CN108588087B - Gene GmLecRK-R for improving disease resistance of plants and application thereof - Google Patents
Gene GmLecRK-R for improving disease resistance of plants and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
Abstract
The invention provides a gene for improving plant disease resistance and application thereof, belongs to the field of plant molecular biology and plant genetic engineering, and relates to a plant-derived disease-resistant gene GmLecRK-R, a recombinant expression vector thereof and application thereof. The gene is derived from soybean, has a nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence of the encoded product is SEQ ID NO. 2. The invention also provides a gene silencing and recombination expression vector, which comprises the gene GmLecRK-R. The gene has a key effect on plant disease resistance, particularly phytophthora resistance. The over-expression of the gene can remarkably promote the disease resistance of soybean and tobacco to different phytophthora, and is an ideal gene for enhancing the disease resistance of plants. The gene is expressed in tobacco by a genetic transformation method to obtain the resistance capability to various pathogenic bacteria, and can also be applied to the aspect of improving the disease resistance of crop breeding.
Description
Technical Field
The invention belongs to the field of plant molecular biology and plant genetic engineering, and particularly relates to a gene for improving plant disease resistance and application thereof.
Background
Currently, research on disease resistance genes in plants has focused on a class of genes encoding receptor proteins containing Nucleotide Binding Sites (NBS) and leucine-rich repeats (LRR)[1-3]. NBS-LRR disease-resistant protein coded by the gene acts in plant cells, and can identify effector molecules secreted by pathogenic bacteria to activate defense reaction of plants, so that infection of the pathogenic bacteria is inhibited. However, the recognition of NBS-LRR type disease-resistant proteins and pathogenic effector molecules in plants follows the hypothesis of "gene-for-gene", i.e., NBS-LRR type disease-resistant proteins can only recognize specific pathogenic effector molecules. Once the effector molecule secreted by the pathogenic bacteria is mutated, the disease-resistant function of the NBS-LRR type disease-resistant protein to the pathogenic bacteria is lost[1-3]. Effector molecules secreted by different races of pathogenic bacteria have diversity, so that NBS-LRR type disease-resistant protein has race specificity on the disease resistance of the pathogenic bacteria. With the continuous change of the composition of the field microspecies, most of genes which are cloned in plants and code NBS-LRR type disease-resistant proteins lose the disease-resistant effect on phytophthora. Therefore, research and development of the gene with broad-spectrum lasting disease resistance to phytophthora are effective ways for improving the disease resistance of crops.
There are a number of cell membrane receptors in plants whose structure includes an extracellular domain, a transmembrane domain and an intracellular domain. The protein participates in identifying a pathogen or a conservative molecular pattern released by a plant in an infection process, and activates the basic defense reaction of the plant, and comprises the following steps: cell necrosis, burst of active oxygen, expression of immune-related gene, and synthesis of secondary metabolite[4]. Several genes encoding cell membrane immunoreceptors have been cloned in plants, such as FLS2, which recognizes bacterial flagellin and activates plant immunity[5]. Flagellin is an important component of bacteria, has important effect on pathogenicity of pathogenic bacteria, and is difficult for pathogenic bacteria to escape recognition of plant cell membrane receptors by modifying such conserved molecules[6]. Due to the fact thatThe plant cell membrane immunoreceptor mediated plant disease resistance has broad spectrum and durability.
Soybean (Glycine max) originates from China and is one of the important grain and oil crops in China. The soybean root rot is a disease with extremely strong damage to soybeans, has large damage area and high damage degree, and is listed as one of the destructive diseases threatening soybean production[7]. Soybean root rot is widely distributed in the major soybean producing areas of the world, and the economic loss caused by the soybean root rot is up to billions of dollars each year around the world [8]. In China, soybean root rot is found in the northeast of the early 90 s[9]The disease is one of the important diseases of soybean production areas in northeast China, Huang-Huai-Hai and southern vegetable soybean production areas, and the production safety of soybeans is seriously threatened. The soybean root rot is mainly caused by Phytophthora sojae (Phytophthora sojae) infection and can cause harm to soybeans in each growth period[2]. Under environmentally favorable conditions, root rot spreads rapidly on infected plants, and its harm can lead to the loss of yield of soybeans. Therefore, the method has important significance for effectively controlling the soybean root rot by improving the disease resistance of crop varieties.
At present, the research of the cell membrane receptor in the plant on the disease resistance of phytophthora is still in the initial stage, and the research of the phytophthora resistance gene of the plant including soybean has important significance on the research of the disease resistance of the plant.
