CN115927364B - Plant disease-resistant related gene RLP53 and application thereof - Google Patents
Plant disease-resistant related gene RLP53 and application thereof Download PDFInfo
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Abstract
The invention discloses a plant disease-resistant related geneRLP53And uses thereof, the genesRLP53The nucleotide sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The invention is realized byedr1Method for screening inhibitor mutant and cloning map position, and related gene for plant disease resistanceRLP53Separation and function identification are carried out, and the first discovery and the demonstration are carried outRLP53The role of the gene in plant resistance to powdery mildew, pseudomonas and oomycetes. In agricultural production, the invention can be applied to the creation of transgenic crops for improving the pathogen resistance of crops.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, in particular to a plant disease resistance related geneRLP53And applications thereof.
Background
Powdery mildew is an important fungal disease, is widely distributed around the world, can infect nearly ten thousand plants including important crops such as wheat, barley and the like, and has serious harm to agricultural production. After the powdery mildew infects plants, the finally produced spores can be covered on the surfaces of leaves, fruits and the like of the plants to form white powder, so that the powdery mildew is called powdery mildew. Powdery mildew can be propagated in a large quantity, so that the growth and development of plants are seriously affected, and even the leaves are withered and dead. Similar to powdery mildew, pseudomonas bacteria and oomycetes can infect a variety of plants, including some important crops, and cause great harm to agricultural production.
In order to combat the invasion of various pathogenic microorganisms, plants have evolved a complex immune system. Plant immunity is generally divided into two layers, basal immunity and disease resistance gene-mediated immunity. Basic immunity recognizes Pathogen-Associated Molecular Signatures (PAMPs) through pattern receptors (Pattern Recognition Receptors, PRRs) located on the cytoplasmic membrane, triggering PAMPs-triggered immune responses, also known as PTI, as the first layer of plant immunity. Pathogenic bacteria have generated new pathogenic mechanisms during long-term evolution in order to inhibit the PTI response, e.g. pathogenic bacteria release effector(s) into plants in different ways, destroying important elements in the PTI response of plants. In turn, some effector agents can be recognized by proteins encoded by resistance genes in plants. This level of immune response is commonly referred to as an Effector-triggered immune response (ETI). Generally PTI has a broad spectrum, while ETI has a higher specificity. Recent studies have found that two levels of resistance work synergistically to enhance plant resistance to pathogenic bacteria.
Early studies found Arabidopsis thalianaedr1The mutant has stronger powdery mildew resistance than wild type plants, and when powdery mildew is infected, induced cell death is generated.EDR1The gene codes for a protein kinase and has kinase activity. Genetic analysis experiments show thatedr1The disease resistance and cell death phenotype of the mutant is dependent on the salicylic acid signaling pathway. However, at present forEDR1The molecular mechanisms that mediate cell death and plant disease-resistant responses remain unclear. In order to find related genes for regulating and controlling plant disease resistance, we screenededr1An inhibitor mutant. We obtained one of the mutants, and by localization and sequencing we obtained the corresponding geneRLP53. Through the functional study of the gene, we find for the first time thatRLP53Has important effect on plant disease resistance reaction. Our results indicate thatRLP53Is an excellent candidate gene for creating crops with improved disease resistance, and has important theoretical value and wide application prospect.
Disclosure of Invention
The invention aims at providing plant disease resistance related genesRLP53And applications thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
plant disease-resistant related geneRLP53The geneRLP53The nucleotide sequence of (2) is shown as SEQ ID NO.1, and the total length of CDS is 2877bp; the plant is Arabidopsis thaliana.
Further toThe above genesRLP53The amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
The plant disease resistance related geneRLP53The application in regulating and controlling the resistance of plants to powdery mildew, pseudomonas and oomycetes; the plant is Arabidopsis thaliana.
The plant disease resistance related geneRLP53The application in cultivating transgenic plants with improved resistance to powdery mildew, pseudomonas and oomycetes; the plant is Arabidopsis thaliana.
The invention has the remarkable advantages that:RLP53the gene is derived from Arabidopsis thaliana, and the related signal path research is clear. And is also provided withRLP53After over-expression, the plant has no obvious influence on the growth and development of the plant, and can be considered to specifically play a role in plant disease resistance reaction.
