CN107022017A - The albumen of cereal cyst nematode Ha 16674, encoding gene and its application - Google Patents
The albumen of cereal cyst nematode Ha 16674, encoding gene and its application Download PDFInfo
- Publication number
- CN107022017A CN107022017A CN201710356377.4A CN201710356377A CN107022017A CN 107022017 A CN107022017 A CN 107022017A CN 201710356377 A CN201710356377 A CN 201710356377A CN 107022017 A CN107022017 A CN 107022017A
- Authority
- CN
- China
- Prior art keywords
- cyst nematode
- nematode
- albumen
- cereal cyst
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Toxicology (AREA)
- Environmental Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the albumen of cereal cyst nematode Ha 16674, encoding gene and its application.A kind of albumen of cereal cyst nematode Ha 16674, its amino acid row SEQ ID NO：Shown in 1.Encode the gene of the albumen of cereal cyst nematode Ha 16674, its cDNA full length nucleotides sequence such as SEQ ID NO：2.Experiment shows that Ha 16674 and Bax co-injection tobacco leafs can suppress the cell death that Bax causes blade.The full length genes of Ha 16674 are overexpressed in arabidopsis, obtain the T3 of stable heredity for transgenic positive plant.The calprotectin Ha 16674 of cereal cyst nematode can reduce the immune defense reaction of arabidopsis, promote pseudomonas syringae to infect.The present invention provides new target drone Ha 16674 for the culture of anti-nematode plant, has good theory directive significance to cultivating the anti-cyst roundworm crop of wide spectrum.
Description
Technical field
The invention belongs to biological technical field, be related to one from cereal cyst nematode gene Ha-16674 gene orders and
Encoding proteins and its application.
Background technology
Wheat cearal cyst nematode (Heterodera avenae) is the gramineous crops such as harm wheat, barley, oat
Important pathogen nematode, at present 37 countries for example the U.S., Canada, Australia, France, England, Norway, Italy,
The generation of the country such as India, Turkey, Syria, Iran (Nicol J, I,Bolat N,et al.The
global importance of the cereal cyst nematode(Heterodera spp.)on wheat and
international approaches to its control[J].Communications in agricultural and
applied biological sciences,2006,72(3):677-686).200 are up in Australian CCN hazard areas
Ten thousand hectares, general production loss is 23-50%, and loss is up to 73-89%, year 70000000 dollars of (Brown of economic loss when serious
R.Cultural practices and their effects on Heterodera avenae and grain yields
of wheat in Victoria,Australia[J].Bulletin OEPP-European and Mediterranean
Plant Protection Organization,1982).The wheat yield loss that cereal cyst nematode is caused in India
47.2%, barley loss reaches as high as 87.2%.
Over nearly 10 years, the Cereal Cyst Nematode of Wheat evil of China's wheat main producing region is on the rise.It is first from 1989
It is secondary Hubei Tianmen find wheat cearal cyst nematode since, at present clear and definite wheat cearal cyst nematode in Henan, river
16 provinces such as north, Beijing, Tianjin, the Inner Mongol, Shandong, Shanxi, Hubei, Anhui, Jiangsu, Ningxia, Qinghai, Tibet and Xinjiang, city,
There is occurrence and distribution autonomous region wheat belt；Occurring area reaches more than 6,000 ten thousand mu, wherein Henan Province's occurring area about 20,000,000
Mu, about 10,000,000 mu of Hebei province, about 20,000,000 mu of Shandong Province, about 6,000,000 mu of Anhui Province, about 4,000,000 mu of Jiangsu Province (Peng Deliang,
The Chinese nematology researchs of kainogenesis Distribution Area [J] of Li Huixia, Wang Xifeng, et al. China wheat cearal cyst nematode,
2008,2:344-345.), and there is the trend of continuous build up.Henan Xuchang and Zhengzhou grave illness field wheat yield are up to 40%
More than, the nematode density of many wheatlands, which is shown, reaches excessive risk index, or even part wheatland is seriously ruined because of state of an illness harm and plants exhausted
Receive.One of the key factor of Cereal Cyst Nematode of Wheat evil as restriction China improving yield of wheat high yield (Deliang P,
NicolJ M,Hongmei L,et al.Current knowledge of cereal cyst nematode(Heterodera
avenae)on wheat inChina[C].Cereal cyst nematodes:status,research and
outlook.Proceedings of the First Workshopof the International Cereal Cyst
Nematode Initiative,Antalya,Turkey,21-23October 2009.,2009:29-34).Now with across
The popularity of area's united reaper, make wheat cearal cyst nematode China's wheat main producing region large area, long-distance communications and
Diffusion, has become the substantial risk venereal disease evil in China North China, northwest and the zonal Wheat Production of the southwestern area of wheat, serious to threaten
The grain security of China.
