CN116041462A - Application of OsGA2 protein, coding gene or related biological material thereof in regulation and control of plant rice blast resistance - Google Patents

Application of OsGA2 protein, coding gene or related biological material thereof in regulation and control of plant rice blast resistance Download PDF

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CN116041462A
CN116041462A CN202211376979.3A CN202211376979A CN116041462A CN 116041462 A CN116041462 A CN 116041462A CN 202211376979 A CN202211376979 A CN 202211376979A CN 116041462 A CN116041462 A CN 116041462A
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osga2
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rice blast
rice
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徐正一
南楠
王婕
于晓明
刘雨同
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Northeast Normal University
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Abstract

The invention relates to the technical field of gene editing, in particular to application of OsGA2 protein, coding gene or biological material related to the OsGA2 protein in regulation and control of plant rice blast resistance. The research of the invention discovers that the OsGA2 protein or the biological material related to the OsGA2 protein has the function of regulating and controlling the resistance of rice blast, and the OsGA2 gene is knocked out to reduce the expression level of the OsGA2 protein, so that the resistance of rice blast can be improved, and the OsGA2 gene is overexpressed to improve the expression level of the OsGA2 protein, so that the resistance of rice blast can be reduced. Through identification and application of the rice blast resistance gene OsGA2, breeders can develop rice blast resistant rice varieties suitable for different rice planting areas in various places, and important guarantee is provided for rice grain yield increase.

Description

Application of OsGA2 protein, coding gene or related biological material thereof in regulation and control of plant rice blast resistance
Technical Field
The invention relates to the technical field of gene editing, in particular to application of OsGA2 protein, coding gene or biological material related to the OsGA2 protein in regulation and control of plant rice blast resistance.
Background
Rice is one of main grain crops in China and is widely planted in various places in China, but the yield of the rice is increased by more than 40% by 2030 in order to meet the increasing grain demands in large population China [1] . This challenge must be overcome by developing high yielding rice varieties that are tolerant to biotic and abiotic stresses [2] . Among biotic stresses, rice blast is the most detrimental threat to high yield of rice [3,4] . The rice blast is one of the most serious fungal diseases which damage the rice production, is a common disease in the rice field, and has the characteristics of long damage time, multiple infection positions, various symptoms and the like [5] . The occurrence of rice blast easily affects the growth process of rice, and further leads to reduced yield and quality of rice. The rice blast is likely to occur in the whole growth period of the rice, and can be classified into seedling blast, leaf blast, festival blast, neck blast, grain blast and the like according to different victims and positions, wherein the common diseases are leaf blast and neck blast, and the influence on the rice yield is serious. Pathogenic bacteria of rice blast have a high propagation speed under a wet condition of 26-28 ℃, and the propagation of the pathogenic bacteria can be inhibited by sunlight irradiation. When rice blast occurs, the diseased plant is usually brown and is mixed with a large amount of gray mold layers, if no measures are taken in time, the rice seedlings gradually curl and die [6] 。
The Gadd45 gene is a completely new gene found in rice, and has not been studied in plants, but the Gadd45 gene in animals has been widely studied. The Gadd45 gene first discovered in animals was identified on an induced basis after being subjected to various stresses associated with growth arrest and was therefore designated growth inhibition and DNA damage inductionGuide type (growth arrest and DNA-damage gene) [7] . The gadd gene was cloned for the first time from Chinese Hamster Ovary (CHO) cells as a subset of transcription factors that are continuously upregulated upon exposure to Ultraviolet (UV) radiation, and in many cases, the gadd gene acts as a growth-blocking and DNA-damaging repair gene that acts as a growth-stopping signal with other DNA damaging agents, including Methyl Methanesulfonate (MMS), hydrogen peroxide and N-acetoxy-2-acetamidofluorobutadiene [8] . Gadd45a is the 45 th member of the 100-fold collection of cDNA clones. Gadd45a responds to a variety of drugs associated with DNA damage, apoptosis, cell cycle checkpoint control, cell damage and other growth regulating processes. Gadd45 proteins are also involved in a variety of cellular processes, often associated with stress signaling and other growth regulation pathways [9] . Gadd45a has been reported to induce in a TM-dependent and protein kinase independent manner following exposure of human cells to Ionizing Radiation (IR) [10] . This induction is regulated by p53 [11] Indeed, gadd45a is the first stress gene found to be regulated by p53 transcription [12] . Gadd45b was originally cloned as a gene expressed after IL-6-induced terminal differentiation and growth arrest of M1D+ myeloid precursor cells. Gadd45g was originally cloned as an early IL-2 response gene in T cells. All three members respond to various environmental factors associated with growth control. These three proteins are highly conserved in metazoans, although there is only one Gadd45 gene in insects, similar to Gadd45g, indicating that this may be an ancestral gene. These proteins are small (18 kDa), highly negatively charged, and localize to the nucleus [13] 。
The Gadd45 gene has various functions in animals as a stress-related gene, but its function as a homologous gene OsGA2 in rice has not been reported in plants.