Reference documents:
1.Dong,S.,Qutob,D.,Tedman-Jones,J.,Kuflu,K.,Wang,Y.C.,Tyler,B.M.,and Gijzen,M.2009.The Phytophthorasojaeavirulencelocus Avr3c encodes a multi-copy RXLR effector with sequence polymorphisms among pathogen strains.PLoS One4(5):e5556.
2.Shan,W.,Cao,M.,Leung,D.,and Tyler,B.M.2004.The Avr1b locus of Phytophthorasojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b.Molecular Plant-Microbe Interactions 17(4):394-403.
3.Vleeshouwers,V.G.A.A.,Raffaele,S.,Vossen,J.H.,Champouret,N.,Oliva,R.,Segretin,M.E.,Rietman,H.,Cano,L.M.,Lokossou,A.,Kessel,G.,et al.2011.Understanding and exploiting late blight resistance in the age of effectors.Annual Review of Phytopathology 49:507-531.
4.Boller,T.,and Felix,G.2009.A renaissance of elicitors:Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.Annual Review of Plant Biology 60:379-406.
5.Sun,Y.,Li,L.,Macho,A.P.,Han,Z.,Hu,Z.,Zipfel,C.,Zhou,J.M.,and Chai,J.2013.Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1immune complex.Science 342(6158):624-628.
6.Naito K.,Taguchi F.,Suzuki T.,Inagaki Y.,Toyoda K.,Shiraishi T.,and Ichinose Y.2008.Amino acid sequence of bacterial microbe-associated molecular pattern flg22is required for virulence.Molecular Plant-Microbe Interactions 21(9):1165-1174.
7.Wrather,J.A.,and Koenning,S.R.2006.Estimates of disease effects on soybeanyields in the United States 2003to 2005.Journal of Nematology 38(2):173-180.
8.Tyler,B.M.2007.Phytophthorasojae:root rot pathogen of soybean and model oomycete.Molecular Plant Pathology 8(1):1-8.
9. shen Chong Yao and Suyan. 1991. Discovery and preliminary research of phytophthora sojae in China. the report on plant pathology 21: 298.
Disclosure of Invention
One of the purposes of the invention is to provide a gene GmLecRK-R.
The second purpose of the invention is to provide a recombinant expression vector containing a GmLecRK-R gene.
The invention also aims to provide application of the gene GmLecRK-R.
The content of the invention is detailed as follows:
the invention provides a gene GmLecRK-R, which is derived from soybean, has a nucleotide sequence shown in SEQ ID NO.1, or has homology of more than 70% with the sequence SEQ ID NO. 1. Preferably, the nucleotide sequence is shown as SEQ ID NO.1, or has more than 80% homology with the sequence SEQ ID NO.1, further preferably, the nucleotide sequence is shown as SEQ ID NO.1, or has more than 85% homology with the sequence SEQ ID NO.1, and more preferably, the nucleotide sequence is shown as SEQ ID NO.1, or has more than 90% homology with the sequence SEQ ID NO. 1.
The invention also provides a protein coded by the gene GmLecRK-R.
The amino acid sequence of the protein coded by the gene GmLecRK-R is SEQ ID NO.2, or the protein which is derived from the SEQ ID NO.2 and can provide plant disease resistance is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the SEQ ID NO. 2.
Using the amino acid sequence encoded by the gene of the present invention, a signal peptide sequence can be designed and artificially added to facilitate expression in plants.
By using the amino acid sequence coded by the gene of the invention, a nucleic acid sequence which is optimized by codons and is favorable for expression in plants can be designed and artificially synthesized.
The invention also provides a recombinant expression vector comprising the gene GmLecRK-R.
The existing plant expression vector can be used for constructing a recombinant expression vector containing the gene GmLecRK-R.
The conventional plant expression vector is preferably a plant transformation plasmid, and may be an expression vector pBin:eGFP, pCambia or pTF101.1, etc.
Preferably, the recombinant expression vector is a vector pBin which a gene GmLecRK-R is inserted into a binary vector pBin:: an eGFP enzyme cutting site KpnI containing a C-terminal eGFP to obtain the gene GmLecRK-R-eGFP.
When the gene is used for constructing a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide; in addition, when a recombinant expression vector is constructed using the gene of the present invention, an enhancer, including a translation enhancer or a transcription enhancer, may also be used.
A transgenic cell line and a recombinant bacterium containing the gene GmLecRK-R.