At present, the preventing and treating of Wheat cyst nematode evil relies primarily on chemical pesticide, on the one hand produces huge pollution to environment,
On the other hand there may be resistance to the action of a drug line insect population, therefore Wheat cyst nematode evil needs green control strategy badly.Seed selection and push away
It is the effective measures for controlling Wheat cyst nematode evil that extensive farming, which plants disease-resistant variety wheat, but disease-resistant variety also has certain lack
Fall into, for example, disease-resistant and high yield phenotype can not be combined.With the development of molecular biology technology, understanding wheat sporangiocyst line in depth
Worm pathogenesis is conducive to the Synthetical prevention strategy of exploitation efficiently, green.Therefore cereal cyst nematode Disease-causing gene, research are excavated
Its pathogenesis, providing target for cultivation resistance plant has highly important practice significance and theory value.
The content of the invention
The present invention provides a kind of to cereal cyst nematode pathogenic related effector Ha-16674 and its encoding proteins,
New target is provided for the cultivation of anti-nematode plant, the culture of confrontation nematode crop has good theory directive significance.
It is an object of the invention to provide a kind of Ha-16674 gene orders from cereal cyst nematode and its encoding proteins.
Another object of the present invention provides a kind of from cereal cyst nematode Ha-16674 gene codes and its encoding proteins
Application.
Above-mentioned purpose is achieved through the following technical solutions：
A kind of cereal cyst nematode Ha-16674 albumen, its amino acid row SEQ ID NO：Shown in 1.
Encode the gene of above-mentioned albumen.
The gene, its cDNA full length nucleotides sequence such as SEQ ID NO：2.
A kind of recombinant vector or trans genie individual, contain said gene sequence.
The skeleton carrier of the recombinant vector is the pGD carriers containing 35S promoter.
Application of the cereal cyst nematode Ha-16674 genes in control of nematode.
Ha-16674 genes suppress plant immune defense reaction, and infecting for nematode is reduced by silence Ha-16674 genes
Rate, Ha-16674 genes can be applied in the preventing and treating of nematodiasis as target.
The present invention has obtained the complete code area 1236bp of Ha-16674 by PCR clones, encodes the egg of 411 amino acid
In vain.
A kind of recombinant vector contains Ha-16674 gene orders, and general plant expression vector, present invention selection contains
The pGD carriers of 35S promoter.A kind of recombinant bacterium, contains above-mentioned recombinant vector.
For recombinant gene expression box, transgenic cell line containing above-mentioned cereal cyst nematode gene Ha-16674 etc.
Protection scope of the present invention should be belonged to.
The present invention obtains a kind of paddy cyst roundworm gene Ha-16674 and its coding egg by substantial amounts of experimental study
In vain, it specify that its function in nematosis.Ha-16674 cDNA total length 1236bp, encode the albumen of 411 amino acid.
Bax injection tobacco leafs can be caused into large area cell death, Ha-16674 and Bax co-injection tobacco leafs, Bax can be suppressed
Cause the cell death of blade.
The present invention collects the wheat root of different time sections after inoculation second instar larvae, centrifuges, obtains with sucrose by the way that root is broken
Infect the polypide of the different growing periods such as rear J2, J3, J4 and white female adult.Utilize Ha-16674 special primers, Realtime-PCR inspections
Survey expression quantity of the Ha-16674 genes in wheat cearal cyst nematode different growing periods.As a result show result Ha-16674 in each age
There is expression phase, is more than 80 times of expression quantity in ovum wherein infecting expression quantity highest in rear second instar larvae.
Ha-16674 full length genes are overexpressed in arabidopsis, obtain the T3 of stable heredity for transgenic positive plant.Ha-
16674 genetically modified plants forms are consistent with wild type, no significant change.It is inoculated with pseudomonas syringae (Pseudomonas
Syringae after), compared with wild type, overexpressing Ha-16674 arabidopsis strengthens P.syringae sensitiveness, explanation
The calprotectin Ha-16674 of cereal cyst nematode can reduce the immune defense reaction of arabidopsis, promote pseudomonas syringae to invade
Dye.
The present invention specify that functions of the Ha-16674 in nematosis.The cell that Ha-16674 can suppress plant is dead
Die, Ha-16674 two age expression quantity after infecting increase, and parasitism is promoted by suppressing the fundamental immunity defense reaction of host.Just
Step determines that Ha-16674 is downright bad by the hypersensitive cell for suppressing plant, so as to regulate and control the fundamental immunity defense reaction of plant.For
Ha-16674 provides theoretical foundation as control of nematode target, also prevents for progressive research nematode effect protein regulation and control host immunity
The mechanism of imperial reaction provides thinking.