Drawings
Fig. 1:RLP53regulating resistance of Arabidopsis thaliana to powdery mildew. (A) A. The phenotype observation of a large amount of inoculated powdery mildew. Inoculating powdery mildew to arabidopsis plants with the size of 4 weeksG. cichoracearumTaking the leaves after 8 days, taking the pictures, and measuring the length of the staff to 1cm. (B) trypan blue staining of leaves. Plant leaves 8 days after inoculation of powdery mildew were stained with trypan blue, after which the growth of powdery mildew spores and death of leaf cells were observed with a microscope and photographed with a scale length of 100 μm. (C) powdery mildew spore count. Leaves were taken 5 days after inoculation of powdery mildew and trypan blue staining was performed, and conidiophores were counted under a microscope. Different lowercase letters represent statistically significant differences (P < 0.05, one-way ANOVA, tukey test).
Fig. 2:RLP53regulating and controlling the resistance of the Arabidopsis thaliana to pseudomonas syringae and oomycetes. (A) Inoculation of 4 week-sized Arabidopsis materialsPtoA DC3000 strain. The concentration of the bacterial liquid is OD 600 = 5×10 -4 Cfu: the number of colonies was counted, and the number of colonies,pad4mutants served as susceptibility controls. Different lowercase letters represent statistically significant differences (P < 0.05, one-way ANOVA, tukey test). (B) Spore statistics were performed 7 days after inoculation of 2 week-old arabidopsis materials with h.a.noco2. 0.1g of the inoculated plant material was weighed, added to 10mL of pre-chilled deionized water, vortexed vigorously, and the spore concentration was counted using a hemocytometer and the units were calculatedNumber of h.a.noco2 spores in weight plant leaves. Different lowercase letters represent statistically significant differences (P < 0.05, one-way ANOVA, tukey test).
Fig. 3:RLP53gene structure, protein sequence and mutation siteRLP53The phenotype of the transgenic plants is overexpressed. (A)RLP53The coding region of the gene does not contain an intron,rlp53-2the mutation site is changed from 200 th base T to A,rlp53-1the mutation type is T-DNA insertion mutation. RLP53 predicts the receptor protein, SP is signal peptide; NT: NT-LRR domain; JM: membrane proximal domain; TM: transmembrane domain; CD, intracellular domain. (B)RLP53Expression, detection after 4 weeks of wild and transgenic Arabidopsis materials are obtainedRLP53Gene transcript levels. (C) expression of RLP53 protein in transgenic plants. Taking 4-week-old leaf blades to extract total protein for immunoblotting detection, detecting RLP53-Myc fusion protein by using alpha-Myc antibody, and displaying loading amount by using Rubiosco protein detected by ponceau dye. Different lowercase letters represent statistically significant differences (P < 0.05, one-way ANOVA, tukey test). (D) Col-0 and RLP53 over-expressed plant material of 4 weeks size. Plant material cultivated for 4 weeks under short sunlight conditions was photographed under the same conditions from the aerial parts, with a scale length of 1cm. (E) inoculation powdery mildew phenotype observation. Inoculating powdery mildew to arabidopsis plants with the size of 4 weeksG. cichoracearumLeaves were taken after 8 days and photographed.pad4Mutants were the susceptibility control. The length of the scale is 0.5cm. (F) powdery mildew spore count. Inoculation of powdery mildewG. cichoracearumLeaves were taken 5 days later and trypan blue stained and the number of conidiophores counted under a microscope.pad4Mutants served as susceptibility controls. Different lowercase letters represent statistically significant differences (P < 0.05, one-way ANOVA, tukey test).
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1rlp53Mutation-inhibiting powdery mildew-resistant phenotypic analysis
(1) Materials and methods
For Arabidopsis thalianaedr1EMS mutagenesis is carried out on the mutant, and the screening of the inhibitor mutant is carried out on the M2 generation of the mutant, thus identifying that the mutant can inhibitedr1Mutants resistant to powdery mildewrlp53 edr1Subsequently, the mutated gene was mapped and designated as a gene based on its encoded proteinRLP53。
Arabidopsis wild type plants Col-0,edr1mutant and method for producing the samerlp53 edr1The mutant was grown in a greenhouse at 22℃for 9 hours and inoculated with powdery mildew after 5 weeks of growth. Disease resistant phenotype was identified 8 days after inoculation, representative leaves were photographed, and representative leaves were trypan blue stained (Trypan Blue Staining) to observe the growth of powdery mildew.
