CN106749591B - From the Mesp1 albumen and its encoding gene of Meloidogyne enterolobii and application - Google Patents
From the Mesp1 albumen and its encoding gene of Meloidogyne enterolobii and application Download PDFInfo
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Abstract
The invention discloses a kind of from the Mesp1 albumen and its encoding gene of Meloidogyne enterolobii and application.The present invention provides the protein that Mesp1 albumen, the amino acid sequence shown in sequence 1 in sequence table or sequence 3 form.The Mesp1 gene of coding Mesp1 albumen also belongs to protection scope of the present invention.The present invention also protects the application of Mesp1 albumen, regulates and controls the parasitic ability of root-knot nematode at least one of following (c1) to (c3): (c1)；(c2) regulate and control the pathogenecity of root-knot nematode；(c3) regulate and control the development of root-knot nematode.The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps: that the substance by the interference Mesp1 gene expression imports the plant that sets out, and obtains genetically modified plants；The genetically modified plants are higher than the plant that sets out to the resistance of root-knot nematode.The present invention has substantial worth for Meloidogyne enterolobii pathogenesis and the preparation of anti-nematode plant.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Mesp1 albumen from Meloidogyne enterolobii and
Its encoding gene and application.
Background technique
Meloidogyne enterolobii (Meloidogyne enterolobii) is a kind of sessile form endoparasitism pathogenic line
Worm is most distributed early in China Hainan, but its host range is extensive, adaptable, and can overcome the resistance of Mi antigen gene, simultaneously
The puncture pasteurella (Pasteuriapenetrans) for not controlled plant root-knot nematode biological and ecological methods to prevent plant disease, pests, and erosion prospect most is parasitic, therefore as
Ear beans root-knot nematode is huge to agricultural production harm, once occurrence of large-area spreads or colonizes, it will cause devastating disaster.Closely
With industry restructuring over year, China's facility cultivation area has been approached 60,000,000 mu, and the suitable temperature of protecting field and the country are frequently
Numerous seedling allocation and transportation invade hinterland for Meloidogyne enterolobii and provide advantage, at present in Beijing, Shandong, Hunan
Etc. ground have found this nematode, this brings significant threat to China's production estimation.
Since there are poor specificities, secondary work for control method seldom to Meloidogyne enterolobii pathogenesis, traditional
With the outstanding problems such as big, preventive effect is limited.RNA interference is engineering plants for nematode resistance band as a kind of new control strategy and technology
Carry out new breakthrough.RNA interferes the major programme of anti-nematode are as follows: building target is that the RNA of nematosis pathogenic related gene is interfered
Rna interference vector is imported and expresses double-stranded RNA (dsRNAs) or siRNA (siRNAs) in plant, taken through lancet by carrier
Food enters in nematode body, causes systemic rnai response, the correlation functions such as nematosis, development, metabolism, movement is caused to go out
Existing obstacle is even dead, so that render transgenic plant realizes the resistance to parasitic nematode.Therefore, Meloidogyne enterolobii is excavated
New gene studies the mechanism of action of its development parasitic to ear beans root-knot nematode, pathogenic, cultivates anti-nematode as target
Plant has great prospect and value.
Summary of the invention
The object of the present invention is to provide a kind of Mesp1 albumen and its encoding gene from Meloidogyne enterolobii and answer
With.
The present invention provides a kind of protein, are obtained from Meloidogyne enterolobii, are named as Mesp1 albumen, are following (a1)
Or (a2) or (a3) or (a4):
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(a2) protein that the amino acid sequence shown in sequence 3 in sequence table forms；
(a3) by (a1) by one or several amino acid residues substitution and/or deletion and/or addition and with root knot line
It the parasitism of worm and/or causes a disease and/or develops the relevant protein as derived from (a1)；
(a4) by (a2) by one or several amino acid residues substitution and/or deletion and/or addition and with root knot line
It the parasitism of worm and/or causes a disease and/or develops the relevant protein as derived from (a2).
In order to make protein in (a3) or (a4) convenient for purifying and detection, can in by sequence table sequence 1 or sequence 3
Shown in amino acid sequence composition protein amino terminal or carboxyl terminal connect the upper label as shown in table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (a3) or (a4) can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological table
It reaches.The encoding gene of above-mentioned (a3) or the protein in (a4) can be by will be shown in sequence 2 in sequence table or sequence 4
The codon of one or several amino acid residues is lacked in DNA sequence dna, and/or the missense of one or several base-pairs of progress is dashed forward
Become, and/or is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
The gene of coding Mesp1 albumen also belongs to protection scope of the present invention.Coding Mesp1 albumen unnamed gene be
Mesp1 gene.