The present invention provides new target drone Ha-16674 for the culture of anti-nematode plant, to cultivating the anti-cyst roundworm crop of wide spectrum
With good theory directive significance.Functions of the Ha-16674 in parasitism is specify that simultaneously, is that cyst roundworm and host are mutual
The research for making mechanism provides new approaches and direction.
Brief description of the drawings
Fig. 1 is Ha-16674 gene electrophoresis results.
Fig. 2 is Ha-16674 cell deaths caused by expression inhibiting Bax in tobacco.A:Bax is individually injected；B:Bax and
Ha-16674 co-injections；C:Bax and empty carrier pGD co-injections；D:Ha-16674 is individually injected.
Fig. 3 is growth expression patterns of the Ha-16674 in different larval instar.Abscissa is the ovum of cereal cyst nematode, two ages,
Two ages, three ages, six different larval instars of four ages and female adult pest after infecting, ordinate are the relative expression quantity of Ha-16674 genes
Fig. 4 strengthens pseudomonas syringae sensitiveness for overexpression Ha-16674 arabidopsis.Col is wildtype Arabidopsis thaliana,
OE Ha-16674 are the arabidopsis of overexpression Ha-16674 genes.
Embodiment
With reference to embodiment, the present invention is described in further detail, and the material used in the present invention is commercially available, sheet
Applicant can provide the public used biomaterial simultaneously.
Embodiment 1:The acquisition of cereal cyst nematode nematode Ha-16674 gene orders
The extraction of 1.1 nematode total serum IgEs
About 100 are taken, 000 second instar larvae after being fully ground in liquid nitrogen, adds 1ml Trizol reagents, room temperature 5min；
200 μ l chloroforms are added, are overturned after fully mixing, room temperature 5min；12000g, centrifuges 15min by 4 DEG C；Supernatant is transferred to centrifuge tube
Afterwards, 600 μ l isopropanols are added, are overturned after fully mixing, room temperature 10min；12000g, centrifuges 15min by 4 DEG C；Supernatant is removed, is added
The ethanol of 1ml 75% washing precipitation, 7500g centrifugation 5min, removes supernatant；75% ethanol washing precipitation is repeated once；Drying at room temperature is sunk
Form sediment；RNase-free water dissolves RNA, is stored in -80 DEG C of refrigerators.Elegans rna reverse transcription synthesizes the chains of cDNA first.
The PCR amplifications of 1.2Ha-16674 gene orders
Design primer Ha-16674F：ATGCTTAGTGCTCCCCCACTG；
Ha-16674R：CTAAAGTTCGTCGTGCTCCTCC.
Amplification system：PrimeSTAR Max Premix (2 ×) 25 μ l, Ha-16674F (10mM) 2 μ l, Ha-16674R
The μ l of (10mM) 2 μ l, cDNA 1, the μ l cumulative volumes of distilled water 20 are mended to 50 μ l；Amplification program：98 DEG C, 5min；98 DEG C, 10s, 56 DEG C,
10s；72 DEG C, 1min, 35 circulations, 72 DEG C, 10min, 16 DEG C of preservations.After PCR amplifications, 1% agarose gel electrophoresis separation
After PCR primer, the step of cutting electrophoretic band uses DNA gel QIAquick Gel Extraction Kit (Promega) to specifications is reclaimed.
The DNA fragmentation of recovery is cloned into pEASY-Blunt Vector, reaction system：PCR primer 0.5-4 μ l, pEASY-Blunt
Vector1 μ l, ddH2O are mended to 5 μ l, are gently mixed, 25 DEG C of reaction 1h.After reaction terminates, it is placed on ice；By connection product thermal shock
Conversion is to bacillus coli DH 5 alpha competent cell, and recon is receiving bacterium colony PCR on the μ g/ml LB flat boards of penicillin K an 50 containing card
After checking, company is sent to be sequenced.Its nucleotide sequence such as SEQ ID NO：Shown in 2, its protein sequence such as SEQ ID NO encoded：1
It is shown.PEASY-Blunt Vector used in the present invention are commercial carrier.
Embodiment 2:Cereal cyst nematode nematode Ha-16674 suppresses cell death
2.1Ha-16674 plant expression vector construction
The end of expressed sequence total length amplification forward primer 5 ' originates in ATG (initiation codon) and connects XhoI restriction enzyme site sequences
Row, reverse primer 3 ' end terminate at TGA (terminator codon) simultaneously-connection BamHI restriction enzyme site sequences.To contain Ha-16674 bases
Because the carrier T of sequence is template, purpose fragment is expanded with high-fidelity enzyme Prime STAR DNA Polymerase.