Reference is made to:
[1]Khush GS(2005)What it will take to feed 5.0billion rice consumers in 2030.Plant Mol Biol 59:1–6.
[2]Selvaraj CI,Nagarajan P,Thiyagarajan K,Bharathi M,Rabindran R(2011)Studies on heterosis and combining ability of well known blast resistant rice genotypes with high yielding varieties of rice(Oryza sativa L.).Int J Plant Breed Genet 5(2):111–129.
[3]Kwon JO,Lee SG(2002)Real-time micro-weather factors ofgrowing field to the epidemics ofrice blast.Res Plant Dis 8:199–206(in Korean,English abstract).
[4]Li YB,Wu CJ,Jiang GH,Wang LQ,He YQ(2007)Dynamic analyses ofrice blast resistance for the assessment of genetic and environmental effects.Plant Breeding 126:541–547.
[5] zheng Chengyou comprehensive control technology for rice blast and its popularization strategy [ J ]. South agriculture, 2022, 16 (4): 66-68.
[6] He Xiaoling the occurrence and control of rice sheath blight and rice blast [ J ]. Guangdong silkworm industry, 2021, 55 (9): 15-16.
[7]Fornace AJ Jr,Nebert DW,Hollander MC,Luethy JD,Papathanasiou M,Fargnoli J,Holbrook NJ(1989)Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.Mol Cell Biol 9:4196–4203.
[8]Fornace AJJ,Alamo IJ,Hollander MC(1988)DNA damage-inducible transcripts in mammalian cells.Proc NatlAcad Sci USA 85:8800–8804.
[9]Gao M,Guo N,Huang C,Song L(2009)Diverse roles ofGADD45alpha in stress signaling.CurrProteinPept Sci 10:388–394.
[10]Papathanasiou MA,Kerr NC,Robbins JH,McBride OW,Alamo IJ,Barrett SF,Hickson ID,Fornace AJJ(1991)Induction by ionizing radiation of the gadd45gene in cultured human cells:lack ofmediation by protein kinase C.Mol Cell Biol11:1009–1016.
[11]Kastan MB,Zhan Q,el-Deiry WS,Carrier F,Jacks T,Walsh WV,Plunkett BS,Vogelstein B,Fornace AJJ(1992)A mammalian cell cycle checkpoint pathway utilizingp53 and GADD45 is defective in ataxia-telangiectasia.Cell 71:587–597.
[12]Hollander MC,Fornace AJ Jr(2002)Genomic instability,centrosome amplifi cation,cell cycle checkpoints and Gadd45a.Oncogene 21:6228–6233.
[13]CretuA,Sha X,Tront J,Hoffman B,Liebermann DA(2009)Stress sensor Gadd45 genes as thera-peutic targets in cancer.CancerTher 7:268–276.
Disclosure of Invention
In order to solve the problems, the invention provides application of OsGA2 protein, coding gene or related biological material in regulating and controlling plant rice blast resistance. The OsGA2 protein provided by the invention has the function of regulating and controlling the resistance of rice blast.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an OsGA2 protein for regulating and controlling plant rice blast resistance, wherein the OsGA2 protein comprises a protein with an amino acid sequence shown as SEQ ID NO. 1.
The invention also provides an OsGA2 gene for encoding the OsGA2 protein in the scheme, and the CDS sequence of the OsGA2 gene comprises a sequence shown as SEQ ID NO. 2.
The invention also provides application of the OsGA2 protein or the biological material related to the OsGA2 protein in the scheme in regulating and controlling plant rice blast resistance.
Preferably, the regulation comprises increasing plant rice blast resistance by negatively regulating the expression level of the OsGA2 protein, or decreasing plant rice blast resistance by positively regulating the expression level of the OsGA2 protein.
Preferably, the biological material comprises one or more of the following:
1) The OsGA2 gene described in the above scheme;
2) A recombinant vector comprising the OsGA2 gene described in the above-mentioned scheme;
3) And (3) negatively regulating the sgRNA expressed by the OsGA2 gene or the recombinant vector in the scheme.
Preferably, the target sequence of the sgRNA comprises one or more of the target sequences shown in SEQ ID No.3 and SEQ ID No. 4.
Preferably, the recombinant vector described in 3) comprises a target sequence and a base vector; the target sequence comprises a target sequence shown as SEQ ID NO.3 or SEQ ID NO. 4.
Preferably, the base vector comprises pYLCRISPR-Cas9PUbi-H.
Preferably, the plant comprises rice.
