CN108588087A - It is a kind of improve disease resistance of plant gene and its application - Google Patents
It is a kind of improve disease resistance of plant gene and its application Download PDFInfo
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Abstract
The present invention provide it is a kind of improve disease resistance of plant gene and its application, belong to molecular biology of plants and genetically engineered plant field, the present invention relates to a kind of plant source disease-resistant gene GmLecRK R and its recombinant expression carrier and applications.The gene source has nucleotide sequence shown in SEQ ID NO.1, the product amino acid sequence of coding is SEQ ID NO.2 in soybean.The present invention also provides a kind of gene silencing and recombinant expression carriers, including gene GmLecRK R of the present invention.The gene pairs disease resistance of plant especially phytophthora resistance has key effect.It is overexpressed the gene conspicuousness and promotes soybean and tobacco to the disease resistance of different phytophthoras, be a kind of gene of ideal enhancing disease resistance of plant.The gene is set to express the resistance capacity for making it obtain to a variety of pathogens in tobacco by the method for genetic transformation, in terms of the improvement of crop breeding disease resistance can also be used in.
Description
Technical field
The invention belongs to molecular biology of plants and genetically engineered plant field, specifically, the present invention relates to one kind to carry
The gene of high disease resistance of plant and its application.
Background technology
Currently, being concentrated mainly on a kind of coding to the research of disease-resistant gene in plant contains nucleotide binding site (NBS)
With the receptor protein gene of full asphalt mixture (LRR)[1-3].The NBS-LRR class ill-resistant proteins of such coded by said gene act on
In plant cell, the defense response of the effector molecule activated plant of pathogen secretion is can recognize that, to inhibit infecting for pathogen.
However the identification of NBS-LRR classes ill-resistant protein and pathogen effector molecule follows " gene-for-gene " hypothesis, i.e. NBS- in plant
LRR classes ill-resistant protein can only identify specific pathogen effector molecule.Once the effector molecule of pathogen secretion morphs,
NBS-LRR classes ill-resistant protein will lose the disease-resistant function of pathogen[1-3].The effector molecule of pathogen difference microspecies secretion is deposited
In diversity, thus NBS-LRR classes ill-resistant protein has Race specificity to the disease resistance of pathogen.As field microspecies form
Continuous variation, the gene for the most of coding NBS-LRR class ill-resistant proteins being cloned into plant all lost to phytophthora
The resistant effect of bacterium.Therefore, research and development have phytophthora the gene of broad spectrum durable disease resistance, are to improve crop resistance
Effective way.
There are a large amount of cell-membrane receptor in plant, structure includes an extracellular domain, a membrane-spanning domain and an intracellular
Domain.The albuminoid participates in the conservative molecular pattern that pathogen or plant are discharged in identification infection processs, the base of activated plant
Plinth defense response, including：Meronecrosis, oxidative burst, the expression of gene involved in immunity and the synthesis of secondary metabolite
Deng[4].The gene of several Codocyte film immunity receptors has been cloned into plant at present, such as can identify the flagellum of bacterium
The FLS2 of protein activation plant immune[5].Flagellin is the important component of bacterium, is played an important roll to Pathogenic,
Pathogen is difficult to escape the identification of plant cell membrane receptor by modifying this kind of standpatter[6].Therefore plant cell membrane is immune
The disease resistance of plant that receptor is mediated has broad spectrum activity and persistence.
Soybean (Glycine max) is important one of the grain and oil crop in China originating from China.Root rot is a kind of
Extremely strong disease destructive to soybean, hazard area is big, extent of the destruction weight, is listed in the destructive disease for threatening Soybean production
One of[7].Root rot is distributed widely in the main soybean producing region in the world, it is annual in the world caused by economic loss it is high
Up to more than ten00000000 dollars[8].In China, root rot is found the beginning of the nineties in the Northeast[9], become northeast, Huang
One of the important disease in Huai-Hai Soybean production area and southern Common beans producing region, the production safety of serious danger side of body soybean.Soybean root rot
Caused by disease is mainly infected by soybean phytophthora (Phytophthora sojae), it can cause to endanger in each breeding time of soybean
Evil[2].In the case where environment is suitable, root rot is spread rapidly on the plant infected, and harm can lead to the exhausted of soybean
Production.Therefore, Crop Improvement variety resistance has great importance to effectively controlling root rot.
The cell-membrane receptor in plant is still at an early stage to the research that phytophthora is disease-resistant at present, and research includes that soybean exists
Interior plant phytophthora resistant gene is of great significance to the research of disease resistance of plant.