Ha-16674F(xho1):CCGCTCGAG ATGCTTAGTGCTCCCCCACTG
Ha-16674R(Pst1):AAAACTGCAG CTAAAGTTCGTCGTGCTCCTCC
Amplification system：PrimeSTAR Max Premix (2 ×) 25 μ l, Ha-16674F (10mM) 2 μ l, Ha-16674R
(10mM) 2 μ l, the μ l of carrier T 1, the μ l cumulative volumes of distilled water 20 are mended to 50 μ l；Amplification program：98 DEG C, 5min；98 DEG C, 10s, 56 DEG C,
10s；72 DEG C, 1min, 35 circulations, 72 DEG C, 10min, 16 DEG C of preservations.PCR primer enters row agarose gel electrophoresis detection, and returns
Receive.Double digestion PCR and pGD plasmid enzyme restriction system：10 × Buffer 3.1 5 μ l, XhoI 0.5 μ l, BamHI 0.5 μ l, DNA or
The μ g of plasmid 2, ultra-pure water is mended to 50 μ l, reacts 1h under the conditions of 37 DEG C, digestion products enter row agarose gel electrophoresis, and reclaim purpose
Fragment.1 μ l, T4DNA Ligase of endonuclease bamhi 10 × Ligation of coupled reaction Buffer 1 μ l, carrier 100ng, purpose
Piece segment DNA 100ng, distilled water is mended to 10 μ l, and overnight, connection product is converted to competent escherichia coli cell for 4 DEG C of coupled reaction
DH5 α, are receiving screening positive clone on penicillin LB flat boards containing 50 μ g/ml cards, after bacterium colony is verified through PCR, are sending company to be sequenced.
The obtained long 1236bp of Ha-16674 gene orders is expanded using Ha-16674 special primers, obtained sequence is sequenced
Containing complete ORFs (ORF), the albumen of 411 amino acid is encoded.
2.2 tobaccos inject transient expression method
The plant expression vector pGD of the sequence containing Ha-16674 is electroporated to Agrobacterium EHA105, and picking monoclonal is in 50 μ
Cultivated in g/ml kanamycins, 50 μ g/ml rifampin liquid LB；220rpm, 28 DEG C of concussion and cultivates, 18h；4000rpm is centrifuged
20min, collects thalline and thalline, concentration OD is resuspended with MES (10mM MgCl2,10mM MES, pH 5.6,150uM AS) buffer solution
=0.7；It is stored at room temperature after 2-3h and injects tobacco；1ml syringes draw bacterium solution, are expelled in 7-8 weeks tobacco leaf；First injection contains
Have after Bax, 24h in injection Bax1 leaf areas injection Ha-166741；3-5d after injection, observes Apoptosis situation.See Fig. 2.
Transient expression in tobacco finds that individually injection Bax can cause tobacco leaf large area cell death, Ha-16674
With Bax co-injection tobacco leafs, cell death is suppressed, therefore Ha-16674 can suppress the cell death that Bax causes blade.
Embodiment 3:Cereal cyst nematode nematode Ha-16674 growth expression patterns
The separation of 3.1 different larval instar larvas
A large amount of second instar larvaes are inoculated in wheat (warm wheat 19) seedling of growth 7 days；Difference 7 days, 20 days and 30 after inoculation
It collection wheat root；Collect the root that infects, with water thoroughly cleaning it is clean after, be cut into as far as possible small fragment；3 times of volumes of addition
Enzyme lysate, 25 DEG C, 180rpm shakes cracking 16h；Residue is separated with sieve, the larva of different larval instar is collected；Residue
2000rpm, is centrifuged after 5min, plus 70% sucrose, suspension residue, and 5-10ml clear water is added dropwise, and 2000rpm centrifuges 5min；Collection is invaded
Two ages, three ages, the nematode of four ages after dye；Female adult pest is directly obtained after inoculation on the root of 40 days.
3.2RNA is extracted and templated synthesis
Two ages, infect after two ages, three ages, four ages, female adult pest and ovum using paramagnetic particle method extract total serum IgE, method reference
Dynabeads mRNA DIRECTTMKit handbooks, reverse transcription synthesis cDNA.
Internal control primer GAPDH-qS1：AGCGGCACAGAACATCATCC；
GAPDH-qAS1：GGTCCTCCGTGTAGCCCAAA；
Ha-16674 specific primers
Ha-166740QF1：CACCGTCAAACACGAGCAGAATA；
Ha-166740QR1：TCTTTGCGTCTGGGTCTGGGATA.