The invention also provides application of the OsGA2 protein or the biological material related to the OsGA2 protein in the scheme in cultivation of transgenic plants with improved rice blast resistance.
The beneficial effects are that:
the invention provides an OsGA2 protein for regulating and controlling plant rice blast resistance, wherein the OsGA2 protein comprises a protein with an amino acid sequence shown as SEQ ID NO. 1. The research of the invention discovers that the OsGA2 protein has the function of regulating and controlling the resistance of rice blast, and can improve the resistance of rice blast by knocking out the OsGA2 gene and reducing the expression level of the OsGA2 protein, and can reduce the resistance of rice blast by overexpressing the OsGA2 gene and improving the expression level of the OsGA2 protein. Through identification and application of the rice blast resistance gene OsGA2, breeders can develop rice blast resistant rice varieties suitable for different rice planting areas in various places, and important guarantee is provided for rice grain yield increase.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of the preparation of OsGA2 mutants and screening of Cas9 free plants, wherein A represents a gene pattern diagram of OsGA2 and the positions of two targets in the genomic sequence of OsGA 2; b represents Sanger sequencing results near the target in two different edited forms of the osga2 homozygous mutant; c represents the use of hygromycin selection to obtain two homozygous mutants osga2-1 and osga2-2 of Cas9 free without CRISPR/Cas9 vector; d represents the protein expression level of OsGA2 in the anaplerotic line plant detected by utilizing FLAG antibody; -60 bp "-" is the length of the 60bp gene ruler;
FIG. 2 shows the lesion phenotype of the osga2 mutant inoculated rice blast S5 (rice blast-S5) strain, wherein A represents the resistance enhancement phenotype of the osga2 mutant against rice blast S5, and B represents the statistics of lesion area using Adobe Photoshop CS Extended 64bit software;
FIG. 3 is a graph showing the preparation of OsGA2OE over-expression plants, wherein A represents the levels of OsGA2 expression in a western blot assay wild type (Kitaake) and three independent OsGA2 over-expression plants (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3); b represents the expression level of OsGA2 in wild type (Kitaake) and over-expressed plants (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3) identified by a real-time quantitative PCR (RT-qPCR) method;
FIG. 4 shows the lesion phenotype of OsGA2OE overexpressing plants inoculated with rice blast S5 (rice blast-S5) strain, wherein A represents the resistance attenuation phenotype of OsGA2OE overexpressing plants against rice blast S5, and B represents the lesion area statistics using Adobe Photoshop CS Extended 64bit software.
Detailed Description
The invention provides an OsGA2 protein for regulating and controlling plant rice blast resistance, wherein the OsGA2 protein comprises a protein with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence shown as SEQ ID NO.1 is specifically as follows:
MAEETPVEAPPAPVLGEPMDLMTALQLVMKKSSAHDGLVKGLREAAKAIEKHAAQLCVLAEDCDQPDYVKLVKALCAEHNVHLVTVPSAKTLGEWAGLCKIDSEGKARKVVGCSCVVVKDFGEESEGLNIVQDYVKSH。
the OsGA2 protein has the function of regulating and controlling the resistance of rice blast, and can improve the resistance of rice blast by knocking out the OsGA2 gene and reducing the expression level of the OsGA2 protein, and can reduce the resistance of rice blast by overexpressing the OsGA2 gene and improving the expression level of the OsGA2 protein.
The invention also provides an OsGA2 gene for encoding the OsGA2 protein in the scheme, wherein the CDS sequence of the OsGA2 gene comprises a sequence shown in SEQ ID NO.2, and the nucleotide sequence shown in SEQ ID NO.2 is specifically as follows: ATGGCGGAAGAGACTCCAGTTGAGGCCCCACCTGCCCCGGTTCTTGGAGAGCCGATGGACCTGATGACTGCTCTGCAGCTTGTGATGAAGAAGTCAAGCGCTCATGATGGGCTCGTGAAGGGGCTCCGTGAGGCGGCCAAGGCCATTGAGAAGCATGCCGCTCAGCTTTGCGTGCTTGCTGAGGACTGTGACCAGCCTGATTATGTCAAGTTGGTCAAGGCACTCTGCGCTGAGCACAATGTTCACCTCGTTACCGTGCCTAGTGCTAAAACTCTTGGCGAGTGGGCAGGGCTTTGCAAGATTGATTCTGAGGGCAAGGCGAGGAAGGTCGTAGGCTGCTCCTGCGTCGTTGTCAAGGACTTCGGTGAAGAGTCAGAGGGCCTCAACATAGTCCAGGACTATGTCAAGTCCCACTAG.
The invention provides an application of the OsGA2 protein or the biological material related to the OsGA2 protein in the scheme in regulating and controlling the rice blast resistance of plants. In the present invention, the plant preferably comprises rice.
