CN116790655A - Gene OsSLM and application thereof - Google Patents
Gene OsSLM and application thereof Download PDFInfo
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Abstract
The invention discloses a gene OsSLM and application thereof. According to the invention, rice lesion mutant slm is obtained through EMS mutagenesis, and after wild type rice lesion mutant slm and mutant leaf blade are treated by fungus chitin and bacterial flagellin, ROS content level is detected, rice blast germ is inoculated artificially, resistance level is identified, expression levels of the mutant and relevant genes of the wild type rice lesion are compared and analyzed by using a fluorescent quantitative PCR technology, genes for controlling rice lesions are cloned and controlled, then genetic transformation is used for verifying gene functions, and finally, a rice immune negative regulation gene OsSLM is successfully separated. Experimental results show that compared with a wild type, the OsSLM function deletion mutant obtained by EMS mutagenesis has extremely improved resistance to rice blast of rice fungal diseases, can be effectively applied to rice disease resistance molecule improvement work, and has a wide development and application prospect.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a gene OsSLM and application thereof.
Background
Plants evolved two layers of the innate immune system to address the injury caused by infection with various pathogenic bacteria (viruses, bacteria and fungi): pathogen-associated molecular pattern-induced immunity (Pathogen-associated molecular pattern triggered immunity, PTI) and Effector-induced immunity (Effector-triggered immunity, ETI). PTI relies on pattern recognition receptors (Pattern recognition receptors, PRRs) on the surface of cell membranes and is generally believed to have weaker but broad-spectrum disease resistance, whereas ETI is stimulated by effector proteins secreted by plant-specific disease resistance proteins (R proteins) to recognize pathogenic bacteria and has stronger race-specific disease resistance. Upon sensing the pathogen infection, the immune system in the plant is rapidly activated, blocking further infection and expansion of the pathogen by rapidly initiating apoptosis (Programmed cell death, PCD) at the site of infection, while transmitting signals to adjacent tissues, inducing resistance in the system. In plants, these two pathways interact to co-combat pathogen infestation.
Rice is one of the most important food crops in the world, and more than 50% of the world population is on rice as the main diet and this proportion is also on an increasing trend year by year. With global warming and frequent natural disasters, maintaining stable and high yield of rice is important to guaranteeing world grain safety. The utilization of the rice autoimmune system to combat diseases and reduce yield losses is one of the more rational and effective measures. The nature of rice lesion mutants is a manifestation of plant Hypersensitive Response (HR) leading to uncontrolled plant PCD. Meanwhile, the defense reaction in most plant disease spot mutants is activated, and the resistance to some plant pathogenic bacteria is obviously enhanced. Therefore, the plant disease spot mutant becomes an ideal material for researching plant PCD and defense reaction, and has important research significance for revealing plant defense pathways, signal regulation networks and the like. With the rise of modern molecular biology, the molecular means is utilized to excavate the disease-resistant gene of the rice, analyze the autoimmune reaction mechanism of the rice, cultivate excellent new disease-resistant varieties, strengthen the disease-resistant capability of the rice, and have important significance for guaranteeing the safe production of the rice.
The defensive response of plants is caused by the recognition of PAMPs by some PPRs on the cell membrane, the PTI response. After PTI is activated, some PRRs may share endocytic motifs, which in the case of activation of a defensive response, transmit an immune signal downstream by endocytosis. After recognition of the signal molecules by the plant's PAMPs receptors, ubiquitination and phosphorylation modifications occur, which are then passed through a sorting process, either by vesicle transport pathways back to the plasma membrane, or transported to vacuoles for degradation, a process of transport mediated by the ESCRT complex is critical for the plant to mount a normal immune response.
The separation and cloning of the disease-resistant related genes is a precondition for researching the disease-resistant mechanism of rice. Meanwhile, compared with the application of the disease-resistant gene, the application of the disease-resistant related gene can provide the plant with wider spectrum and long-acting resistance, and the disease resistance of the rice variety is improved by inhibiting the function of the disease-resistant negative regulation factor related gene, so that the disease resistance of the plant is further enhanced, and the disease resistance spectrum of the plant is widened.
Disclosure of Invention
The invention aims to provide a gene OsSLM and application thereof.
In order to achieve the aim of the invention, in a first aspect, the invention provides application of a gene OsSLM in regulating and controlling rice disease resistance.
Further, the modulation is negative modulation.