3.3 real-time quantitative PCR
Reagent uses Takara companies SYBR Green Realtime PCR Master Mix, and instrument uses ABI7500.
Amplification system：2×SYBR Green Mix 20μl；The μ l of 22 μ l, cDNA templates of μ l, Prime2 (10mM) of Prime1 (10mM) 1,
The μ l of cumulative volume 25.Amplification program：95℃30sec；95 DEG C of 15sec, 60 DEG C of 15sec, 72 DEG C of 45sec, 95 DEG C of 15sec,
40cycle；60℃60sec；95℃15sec；60℃30sec.It is more than experiment in triplicate.
Using Ha-16674 specific primers, Realtime-PCR detects Ha-16674 genes in wheat cearal cyst nematode
The expression quantity of different growing periods.As a result show that result Ha-16674 has expression in each age, wherein infecting in rear second instar larvae
Expression quantity highest, hence it is evident that higher than other ages.
The arabidopsis of embodiment 4, which overexpresses Ha-16674, to be strengthened pseudomonas syringae sensitiveness
4.1 inflorescence infusion methods obtain transgenic arabidopsis：
Plant expression vector pYBA1143:Ha-16674 structure:
Primer：Ha-16674F1(Pst1):AAAACTGCAG ATGCTTAGTGCTCCCCCACTG
Ha-16674R1(xho1):CCGCTCGAGCTAAAGTTCGTCGTGCTCCTCC
Amplification system：PrimeSTAR Max Premix (2 ×) 25 μ l, Ha-16674F1 (10mM) 2 μ l, Ha-16674R1
(10mM) 2 μ l, the μ l of carrier T 1, the μ l cumulative volumes of distilled water 20 are mended to 50 μ l；Amplification program：98 DEG C, 5min；98 DEG C, 10s, 56 DEG C,
10s；72 DEG C, 1min, 35 circulations, 72 DEG C, 10min, 16 DEG C of preservations.PCR primer enters row agarose gel electrophoresis detection, and returns
Receive.Double digestion PCR and pYBA1143 plasmid enzyme restriction system：10 × Buffer 3.1 5,0.5 0.5 μ l of μ l, BamHI of μ l, XhoI,
The DNA or μ g of plasmid 2, ultra-pure water is mended to 50 μ l, reacts 1h under the conditions of 37 DEG C, digestion products enter row agarose gel electrophoresis, and return
Receive purpose fragment.The μ l of 1 μ l, T4DNA Ligase of endonuclease bamhi 10 × Ligation of coupled reaction Buffer 1, carrier
100ng, purpose fragment DNA 100ng, distilled water are mended to 10 μ l, and overnight, connection product is converted to Escherichia coli for 4 DEG C of coupled reaction
Competent cell DH5 α, are receiving screening positive clone on penicillin LB flat boards containing 50 μ g/ml cards, after bacterium colony is verified through PCR, are sending
Company is sequenced.
pYBA1143:Ha-16674 is transferred to after Agrobacterium GV3101, is chosen monoclonal and is shaken bacterium；Agrobacterium containing recombinant plasmid
Bacterium solution is with 1:200 are transferred to and receive the μ g/ml of penicillin 50 and 50 μ g/ml rifampin LB culture mediums containing card, 220rpm, and 28 DEG C are shaken overnight
Swing culture；4000rpm centrifuges 15min, abandoning supernatant to bacterium solution at room temperature；Thalline is resuspended in 5% sucrose solution, adds 0.03%
SilwetL77, it is well mixed that conversion fluid is made；Fruit pod on Arabidopsis plant to be transformed is cut off, inflorescence is left；By arabidopsis
Plant inflorescence dips in about 30s in bubble conversion fluid down；Plant after conversion keeps humidity, places in the dark after 16h, and 16h illumination/
8h dark culturings；Conversion is repeated once after one week, with high conversion；After transformed plant is ripe, sowing sub- T1 generations, by obtained kind
Son is carried out after surface sterilization with 1% mercuric chloride, is sowed on the MS culture mediums containing 50 μ g/ml, screens resistance seedling；Seed is trained in resistance
Support base on grow two weeks after, by have card receive penicillin resistance Arabidopsis thaliana Seedlings be transplanted in soil continue cultivate harvest seed.
Ha-16674 full length genes are building up to plant expression vector pYBA1143, overexpressed in arabidopsis, obtain steady
The T3 of fixed heredity is for transgenic positive plant.Ha-16674 genetically modified plants forms are consistent with wild type, no significant change.