In the present invention, the regulation preferably includes increasing the resistance to rice blast of a plant by negatively regulating the expression level of the OsGA2 protein, or decreasing the resistance to rice blast of a plant by positively regulating the expression level of the OsGA2 protein.
In the present invention, the biomaterial preferably includes one or more of the following:
1) The OsGA2 gene described in the above scheme;
2) A recombinant vector comprising the OsGA2 gene described in the above-mentioned scheme;
3) And (3) negatively regulating the sgRNA expressed by the OsGA2 gene or the recombinant vector in the scheme.
In the present invention, the recombinant vector of the OsGA2 gene in the above-mentioned scheme preferably comprises a recombinant vector capable of overexpressing the OsGA2 gene, the recombinant vector capable of overexpressing the OsGA2 gene preferably comprises a basic vector preferably comprising a pCsV1300-3 xFLAG vector and a coding sequence of the OsGA1 protein, and the coding sequence of the OsGA1 protein is preferably located between the XbaI and BamHI sites of the pCsV1300-3 xFLAG vector. The recombinant vector capable of over-expressing the OsGA2 gene can improve the expression level of the OsGA2 protein and reduce the resistance of rice blast.
The pCsV1300-3 xFLAG vector of the present invention is preferably constructed by ligating a 3 xFLAG sequence after the BamHI site of the pCsV1300 vector, and the specific method preferably comprises: homologous primers were designed based on FLAG tag sequence (ATGGACTACAAAGACCATGATGGAGACTATAAGGATCACGACATCGATTACAAGGACGATGACGATAAG, SEQ ID NO. 11): FLAG-F:5'-ctcggtaccggatccATGGACTACAAAGACCATGATGGAGACTATAAGGATCACGACATCGATTACAAGGACGATGACGATAAG-3', SEQ ID NO.12 and FLAG-R:5'-gaacgatcgggaattCTTATCGTCATCGTCCTTGTAATCGATGTCGTGATCCTTATAGTCTCCATCATGGTCTTTGTAGTCCAT-3', SEQ ID No.13, wherein, of the two primer sequencesThe lower case sequence is the homology arm. 1. Mu.L of each of the primers FLAG-F and FLAG-R (the working concentration of both primers was 100. Mu.M) was placed in 48. Mu.L of ddH 2 O, mixing and then placing the mixture in boiling water until the boiling water is cooled to room temperature, namely annealing the single-stranded primer into a double-stranded short DNA fragment with a homology arm. The obtained double-stranded short DNA fragment and the pCsV1300 vector obtained by BamHI digestion are subjected to homologous recombination to obtain the pCsV1300-3 xFLAG vector. The pCsV1300 vectors of the invention are reported in the prior art, see in particular (ADNA Methylation Reader-Chaperone Regulator-Transcription Factor Complex Activates OsHKT1;5Expression during Salinity Stress,2020.09).
In the present invention, the target sequence of the sgRNA preferably includes the target sequence shown in SEQ ID No.3 and/or SEQ ID No.4, and the nucleotide sequence of the target sequence shown in SEQ ID No.3 is specifically as follows: CCGATGGACCTGATGACTGCTC (PAM site for the last three bases); the nucleotide sequence of the target sequence shown in SEQ ID NO.4 is specifically as follows: GCTCGTGAAGGGGCTCCGTGAGG (PAM site for the last three bases).
In the present invention, the recombinant vector for negative regulation of the expression of the OsGA2 gene in the above-described scheme preferably comprises a target sequence and a base vector; the target sequence preferably comprises the target sequence shown in SEQ ID NO.3 or SEQ ID NO. 4; the base vector preferably comprises pYLCRISPR-Cas9PUbi-H.
The sgRNA or recombinant vector for negative regulation of the expression of the OsGA2 gene can reduce the expression of the OsGA2 gene and the expression level of the OsGA2 protein, thereby improving the rice blast resistance. Through identification and application of the rice blast resistance gene OsGA2, breeders can develop rice blast resistant rice varieties suitable for different rice planting areas in various places, and important guarantee is provided for rice grain yield increase.
The invention also provides application of the OsGA2 protein or the biological material related to the OsGA2 protein in the scheme in cultivation of transgenic plants with improved rice blast resistance.
In the present invention, the transgenic plant preferably comprises transgenic rice.
For further explanation of the present invention, the application of the OsGA2 protein, the coding gene or the biological material related thereto provided by the present invention to the regulation of rice blast resistance of plants will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of OsGA2 gene mutant by CRISPR/Cas9 gene editing technology
Two different targets were selected by the target gene OsGA2 sequence using http:// skl. Scau. Edu. Cn/website, the target positions are shown as A in FIG. 1, wherein the target 1 (target 1) nucleotide sequence is: CCGATGGACCTGATGACTGCTC (SEQ ID NO.3, starting three bases as PAM site); the target 2 (target 2) nucleotide sequence is: GCTCGTGAAGGGGCTCCGTGAGG (SEQ ID NO.4, last three bases are PAM sites).