Mesp1 gene is specially following (b1) or (b2) or (b3) or (b4):
(b1) code area DNA molecular as shown in sequence 2 in sequence table；
(b2) code area DNA molecular as shown in sequence 4 in sequence table；
(b3) hybridize and encode DNA points of Mesp1 albumen with (b1) above or (b2) described DNA molecular under strict conditions
Son；
(b4) there is 90% or more homology with above (b1) or (b2) DNA molecular and encodes the DNA of Mesp1 albumen
Molecule.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS, in DNA or RNA
Hybridize at 65 DEG C in hybrid experiment and washes film.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue or recombination containing Mesp1 gene
Bacterium all belongs to the scope of protection of the present invention.
The recombinant expression carrier of Mesp1 gene can be contained with existing expression vector establishment.The expression vector includes double
First agrobacterium vector and the carrier etc. that can be used for micropellet bombardment.It, can be in its turn when recombinant expression carrier gene constructed using Mesp1
Any enhanced, composing type, organizing specific type or inducible promoter are added before recording initiation nucleotide, they can individually make
It is used in combination with or with other promoters；In addition, enhancing also can be used when recombinant expression carrier gene constructed using Mesp1
Son, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region rises
Beginning codon etc., but must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control
The source of signal and initiation codon be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come
From transcription initiation region or structural gene.For the ease of identifying and screening, expression carrier used thereof can be processed, table is such as added
Up to the gene for the enzyme or luminophor that can produce color change, resistant antibiotic marker or anti-chemical reagent mark
Remember gene etc..Consider from transgenosis safe, any selected marker can be not added.
The recombinant expression carrier is recombinant plasmid pGWB402-Mesp1.
Recombinant plasmid pGWB402-Mesp1 are as follows: using expression vector pGWB402 as skeleton, there is Mesp1 gene.
The construction method of recombinant plasmid pGWB402-Mesp1 specifically comprises the following steps:
(1) it constructs following recombinant plasmid pJLsmart-Mesp1: being inserted in I restriction enzyme site of Sma of entry vector pJLsmart
Enter Mesp1 gene；
(2) recombinant plasmid pJLsmart-Mesp1 and expression vector pGWB402 are taken, LR recombining reaction is carried out, is recombinated
Plasmid pGWB402-Mesp1.
The present invention also protects the application of Mesp1 albumen, is at least one of following (c1) to (c3):
(c1) regulate and control the parasitic ability of root-knot nematode；
(c2) regulate and control the pathogenecity of root-knot nematode；
(c3) regulate and control the development of root-knot nematode.
The present invention also protects the interference carrier for inhibiting Mesp1 gene expression.The interference carrier is concretely: having special
The recombinant plasmid of DNA fragmentation: the specific DNA fragment from upstream to downstream successively include such as lower curtate: DNA molecular a, interval sequence
Column and DNA molecular b.DNA molecular a is as shown in the sequence 7 of sequence table.The 1st to 300 core of sequence 8 of DNA molecular b such as sequence table
Shown in thuja acid.The 1st to 300 nucleotide of DNA molecular b and DNA molecular a reverse complemental.The interference carrier more specifically may be used are as follows:
Have between II site DNA molecular a, Pst I and BstE using carrier pCAMBIA3301 as skeleton, between I site Nco I and Xho and has
There is DNA molecular b.
The construction method of interference carrier specifically comprises the following steps:
(1) it constructs following recombinant plasmid I: being skeleton carrier with carrier pSAT 5, be inserted between I site Nco I and Xho
DNA molecular first (DNA molecular first is double chain DNA molecule shown in the sequence 7 of sequence table), is inserted between I site Pst I and Kpn
DNA molecular second (DNA molecular second is double chain DNA molecule shown in the sequence 8 of sequence table)；
(2) following recombinant plasmid II is constructed
1. taking recombinant plasmid I, double digestion is carried out with restriction enzyme Nco I and BstE II, recycles small fragment；
2. taking carrier pCAMBIA3301, double digestion is carried out with restriction enzyme Nco I and BstE II, recycles carrier bone
Frame.
3. 1. small fragment that step obtains is connect with 2. carrier framework that step obtains, (the interference of recombinant plasmid II is obtained
Carrier).