Bibliography：
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, Su Yanchun.1991.The discovery of Chinese soybean phytophthora germ and Primary Study Plant Pathologies 21:
298。
Invention content
One of the objects of the present invention is to provide a kind of gene GmLecRK-R.
The second purpose of the present invention is to provide a kind of recombinant expression carriers containing GmLecRK-R genes.
The three of the object of the invention are to provide the application of gene GmLecRK-R.
Specifically details are as follows for the content of present invention：
The present invention provides a kind of gene GmLecRK-R, and the gene source is in soybean, nucleotide sequence such as SEQ ID NO.1
It is shown, or there is 70% or more homology with sequence SEQ ID NO.1.Preferably, nucleotide sequence such as SEQ ID NO.1 institutes
Show, or there is 80% or more homology with sequence SEQ ID NO.1, it is further preferred that nucleotide sequence such as SEQ ID NO.1
It is shown, or there is 85% or more homology with sequence SEQ ID NO.1, it is furthermore preferred that nucleotide sequence such as SEQ ID NO.1 institutes
Show, or there is 90% or more homology with sequence SEQ ID NO.1.
The present invention also provides a kind of protein of gene GmLecRK-R codings.
The protein of gene GmLecRK-R codings of the present invention, amino acid sequence are SEQ ID NO.2 or SEQ
The amino acid sequence of ID NO.2 passes through the substitution of one or several amino acid residues and/or lacks and ors add and can provide plant
The protein derived from SEQ ID NO.2 of object disease resistance.
Using the amino acid sequence of the gene code of the present invention, it can design and manually add signal peptide sequence to be conducive to
Expression in plant.
Using the amino acid sequence of the gene code of the present invention, it can design and be conducive to artificial synthesized codon optimization
The nucleic acid sequence expressed in plant.
The present invention also provides a kind of recombinant expression carriers including the gene GmLecRK-R.
The recombinant expression carrier of the gene GmLecRK-R can be contained with existing plant expression vector construction.
Existing plant expression vector is preferably plant transformation plasmid, can be expression vector pBin::EGFP, pCambia or
PTF101.1 etc..
Preferably, the recombinant expression carrier is that gene GmLecRK-R is inserted into the double base containing the ends C- eGFP to carry
Body pBin::The carrier pBin obtained in eGFP restriction enzyme sites KpnI::GmLecRK-R-eGFP.
When using the gene constructed recombinant expression carrier, any type enhancing can be added before its transcription initiation nucleotide
Type promoter or constitutive promoter；In addition, when using gene constructed recombinant expression carrier of the invention, enhancing also can be used
Son, including translational enhancer or transcriptional enhancer.
Transgenic cell line and recombinant bacterium containing gene GmLecRK-R described above.
The primer pair of the overall length or any segment that expand the gene GmLecRK-R also belongs to protection scope of the present invention.
A kind of transformant is imported by the recombinant expression carrier obtained by host cell, and the host cell is preferably big
Coli cell or agrobatcerium cell.
For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be carried out
Processing can generate the enzyme of color change or the gene of luminophor, resistant as the coding that can be expressed in plant is added
Antibiotic marker or anti-chemical reagent label etc..
The present invention also provides the protein or recombinant expression carrier of the gene GmLecRK-R or its coding or transformant
Application in improving plant immune resistance or disease resistance.
The present invention also provides the protein or recombinant expression carrier of the gene GmLecRK-R or its coding or transformant
Application in plant improves pathogenic bacteria immune resistance or improves disease caused by Genes For Plant Tolerance pathogenic bacteria.Especially improved in plant
Application in disease caused by phytophthora immune resistance or raising Genes For Plant Tolerance phytophthora.
The present invention also provides the protein or recombinant expression carrier of the gene GmLecRK-R or its coding or transformant to exist
Application in plant breeding.
The present invention also provides the protein or recombinant expression carrier of the soybean gene GmLecRK-R or its coding or turn
Change body obtained after importing plant notable disease resistance/or increase yield kind in application, preferably importing soybean, tobacco, kind
Eggplant or potato enhanced disease resistance and/or increased yield kind in application.
Beneficial effects of the present invention：
The protein of gene GmLecRK-R codings of the present invention enhances plant to phytophthora disease resistance.The mistake in plant
Expression does not interfere with plant growth character, especially has broad spectrum activity to the identification of phytophthora, can significantly increase plant to phytophthora
The disease resistance of bacterium in terms of the present invention can be used in the improvement of crop breeding disease resistance, is expected to the disease resistance for improving plant to epidemic disease,
To achieve the purpose that volume increase subtracts medicine.