Candidate target sequences target 1 and target 2 were inserted into the intermediate vector pYLsgRNA-LacZ-OsU a, respectively, and then the fragments were inserted into the final vector pYLCRISPR-Cas9PUbi-H by Golden Gate cloning using BsaI cleavage sites (specific construction procedure references: ma X, zhang Q, zhu Q, et al A robustCRISPR/Cas 9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant,2015, 8:1274-1284.).
The successfully constructed CRISPR/Cas9 vector is transformed into wild rice Kitaake callus, and positive transgenic plants are obtained through hygromycin screening, specifically: plants capable of growing on hygromycin-containing medium are positive transgenic plants (transformation methods and screening process references: toki S, haraN, ono K, et al early infection of scutellum tissue with Agrobacterium allows high-speed transformation office plant Journal,2006, 47:969-976.).
2. Molecular characterization of the edited form of the obtained positive transgenic plant
The genomic DNA of rice leaves was used as a template, PCR amplification was performed using the following identification primers, sanger sequencing was performed on the amplified products, and the mutant edited form was analyzed.
The osga2 mutant identification primers were: osGA2-crispr-Check-F (SEQ ID NO. 5): GCGAGTTGTTCTGGCTGAG; osGA2-crispr-Check-R (SEQ ID NO. 6): CGCCTTGCCCTCAGAAT.
The PCR amplification system was formulated according to the instructions for 2 XEs TaqMastermix (Dye), specifically: 2 XEs Taq Master mix (Dye) 10. Mu.L, osGA2-crispr-Check-F (working concentration 10. Mu.M) 0.8. Mu.L, osGA2-crispr-Check-R (working concentration 10. Mu.M) 0.8. Mu.L, DNA template 1. Mu.g, ddH 2 O was added to 20. Mu.L.
The PCR reaction system is as follows: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 15s, and cycling for 35 times; extending at 72℃for 2min.
As a result, as shown in FIG. 1B, two independent CRISPR homozygous mutant lines of osga2 were obtained, designated as osga2-1 and osga2-2, respectively. In OsGA2-1, 4 bases (GACC) were deleted 58-61 bp downstream of the ATG of the initiation codon of the OsGA2 gene, and a frame shift mutation was generated, resulting in premature formation of a stop codon 58-60 bp. In OsGA2-2, 1 base C was deleted 126bp downstream of the initiation codon ATG of the OsGA2 gene, and a frame shift mutation was generated, resulting in the premature formation of a stop codon at 331 to 333 bp.
To exclude the effect of Cas9 gene in progeny plants, seeds of homozygous mutant plants were screened on 1/2MS medium containing 50mg/L hygromycin, and seeds that could not grow on this medium (seeds that could not germinate) were Cas9 free homozygous mutants that did not contain CRISPR/Cas9 vector as the subsequent experimental material, as shown in C in fig. 1.
3. Construction of the background of the osga2 mutant of the anaplerotic plants Com#2-1 and Com#2-2
To further confirm whether the OsGA2 mutant phenotype was due to the loss of the OsGA2 gene. According to the website https:// phytozome-next.jgi.doe.gov/query of OsGA2 genome information, a region 1-2000 bp upstream of an initiation codon ATG of an OsGA2 gene (OsKitaake 07g080700.1) is selected as a promoter to drive and express a CDS sequence of the OsGA2 gene fused with a FLAG tag, an OsGA2pro::2×FLAG-OsGA2 recombinant plasmid (OsGA 2pro represents a selected promoter) taking pCAMBIA1302 as a carrier skeleton is constructed, and transformed into homozygous mutant OsGA2-1 callus containing Cas9 free, and the homozygous mutant can grow on a 1/2MS medium containing 50mg/L hygromycin, namely the transformed callus is differentiated into a complementation line plant taking the OsGA2 mutant as a background, and the serial numbers are Com#2-1 and Com#2-2. And then, carrying out Westernblot to detect the protein expression level of the OsGA2 in the anaplerotic line plant by using FLAG antibody, and taking alpha-H3 as a control.
As a result, as shown in D in FIG. 1, it was found that the target band of FLAG-OsGA2 could be detected in the anaplerotic plants Com#2-1 and Com#2-2.
4. Wild type (Kitaake), mutant (osga 2-1 and osga 2-2) and anaplerotic line (Com#2-1 and Com#2-2) were inoculated with the pathogenic phenotype of rice blast S5 strain
Inoculating bacterial solutions of rice blast S5 (rice blast-S5) into wild type Kitaake, mutant osga2-1 and osga2-2, and anaplerotic line Com#2-1 and Com#2-2 respectively, scratching 3 wounds on each leaf of each material by using sterile forceps, adding 10 mu L of bacterial solution into each wound, wherein the bacterial number of the bacterial solution is 2×10 5 The onset of rice blast was observed 7 days after inoculation at 3 spores/mL of each material (see Lu F, wang H, wang S, et al, enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor TurectoreEFR. Journal ofIntegrative Plant biology.2015,57, 641-652.).