4.2 Ha-16674 transfer-gen plants are inoculated with pseudomonas syringae
PstDC3000 is containing 100mg/l rifampins, and 50mg/l cards receive penicillin, 25mg/l streptomysins 5-12mg/l tetra-
The flat lining outs of KBM of ring element, 28 DEG C of culture 2d, select monoclonal and are inoculated in 20ml KBM fluid nutrient mediums and shake bacterium, 28 DEG C of mistakes
Night；4000rpm centrifugations 5min collects thalline, with isometric 10mM MgCl2Wash 2 times；Use 10mM MgCl2It is diluted to OD600=
0.001 (equivalent to 106cfu/ml)；The arabidopsis of the non-bolting of selection, the light green and blade that is fully deployed, injects bacterium solution.
It is inoculated with after pseudomonas syringae (Pseudomonas syringae), compared with wild type, overexpresses Ha-
16674 arabidopsis strengthens P.syringae sensitiveness, and plan can be reduced by illustrating the Ha-16674 of wheat cearal cyst nematode
The immune defense reaction of southern mustard, promotes pseudomonas syringae to infect.
SEQUENCE LISTING
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>Cereal cyst nematode Ha-16674 albumen, encoding gene and its application
<130>PP17068-ZWB
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 411
<212> PRT
<213>Cereal cyst nematode Ha-16674 albumen
<400> 1
Met Leu Ser Ala Pro Pro Leu Leu Leu Leu Ala Leu Ala Gly Leu Val
1 5 10 15
Ser Val His Ala Glu Val Phe Phe Lys Glu Glu Phe Ser Asp Asp Asn
20 25 30
Trp Ser Asp Arg Trp Ile Thr Ser Lys His Lys Asp Asp Tyr Gly Lys
35 40 45
Phe Glu Leu Ser His Gly Lys Phe Phe Gly Asp Lys Ile Arg Asp Gln
50 55 60
Gly Leu Lys Thr Ser Gln Asp Ala Arg Phe Tyr Ala Ile Ser Ala Lys
65 70 75 80
Phe Pro Lys Lys Phe Ser Asn Lys Gly Lys Pro Leu Val Val Gln Phe
85 90 95
Thr Val Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Leu Lys
100 105 110
Leu Met Ala Ser Asp Ile Asn Gln Glu Asp Phe His Gly Glu Thr Pro
115 120 125
Tyr His Val Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys
130 135 140
Val His Leu Ile Leu Gly Tyr Lys Gly Lys Asn Tyr Leu Val Lys Lys
145 150 155 160
Asp Ile Arg Cys Lys Asp Asp Glu Leu Ser His Leu Tyr Thr Leu Ile
165 170 175
Leu Ser Pro Asp Asn Thr Tyr Glu Val Gln Ile Asp Gly Glu Lys Val
180 185 190
Glu Gly Gly Glu Leu Glu Ala Asp Trp Asp Leu Leu Pro Ala Lys Lys
195 200 205
Ile Lys Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Asp Lys Glu
210 215 220
Tyr Ile Asp Asp Pro Asp Asp Lys Lys Pro Glu Asp Trp Glu Lys Pro
225 230 235 240
Glu His Ile Pro Asp Pro Asp Ala Lys Lys Pro Asp Asp Trp Asp Asp
245 250 255
Glu Met Asp Gly Asp Trp Glu Pro Pro Met Ile Asp Asn Pro Glu Tyr
260 265 270
Lys Gly Thr Trp Lys Pro Lys Gln Ile Lys Asn Pro Asn Tyr Lys Gly
275 280 285
Lys Trp Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Gln Pro Asp Asp
290 295 300
Asp Leu Tyr Leu Arg Glu Asn Trp Gly Ala Ile Gly Ile Asp Ile Trp
305 310 315 320
Gln Val Lys Ser Gly Thr Ile Phe Asp Asn Ile Leu Leu Ala Asp Ser
325 330 335
Val Glu Asp Ala Lys Lys His Ala Ala Asp Thr Phe Asp Gln Leu Lys
340 345 350
Glu Ala Glu Lys Lys Gln Lys Glu Ala His Asp Glu Glu Glu Arg Lys
355 360 365
Lys Phe Glu Glu Glu Glu Lys Lys Arg Lys Glu Glu Glu Glu Lys Lys
370 375 380
Ser Thr Lys Asp Glu Glu Glu Pro Glu Glu Asp Glu Glu Asp Lys Lys
385 390 395 400
Lys Val Glu Glu Glu Glu Glu His Asp Glu Leu
405 410
<210> 2
<211> 1236
<212> DNA
<213>The Ha-16674 genes of cereal cyst nematode
<400> 2
atgcttagtg ctcccccact gttattgtta gccttggccg gcctcgtctc cgtccatgcg 60
gaggtcttct ttaaggagga gttttctgat gacaattgga gtgatcgctg gatcacatct 120
aagcacaaag acgactacgg gaagttcgag ttgtctcatg ggaagttttt cggagacaaa 180
attcgggatc aaggcctgaa aacgagccag gacgcacgat tctatgccat ctctgccaaa 240
ttcccgaaga agttcagcaa caaggggaag cctttggttg tgcagttcac cgtcaaacac 300
gagcagaata tcgactgtgg tggaggctac ctgaagctta tggcttcgga cattaaccag 360
gaggactttc atggcgagac gccttaccat gtgatgtttg gcccggacat ttgcggtcct 420
