a vector backbone fragment of about 16000bp in length and an associated expression cassette fragment of about 1300bp in length are obtained. The PYLCRISPR/Cas9Pubi-H-OsBRH1 plasmid is transformed into agrobacterium EHA105, and positive clones are selected and stored in a refrigerator at-80 ℃ for standby.

Example 3

The embodiment provides a method for constructing a PUN1301-OsBRH1 vector, which comprises the following steps:

Material preparation: osBRH1 overexpressing material and wild rice variety thereof, nipponbare (used as a pathogenic control inoculated with Pyricularia oryzae), overexpressing vector: PUN1301.

(1) Double enzyme cutting PUN1301 carrier, the reaction condition is 37 ℃ for 30min. The reaction system is shown in Table 11:

table 11PUN1301 double enzyme cutting system

(2) Designing a joint primer with sequences consistent with two ends of the PUN1301 after double digestion, wherein the PUN1301-OsBR H1-F/PUN 1301-OsBRH-R (enzyme digestion site is reserved), using OsBRH1 as a template, and using TOYOBO company high-fidelity enzyme, wherein the reaction conditions are as follows: pre-denaturation: carrying out at 94 ℃ for 3min; denaturation: annealing at 94℃for 10 s: extension was performed at 55℃for 15 s: 20s at 68℃for 30 cycles; finally, extending at 68 ℃ for 5min; preserving at 4 ℃. The reaction system was consistent with the previous total amount of 50. Mu.L. After electrophoresis, the gel is recovered to obtain OsBR H fragments with two homologous sequence joints after PUN1301 enzyme digestion. The ligation was carried out by the method of In-Fusion at 50℃for 18min. The reaction system is shown in Table 12:

Table 12PUN1301-OsBRH1 connecting System

(3) And (3) converting the ligation reaction connection solution into escherichia coli DH5 alpha competent cells, and selecting a bacterial solution for identification. The OsBRH1 was identified to be successfully incorporated into the PUN1301 vector. Plasmids were extracted with plasmid extraction kit (SanPrep column type plasmid extraction kit) from Shanghai Biotechnology. The PUN1301-OsBRH1 plasmid is transformed into agrobacterium EHA105, and positive clones are selected and stored in a refrigerator at-80 ℃ for standby.

Example 4

The present example provides a method for agrobacterium-mediated genetic transformation of rice (a flow chart of genetic transformation of rice is shown in fig. 5), comprising the steps of:

(1) Mature embryo callus induction, soaking seeds with 75% ethanol, sterilizing for 1min,2.5% sodium hypochlorite (50 mL with 1 drop of Tween-20) for 15min, slightly shaking for 10min, soaking with 2.5% sodium hypochlorite for 15min, washing with sterile water for 5 times, each time for 5min, sucking excessive sterile water with sterile filter paper, inoculating to callus induction culture medium N6D (N6 +100mg/L inositol +2mg/L, 4-D +2.878g/L proline +300mg/L acid hydrolyzed casein 30g/L sucrose, pH5.8), placing 30-40 granules in each dish, and culturing at 28-30deg.C. And then the callus is stripped, after a week of culture, the callus is carefully separated from the seeds and the buds by forceps and is transferred to a new callus induction medium for continuous culture for 3 days after most of the seeds grow yellowish compact callus.

(2) Agrobacterium is infected, agrobacterium is scratched and activated, monoclonal shaking is selected and identified, 50 mu L of bacterial liquid is taken in 50mLYEB+Km+Rif culture medium, shaking is carried out for 200 times at 28 ℃ overnight until OD ₆₀₀ =0.5 (0.2) is about, centrifugation is carried out for 10min at 4100rpm and 4 ℃, the bacterial body is resuspended in an equal volume of AAM culture medium, as (acetosyringone) is added to 100 mu M, and the bacterial body is placed on ice for more than 1h for standby. Placing the callus with better growth vigor in a sterile tissue culture bottle, pouring engineering bacteria suspension, slightly shaking, soaking for 10min, pouring out the redundant bacteria liquid, sucking the redundant bacteria liquid around the callus with sterile filter paper, transferring to a co-culture medium (N6D+100 mu MAs30g/L sucrose, pH 5.8), carrying out dark culture at 25 ℃ for 3 days on each dish for 30-40.

(3) Washing bacteria, namely placing the infected callus into a sterile tissue culture bottle, washing the infected callus with sterile water for 8-9 times, wherein the time is not counted for the first 4 times, and the time is 4-5 minutes later until the sterile water is not obviously turbid, and carefully transferring the washed callus into a new sterile tissue culture bottle; washing with 500mg/L carbenicillin water three times, the first 10min, and the latter two times each 15min; washing with sterile water for 4-5min for 2-3 times.