This research finds that GmLecRK-R has soyabean phytophthora and Phytophthora nicotianae to the analysis of cell-membrane receptor in soybean
There is disease-resistant function.It is inoculated with soyabean phytophthora on the soybean hairy root for being overexpressed GmLecRK-R genes, the soyabean phytophthora colonized
Conspicuousness is measured to reduce.GmLecRK-R gene pairs soybean disease resistances play very important effect, and soybean is as a kind of important warp
The Typical Representative of Ji crop and legume can drive many other plant cell membranes solidifying the research of GmLecRK-R in soybean
The correlative study of the plain receptor protein kinase of collection.In addition be overexpressed in tobacco leaf the gene can conspicuousness enhance tobacco to tobacco
The disease resistance of phytophthora, caused by scab all conspicuousness reduce, show GmLecRK-R to different phytophthoras have broad-spectrum disease resistance work
With.These researchs will preferably illustrate plant to the disease-resistant function of phytophthora, can be provided for resistant gene engineering breeding outstanding
Disease-resistant gene resource.
Description of the drawings
Fig. 1 pBin::GmLecRK-R-eGFP genetically engineered soybean hairy radixin detection of expression.Western blot detections
The expression quantity of GmLecRK-R-eGFP, detection antibody are anti-GFP.
Fig. 2 pBin::EGFP and pBin::It is generated after GmLecRK-R-eGFP genetically engineered soybeans inoculation soyabean phytophthora
Egg spore.A, soybean hairy root generate the symptom of egg spore.B generates the quantity statistics of egg spore on soybean hairy root.
Fig. 3 pBin::Protein expression detects in GmLecRK-R-eGFP transgene tobaccos.
Fig. 4 pBin::EGFP and pBin::GmLecRK-R-eGFP transgene tobaccos inoculation Phytophthora nicotianae Breda shows after 3 days
Disease symptom.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Primer is by Nanjing Genscript Biotechnology Co., Ltd. involved by the embodiment of the present invention
Synthesis.
The clone of embodiment 1.GmLecRK-R genes and Sequence structure analysis
Soybean and rich -47 seed are directly sowed in the flowerpot equipped with Nutrition Soil, greenhouse (21-23 DEG C, 14h illumination/10h
It is dark) culture, take extraction of the 2 week old plant for RNA.
Total RNAs extraction：Using soybean leaves as material, the extracting of total serum IgE using Omega companies RNA extracts kits according to
Illustrate to operate, its rna content and quality are detected with spectrophotometer.
Reverse transcription generates the first chain：0.7 μ g RNA are taken to make template, according to Takara companies PrimeScript reverse transcriptase
Matched reagent operation instruction carries out cDNA synthesis, is settled to 20uL reactions.Take suitable reverse transcription product for subsequent gene
Clone PCR.
Using the first chains of cDNA as RT-PCR templates, conventional method is PCR, expands GmLecRK-R genetic fragments or overall length
Gene：
PCR amplification primer sequence：
Sense primer：SEQ ID NO.4
(5’-TTACGAACGATAGCCGGTACCATGTCTCATTCCAAAACCCCCG-3’)
Downstream primer：SEQ ID NO.5
(5 '-GCTCACCATCCCGGGGGTACCAACCGGAGGAGGGGAGGGTTGC-3 '),
50uL reaction systems are, wherein 5 × buffer 10uL, 2.5mM dNTPs 4uL, Takara Primer STAR
Taq enzyme 0.5uL, template cDNA 1uL, adds water to 50uL；PCR amplification program is 98 DEG C of pre-degenerations 2 minutes, and 98 DEG C are denaturalized 30 seconds,
58 DEG C are annealed 10 seconds, and 72 DEG C extend 2 minutes, recycle 40 times, and last 72 DEG C extend 10 minutes；Electrophoresis point is carried out on Ago-Gel
From the dyeing photograph of, ethidium bromide (EB), record is as a result, simultaneously gel extraction GmLecRK-R PCR products.