Wherein, the gene OsSLM is:
i) SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b)
ii) SEQ ID NO:1 by substitution, deletion and/or addition of one or more nucleotides and expressing the same functional protein; or (b)
iii) Under stringent conditions with SEQ ID NO:1 and expressing the same functional protein in a solution of 0.1 XSSPE containing 0.1% SDS or 0.1 XSSC containing 0.1% SDS at 65℃and washing the membrane with the solution; or (b)
iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.
The disease resistance of the invention is rice blast resistance. For example, the pathogenic bacteria is pyriform (Magnaporthe grisea Barr.).
In a second aspect, the present invention provides a method for improving disease resistance of rice, which uses genetic engineering means to perform site-directed mutagenesis on rice gene OsSLM, so that the gene function is deleted, thereby improving disease resistance of rice.
Furthermore, site-directed mutagenesis was performed on the rice gene OsSLM, resulting in a deletion of 4bp (GGGG) in the fifth exon of the gene, and finally, frame shift mutation resulted in premature termination of the translation of the amino acid at codon 192 (SEQ ID NO: 4).
In a third aspect, the present invention provides an OsSLM protein mutant which is:
(a) Consists of SEQ ID NO:4, and a protein consisting of an amino acid sequence shown in the formula (4); or (b)
(b) SEQ ID NO:4, and a protein derived from (a) which has the same function and is obtained by substituting, deleting or adding one or more amino acids.
In a fourth aspect, the invention provides a gene encoding the protein mutant.
In a fifth aspect, the present invention provides biological materials comprising the genes, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.
In a sixth aspect, the present invention provides the use of said gene or a biological material comprising said gene for increasing disease resistance in rice.
Further, the application includes:
1) Allowing the plant to contain said gene; or (b)
2) Allowing the plant to express the protein mutant.
In a seventh aspect, the present invention provides the use of transgenic rice obtained according to the method or the use in plant breeding.
Methods of breeding include, but are not limited to, transgenesis, crosses, backcrosses, selfing, or asexual propagation.
According to the invention, rice lesion mutant slm is obtained through EMS mutagenesis, and after wild type medium-speed rice 8015 and mutant leaves are treated by fungus chitin and bacterial flagellin, the ROS content level is detected, rice blast bacteria are inoculated artificially, the resistance level is identified, the expression levels of the mutant and the relevant genes of the wild type medium-speed rice are compared and analyzed by using a fluorescent quantitative PCR technology, the genes for controlling rice lesions are cloned and controlled, then the gene function is verified through genetic transformation, and finally, a rice immune negative regulation gene-OsSLM is successfully separated. Experimental results show that compared with a wild type, the OsSLM function deletion mutant obtained by EMS mutagenesis has extremely improved resistance to rice blast of rice fungal diseases, can be effectively applied to rice disease resistance molecule improvement work, and has a wide development and application prospect.
Drawings
FIG. 1 is a graph comparing the phenotype of programmed cell death of wild-type Zhonghui 8015 and mutant slm in a preferred embodiment of the invention. a-b are the spot-like phenotypes of plants (a) and leaves (b) at tillering stage (60 days after sowing). c-d heading stage plants (c) and leaf (d) type plaque phenotype.
FIG. 2 is a graph showing comparison of the results of the identification of the resistance of the artificial inoculation of Pyricularia oryzae in the preferred embodiment of the present invention. (a) is a leaf spot phenotype comparison graph of inoculated rice blast fungus, (b) is a leaf spot area comparison graph of inoculated rice blast fungus, and (c) is a leaf spot biomass comparison graph of inoculated rice blast fungus.
FIG. 3 is a graph showing the relative expression levels of 8 defense-related genes (OsNPR 5, osNPR1, osPR1a, osPR1b, osJAZ1, osPR10, osWRKY45 and OsPAL 1), wherein (a) is the tillering stage and (b) is the heading stage.
FIG. 4 is a graph showing the dynamic analysis of ROS accumulation in a preferred embodiment of the present invention. (a) A graph of ROS dynamic analysis after chitin treatment, and (b) a graph of ROS accumulation dynamic analysis after Flg22 treatment.
FIG. 5 shows the mutation pattern of OsSLM according to the preferred embodiment of the present invention. (a) the structure and mutation pattern of the OsSLM gene. (b) analysis of OsSlM expression levels in WT and slm. (c) amino acid sequence analysis of OsSLM and OsSLM.
FIG. 6 is a graph showing the phenotype comparison of the transgenic complementary materials in the preferred embodiment of the present invention. (a) is a whole plant comparison graph, (b) is a single leaf comparison graph, (c) transcript levels of OsSLM at WT, slm and complementary plants, (d) WT, slm and complementary plant leaf DAB staining, (e) rice blast resistance identification of wild type, mutant and complementary plants.