(4) Resistant callus is selected, the washed callus is transferred to a selection medium N6SC II (N6 D+400mg/LCb (carboxin) +50mg/LHyg g/L sucrose, pH 5.8), 20-30 calli are cultured for 2 weeks at 28-30 ℃, then N6SC II is replaced for 2 weeks, and then N6SC II is replaced for 2-3 weeks until new callus is grown.

(5) Callus buds are induced, newly grown resistant callus is picked out, transferred to a bud induction culture medium RE (MS+2mg/LKT+0.02 mg/LNAA+2g/L acid hydrolysis casein 30g sucrose, 30g sorbitol pH5.8), cultured for 2 weeks (partial callus is blackened, partial adventitious roots are grown and partial green bud spots are grown) at the temperature of 30 ℃ for 10-20 per dish, and the callus with better growth condition is subcultured with the new bud induction culture medium for 1-2 weeks until a plurality of green buds are grown.

(6) Rooting, transferring the seedlings with a few calli into MS culture medium, and culturing at 30deg.C under light for 1-2 weeks until most of the roots grow.

(7) And (3) hardening and transplanting, taking out the seedlings from the culture medium, flushing the culture medium by clear water, transferring the seedlings to a test tube filled with sterile water for culturing for one week at 30 ℃, and transferring the seedlings into a potted plant or a field.

Extracting transformed plants for molecular identification, designing hygromycin primer hygF (SEQ ID NO. 7): CCGGAAGTG CTTGACATTGG, hygR (SEQ ID NO. 8): GCCGAATTAATTCGGGG, amplifying a section of hygromycin gene of the transgenic plant, and amplifying a 1035bp fragment as a positive plant. Collecting T0 positive transgenic plant seeds, picking young leaves to extract DNA after sowing, and carrying out mutation detection of target sites, wherein primers of PCR of the target sites are OsBRH-TC-F (SEQ ID NO. 9): ATGTCGAAGAAAATTGTGGT, OSBRH-TC-R (SEQ ID NO. 10): TCAGCAAATGGCGCACGAGT A489 bp fragment was amplified and sequenced.

(8) Meanwhile, RNA is extracted from young leaves, and after inversion into CDNA, RT-qPCR is performed to measure OsBRH relative expression quantity. The primers for QPCR were OsBRH1-Q-F (SEQ ID NO. 13): GCATGACAACAAGG ACAAGCA, osBRH1-Q-R (SEQ ID NO. 14): CGAAATGGCGTCGATTCCGA.

Referring to FIG. 6, it is known that the frame shift mutant plants are homozygous knockout plants, i.e., the seeds of M- (1-5) rice are homozygous Cas9 knockout mutants of OsBRH gene.

Example 5

This example provides rice Cas9 knockout mutants and rice blast resistance identification of overexpressed plants, comprising the steps of:

(1) Appropriate amounts of the mutant and overexpressing material seeds, wild rice seeds and CO39 control rice seeds obtained in examples 3 and 4 were immersed in water and germinated in an incubator at 30℃for 2-3 d. Then sowing the rice seeds with the germination acceleration into small flowerpots (diameter of 10 cm) filled with soil, 8-10 rice seeds are planted in each pot, 3 repeats are arranged for each treatment, and the rice seedlings are cultivated in a greenhouse with illumination intensity of 10000Lx, light period of 14h illumination/10 h darkness and 28 ℃ until the rice grows to three leaves or four leaves.

(2) Culturing activated rice blast fungus in rice bran culture medium for 8-10 d until hypha is fully spread on a culture dish, scraping a layer of hypha on the surface of the culture medium, culturing for 3-5 d under the condition of 60% illumination to enable the rice blast fungus to produce spores, washing the spore-producing rice bran plate with 3-5 mL of sterilizing water containing 0.02% Tween-20, filtering the washing liquid with single-layer mirror wiping paper, and counting the filtrate with a cell counting plate to enable the final concentration of spore suspension to be 1.0x10 ⁵～2.0×10⁵/mL. The diluted spore suspension is uniformly sprayed on the rice leaves by a high-pressure atomizer, and 15mL of spore suspension is sprayed on each rice leaf repeatedly. And (3) placing the inoculated seedlings in an inoculation room with the humidity higher than 90% and the temperature of 26 ℃ for dark culture for 24 hours, changing the photoperiod to 12h illumination/12 h darkness, and continuing to culture for 7 days.