A primer pair for amplifying the full length or any fragment of the gene GmLecRK-R also belongs to the protection scope of the invention.
A transformant obtained by introducing the recombinant expression vector into a host cell, preferably an Escherichia coli cell or an Agrobacterium cell.
In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, which are expressed in plants, antibiotic markers having resistance or chemical resistance markers, etc.
The invention also provides application of the gene GmLecRK-R or the protein coded by the gene GmLecRK-R or the recombinant expression vector or the transformant in improving the plant immune resistance or disease resistance.
The invention also provides application of the gene GmLecRK-R or the protein coded by the gene GmLecRK-R or the recombinant expression vector or the transformant in improving the immunity of pathogenic bacteria of plants or improving the resistance of the plants to diseases caused by pathogenic bacteria. In particular to the application of the plant in improving the immunity resistance of phytophthora or improving the resistance of the plant to diseases caused by the phytophthora.
The invention also provides application of the gene GmLecRK-R or the protein coded by the gene GmLecRK-R or a recombinant expression vector or a transformant in plant breeding.
The invention also provides application of the soybean gene GmLecRK-R or the protein coded by the same or a recombinant expression vector or a transformant in obtaining varieties with remarkable disease resistance and/or yield increase after being introduced into plants, preferably in obtaining varieties with enhanced disease resistance and/or yield increase after being introduced into soybeans, tobacco, tomatoes or potatoes.
The invention has the beneficial effects that:
the protein coded by the gene GmLecRK-R enhances the disease resistance of plants to phytophthora. The plant growth traits are not influenced by overexpression in plants, the broad-spectrum property is particularly realized on the identification of phytophthora, the disease resistance of the plants to the phytophthora can be obviously enhanced, the plant growth promoter can be applied to the aspect of improving the disease resistance of crop breeding, and the disease resistance of the plants to the phytophthora is hopefully improved, so that the purposes of increasing yield and reducing pesticide consumption are achieved.
The study finds that the GmLecRK-R has disease resistance on phytophthora sojae and phytophthora nicotianae through the analysis of cell membrane receptors in soybeans. The soybean hairy roots over expressing the GmLecRK-R gene are inoculated with the phytophthora sojae, and the amount of the colonized phytophthora sojae is obviously reduced. The GmLecRK-R gene plays a very important role in soybean disease resistance, and as a typical representative of important commercial crops and leguminous plants, the research on the GmLecRK-R in soybeans can drive the related research on many other plant cell membrane lectin receptor protein kinases. In addition, the over-expression of the gene in the tobacco leaves can remarkably enhance the disease resistance of tobacco to phytophthora nicotianae, and the disease spots caused by the over-expression are remarkably reduced, which shows that the GmLecRK-R has broad-spectrum disease resistance to different phytophthora nicotianae. The research can better clarify the disease resistance function of the plant to phytophthora, and can provide excellent disease resistance gene resources for breeding of disease resistance gene engineering.
Drawings
FIG. 1 pBin GmLecRK-R-eGFP transgenic soybean hairy root protein expression detection. And detecting the expression quantity of the GmLecRK-R-eGFP by using Western blot, wherein the detection antibody is anti-GFP.
FIG. 2 pBin:: eGFP and pBin:: GmLecRK-R-eGFP transgenic soybean oospores generated after phytophthora sojae inoculation. And A, oospore generation of soybean hairy roots. And B, counting the number of oospores generated on the soybean hairy roots.
FIG. 3 pBin protein expression assay in GmLecRK-R-eGFP transgenic tobacco.
FIG. 4 pBin:: eGFP and pBin:: GmLecRK-R-eGFP transgenic tobacco shows the onset symptoms after 3 days of inoculation of Phytophthora nicotianae.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The primer related to the embodiment of the invention is synthesized by Nanjing Kingsrei Biotechnology GmbH.
Example 1 cloning and sequence Structure analysis of the GmLecRK-R Gene
Directly sowing semen glycines and Feng-47 seed in a flowerpot filled with nutrient soil, culturing in greenhouse (21-23 deg.C, 14 hr light/10 hr dark), and collecting 2 weeks old plant for extracting RNA.
Total RNA extraction: the soybean leaves are taken as a material, the total RNA extraction is carried out by adopting an Omega RNA extraction kit according to the instructions, and the RNA content and quality are detected by a spectrophotometer.
Reverse transcription to generate the first strand: mu.g of RNA was used as a template, and cDNA synthesis was carried out according to the instructions of the kit for PrimeScript reverse transcriptase of Takara, Inc., and the volume was adjusted to 20 uL. Appropriate amounts of the reverse transcription products were taken for subsequent gene cloning PCR.