To quantitatively analyze the resistance of plants, the plants were inoculated with powdery mildew after growing for 4-5 weeks under the same conditions, and after 5 days of inoculation, leaves were trypan blue stained and monospore conidiophore counts were performed under a microscope. Statistical analysis was performed on the count results of 20-30 monospores.
(2) Results and analysis
After 8 days of powdery mildew inoculation, a large amount of powdery mildew is generated on the surface of the leaves of the wild Col-0edr1The leaf of the mutant only supports the growth of small amounts of powdery mildew and significant cell death occurs. Whilerlp53 edr1The mutant did not develop and did not develop on the leaf surface after 8 days of powdery mildew inoculationedr1Similar cells died, but similar to wild type Col-0, a large number of spores were produced (fig. 1A). To further observe the powdery mildew phenotype, leaves were trypan blue stained (fig. 1, B), as can be seen,edr1obvious cell death was seen after staining of the mutants and only small amounts of spores were produced, whereas wild type andrlp53 edr1neither double mutation causes cell death, but rather produces a large number of hyphae and spores. Quantitative analysis showsedr1The number of conidia is significantly lower than that of wild type Col-0rlp53Inhibition ofedr1Resistance phenotype of (FIG. 1C), these junctionsFruit showsedr1Resistance to powdery mildew and powdery mildew-induced cell deathRLP53。
Example 2rlp53Phenotypic analysis of mutants to inhibit other pathogenic bacteria
(1) Materials and methods
Arabidopsis wild type plants Col-0,edr1the mutant is used for the preparation of the mutant,rlp53 edr1plants were grown in a greenhouse at 22℃for 9 hours with light for 2 weeks. Mutant of susceptibility pad4Oomycetes grown for 7 daysH. a. Noco2) As a bacterial source, the oomycete spores are diluted to 5 multiplied by 10 by vortex shaking with water 4 /ml. Spores were sprayed evenly onto the surface of 2 week-sized plant leaves using a watering can. The seedling tray is covered with a transparent cover, the periphery of the seedling tray is sealed by an adhesive tape for moisture preservation, the seedling tray is placed in an incubator at the temperature of 16 ℃ and the relative humidity of 90%, the disease-resistant phenotype is observed after 7 days, and spore counting is carried out by a blood cell counting plate.
Pseudomonas syringae (Pto DC 3000) was streaked on KB solid medium containing the corresponding resistance for 12 h with 10 mM MgCl 2 Bacteria grown on the medium were collected and diluted by gradient to a concentration of OD 600 =5×10 -4 . Bacteria were injected into the back of 4 week-sized arabidopsis leaves and sampled after injection 3 h as a 0 day control. Taking out the blades by using a puncher. Mu.l of 10 mM MgCl was added 2 Fully grinding the blade, and diluting the obtained blade homogenate to 10 -1 And 10 -2 The colonies on the medium were counted after plating KB plates at 28℃for 2-3 days at both concentrations. Leaves 3 days after infestation were again sampled with a punch and bacteria counted.
(2) Results and analysis
For Pseudomonas syringae, the quantitative results show (FIG. 2A),rlp53 edr1mutants are more susceptible than the wild type, supporting significantly higher numbers of bacterial growth than the wild type. Indicating thatRLP53Participating in Pseudomonas syringaePtoDisease resistance of DC 3000.
The results of the statistics on oomycetes are shown (figure 2B),rlp53 edr1in the mutant, spore growth of a large number of oomycetes is supported, and the number of spores is higher than that of wild typeType, indicateRLP53Is involved in the disease resistance to oomycetes.