The present invention also protects the RNA (RNA interfering) for inhibiting Mesp1 gene expression.The sequence 9 of RNA interfering such as sequence table
And/or as shown in the sequence 10 of sequence table.
The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps: the interference carrier or institute
It states RNA interfering and imports the plant that sets out, obtain genetically modified plants；The genetically modified plants go out the resistance of root-knot nematode higher than described in
Send out plant.The plant that sets out is monocotyledon or dicotyledon.The plant concretely arabidopsis that sets out, such as brother
Rival Asia Arabidopsis thaliana ecotype.
The present invention is also protected for inhibiting the substance of Mesp1 gene expression preparing the application in product；The product function
For inhibition root-knot nematode to the parasitism of plant and/or root-knot nematode can be inhibited to the pathogenic of plant and/or inhibit root-knot nematode hair
It educates.The substance concretely interference carrier or the RNA interfering for inhibiting Mesp1 gene expression.The plant is single
Cotyledon plant or dicotyledon.The plant concretely arabidopsis, such as Columbia ecotype arabidopsis.
The present invention is also protected for inhibiting the substance of Mesp1 protein active preparing the application in product；The product function
For inhibition root-knot nematode to the parasitism of plant and/or root-knot nematode can be inhibited to the pathogenic of plant and/or inhibit root-knot nematode hair
It educates.The plant is monocotyledon or dicotyledon.The plant concretely arabidopsis, such as Columbia ecotype
Arabidopsis.
Any description above root-knot nematode concretely Meloidogyne enterolobii.
Esophageal gland plays a significant role in nematode and host's interaction pathogenic course.Most parasitism pathogenic related genes exist
Nematode esophageal gland expression, coding generates secretory protein, then punctures and inject via lancet and enter in plant cell, plays parasitic cause
The relevant sophisticated functions of disease.
Mesp1 gene provided by the invention is expressed in sub- abdomen esophageal gland from the point of view of tissue specificity, is come from temporal
See that the four-age larva period expression quantity in Meloidogyne enterolobii is higher.Transient expression Mesp1 albumen can be on tobacco leaf
Inhibit to promote the programmed cell death (PCD) that apoptosis protein BAX induces by mouse.Arabidopsis mediate RNAi experiments have shown that, interference
Nematode Mesp1 gene expression can be substantially reduced the normal development of Pathogenicity and nematode, show this gene and enterolobium cyclocarpum root
It ties nematosis and is an important target gene in plant intracorporal development correlation.
The present invention has substantial worth for Meloidogyne enterolobii pathogenesis and the preparation of anti-nematode plant.
Detailed description of the invention
Fig. 1 is the result of embodiment 2.
Fig. 2 is the result of embodiment 3.
Fig. 3 is the result of embodiment 4.
Fig. 4 is the root knot number in embodiment 5 on average every plant of plant.
Fig. 5 is the nematode count in embodiment 5 in the root of average every plant of plant.
Fig. 6 is the nematode photo after being inoculated with Meloidogyne enterolobii 20 days in embodiment 5 in plant root.
Specific embodiment
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.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Refer to that the document of " Meloidogyne enterolobii " is as follows: [title]: MELOIDOGYNE-ENTEROLOBII N-SP
(MELOIDOGYNIDAE),A ROOT-KNOT NEMATODE PARASITIZING PACARA EARPOD TREE IN
CHINA；[author]: YANG, B.；EISENBACK,J.D.；[source]: JOURNAL OF NEMATOLOGY, 1983,15 (3),
381-391。
Entry vector pJLsmart (" entry vector pJLSmart " i.e. in document): bibliography: Johannes
Mathieu, NormanWarthmann, Frank Ku ¨ ttner, and Markus Schmid；Export of FT Protein
fromPhloem Companion Cells Is Sufficientfor Floral Induction in Arabidopsis；
Current Biology 17,1055–1060,June 19,2007。
Expression vector pGWB402: bibliography: Tsuyoshi NAKAGAWA etc.；Improved Gateway Binary
Vectors:High-Performance Vectors for Creation of Fusion Constructs in
Transgenic Analysis of Plants；Biosci.Biotechnol.Biochem.,71(8),2095-2100,
2007。
Carrier pSAT 5 (" pSAT5.nosP.RNAi " i.e. in document): bibliography: MeryDafny-Yelin,
Sang-Min Chung,Ellen L.Frankman,and TzviTzfira；pSAT RNA Interference Vectors:
A Modular Series forMultiple Gene Down-Regulation in Plants；Plant Physiology,
December 2007,Vol.145,pp.1272–1281。
Tobacco used in embodiment is this life cigarette.