Detailed Description
The invention aims to provide a rice immune negative regulation protein OsSLM related to rice disease resistance, a coding gene and application thereof.
The invention adopts the following technical scheme:
the invention provides a rice immune negative regulation protein OsSLM, which is:
(a) Consists of SEQ ID NO:4, and a protein consisting of an amino acid sequence shown in the formula (4); or (b)
(b) SEQ ID NO:4, and a protein derived from (a) which has the same function and is obtained by substituting, deleting or adding one or more amino acids.
The present invention provides a gene encoding the above protein.
Further, the genome gene sequence of the OsSLM encoding the rice immune negative regulatory protein is shown as (A) or (B):
(A) SEQ ID NO:1, and a nucleotide sequence shown in the specification;
(B) A mutant gene, allele or derivative which is produced by adding and/or substituting and/or deleting one or more nucleotides in the nucleotide sequence defined in (A) and which encodes a protein having the same function.
Further, the cDNA sequence for encoding the rice immune negative regulation protein OsSLM is shown in (C) or (D):
(C) SEQ ID NO:2, a nucleotide sequence shown in seq id no;
(D) A cDNA which is produced by adding and/or substituting and/or deleting one or more nucleotides in the nucleotide sequence defined in (C) and can encode a protein having the same function.
The protein and the gene can be applied to the improvement of rice disease resistance molecules.
The invention also provides a mutant protein of the rice immune negative regulation protein OsSLM for improving the disease resistance of rice, and a coding gene, an expression vector, a transformant and application thereof.
Further, the amino acid sequence of the mutant protein of the rice immune negative regulation protein OsSLM is shown as SEQ ID NO: 4.
The invention also provides a gene for encoding the protein.
The invention also provides an expression vector or transformant containing the gene.
The invention also provides application of the protein, the gene or the expression vector and the transformant in improving the disease resistance of rice.
Further, the disease resistance is rice blast resistance.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Fungal Chitin, bacterial flagellin, flg22, used in the examples below, were purchased from Santa Cruz and guangzhou zimuth biosystems, respectively.
The term PAMP: pathogen-associated molecular pattern (Pathogen-associated molecular patterns).
EXAMPLE 1 Slm mutant acquisition and identification of apoptosis phenotypes
And (3) recovering 8015 (wild type, WT) in the indica rice restorer is subjected to EMS mutagenesis screening to obtain a part of lesion-like mutants. Under natural field environment conditions, about 60 days after sowing and transplanting, reddish brown lesions appear at the base of the main veins of the leaves, and the mutant is named slm (spotted leaf midrib). As rice grows, the lesions on the main vein develop upward and spread to both sides of the main vein until the heading stage spreads to the whole leaf (fig. 1).
EXAMPLE 2 identification of rice blast resistance
The rice blast inoculation adopts a punching method. Sowing wild Zhonghui 8015 and mutant slm seeds into cylindrical culture pots with the diameter of 15cm and the height of 15cm filled with sterilized nutrient soil after seed soaking and germination accelerating, and culturing 5 plants in each pot at constant temperature for 12 hours under illumination and 12 hours in the dark at 26 ℃ in an artificial greenhouse until the tillering stage. When the mutant type lesion phenotype was produced, a round wound with a diameter of 1.5mm was artificially made on the surface of the inverted trilobal leaf by using a punch, and 10. Mu.l of the prepared spores were sucked to a concentration of about 10 5 A physiological race of Pyricularia oryzae 12-144-1-1 (see Zhang Y, liu Q, zhang Y, chen Y, yu N, cao Y, zhan X, cheng S, cao L.LMM24 Encodies Receptor-Like Cytoplasmic Kinase 109,Which Regulates Cell Death and Defense Responses in Rice.Int J Mol Sci.2019 Jul 2;20 (13): 3243.Doi:10.3390/ijms20133243.PMID:31269643; PMCID: PMC 6651581.) was dropped onto the wound and fixed with a transparent adhesive tape, the light and temperature were kept unchanged in a climatic chamber, the humidity was adjusted to 80% to promote the onset of the culture, and the spot size was investigated after 14 days and sampled. DNA of rice leaves and rice blast fungus at the inoculation position is extracted, a fluorescent quantitative PCR technology is applied, rice action is used as an internal reference gene, and DNA of a rice blast fungus gene Pot2 (accession number on NCBI: CP 050928) is relatively quantified, so that the ratio of biomass of the rice leaves and the rice blast fungus is represented. The amplification primers were as follows (5 '-3'):
OsActin-F：CAGGCCGTCCTCTCTCTGTA
OsActin-R：AAGGATAGCATGGGGGAGAG
MoPot2-F：ACGACCCGTCTTTACTTATTTGG
MoPot2-R：AAGTAGCGTTGGTTTTGTTGGAT
compared with wild type strain No. 8015, the mutant slm has obviously improved resistance to Pyricularia oryzae physiological race 12-144-1-1, the lesion area is obviously smaller than that of the wild type strain, and the propagation and amplification speed of Pyricularia oryzae in the mutant blade is obviously and effectively inhibited (figure 2).