FIG. 7 is a graph showing the comparison of leaf area and leaf spot area after inoculating rice blast fungus 7d with wild type CO39 rice of OsBRH gene mutant plant of example 4 of the present invention; FIG. 7 is a graph showing the comparison of leaf area and leaf spot area after the OsBRH gene over-expression plant and its wild rice of example 4 of the present invention are inoculated with Pyricularia oryzae 7 d;

The area of lesions of inoculated rice was analyzed using ImageJ software. Referring to FIG. 7, it can be seen from FIGS. 7a and b that the wild type rice is substantially free of disease and almost free of lesions. The rice variety CO39 has the most serious disease and the largest disease spot expansion, has typical clostridial disease spots, has white middle and brown edge, accords with the characteristics of disease incidence grade 5, and has the disease spot area of 40.29 percent. The leaf of OsBRH1 mutant also has the disease spots, which accords with the 4-grade disease-sensing characteristic, but the disease spots are less than CO39, and the disease spot area reaches 21.88%, which shows that the rice blast resistance of the wild type can be reduced after OsBRH gene knockout. Meanwhile, as can be seen from FIGS. 7c and d, the disease condition of the rice variety Nipponbare is serious, and the disease area reaches 25.01%. Leaves of plants overexpressing OsBRH gene in Japanese sunny are basically free of disease spots, and the area of the disease spots is only 10.11 percent, so that the disease resistance characteristic of level 2 is achieved. The above results further confirm that the rice OsBRH gene has a rice blast resistance function.

Example 6

This example provides molecular mechanism identification of rice Cas9 knockout mutants and overexpressing plants, comprising the steps of:

(1) Appropriate amounts of mutants and over-expression material of 24h and 48h of Pyricularia oryzae in example 5, wild type rice, and leaves of the CO39 control rice plants were taken. Total RNA was extracted according to TaKaRaMiniBESTPlantRNAExtractionKit procedure. The total RNA was then inverted to cDNA according to the TAKARAPRIMESCRIPT ^TM RTMasterMix protocol.

(2) Designing a primer OsPR10a-F: CGCCGCAAGTCATGTCCTAA and OsPR10a-R: GCCATAGTAGCCATCCACGA, OSPR3-F: CCCACATACTGCGAGCCCAA and OsPR3-R: GTCATCCAGAACCAGAACGCC. And detecting the relative expression quantity of OsPR10a and OsPR3 by RT-qPCR. RT-qPCR Using TaKaRaPremixExTaq ^TM II (RR 820L). And (3) adopting a reaction system of 12 mu L, taking the rice endogenous Actin gene action as an internal reference gene, and repeating each sample for 4 times. The 12. Mu.L reaction system comprises: 1 mu L cDNA, 6.25 mu L2XTBGreen PremixExTaq II, 4.25 mu LH2O. The reaction procedure for RT-qPCR was set as follows: 95 ℃/5min;95 ℃/5sec;60 ℃/30sec;39cycles.

Referring to FIG. 8, it can be seen from FIG. 8a that OsPR10a has significantly higher wild-type expression levels than OsBRH1-Cas9 knockout mutants; plants were significantly higher than wild type at over-expression OsBRH. As can be seen from fig. 8b, osPR3 expressed significantly higher than OsBRH1-Cas9 knockout mutants in wild type; plants were significantly higher than wild type at over-expression OsBRH. OsBRH 1A is described as being capable of improving the expression of defense related genes OsPR10a and OsPR3 for disease resistance.

Example 7

This example provides molecular mechanism identification of rice Cas9 knockout mutants and overexpressing plants, comprising the steps of:

(1) A proper amount of 2g of rice leaf, which is infected with the mutant and the over-expression material of rice blast fungus 7d in example 5, was taken. Adding pre-cooled acetone and a little quartz sand at 4 ℃ according to the ratio of the material to the extractant of 1:1, grinding into homogenate, transferring into a centrifuge tube 3000r/min, centrifuging for 10min, discarding residues, and obtaining supernatant as a sample extracting solution.

(2) 1Ml of the sample extract was pipetted, 5% titanium sulfate and concentrated ammonia were added, and after precipitation had occurred, the mixture was centrifuged at 3000rpm/min for 10min, and the supernatant was discarded. The precipitate is repeatedly washed with acetone for 3 to 5 times until the plant pigment is removed. To the washed precipitate, 5ml of 2mol sulfuric acid was added, after complete dissolution, the precipitate was carefully transferred to a 10ml volumetric flask, and the centrifuge tube was rinsed several times with distilled water, the washes were combined and then fixed to a 10ml scale, and colorimetry was performed at 415nm wavelength.