As shown in FIG. 2A, the leaves of wild type Kitaake inoculated with rice blast S5 showed small areas of yellow lesions, the leaves of mutants osga2-1 and osga2-2 showed little yellow lesions at the inoculation sites, and the leaves of the anaplerotic lines Com#2-1 and Com#2-2 showed small areas of yellow lesions at the inoculation sites. Vaccination experiments the same results were obtained in two independent vaccination experiments using two independent mutant lines, the pictures showing several leaves randomly selected in the results.
5. Statistics of lesion areas of wild type (Kitaake), mutant (osga 2-1 and osga 2-2) and anaplerotic line (Com#2-1 and Com#2-2) inoculated rice blast S5 strain
The pictures of rice leaves showing yellow lesions after inoculation of the strains of rice blast S5 with wild type (Kitaake), mutant (osga 2-1 and osga 2-2) and the anaplerotic line (Com #2-1 and Com # 2-2) were opened with Adobe Photoshop CS Extended 64bit software, the position of yellow lesions selected by the magnetic lasso tool was selected, the histogram panel was opened in the window, and the pixels were found to give pixels of the selected area according to the formula: 1 square inch = 72 x 72 pixels = 5184 pixels, 1 square inch = 0.00064516 square meters, and the area of the selected yellow spot area is finally calculated. The statistical result of the lesion area ratio is the data statistics of 3 leaves of each material, the data are experimental average value + -SD, 3 biological replicates, t test, and the P value is less than 0.01. The statistical results are shown in fig. 2B and table 1.
TABLE 1 lesion area ratio of different materials (%)
Group of Blade 1 Blade 2 Blade 3
Kitaake 16.17 14.81 15.43
osga2-1 5.13 4.27 4.86
osga2-2 4.93 3.75 4.38
Com#2-1 15.13 14.52 14.79
Com#2-2 14.61 16.32 15.79
As can be seen from B in FIG. 2 and Table 1, the lesion areas of the leaves of the mutants osga2-1 and osga2-2 were significantly smaller than those of the leaves of the wild-type Kitaake and the make-up materials Com#2-1 and Com#2-2.
Example 2
Construction of OsGA2 Gene overexpression plant OsGA2OE
Leaf RNA of rice variety Kitaake is extracted and reverse transcribed into cDNA. The cDNA is used as a template, and SEQ ID NO.7 (5'-ATGGCGGAAGAGACTCCAGTTGA-3') and SEQ ID NO.8 (5'-GTGGGACTTGACATAGTCCTGGACTA-3') are used as primers for amplification, and a PCR amplification system is prepared according to the use instruction of 2X plant Master mix, and specifically comprises the following steps: 2 Xplanta Mastermix 25. Mu.L, upstream primer (10. Mu.M) 2. Mu.L, downstream primer (10. Mu.M) 2. Mu.L, cDNA template 400ng, ddH 2 O was added to 50. Mu.L. The PCR reaction system is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, extension at 72℃for 60s, and circulation 32 times; the PCR product of 414bp is obtained after the PCR product is fully extended for 5min at 72 ℃. The PCR product has nucleotides 1 to 414 of the sequence shown in SEQ ID NO. 2.
The recovered product is used as a template, homologous recombination primers (SEQ ID NO.9:5'-tctatcgattctagaATGGCGGAAGAGACTCCAGTTGA-3' and SEQ ID NO.10: 5'-gtagtccatggatccGTGGGACTTGACATAGTCCTGGACTA-3') are used for carrying out a second PCR amplification, and a second PCR amplification system is prepared according to the use instruction of 2X plant Master mix, and specifically comprises the following steps: 2 Xplanta Mastermix 12.5. Mu.L, 1. Mu.L of upstream primer (10. Mu.M), 1. Mu.L of downstream primer (10. Mu.M), template10ng，ddH 2 O was made up to 25. Mu.L. The second round PCR reaction system is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s and extension at 72℃for 60s (e.g.primer Tm. Gtoreq.72℃, annealing step can be deleted, extension step can be performed directly), and the cycle is repeated 32 times; extending at 72 deg.C for 5min.