Electrophoretic band is recycled with Agarose Gel DNA Purification Kit (TaKaRa).Gel extraction
GmLecRK-R PCR product according to ClonExpress II One Step Cloning Kit (Vazyme) according to illustrating to grasp
It is connected to the pBin of KpnI digestions::PBin is obtained on eGFP carriers::GmLecRK-R-eGFP plasmids convert Escherichia coli sense
By state cell GM109, LB (receiving 50ug/mL containing card) tablet is applied, bacterium colony PCR verifications three clones of picking after 37 DEG C of cultures 16 hours
Plasmid is extracted according to plasmid extraction kit operation (Takara), send Nanjing Jin Sirui companies to be sequenced, sequence such as SEQ ID
NO.1.It is sequenced in the electroporated K599 or GV3101 to Agrobacterium of correct plasmid, applies LB and (receive 50ug/mL containing card, streptomysin
50ug/mL) tablet, bacterium colony PCR verification picking correctly clone progress subsequent experimental after 30 DEG C of cultures 48 hours.
GmLecRK-R genes are expressed in 2. Soybean Root Hairs of embodiment：
Specific steps are summarized as follows：
1) soybean closes rich -47 plantation：
Soybean is closed to rich -47 seed to be uniformly sowed in the flowerpot equipped with vermiculite, initial stage is covered flowerpot with black plastic film
Flowerpot is placed in culture at dark by lid, after etiolated seedling is uniformly grown, is opened black plastic film, is removed kind of a skin, watering (about 3
It was by 4 days), it is placed in suitable for cultivating (half to 2 days about 1 day) under illumination, i.e., the cotyledon of six day age plant can be used for testing.
2) Agrobacterium is cultivated
Picking transfects pBin from tablet respectively::GmLecRK-R-eGFP carriers and pBin::The root of hair agriculture of eGFP carriers
Bacillus K599 single bacterium colonies are inoculated into 2mL LB liquid mediums (card receive 50ug/mL, streptomysin 50ug/mL) in constant temperature respectively
30 DEG C on shaking table, 200rpm overnight incubations.The K599 agrobacterium rhizogenes liquid 4500rpm being incubated overnight is centrifuged 3 minutes and collects bacterium
Body.With buffer solution (component：10mM 2-[N-morpholino]ethanesulfonic acid,10mM MgCl2,100μM
Acetosyringone pH 5.6) thalline were collected by centrifugation after suspension bacteria liquid.After repetition washes 3 times, bacterium solution is diluted with buffer solution, it will
Bacterium solution OD600 values are adjusted to 0.4.
3) hairy root converts
A, soybean cotyledon culture medium is down flat plate, and (+0.8% agar of MS+20g/L sucrose+cephalothin 120ug/mL+ carboxylics benzyl is green
Mycin 120ug/mL).
B, soybean cotyledon sterilizing.Soybean cotyledon is removed in petiole base, the plant that surface vermiculite is placed on sterilizing is washed away with water
It in object tissue culture flasks, is sterilized 30 seconds with 70% alcohol first, secondly with 10%, the sodium hypochlorite submergence containing 0.5% effective chlorine is gone out
Bacterium 15 minutes is finally rinsed 5 times with sterile tap water, sodium hypochlorite is rinsed well.
C, soybean cotyledon wound.Sterile gloves are put in super-clean bench, and soybean cotyledon petiole portion is cut with sterilizing scalpel
Position, and among cotyledon lower epidermis a wound is dug out by petiole position.
Soybean cotyledon epicuticle Jing Guo wound is affixed on culture medium and placed, with 1mL injector for medical purpose in wound by d downward
Appropriate bacterium solution in upper drop forms the vesicle slightly swelled.
Culture dish is sealed up sealed membrane by e, is placed under 25 DEG C of dark conditions and is cultivated, or in dark and illumination alternation condition
Lower culture.
4) Preliminary Identification of hairy root
A, in incubation, brown (couple of days) can be become by connecing the wound at bacterium first, then again intermediate or entire
Wound grows callus, and callus is in granular form or is linked to be sheet (starting to grow for one week or so), and callus needs again
Hairy root is grown by one to two week.
The cotyledon for growing hairy root is placed in fluorescence microscopy under the microscope, the hairy that will have green fluorescence on culture dish by b
Root marks.
C, the cotyledon switching 15cm culture dishes for choosing the hairy root for having green fluorescence are enlarged culture, pay attention to observing hairy
The upgrowth situation of root, until hairy root long can carry out subsequent experimental after going out lateral root.
The culture of 3. phytophthora of embodiment
Genetically engineered soybean phytophthora with red fluorescence is inoculated into (G418 50ug/ on solid V8 culture medium flat plates
ML it), or by Phytophthora nicotianae Breda is inoculated on solid V8 culture medium flat plates.The V8 culture medium flat plates of inoculation are placed in 25 DEG C of dark conditions
Lower culture can be used to subsequent experimental in 5 days.