(3) And (3) making a standard curve: 7 centrifuge tubes of 10ml were taken, numbered sequentially and reagents were added as in Table 13. After the precipitate was completely dissolved, it was carefully transferred to a 10ml volumetric flask, the centrifuge tube was rinsed a small number of times with distilled water, the washes were combined and then fixed to a 10ml scale, and colorimetry at 415nm wavelength.

TABLE 13 configuration table for measuring H ₂O₂ concentration standard curve

Referring to FIG. 9, on day 7 of infection with Guy11, H ₂O₂ accumulated more in the leaves of the wild type plants than in the mutant plants; overexpression OsBRH1 also increased H ₂O₂ accumulation in the leaf.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1. A rice blast resistance gene OsBRH1 for efficiently utilizing resources is characterized in that the nucleotide sequence of OsBRH is shown as SEQ ID NO. 1.

2. The protein encoded by rice blast resistance gene OsBRH gene of claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.

3. A recombinant vector comprising the rice blast resistance gene OsBRH according to claim 1.

4. A recombinant engineering bacterium comprising the rice blast resistance gene OsBRH according to claim 1.

5. A method for constructing a OsBRH gene knockout mutant plant, characterized in that the rice blast resistant gene OsBRH1 gene construction in rice is knocked out by constructing a knocked-out expression vector, comprising the following steps:

s1: cloning a novel rice blast resistance gene OsBRH to a PYLCRISPR/Cas9Pubi-H vector to obtain an expression vector;

S2: infecting rice with the expression vector by using an agrobacterium transformation method to obtain a mutant plant with the gene knocked out;

s3: after the gene knockout mutant plant seeds are cultured to the heart of three leaves, spore suspension of rice blast fungus is inoculated, and after 7d of culture, leaf spot areas are observed, recorded and analyzed.

6. The method for constructing a mutant plant according to claim 5, further comprising the step of molecular identification of the DNA of the mutant plant with the gene knocked out, specifically comprising the steps of:

Designing hygromycin primers hygF and hygR, and amplifying a section of hygromycin gene of the gene knocked-out mutant plant to obtain a 1035bp fragment which is a positive plant; collecting seeds of positive plants, and extracting DNA from young leaves after sowing to perform mutation detection of target sites;

designing a primer OsBRH-TC-F, osBRH1-TC-R containing a target site fragment PCR, amplifying a fragment with the size of 500bp, and carrying out sequencing analysis, wherein the sequencing result is that the plant with the frame shift mutation is a homozygous knockout plant, and the offspring is that the homozygous Cas9 knockout mutant plant.

7. The method for constructing a mutant plant according to claim 5, wherein the spore suspension of Pyricularia oryzae is inoculated in S3, and after 7d of culture, leaf spot areas are observed, recorded and analyzed, and the method comprises the following specific steps:

Spraying and inoculating spore suspension of 1.0X10 ⁵～2.0×10⁵/mL Pyricularia oryzae by using a high pressure atomizer, culturing for 7d in an inoculation room, observing leaf spot, taking a photograph based on the second young leaf, recording, and analyzing the area of the spot by using imageJ software.

8. A method for constructing OsBRH gene over-expression plants, which is characterized in that the rice blast resistance gene OsBRH gene as defined in claim 1 in rice is over-expressed by constructing an over-expression vector, comprising the following steps:

s1: cloning a novel rice blast resistance gene OsBRH to a PUN1301 vector to obtain an overexpression vector;

S2: infecting rice with the expression vector by using an agrobacterium transformation method to obtain a mutant plant with the gene knocked out;

s3: culturing the seeds of the gene over-expression plants to three leaves and one heart, inoculating spore suspension of rice blast fungus, culturing for 7 days, and observing, recording and analyzing leaf spot areas.

9. The method for constructing an over-expressed plant according to claim 8, further comprising the step of molecular identification of the over-expressed plant, specifically comprising the steps of:

Designing hygromycin primers hygF and hygR, and amplifying a section of hygromycin gene of the plant with the gene over-expressed, wherein the amplified 1035bp fragment is a positive plant;

Collecting seeds of positive plants, picking young leaves after sowing to extract RNA, reversing the extracted RNA into CDNA, and then carrying out RT-qPCR to detect the relative expression quantity of gene OsBRH 1.