The discovery of embodiment 1, Mesp1 albumen and Mesp1 gene
1, the Tomato Root System of inoculation Meloidogyne enterolobii 45-60 days is taken, picking pieces of an egg are hatched in sterile water after cleaning
3 days, fresh second instar larvae about l0-20 μ L is collected by centrifugation.
2, liquid nitrogen frozen, tissue grinder's disrupted sample extract total serum IgE with Trizol (Invitrogen) method, use
The genomic DNA mixed in Recombinant DNase I (Takara) digestion total serum IgE, then uses Super ScriptTM
III Reverse Transcriptase Kit (Invitrogen) carries out reverse transcription, obtains Meloidogyne enterolobii second instar larvae
cDNA。
3, using cDNA as template, Mesp1 gene is expanded using upstream primer Mesp1_F and downstream primer Mesp1_R.
Upstream primer Mesp1_F:5 '-ATGTCCATCTTCCTTACTTCTGCTC-3 '；
Downstream primer Mesp1_R:5 '-TCACATTTTCATAGTACAAGCCTTC-3 '.
Amplification system: ddH214.8 μ L, 5 × HF PCR buffer of O, 5.0 μ L, 2.5mM dNTPs, 2.0 μ L,
Mesp1_F 1.0 μ L, Mesp1_R 1.0 μ L, cDNA 1.0 μ L, Phusion DNA polymerase (NEB) 0.2 μ L, it is overall
It is 25.0 μ L.
Amplification condition: 94.0 DEG C of initial denaturation 5min, 94.0 DEG C of denaturation 30s, 58.0 DEG C of annealing 30s, 72.0 DEG C extend
1min, totally 35 recycle, and reaction is terminated after 72.0 DEG C of heat preservation 10min.
4, whole amplified productions are taken, 6 × loading buffer is added, then carries out 0.8% agarose gel electrophoresis, adopts
It with GenStar plastic recovery kit recovery product, is connect with pMD18-T (Takara) carrier, Transformed E .coliDH5 α competence
Cell carries out PCR with aforementioned primer and identifies to obtain positive colony, and positive colony measures extension increasing sequence with carrier universal primer.
Sequencing result shows open reading frame shown in the sequence 2 in amplified production with sequence table, polynucleotide
Protein shown in sequence 1.The sequence 1 of sequence table is named as Mesp1 albumen, its encoding gene is named as Mesp1 base
Cause.
The inhibiting effect that embodiment 2, Mesp1 react plant immune
One, construction recombination plasmid
1, entry vector pJLsmart is taken, digestion is carried out with restriction endonuclease sma I, obtains linearized vector.
2, the linearized vector that double chain DNA molecule shown in the sequence of sequence table 4 is obtained with step 1 is attached, is obtained
To recombinant plasmid pJLsmart-Mesp1.According to sequencing result, structure description is carried out such as to recombinant plasmid pJLsmart-Mesp1
Under: double chain DNA molecule shown in the sequence 4 of sequence table is inserted in I restriction enzyme site of Sma of entry vector pJLsmart.Sequence
Protein shown in the sequence 3 of double chain DNA molecule polynucleotide shown in the sequence 4 of table.
3, recombinant plasmid pJLsmart-Mesp1 and expression vector pGWB402 are taken, LR recombining reaction is carried out, obtains recombination matter
Grain pGWB402-Mesp1.According to sequencing result, structure is carried out to recombinant plasmid pGWB402-Mesp1 and is described as follows: carried with expressing
Body pGWB402 is skeleton, double chain DNA molecule shown in the sequence 4 with sequence table.
The double chain DNA molecule shown in the sequence 5 of sequence table replaces double chain DNA molecule shown in the sequence 4 of sequence table, presses
It is operated according to above step, obtains recombinant plasmid pGWB402-eGFP.Double-stranded DNA molecule shown in the sequence 5 of sequence table is
EGFP gene.According to sequencing result, structure is carried out to recombinant plasmid pGWB402-eGFP and is described as follows: with expression vector
PGWB402 is skeleton, double chain DNA molecule shown in the sequence 5 with sequence table.