The second round of PCR products were recovered and subjected to homologous recombination with pCsV1300-3 xFLAG vector backbone obtained by digestion with XbaI and BamHI. Wherein the pCsV1300-3 xFLAG vector is obtained by modifying the pCsV1300 vector skeleton at the original BamHI site and then ligating the modified pCsV1300 vector skeleton into the 3 xFLAG sequence, and the specific method is as follows: homologous primers were designed based on the FLAG tag sequence (SEQ ID NO. 11): FLAG-F: SEQ ID NO.12 and FLAG-R: SEQ ID NO. 13. 1. Mu.L of each of the primers FLAG-F and FLAG-R (the working concentration of both primers was 100. Mu.M) was placed in 48. Mu.L of ddH 2 O, mixing and then placing the mixture in boiling water until the boiling water is cooled to room temperature, namely annealing the single-stranded primer into a double-stranded short DNA fragment with a homology arm. The obtained double-stranded short DNA fragment and the pCsV1300 vector obtained by BamHI digestion are subjected to homologous recombination to obtain the pCsV1300-3 xFLAG vector. The pCsV1300 vectors of the invention are reported in the prior art, see in particular (A DNA Methylation Reader-Chaperone Regulator-Transcription Fact or Complex Activates OsHKT1;5Expression during Salinity Stress,2020.09).
After the second round of PCR recovery was obtained, the PCR product was inserted between XbaI and BamHI sites of the linearized pCsV1300-3 xFLAG vector using 2 xClonExpress Mix recombinase to obtain the final vector pCsV1300-OsGA2-3 xFLAG.
The homologous recombination reaction system is as follows: the second PCR recovery product was 18ng, linearized pCsV1300-3 xFLAG vector 60ng (the molar ratio of the second PCR recovery product to linearized pCsV1300-3 xFLAG vector was 3:1), 2 xClonExpress Mix 5. Mu.L, ddH 2 O was added to 10. Mu.L.
After sequencing, the recombinant vector is an OsGA2 over-expression vector, wherein the sequence of SEQ ID NO.2 is inserted between XbaI and BamHI cleavage sites of a pCsV1300-3 xFLAG vector from nucleotide 1 to nucleotide 414.
The pCsV1300-OsGA2-3 xFLAG over-expression vector which is successfully constructed is transformed into wild rice Kitaake callus, and positive transgenic plants are obtained through hygromycin screening, specifically: plants capable of growing on hygromycin-containing medium are positive transgenic plants (transformation methods and screening process references: toki S, hara N, ono K, et al early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice plant Journal,2006, 47:969-976.).
2. Molecular characterization of the Positive transgenic plants obtained above
Cutting fresh tender leaves of a third plant with the top down, placing the fresh tender leaves in a precooled 2mL centrifuge tube with steel balls, rapidly placing the centrifuge tube into liquid nitrogen for freezing, and fully grinding the centrifuge tube into powder by a sample grinder. And then adding 200 mu L of IPbuffer lysate into the ground sample, fully lysing the sample, centrifuging, and boiling the sample. Western blot was performed using the alpha-FLAG antibody to detect the protein expression level of OsGA2 in wild type (Kitaake) and over-expressed plants (OsGA 2 OE), with alpha-H3 as a control.
As a result, three overexpression lines (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3) having higher protein expression levels of OsGA2 were selected from the three overexpression lines (OsGA 2 OE) using wild-type (Kitaake) as a negative control, as shown in FIG. 3A. And the expression level of OsGA2 in transgenic plants was identified by a real-time quantitative PCR (RT-qPCR) method, as shown in B in FIG. 3 and Table 2, and the expression level of OsGA2 was significantly increased in three transgenic lines compared to wild-type Kitaake.
TABLE 2 expression levels of OsGA2 from different strains
Group of Plant 1 Plant 2 Plant 3
Kitaake 1.008 0.995 1.037
OsGA2OE-1 24.853 25.256 25.126
OsGA2OE-2 31.192 30.757 30.653
OsGA2OE-3 26.783 27.515 27.128
3. Observation of the pathogenic phenotype of the OsGA2 overexpressing Strain inoculated with the Rice blast S5 Strain
Dried seeds of wild type (Kitaake) and over-expressed (OsGA 2 OE) harvested at the same place in the same year are placed in a sterilized conical flask, a proper amount of sodium hypochlorite solution (available chlorine is more than or equal to 8 percent) is added, the seeds are soaked for 10 minutes, then the seeds are washed for 4 to 5 times by distilled water, and the washed seeds are placed in a glass culture dish filled with wet filter paper in an incubator (the whole experiment ensures that the filter paper is wet). The culture conditions are as follows: the relative humidity is 65 percent at 28 ℃, the light is irradiated for 10 hours, the light is dark for 14 hours, and the light intensity is 200 mu M, photons, M -2 ·s -1 。
To further investigate the function of OsGA2 in rice blast resistance, three independent overexpressing strains selected OsGA2 over express (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3) were cultured to a trefoil-heart stage, and a bacterial suspension of rice blast S5 (rice blast-S5) was inoculated on the upper surfaces of leaves of wild-type Kitaake and the overexpressing strains of rice (inoculation method and amount refer to step 4 in example 1), and the onset symptoms of rice blast were observed 7 days after inoculation.