4. hairy root of embodiment infects
In the fluorescence microscopy hairy root after expanding and cultivating under the microscope, the hairy root of green fluorescence is filtered out, with cutting
Knife, which is cut to be placed in water, impregnates moisturizing, and washes away the MS solid mediums adhered on hairy root simultaneously.Cut 2.5 × 4.5cm
Size filter paper soaks, and filter paper one end is laid on glass slide, by the hairy root proper alignment that even thickness, upgrowth situation are uniform
It is placed on filter paper, towards glass slide frosted glass side and the tip of a root is allowed to stretch out filter paper tip of a root side, all tips of a root is kept to stretch as possible
The distance for going out filter paper is consistent.Every glass slide places 10 or so hairy roots for infecting.By the soybean with red fluorescence
Phytophthora cuts the rectangle mycelia block of adjoining dimensions, will be overlying on all hairy root tips of a root on glass slide on one side with mycelia
On, it is placed on hairy root with the other end of the filter paper soaked.All glass slides are placed in the 15cm circular filter papers for being lined with moistening
In 15cm sky culture dishes, it is put under 25 DEG C of dark conditions and cultivates.The soybean hairy root that inoculation soyabean phytophthora infects 2 days is taken to be placed in
Disease symptom observation is carried out under fluorescence microscope, as a result shows that phytophthora is infected in the soybean hairy root for being overexpressed GmLecRK-R
The egg spore quantity of upper generation substantially reduces (Fig. 2), shows that overexpression GmLecRK-R significantly increases soybean and resists to soybean phytophthora
Characteristic of disease.
EGFP and GmLecRK-R-eGFP is overexpressed in 5. tobacco of embodiment
1) culture of Agrobacterium
Picking transfects pBin from tablet respectively::GmLecRK-R-eGFP carriers and pBin::The GV3101 of eGFP carriers
Single bacterium colony and silencing suppressor P19 are inoculated into 2mL LB liquid mediums (card receive 50ug/mL, rifampin 50ug/mL) respectively
In 30 DEG C on constant-temperature table, 200rpm overnight incubations.The agrobacterium liquid 4500rpm being incubated overnight is centrifuged 3 minutes and collects thalline.
With buffer solution (component：10mM 2-[N-morpholino]ethanesulfonic acid,10mM MgCl2,100μM
Acetosyringone pH 5.6) thalline were collected by centrifugation after suspension bacteria liquid.After repetition washes 3 times, bacterium solution is diluted with buffer solution, it will
Containing the pBin that has illicit sexual relations::GmLecRK-R-eGFP carriers and pBin::EGFP bacterium solutions carry out 1 with P19:1 mixes to OD600 values 0.6.
2) tobacco expressed eGFP and GmLecRK-R-eGFP.The Agrobacterium prepared is injected into tobacco leaf with syringe
In, tobacco is in greenhouse (21-23 DEG C, 14h illumination/10h dark) culture after injection.
3) being overexpressed GmLecRK-R-eGFP conspicuousnesses enhances tobacco to phytophthora disease resistance
Inject blade inoculation Phytophthora nicotianae two days later.Disease symptom observation (Fig. 4), film recording knot are carried out in inoculation within 3 days
Fruit.Compared with negative control, there is scab conspicuousness after being inoculated with Phytophthora nicotianae Breda in the tobacco for being overexpressed GmLecRK-R-eGFP
Reduce, these results, which confirm, is overexpressed the disease resistance that GmLecRK-R-eGFP conspicuousnesses improve tobacco to different phytophthoras.