And (3) taking the first cDNA chain as an RT-PCR template, performing PCR by a conventional method, and amplifying a GmLecRK-R gene fragment or a full-length gene:
PCR amplification primer sequence:
an upstream primer: SEQ ID NO.4
(5’-TTACGAACGATAGCCGGTACCATGTCTCATTCCAAAACCCCCG-3’)
A downstream primer: SEQ ID NO.5
(5’-GCTCACCATCCCGGGGGTACCAACCGGAGGAGGGGAGGGTTGC-3’)，
The 50uL reaction system, in which 5 XBuffer 10uL, 2.5mM dNTPs 4uL, Takara Primer STAR Taq enzyme 0.5uL, template cDNA 1uL, water to 50 uL; the PCR amplification program comprises pre-denaturation at 98 ℃ for 2 minutes, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 10 seconds, extension at 72 ℃ for 2 minutes, circulation for 40 times, and final extension at 72 ℃ for 10 minutes; the PCR products of GmLecRK-R were recovered by electrophoresis on agarose gels, photographed by Ethidium Bromide (EB) staining, and the results recorded and excised.
The electrophoretic band was recovered with an Agarose Gel DNA Purification Kit (TaKaRa). PCR products of GmLecRK-R recovered from the gel cutting were ligated to KpnI digested pBin:: eGFP vector according to the protocol of Clonexpress II One Step Cloning Kit (Vazyme) to obtain pBin:: GmLecRK-R-eGFP plasmid, E.coli competent cell GM109 was transformed, LB (containing 50ug/mL) plates were applied, after 16 hours of incubation at 37 ℃ colony PCR was verified, three clones were picked up and plasmid extracted according to the plasmid extraction Kit protocol (Takara) and sent to Nanjing Kingsry for sequencing with the sequence shown as SEQ ID No. 1. The plasmid with correct sequencing is transformed into agrobacterium K599 or GV3101 by electric shock, coated with LB (containing 50ug/mL kanamycin and 50ug/mL streptomycin) plate, cultured at 30 ℃ for 48 hours, and then colony PCR is verified to pick correct clone for subsequent experiments.
Example 2 expression of the GmLecRK-R gene in Soybean root hairs:
the concrete steps are briefly described as follows:
1) planting soybean Hefeng-47:
uniformly sowing seeds of the soybean Hefeng-47 in a flowerpot filled with vermiculite, covering the flowerpot with a black plastic film or placing the flowerpot in the dark for culturing at the initial stage, after yellow seedlings grow uniformly, uncovering the black plastic film, removing seed coats, watering (about 3 days to 4 days), placing the flowerpot in proper illumination for culturing (about 1 day and half to 2 days), namely, cotyledons of plants with the age of six days can be used for experiments.
2) Agrobacterium culture
Single colonies of Agrobacterium rhizogenes K599 transfected with pBin:: GmLecRK-R-eGFP vector and pBin:: eGFP vector were picked from the plates, and inoculated into 2mL LB liquid medium (50 ug/mL Kanna, 50ug/mL streptomycin) respectively, and cultured overnight at 200rpm at 30 ℃ on a constant temperature shaker. The overnight cultured K599 rhizogenes Agrobacterium solution was centrifuged at 4500rpm for 3 minutes to collect the cells. Buffer (composition: 10mM 2- [ N-morpholino)]ethanesulfonic acid,10mM MgCl 2100 μ M acetosyringone pH 5.6) suspension of the bacterial solution and centrifugation to collect the cells. After repeated washing for 3 times, the bacterial solution was diluted with a buffer solution, and the OD600 value of the bacterial solution was adjusted to 0.4.
3) Hairy root transformation
a, pour-plate soybean cotyledon medium (MS +20g/L sucrose + 0.8% agar + Thiosporin 120ug/mL + carbenicillin 120 ug/mL).
And b, sterilizing the soybean cotyledon. Removing soybean cotyledon from the base of petiole, washing off vermiculite on the surface with water, placing in a sterilized plant tissue culture bottle, sterilizing with 70% alcohol for 30 s, soaking and sterilizing with 10% sodium hypochlorite containing 0.5% effective chlorine for 15 min, washing with sterilized tap water for 5 times, and washing with sodium hypochlorite.