Example 3RLP53Cloning and identification
(1) Materials and methods
To clone the repressor gene, we willrlp53edr1The mutant is hybridized with an Arabidopsis wild Landsberg ecological plant, and the obtained F1 generation plant is selfed to generate an F2 generation population. Selection of plants from F2 generationedr1Mutant but is associated withrlp53edr1 40 individuals with similar mutant phenotypes were genotyped with coarse localization markers (http:// signal. Salk. Edu/genome/SSLP_info/SSLPsorded. Html) evenly distributed on the Arabidopsis genome. The results of the coarse positioning indicate thatRLP53Is located on chromosome 5. Subsequently, we designed a new molecular marker, genotype the F2 plants, and identify themRLP53Positioned between NGA139 and PHYC. The mutant was then subjected to genomic sequencing to find the mutant gene (fig. 3A).
(2) Results and analysis
We analyzed the sequencing results and found that it was found thatRLP53(AT 5G 27060) this gene has a base change from T to A, resulting in a change AT amino acid 67 (I67N) in the protein sequence (FIG. 3A).
Example 4 overexpressionRLP53Phenotypic analysis of transgenic Arabidopsis inoculated powdery mildew
(1) Materials and methods
Will beRLP53The full-length CDS sequence is connected into a pSuper1300-Myc vector to construct a 35S promoter driverRLP53The Coding (CDS) sequence expression vector 35S: RLP53-Myc, the transformation of Arabidopsis thaliana, the selection of antibiotics to obtain homozygous transgenic lines, the detection of real-time quantitative PCR, and the finding of the homozygous transgenic linesRLP53Transgenic lines with significantly increased levels of gene expression.
The wild-type Col-0 was subjected to the procedure, RLP53the over-expression transgenic plants are planted in a greenhouse at 22 ℃ for 9 hours, and are inoculated with powdery mildew after growing for 4 weeks. And (3) carrying out powdery mildew disease resistance phenotype identification 5 days after inoculation, carrying out trypan blue staining on infected leaves, photographing, and observing the growth condition of powdery mildew on the leaves.
To quantitatively analyze the resistance of plants, leaves of wild-type and mutant plants inoculated with powdery mildew were counted under a microscope for single spore conidiophores and subjected to statistical analysis, and the experiment was repeated at least 3 times.
(2) Results and analysis
Quantitative PCR detection results show that the expression is excessiveRLP53In the transgenic lines of (a) and (b),RLP53the gene expression level of (a) was significantly higher than that of the wild type plant (fig. 3B); and these two are overexpressedRLP53High amounts of RLP53 protein are also accumulated in the transgenic lines of (a) of (b) (fig. 3C); under normal condition growth, overexpressionRLP53Plants of the transgenic lines of (a) grew normally, similar to the wild type (fig. 3D); after inoculation of powdery mildew, overexpressionRLP53The number of monospore conidiophores of the transgenic lines of (a) was significantly lower than that of the wild type, indicating overexpressionRLP53Resistance of plants to powdery mildew can be enhanced (fig. 3E, fig. 3F).
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (2)
1. The application of the over-expressed plant disease-resistant related gene RLP53 in improving the resistance of plants to powdery mildew is characterized in that: the nucleotide sequence of the gene RLP53 is shown as SEQ ID NO.1, and the total length of CDS is 2877bp; the amino acid sequence of the protein coded by the gene RLP53 is shown as SEQ ID NO. 2; the plant is Arabidopsis thaliana; the powdery mildew is powdery mildew bisporus (Golovinomyces cichoracearum).
2. The application of the over-expressed plant disease-resistant related gene RLP53 in cultivating transgenic plants with improved powdery mildew resistance is characterized in that: the nucleotide sequence of the gene RLP53 is shown as SEQ ID NO.1, and the total length of CDS is 2877bp; the amino acid sequence of the protein coded by the gene RLP53 is shown as SEQ ID NO. 2; the plant is Arabidopsis thaliana; the powdery mildew is powdery mildew bisporus (Golovinomyces cichoracearum).
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| Tabata,S.，等.Arabidopsis thaliana receptor like protein 53 (RLP53), mRNA.Genbank登录号：NM_00134399.1.2019,参见全文. * |
| The receptor-like protein53 immune complex associates with LLG1 to positively regulate plant immunity;Renjie Chen，等;Journal of Integrative Plant Biology;第64卷(第9期);第1833-1846页 * |
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