EXAMPLE 3 defense-related Gene expression analysis
Wild Zhonghui 8015 and mutant slm are planted in the test field of China paddy research institute, and normal field management is performed. 3 single plants with consistent growth vigor are selected in the tillering stage and the heading stage, the tip parts (about 1/3 of the total length of the leaves) of the three leaves are respectively taken, total RNA is extracted by using an RNAprep Pure plant total RNA extraction kit of Beijing Tiangen Biochemical technology Co., ltd, a cDNA first strand is synthesized by using an Toyo-yo First strand cDNA Synthesis Kit Rever Tra Ace kit, a ChamQ SYBR qPCR Master Mix kit of Nuo-NJ company is selected for fluorescence quantitative PCR, and the operation steps are referred to the specification. The primers used were as follows (5 '-3'):
qOsPR5 CTTCTGCCCATAATGCATCATCTGATTATCGATCAAGGTGTCGT
qOsNPR1 CACTGCACTACGCCGTCGAACTCTCTTCGCCTCGCAGCAA
qOsPOX GCTCCAAGGTGAACTCCTAATTATATGGGTATATGTGGTGTGGC
qOsPR1a CGTGTCGGCGTGGGTGTGGCGAGTAGTTGCAGGTGATG
qOsPR1b CATTGCTTTGGCCATGGTAGGAACCCCAGAAGAGGTTCTC
qOsJAZ1 GCGCTCCCGGAGArGCCGATTTCGCTCGTTGTCGTGATCCTGT
qOsPR10CTCAAGATGATCGAGGACTACCAGAAAGGCACATAAACACAACC
qOsWRKY4 GCCGACGACCAGCACGATCACC
5 ACGAGCCGACGCCGCCCTC
qOsAOS2 AAGCTGCTGCAATACGTGTACTGGCGACGAGCAACAGCCTTCCG
qOsPAL1 GACCCTGTATTTTCTTCGTTCGAGTAGCAATACTTTCACCCCAA
the results show that 8 defending related genes are up-regulated in the mutant, and the up-regulation times are different from 3-400 times. Thus further demonstrating the negative regulation of the mutant gene during the course of immune stress (FIG. 3).
Example 4 dynamic analysis of ROS accumulation after PAMP treatment
Material planting method referring to the rice blast inoculation part in example 2, WT and slm leaves were sown and transplanted for about 60 days, punched and sampled with a 0.5cm aperture puncher, the main vein was avoided, and the sampled articles were left in darkness in double distilled water overnight to eliminate ion penetration and defending reaction due to physical damage. Three leaves were randomly picked up with forceps, blotted on clean filter paper, then carefully picked up and placed in a 1.5mL centrifuge tube, and 1. Mu.L of streptavidin, 100. Mu.L of a protease streptavidin-HRP substrate, 1. Mu.L of statin (800 nM) or 1. Mu.L of Flg22 (10. Mu.M), and 1. Mu.L of double distilled water were added in sequence as controls (Mock). The tube was placed in a Glomax (Promega, E5311) instrument to determine the amount of luminescence, once for 10s, for a total of 20min. As shown in FIG. 4, mutant slm was more responsive to both statin and Flg22, and RO5S (reactive oxygen species) accumulation rate and peak were significantly higher than that of wild-type strain Hei 8015.
EXAMPLE 5 cloning of OsSLM Gene
Hybridization with slm as female parent and WT as male parent to obtain F 1 Bagging and selfing to obtain F 2 Isolating the population. 30 strains of F with the disease-like spot phenotype are taken 2 DNA is extracted from the single plant, and the DNA is uniformly mixed in the same proportion to construct a mutant mixed pool. The same method is used for constructing a WT pool, carrying out library construction sequencing, carrying out gene annotation on candidate sites by using ANNOVAR, and preferentially selecting genes at which the sites causing frame shift mutation or non-synonymous mutation or alternative splicing are located as candidate genes. Based on the distribution of SNP and InDel, and the functional annotation of the genome of reference rice variety 9311, it was found that only InDel deletion (-4 bp, GGGG) on gene BGIOSGA018999 (LOC_Os05g 01250) on chromosome 5 resulted in frame shift mutation. Loc_os05g01250 was therefore listed as a candidate gene for analysis.