Embodiment 6.GmLecRK-R albumen cumulative amounts detect
Collect the detection of the soybean hairy root or tobacco leaf progress albumen cumulative amount of expression GmLecRK-R-eGFP.It will receive
(group is divided into grinding addition protein extract after the hairy root or tobacco leaf liquid nitrogen flash freezer of collection：Group is divided into：150mM NaCl,
50mM Tris-HCl pH 7.5,10mM EDTA (ethylenedia-minetetraacetic acid), 1.0% (v/v)
1.0% (v/v) protease inhibitor of NP-40,1mM phenylmethylsulfonyl fluoride, and
Cocktail) mixing is placed in 30 minutes on ice.18000g is collected by centrifugation supernatant 80uL and 5 times of albumen loading buffers of 20uL is added
Liquid boiling water bath 10 minutes after mixing.20uL samples are taken to be separated by electrophoresis on PAGE gel, it is small that 120V runs glue 1.5
When.Protein sample is gone on pvdf membrane after reaction, sealer is incubated with 5%PBST milk.It is added 1:5000 diluted GFP
Primary antibody (Abmart) with PBST washes film 5 minutes three times after being incubated 2 hours, is then added 1:10000 diluted mouse it is anti-(LI-COR,
Irdye 800,926-32210) be incubated 30 minutes after with PBST wash film 5 minutes three times, sweep film the result shows that in genetically engineered soybean
In hairy root and tobacco leaf, LecRK-R-eGFP can normal expression synthetic proteins (Fig. 1 and Fig. 3).
To sum up, it can be seen that gene GmLecRK-R-eGFP has crucial resistant effect to the anti-phytophthora of tobacco.It is overexpressed
The gene conspicuousness promotes tobacco to the disease resistance of different phytophthoras, is a kind of gene of ideal enhancing disease resistance of plant.It is logical
The method for crossing genetic transformation makes the gene express the resistance capacity for making it obtain to a variety of pathogens in tobacco, so as to carry
High crop field resistance.
Sequence table
<110>Agricultural University Of Nanjing
<120>It is a kind of improve disease resistance of plant gene and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2007
<212> DNA
<213>Soybean and rich -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 and rich -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 (10)
1. a kind of gene GmLecRK-R, nucleotide sequence has as shown in SEQ ID NO.1, or with sequence SEQ ID NO.1
70% or more homology.
2. the protein that gene GmLecRK-R described in claim 1 is encoded.
3. protein according to claim 2, it is characterised in that its amino acid sequence is SEQ ID NO.2 or SEQ ID
The amino acid sequence of NO.2 passes through the substitution of one or several amino acid residues and/or lacks and ors add and can provide plant
The protein derived from SEQ ID NO.2 of disease resistance.
4. containing the recombinant expression carrier of gene GmLecRK-R, transgenic cell line or recombinant bacterium described in claim 1.
5. expanding the overall length of gene GmLecRK-R or the primer pair of any segment described in claim 1.
6. a kind of transformant is imported by the recombinant expression carrier described in claim 4 obtained by host cell, the host cell
It is preferred that Bacillus coli cells or agrobatcerium cell.
It is carried 7. being recombinantly expressed described in protein, claim 4 described in gene GmLecRK-R, claim 2 described in claim 1
Transformant described in primer pair or claim 6 described in body, transgenic cell line or recombinant bacterium, claim 5 is exempted from raising plant
Application in epidemic disease resistance or disease resistance.
It is carried 8. being recombinantly expressed described in protein, claim 4 described in gene GmLecRK-R, claim 2 described in claim 1
Transformant described in primer pair or claim 6 described in body, transgenic cell line or recombinant bacterium, claim 5 is improved in plant and is caused
Application in disease caused by germ immune resistance or raising Genes For Plant Tolerance pathogenic bacteria；It is anti-especially phytophthora bacterial immunity to be improved in plant
Property or improve application caused by Genes For Plant Tolerance phytophthora in disease.
It is carried 9. being recombinantly expressed described in protein, claim 4 described in gene GmLecRK-R, claim 2 described in claim 1
Transformant is in plant breeding described in primer pair or claim 6 described in body, transgenic cell line or recombinant bacterium, claim 5
Application, especially obtained after importing plant notable disease resistance/or increase yield kind in application.
10. application according to claim 9, it is characterised in that the plant is soybean, tobacco, tomato or potato.
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Cited By (6)
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| CN110713528A (en) * | 2019-10-30 | 2020-01-21 | 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 |
| CN110938118A (en) * | 2019-12-17 | 2020-03-31 | 南京农业大学 | Plant immune activation protein PC2 secreted by phytophthora infestans and application thereof |
| CN112143746A (en) * | 2020-09-30 | 2020-12-29 | 南京农业大学 | Gene GmAP5 for improving disease resistance of plants and application thereof |
| CN112626080A (en) * | 2020-12-23 | 2021-04-09 | 河南大学 | R gene for controlling soybean-rhizobium matching property, protein and application thereof |
| CN113151320A (en) * | 2021-03-22 | 2021-07-23 | 华中农业大学 | Potato StLecRK-VI.1 and StTET8 genes and application thereof in improvement of late blight resistance |
| 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 |
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| 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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