ggcaccaaaa aagttcacct cattctcggc tacaaaggca aaaactatct ggtcaagaag 480
gacattcggt gcaaggatga cgaactcagc catctgtaca cgctgatctt gagcccggac 540
aacacttacg aggttcaaat tgacggggag aaggtcgagg gcggcgaatt ggaggccgac 600
tgggacctgc tgccggcgaa gaaaatcaag gaccccgacg cgaagaagcc ggaggactgg 660
gacgacaagg agtacatcga cgacccggac gacaaaaagc cggaagactg ggaaaagccg 720
gaacatatcc cagacccaga cgcaaagaag ccggatgact gggacgacga aatggacggg 780
gactgggagc cgccgatgat cgacaacccg gaatacaaag gcacatggaa gccgaaacag 840
atcaaaaacc ccaactacaa gggcaagtgg atccatcccg agatcgacaa ccccgagtac 900
cagccggacg acgacctcta cttgcgagag aactggggcg ccatcggaat tgacatttgg 960
caggtcaagt cggggaccat cttcgacaac attttgttag ccgattctgt ggaggatgcc 1020
aaaaagcacg cggcggacac tttcgaccaa ttgaaagagg cggagaagaa gcagaaggaa 1080
gcacatgacg aggaggagcg caagaagttc gaggaggaag agaagaagcg gaaggaggag 1140
gaggagaaga agtcgacaaa ggacgaagag gagccggaag aggacgagga ggacaagaag 1200
aaggtggagg aagaggagga gcacgacgaa ctttag 1236
Claims (6)
1. a kind of cereal cyst nematode Ha-16674 albumen, its amino acid row SEQ ID NO：Shown in 1.
2. encode the gene of the cereal cyst nematode Ha-16674 albumen described in claim 1.
3. gene, its cDNA full length nucleotides sequence such as SEQ ID NO according to claim 2：2.
4. a kind of recombinant vector or trans genie individual, the sequence containing gene described in claim 1 or 2.
5. recombinant vector according to claim 4, its skeleton carrier is the pGD carriers containing 35S promoter.
6. application of the cereal cyst nematode Ha-16674 genes in control of nematode described in Claims 2 or 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710356377.4A CN107022017A (en) | 2017-05-16 | 2017-05-16 | The albumen of cereal cyst nematode Ha 16674, encoding gene and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710356377.4A CN107022017A (en) | 2017-05-16 | 2017-05-16 | The albumen of cereal cyst nematode Ha 16674, encoding gene and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107022017A true CN107022017A (en) | 2017-08-08 |
Family
ID=59528865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710356377.4A Pending CN107022017A (en) | 2017-05-16 | 2017-05-16 | The albumen of cereal cyst nematode Ha 16674, encoding gene and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107022017A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108727483A (en) * | 2018-05-25 | 2018-11-02 | 中国农业大学 | The HaGLAND5 albumen and its encoding gene of cereal cyst nematode and application |
| CN111187774A (en) * | 2020-03-17 | 2020-05-22 | 苏州市外来有害生物防控技术中心 | Caulnet protein gene of meloidogyne pseudograminis and application thereof |
| CN111690049A (en) * | 2020-07-29 | 2020-09-22 | 青岛农业大学 | Gene G20E03, protein coded by same and application thereof in improving soybean cyst nematode resistance of tobacco plants |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070271630A1 (en) * | 2005-02-24 | 2007-11-22 | Boukharov Andrey A | Methods for genetic control of plant pest infestation and compositions thereof |
| CN102234651A (en) * | 2010-04-23 | 2011-11-09 | 中国科学院成都生物研究所 | Nucleotide sequence of gene for resistance to Cereal cyst nematode, Heterodera avenae and application thereof |
| CN104178490A (en) * | 2014-08-18 | 2014-12-03 | 中国科学院成都生物研究所 | Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and vector and application thereof |
-
2017
- 2017-05-16 CN CN201710356377.4A patent/CN107022017A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070271630A1 (en) * | 2005-02-24 | 2007-11-22 | Boukharov Andrey A | Methods for genetic control of plant pest infestation and compositions thereof |
| US20090188005A1 (en) * | 2005-02-24 | 2009-07-23 | Boukharov Andrey A | Methods for genetic control of plant pest infestation and compositions thereof |
| CN102234651A (en) * | 2010-04-23 | 2011-11-09 | 中国科学院成都生物研究所 | Nucleotide sequence of gene for resistance to Cereal cyst nematode, Heterodera avenae and application thereof |