To verify if the candidate gene LOC_OsO5g01250 has mutation in slm, the full-length sequences of LOC_Os05g01250 genomes of WT and slm were PCR amplified with the gene sequence annotated by RGAP (http:// price. Uga. Edu /) website as a template, and sequenced and aligned. The results indicate that a deletion mutation of 4bp (GGGGGG) occurs at the junction of the fifth exon end and the intron of the slm LOC_Os05g01250 gene. To verify if the mutation caused a frame shift in the coding sequence, RNA from WT and slm was extracted, the CDS sequence of LOC_Os05g01250 was amplified by reverse transcription, sequencing alignment was followed by discovery that the mutation caused a fifth exon deletion of 4bp (GGGGGG), and finally the frame shift mutation resulted in premature termination of translation of the amino acid at codon 192 (SEQ ID NO: 4). The level of transcription of the gene in vivo in rice after mutation was verified by qRT-PCR method, and as a result, it was found that the level of transcription of the gene was extremely significantly reduced in slm (FIG. 5).
Example 6 transgenic complementation verification of OsSLM Gene function
To verify if the plaque-like phenotype resulted from the OsSLM mutation, 3.7Kb from the WT including the complete coding region of the OsSLM gene, 2.5Kb before the start codon, 1.6Kb after the stop codon, and a total of 6.8Kb sequence were inserted into the complementary vector pCAMBIA1300 vector, and the recombinant plasmid was transferred into slm callus by agrobacterium infection. Under the natural field environment condition, all T 1 Positive transgenic plants showed consistent growth and development with WT, with no appearance of the plaque-like phenotype throughout the growth cycle. Subsequently, the transcript level of OsSLM in the complementation transgenic plant was examined, and as a result, it was found that the transcript level of OsSLM in the complementation transgenic plant was recovered. The leaves of the heading stage plants were then DAB stained as shown in FIG. 6, T 1 Leaves of the generation of complementing plants did not show brown precipitate in slm, H 2 O 2 The level was restored to the normal level of WT. In contrast, the rice blast resistance test results showed that the rice blast resistance of the complemented plants was also restored to a level comparable to that of the wild type. The above results demonstrate that both the slm-like plaque phenotype and the significantly enhanced rice blast resistance are caused by the functional loss of OsSLM.
The result fully shows that the OsSLM is a resistance related protein and mainly plays a negative regulation role in the rice immune stress reaction process. With the development of CRISP genome site-directed editing technology and the gradual maturation of other molecular biology technical means, the gene can be effectively applied to rice resistance molecule improvement in the future, and has great development and utilization values and significance.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one 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.
Claims (10)
1. Application of gene OsSLM in regulating and controlling rice disease resistance;
the regulation is negative regulation;
wherein, the gene OsSLM is:
i) SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b)
ii) SEQ ID NO:1 by substitution, deletion and/or addition of one or more nucleotides and expressing the same functional protein; or (b)
iii) Under stringent conditions with SEQ ID NO:1, under stringent conditions in a solution of 0.1 XSSPE containing 0.1% SDS or 0.1 XSSC containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)
iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.
2. The use according to claim 1, wherein the disease resistance is resistance to rice blast.
3. The method for improving the disease resistance of the rice is characterized in that a genetic engineering means is utilized to carry out site-directed mutagenesis on a rice gene OsSLM, so that the gene function is deleted, thereby improving the disease resistance of the rice;
locking the disease resistance to rice blast;
wherein the definition of the gene OsSLM is as described in claim 1.
An osslm protein mutant characterized in that it is:
(a) Consists of SEQ ID NO:4, and a protein consisting of an amino acid sequence shown in the formula (4); or (b)
(b) SEQ ID NO:4, and a protein derived from (a) which has the same function and is obtained by substituting, deleting or adding one or more amino acids.
5. A gene encoding the mutant protein of claim 4.
6. A biological material comprising the gene according to claim 5, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.
7. Use of the gene of claim 4 or the biological material of claim 5 for improving disease resistance of rice.
8. The application according to claim 7, characterized in that it comprises:
1) Allowing the plant to contain said gene; or (b)
2) Allowing the plant to express the protein mutant.
9. Use of transgenic rice obtainable according to the method of claim 4 or the use of claim 7 or 8 in plant breeding.
10. The use according to claim 9, wherein the breeding method comprises transgenesis, crossing, backcrossing, selfing or asexual reproduction.
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