And c, soybean cotyledon wound. Putting sterile gloves in a super clean bench, cutting the petiole part of the soybean cotyledon with a sterile scalpel, and digging a wound in the middle of the lower epidermis of the cotyledon close to the petiole part.
d, sticking the upper surface skin of the wounded soybean cotyledon downwards to a culture medium, placing, and dripping a proper amount of bacterial liquid on the wound by using a 1mL medical injector to form a slightly raised small blister.
e, sealing the culture dish with a sealing film, and culturing at 25 ℃ in the dark or under the alternate conditions of dark and light.
4) Preliminary identification of hairy roots
During the culture process, firstly, the wound at the inoculation position becomes brown (two or three days), then, callus grows in the middle or the whole wound, the callus is granular or is connected into sheets (the callus starts to grow in about one week), and the callus needs to grow hairy roots in one to two weeks.
And b, observing the cotyledons with the hairy roots under a fluorescence microscope, and marking the hairy roots with green fluorescence on a culture dish.
And c, selecting cotyledons of the green fluorescent hairy roots, transferring the cotyledons to a 15cm culture dish for amplification culture, observing the growth condition of the hairy roots, and performing subsequent experiments after the hairy roots grow to form lateral roots.
Example 3 cultivation of Phytophthora
Transgenic phytophthora sojae with red fluorescence was inoculated onto solid V8 medium plates (G41850 ug/mL), or phytophthora nicotianae was inoculated onto solid V8 medium plates. The inoculated V8 culture medium plate is placed in dark condition at 25 ℃ for 5 days and then can be used for subsequent experiments.
Example 4 infection of hairy roots
Observing the hairy roots after the enlarged culture under a fluorescence microscope, screening out the hairy roots with green fluorescence, shearing the hairy roots by using scissors, soaking the sheared hairy roots in water for moisturizing, and washing off the MS solid culture medium attached to the hairy roots. Cutting filter paper with the size of 2.5 multiplied by 4.5cm to soak, paving one end of the filter paper on a glass slide, arranging hairy roots with uniform thickness and uniform growth condition on the filter paper in order, enabling one side of the root tip to face one side of the glass slide wool glass and enabling the root tip to extend out of the filter paper, and keeping the distance that all the root tips extend out of the filter paper consistent as far as possible. Approximately 10 hairy roots were placed per slide for infection. Cutting Phytophthora sojae with red fluorescence into rectangular mycelium blocks with similar size, covering the root tips of all hairy roots on the glass slide with the side with mycelium, and covering the hairy roots with the other end of the soaked filter paper. All slides were placed in 15cm empty petri dishes padded with wetted 15cm round filter paper and incubated at 25 ℃ in the dark. The soybean hairy roots inoculated with the phytophthora sojae and infected for 2 days are placed under a fluorescence microscope for disease symptom observation, and the result shows that the number of oospores generated by phytophthora sojae infected on the soybean hairy roots over-expressing GmLecRK-R is obviously reduced (figure 2), which indicates that the disease resistance of soybeans to phytophthora sojae is obviously enhanced by over-expressing GmLecRK-R.
Example 5 overexpression of eGFP and GmLecRK-R-eGFP in tobacco
1) Cultivation of Agrobacterium
The transfected pBin:: GmLecRK-R-eGFP vector and pBin:: eGFP vector single GV3101 colony and suppressor of silencing P19 were picked from the plates, and inoculated into 2mL LB liquid medium (50 ug/mL of Carna, 50ug/mL of rifampicin) at 30 ℃ on a constant temperature shaker and cultured overnight at 200 rpm. The overnight cultured Agrobacterium solution was centrifuged at 4500rpm for 3 minutes to collect the cells. Buffer (composition: 10mM 2- [ N-morpholino)]ethanesulfonic acid,10mMMgCl 2100 μ M acetosyringone pH 5.6) suspension of the bacterial solution and centrifugation to collect the cells. After repeated washing for 3 times, the bacterial solution was diluted with buffer solution and mixed 1:1 with P19 to obtain an OD600 value of 0.6, wherein the vector contains pBin GmLecRK-R-eGFP and the bacterial solution contains pBin eGFP.
2) Tobacco expresses eGFP and GmLecRK-R-eGFP. Injecting the prepared agrobacterium into tobacco leaves by using an injector, and culturing the tobacco after injection in a greenhouse (21-23 ℃, 14h light/10 h dark).