| CN104178490A (en) * | 2014-08-18 | 2014-12-03 | 中国科学院成都生物研究所 | Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and vector and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: ACY01917.1: "calreticulin 1 [Radopholus similis]", 《GENBANK》 * |
| 刘世名: "大豆的孢囊线虫抗性研究新进展", 《中国科学》 * |
| 李娟: "农作物重要病原线虫生物防控的研究进展", 《生命科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108727483A (en) * | 2018-05-25 | 2018-11-02 | 中国农业大学 | The HaGLAND5 albumen and its encoding gene of cereal cyst nematode and application |
| CN111187774A (en) * | 2020-03-17 | 2020-05-22 | 苏州市外来有害生物防控技术中心 | Caulnet protein gene of meloidogyne pseudograminis and application thereof |
| CN111187774B (en) * | 2020-03-17 | 2023-04-14 | 苏州市外来有害生物防控技术中心 | Caulnet protein gene of meloidogyne pseudograminis and application thereof |
| CN111690049A (en) * | 2020-07-29 | 2020-09-22 | 青岛农业大学 | Gene G20E03, protein coded by same and application thereof in improving soybean cyst nematode resistance of tobacco plants |
| CN111690049B (en) * | 2020-07-29 | 2022-08-19 | 青岛农业大学 | Gene G20E03, protein coded by same and application of gene in improving soybean cyst nematode resistance of tobacco plants |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yeh et al. | Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura | |
| CN103718895B (en) | The method of Control pests | |
| WO2015067194A1 (en) | Method for controlling pests | |
| CN103719137B (en) | Pest control method | |
| CN102972243B (en) | Method for controlling pests | |
| CN103039494A (en) | Method for controlling pests | |
| CN112175988B (en) | Application of cucumber phloem lectin CsPL1 in resisting melon epidemic disease | |
| CN107022017A (en) | The albumen of cereal cyst nematode Ha 16674, encoding gene and its application | |
| CN108892721B (en) | Chrysalid pteromalid venom Kazal-type serine protease inhibitor PpSPI20 protein and application | |
| CN106497966A (en) | The purposes of insecticidal proteins | |
| CN104886111B (en) | Purpose of insecticidal protein | |
| CN112175987A (en) | Application of cucumber light-harvesting chlorophyll a/b binding protein CsPS1 in resisting melon epidemic disease | |
| CN103172716B (en) | Heat-resistant plant gene and application thereof | |
| CN101503693B (en) | Halimodendron halodendron ERF transcription factor cDNA sequence, expression vector and use thereof | |
| CN104920425B (en) | The purposes of insecticidal proteins | |
| CN112175986B (en) | Application of cucumber 6-phosphogluconolactonase CsPmR1 in resisting melon epidemic disease | |
| CN114438118A (en) | Method for efficiently expressing Bt protein Cry56Aa1 in rice and corn to resist spodoptera frugiperda | |
| CN109468336A (en) | Brown paddy plant hopper protein phosphatase gene NlPP1, albumen and its dsRNA and application | |
| CN102191253B (en) | Plant disease-resistant regulation and control gene UEP and application thereof | |
| CN114717256A (en) | Method for efficiently expressing Bt egg Cry2Ag1 resistance spodoptera frugiperda in rice | |
| CN108822210B (en) | Chrysalid pteromalid venom Kazal-type serine protease inhibitor PpSPI24 protein and application | |
| CN112175967B (en) | PEN1 gene for enhancing plant resistance to lepidoptera pests and application thereof | |
| Li et al. | Characterization of the psoRPM1 gene for resistance to root-knot nematodes in wild myrobalan plum (Prunus sogdiana) | |
| CN106749566A (en) | The method that insecticidal proteins are combined and its manage insect-resistant | |
| CN103146711B (en) | Artificially synthesized aphid-resistant gene ASGNA (artificial synthetic galanthus nivalis agglutinin), as well as synthesizing method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170808 |
|
| WD01 | Invention patent application deemed withdrawn after publication |