As shown in FIG. 4A, leaves of wild type Kitaake inoculated with rice blast S5 showed small-area yellowing lesions, and leaves of overexpressed lines (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3) showed large-area yellowing lesions at the inoculation sites. Inoculation experiments the same results were obtained in two independent inoculation experiments using three independent overexpression lines, the pictures showing several leaves randomly selected in the results.
4. Statistics of lesion area of rice blast S5 strain inoculated with OsGA2 overexpression strain
The method comprises the steps of opening a rice leaf picture of a yellow spot after inoculating an OsGA2 over-expression strain to a strain of rice blast S5 by using Adobe Photoshop CS Extended 64bit software, selecting a position of the yellow spot by a magnetic lasso tool, opening a histogram panel in a window, finding pixels to obtain pixels of a selected area, and obtaining the pixels of the selected area according to the formula: 1 square inch = 72 x 72 pixels = 5184 pixels, 1 square inch = 0.00064516 square meters, and the area of the selected yellow spot area is finally calculated. The statistical result of the lesion area ratio is the data statistics of 3 leaves of each material, the data are experimental average value + -SD, 3 biological replicates, t test, and the P value is less than 0.01. The statistical results are shown in fig. 4B and table 3.
TABLE 3 lesion area ratio of different materials (%)
Group of Blade 1 Blade 2 Blade 3
Kitaake 14.1 15.2 14.9
OsGA2OE-1 89.6 88.1 88.7
OsGA2OE-2 92.3 93.8 92.8
OsGA2OE-3 91.6 94.3 93.5
As can be seen from FIG. 4B and Table 3, the lesion area of the leaves of the OsGA2 overexpressing lines (OsGA 2OE-1, osGA2OE-2 and OsGA2 OE-3) was significantly larger than that of the leaves of the wild-type Kitaake material.
In conclusion, the invention proves that the OsGA2 protein or the biological material related to the OsGA2 protein has the function of regulating and controlling the resistance of rice blast, the expression level of the OsGA2 protein is reduced by knocking out the OsGA2 gene, the resistance of the rice blast can be improved, the OsGA2 gene is over-expressed, the expression level of the OsGA2 protein is improved, and the resistance of the rice blast can be reduced. Through identification and application of the rice blast resistance gene OsGA2, breeders can develop rice blast resistant rice varieties suitable for different rice planting areas in various places, and important guarantee is provided for rice grain yield increase.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. An OsGA2 protein for regulating and controlling plant rice blast resistance is characterized in that the OsGA2 protein comprises a protein with an amino acid sequence shown as SEQ ID NO. 1.
2. An OsGA2 gene encoding the OsGA2 protein of claim 1, wherein the CDS sequence of the OsGA2 gene comprises the sequence shown in SEQ ID No. 2.
3. Use of an OsGA2 protein or a biological material related thereto as claimed in claim 1 for modulating rice blast resistance in plants.
4. The use according to claim 3, wherein the regulation comprises increasing the resistance to rice blast of a plant by negatively regulating the expression level of an OsGA2 protein or decreasing the resistance to rice blast of a plant by positively regulating the expression level of an OsGA2 protein.
5. The use according to claim 3, wherein the biological material comprises one or more of the following:
1) The OsGA2 gene of claim 2;
2) A recombinant vector comprising the OsGA2 gene of claim 2;
3) An sgRNA or recombinant vector that negatively regulates the expression of the OsGA2 gene of claim 2.
6. The use according to claim 5, wherein the target sequence of sgRNA comprises one or more of the target sequences shown in SEQ ID No.3 and SEQ ID No. 4.
7. The use according to claim 5, wherein the recombinant vector of 3) comprises a target sequence and a base vector; the target sequence comprises a target sequence shown as SEQ ID NO.3 or SEQ ID NO. 4.
8. The use of claim 7, wherein the base vector comprises pYLCRISPR-Cas9PUbi-H.
9. The use according to any one of claims 3 to 8, wherein the plant comprises rice.
10. Use of an OsGA2 protein or biological material related thereto as claimed in claim 1 for the cultivation of transgenic plants having increased resistance to rice blast.
CN202211376979.3A 2022-11-04 2022-11-04 Application of OsGA2 protein, coding gene or related biological material thereof in regulation and control of plant rice blast resistance Pending CN116041462A (en)

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Publication number Priority date Publication date Assignee Title
CN116064648A (en) * 2022-11-04 2023-05-05 东北师范大学 Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064648A (en) * 2022-11-04 2023-05-05 东北师范大学 Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance
CN116064648B (en) * 2022-11-04 2024-02-02 东北师范大学 Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance

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