3) Remarkably enhancing disease resistance of tobacco to phytophthora by over-expressing GmLecRK-R-eGFP
The leaves were inoculated with phytophthora nicotianae two days after injection. Disease symptoms were observed 3 days after inoculation (fig. 4) and the results were recorded photographically. Compared with a negative control, the tobacco over-expressing the GmLecRK-R-eGFP has remarkably reduced lesion spots after being inoculated with the phytophthora nicotianae, and the results prove that the disease resistance of the tobacco to different phytophthora nicotianae is remarkably improved by over-expressing the GmLecRK-R-eGFP.
Example 6 detection of cumulative amount of GmLecRK-R protein
And collecting soybean hairy roots or tobacco leaves expressing GmLecRK-R-eGFP for detecting the protein accumulation amount. The collected hairy roots or tobacco leaves were liquid nitrogen-frozen and ground, and then protein extract (consisting of 150mM NaCl,50mM Tris-HCl pH 7.5,10mM EDTA (ethylene-methacrylic acid), 1.0% (v/v) NP-40,1mM phenylmethylisulfonyl fluoride, and 1.0% (v/v) protease inhibitor cocktail was added and mixed well on ice for 30 minutes. 18000g, centrifuging, collecting supernatant, adding 20uL of 5 times protein loading buffer solution, mixing, and boiling in water bath for 10 min. 20uL samples were run on SDS-PAGE gels for 1.5 h at 120V. After the reaction, the protein sample was transferred to PVDF membrane, and the membrane was sealed by incubating with 5% PBST milk. After 2 hours of incubation with 1:5000 dilution of GFP primary antibody (Abmart) three times 5 minutes after membrane washing with PBST, followed by 30 minutes of incubation with 1:10000 dilution of murine antibody (LI-COR, irdye 800, 926-.
In conclusion, the gene GmLecRK-R-eGFP has a key disease-resistant effect on phytophthora nicotianae. The over-expression of the gene can remarkably promote the disease resistance of tobacco to different phytophthora, and is an ideal gene for enhancing the disease resistance of plants. The gene is expressed in tobacco by a genetic transformation method to obtain the resistance capability to various pathogenic bacteria, thereby improving the disease resistance of crops in the field.
Sequence listing
<110> Nanjing university of agriculture
<120> gene for improving disease resistance of plants and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2007
<212> DNA
<213> Soybean Hefeng-47 (Hefeng 47)
<400> 1
atgtctcatt ccaaaacccc cgctgccggc gaaactctat tttccggcac ggcgttcctg 60
attctcctcc acctctttct cttccttact cccgcacttt cccttgactt cctattcaac 120
tccttcgccg gcgtcaccaa cctcactctc atcaaagacg ctcgcgtcga cgcctccgtc 180
atccgaatga acaacgactc caatcagtac tcctacggcc gcgccttcta ccccgtcaaa 240
attcccatgc tcaaaacaaa cacctccaat aactcctctt ccatttcttc cttctccact 300
tcctttgtct tctccatctt gccgcagatc tctaccagcc ccggcttcgg cctcgccttc 360
gtcctctcca acaccaccga ccctcccggc gccatcgcca gccagtactt cggcctattc 420
accaacgcaa cctccccttc cgttttcccc ctcgtcgccg tcgaattcga taccggccgc 480
aaccccgagt tcaacgacat cgacgacaac cacatcggaa tcgacctcaa caacatcgag 540
tccataaacg ccaccactgc cggctacttc aactcctccg gcgccttcgt gccggtgcgc 600
atgcgcaccg gccagaacat ccacgcctgg atcgacttcg acggcgagaa tctcgagttc 660
aacgtaaccg tcgcgccaat cggcgtttcg cgccctacga aacctactct tcggtatcag 720
aatcccgcca tagctgacta cgtgtccagt aacatgtacg tagggttttc cgcttcgaaa 780
acgaactgga tcgaggcgca gagagttctc gcatggagct tcagcgattc aggacctgca 840
agggagctca acacaacgaa tttaccagtt tttgaactag aatcgtcttc ttcctcgctt 900
tctaacggcg caatagcggg catcgtcatc ggttctttta tttttgttct tatatgcgct 960
tctggttttt acttatggtg gcgaatgaac aaagcgaacg aggaagaaga cgagatcgaa 1020
gactgggagc tagagtactg gccgcacaga ttttcctacg aggaactaag ttacgcgaca 1080
ggggaatttc ggaaggagat gctgttaggt tcgggagggt tcgggagagt gtacaaagga 1140
acattgccta acaacacgga aattgcggtg aagtgcgtga accacgattc aaagcaaggg 1200
ttgcgtgaat tcatggcgga gatttcaagc atggggaggc ttcagcacaa gaacttggtt 1260
caaatgagag gatggtgcag aaaggggaac gagcttttgc tggtttatga ttacatgcca 1320
aacgggagtc tcaacaagtg ggttttcgat aagtccgaca aggttttagg gtgggagcaa 1380
cgccgtcgta tacttgtcga cgtggcagag gggcttaact accttcacca cggttgggac 1440
caggttgtta ttcatagaga tattaaatcg agcaacattc tgttggacgc cgacatgaga 1500
gggagattag gggactttgg tctggccaag ctttacacgc acggggaggt tcccaacacc 1560
acgcgtgtgg tggggacgtt gggctacttg gcgccggagc tggccacggt ggcggccccc 1620
acttcggcga ccgacgtcta cagcttcggg gtggtgctgc tggaggtggc gtgcggtagg 1680
cggccgatag agacgtcggt ggcagaggag gaggtggtgc tcattgattg ggtcagggag 1740
ctgtacgcga aggggtgcgc gcgtgaggct gcggatttga ggattagagg ggagtacgat 1800
gagggagatg tggagatggt gttgaagcta gggttggctt gttgccaccc tgatcctcag 1860
aggagaccca ccatgaagga ggtcgttgcg cttctcttgg gagaggaccc gccggaggca 1920
cccggaaaag tcttgtccga tttggttcgc ggtggcgagg attccgacga ggccgcgcct 1980
ttgcaaccct cccctcctcc ggtttga 2007
<210> 2
<211> 668
<212> PRT
<213> Soybean Hefeng-47 (Hefeng 47)
<400> 2
Met Ser His Ser Lys Thr Pro Ala Ala Gly Glu Thr Leu Phe Ser Gly
1 5 10 15
Thr Ala Phe Leu Ile Leu Leu His Leu Phe Leu Phe Leu Thr Pro Ala
20 25 30
Leu Ser Leu Asp Phe Leu Phe Asn Ser Phe Ala Gly Val Thr Asn Leu
35 40 45
Thr Leu Ile Lys Asp Ala Arg Val Asp Ala Ser Val Ile Arg Met Asn
50 55 60
Asn Asp Ser Asn Gln Tyr Ser Tyr Gly Arg Ala Phe Tyr Pro Val Lys
65 70 75 80
Ile Pro Met Leu Lys Thr Asn Thr Ser Asn Asn Ser Ser Ser Ile Ser
85 90 95
Ser Phe Ser Thr Ser Phe Val Phe Ser Ile Leu Pro Gln Ile Ser Thr
100 105 110
Ser Pro Gly Phe Gly Leu Ala Phe Val Leu Ser Asn Thr Thr Asp Pro
115 120 125
Pro Gly Ala Ile Ala Ser Gln Tyr Phe Gly Leu Phe Thr Asn Ala Thr
130 135 140
Ser Pro Ser Val Phe Pro Leu Val Ala Val Glu Phe Asp Thr Gly Arg
145 150 155 160
Asn Pro Glu Phe Asn Asp Ile Asp Asp Asn His Ile Gly Ile Asp Leu
165 170 175
Asn Asn Ile Glu Ser Ile Asn Ala Thr Thr Ala Gly Tyr Phe Asn Ser
180 185 190
Ser Gly Ala Phe Val Pro Val Arg Met Arg Thr Gly Gln Asn Ile His
195 200 205
Ala Trp Ile Asp Phe Asp Gly Glu Asn Leu Glu Phe Asn Val Thr Val
210 215 220
Ala Pro Ile Gly Val Ser Arg Pro Thr Lys Pro Thr Leu Arg Tyr Gln
225 230 235 240
Asn Pro Ala Ile Ala Asp Tyr Val Ser Ser Asn Met Tyr Val Gly Phe
245 250 255
Ser Ala Ser Lys Thr Asn Trp Ile Glu Ala Gln Arg Val Leu Ala Trp
260 265 270
Ser Phe Ser Asp Ser Gly Pro Ala Arg Glu Leu Asn Thr Thr Asn Leu
275 280 285
Pro Val Phe Glu Leu Glu Ser Ser Ser Ser Ser Leu Ser Asn Gly Ala
290 295 300
Ile Ala Gly Ile Val Ile Gly Ser Phe Ile Phe Val Leu Ile Cys Ala
305 310 315 320
Ser Gly Phe Tyr Leu Trp Trp Arg Met Asn Lys Ala Asn Glu Glu Glu
325 330 335
Asp Glu Ile Glu Asp Trp Glu Leu Glu Tyr Trp Pro His Arg Phe Ser
340 345 350
Tyr Glu Glu Leu Ser Tyr Ala Thr Gly Glu Phe Arg Lys Glu Met Leu
355 360 365
Leu Gly Ser Gly Gly Phe Gly Arg Val Tyr Lys Gly Thr Leu Pro Asn
370 375 380
Asn Thr Glu Ile Ala Val Lys Cys Val Asn His Asp Ser Lys Gln Gly
385 390 395 400
Leu Arg Glu Phe Met Ala Glu Ile Ser Ser Met Gly Arg Leu Gln His
405 410 415
Lys Asn Leu Val Gln Met Arg Gly Trp Cys Arg Lys Gly Asn Glu Leu
420 425 430
Leu Leu Val Tyr Asp Tyr Met Pro Asn Gly Ser Leu Asn Lys Trp Val
435 440 445
Phe Asp Lys Ser Asp Lys Val Leu Gly Trp Glu Gln Arg Arg Arg Ile
450 455 460
Leu Val Asp Val Ala Glu Gly Leu Asn Tyr Leu His His Gly Trp Asp
465 470 475 480
Gln Val Val Ile His Arg Asp Ile Lys Ser Ser Asn Ile Leu Leu Asp
485 490 495
Ala Asp Met Arg Gly Arg Leu Gly Asp Phe Gly Leu Ala Lys Leu Tyr
500 505 510
Thr His Gly Glu Val Pro Asn Thr Thr Arg Val Val Gly Thr Leu Gly
515 520 525
Tyr Leu Ala Pro Glu Leu Ala Thr Val Ala Ala Pro Thr Ser Ala Thr
530 535 540
Asp Val Tyr Ser Phe Gly Val Val Leu Leu Glu Val Ala Cys Gly Arg
545 550 555 560
Arg Pro Ile Glu Thr Ser Val Ala Glu Glu Glu Val Val Leu Ile Asp
565 570 575
Trp Val Arg Glu Leu Tyr Ala Lys Gly Cys Ala Arg Glu Ala Ala Asp
580 585 590
Leu Arg Ile Arg Gly Glu Tyr Asp Glu Gly Asp Val Glu Met Val Leu
595 600 605
Lys Leu Gly Leu Ala Cys Cys His Pro Asp Pro Gln Arg Arg Pro Thr
610 615 620
Met Lys Glu Val Val Ala Leu Leu Leu Gly Glu Asp Pro Pro Glu Ala
625 630 635 640
Pro Gly Lys Val Leu Ser Asp Leu Val Arg Gly Gly Glu Asp Ser Asp
645 650 655
Glu Ala Ala Pro Leu Gln Pro Ser Pro Pro Pro Val
660 665
Claims (2)
1, SEQ ID NO.1GmLecRK-ROr the protein shown in SEQ ID NO.2 is applied to improving the immunity resistance of phytophthora or improving the resistance of the plant to diseases caused by phytophthora; the plant is tobacco or soybean.
2, SEQ ID NO.1GmLecRK-ROr the protein shown in SEQ ID NO.2 is applied to the cultivation of phytophthora immunity resistant plants; the plant is tobacco or soybean.
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| CN201810465038.4A CN108588087B (en) | 2018-05-16 | 2018-05-16 | Gene GmLecRK-R for improving disease resistance of plants and application thereof |
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| CN108588087B true CN108588087B (en) | 2022-06-03 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588087B (en) * | 2018-05-16 | 2022-06-03 | 南京农业大学 | Gene GmLecRK-R for improving disease resistance of plants and application thereof |
| CN110713528B (en) * | 2019-10-30 | 2021-06-11 | 中国科学院东北地理与农业生态研究所 | Application of soybean phytophthora root rot resistant related gene GmLMM1 |
| CN110938118B (en) * | 2019-12-17 | 2022-04-29 | 南京农业大学 | Plant immune activation protein PC2 secreted by phytophthora infestans and application thereof |
| CN112143746B (en) * | 2020-09-30 | 2022-04-29 | 南京农业大学 | Gene GmAP5 for improving disease resistance of plants and application thereof |
| CN112626080B (en) * | 2020-12-23 | 2022-07-01 | 河南大学 | R gene for controlling soybean-rhizobium matching property, protein and application thereof |
| CN113151320B (en) * | 2021-03-22 | 2022-06-28 | 华中农业大学 | Potato StLecRK-VI.1 and StTET8 genes and application thereof in improvement of late blight resistance |
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