The double chain DNA molecule shown in the sequence 6 of sequence table replaces double chain DNA molecule shown in the sequence 4 of sequence table, presses
It is operated according to above step, obtains recombinant plasmid pGWB402-BAX.Double-stranded DNA molecule shown in the sequence 6 of sequence table is
BAX gene (BAX gene encoding murine shown in sequence 6 promotees apoptosis protein).According to sequencing result, to recombinant plasmid
PGWB402-BAX carries out structure and is described as follows: double shown in the sequence 6 with sequence table using expression vector pGWB402 as skeleton
Ssdna molecule.
Two, preparation and reorganization Agrobacterium
Buffer suspension liquid: solvent pH5.6,10mM MES buffer, MgCl containing 10mM2With 0.2mM AS.
PGWB402-Mesp1 is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium, it is then outstanding with buffer suspension liquid
Floating recombinational agrobacterium, obtains bacteria suspension, is named as Mesp1 bacteria suspension (OD600nm=0.4).
PGWB402-eGFP is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium, it is then outstanding with buffer suspension liquid
Floating recombinational agrobacterium, obtains bacteria suspension, is named as eGFP bacteria suspension (OD600nm=0.4).
PGWB402-BAX is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium, is then suspended with buffer suspension liquid
Recombinational agrobacterium obtains bacteria suspension, is named as BAX bacteria suspension (OD600nm=0.4).
Three, the inhibiting effect that Mesp1 reacts plant immune
1, tobacco leaf is taken, two injection orifices of first row (injection orifice 1 and injection orifice 4) close to petiole inject buffer suspension
Two injection orifices (injection orifice 2 and injection orifice 5) of liquid (50 microlitres of every hole), an intermediate row inject Mesp1 bacteria suspension (every hole 50
Microlitre), two injection orifices (injection orifice 3 and injection orifice 6) of bottom row inject eGFP bacteria suspension (50 microlitres of every hole), and standing is incubated
It educates for 24 hours.
2, after completing step 1, on the right side of the vein in three injection orifices (injection orifice 4, injection orifice 5 and injection orifice 6) of tandem
It injects in BAX bacteria suspension (50 microlitres of every hole), stationary incubation 5 days.
3, after completing step 2, the downright bad situation of tobacco leaf is observed.
The result is shown in Figure 1.There is not dark brown allergy in the position of co-injection Mesp1 bacteria suspension and BAX bacteria suspension on blade
Property necrosis symptom, there is obvious dark brown hypersensitive necrosis disease in the position of co-injection buffer suspension liquid and BAX bacteria suspension on blade
Shape, there is obvious dark brown hypersensitive necrosis symptom in the position of co-injection eGFP bacteria suspension and BAX bacteria suspension on blade, on blade
It is individually injected on the independent position for injecting Mesp1 bacteria suspension, blade and individually injects thallus on the part and blade of eGFP bacteria suspension
The position of suspension does not occur any hypersensitive necrosis symptom.
It carries out five repetitions to test, as a result unanimously.
The result shows that BAX causes apoptosis, and Mesp1 can inhibit the apoptotic as caused by BAX dead
It dies, and then shows that Mesp1 takes part in the process for inhibiting plant immune reaction.
The tissue positioning of embodiment 3, Mesp1 gene
With DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) object
Ear beans root-knot nematode second instar larvae carries out hybridization in situ experiment.
As a result see Fig. 2.Results of hybridization shows that antisense probe has hybridization signal, and hybridization signal is located at sub- abdomen esophageal gland, shows
Mesp1 gene may be sub- abdomen esophageal gland cell expression.
The express spectra of embodiment 4, Mesp1 gene
Mesp1 gene is analyzed in each puberty (ovum, parasitism of Meloidogyne enterolobii using real-time fluorescence quantitative PCR
Preceding second instar larvae, parasitic stage second instar larvae, third-instar larvae, four-age larva and mature female adult) differential expression situation.
The primer that real time fluorescent quantitative uses is as follows:
Mesp1-qRT-F:5'-GCTTGATCACGCGGATACCA-3'；
Mesp1-qRT-R:5'-GCAATCGCCTGAACCCTCTG-3'.
Using tubulin as reference gene, biology three times is carried out respectively and repeats to test, using 2-△△CtMethod analyzes result.
As a result see Fig. 3.The results show that the expression quantity relative to ovum period, four ages expression of the Mesp1 gene after infecting
Extremely significant up-regulation is measured, up-regulation multiple is 5.62 times.Mesp1 gene may participate in the maintenance of giant cell.
Embodiment 5, the building of interference carrier and its application in the plant for cultivating anti-nematosis
One, the building of interference carrier
1, construction recombination plasmid I
It is skeleton carrier with carrier pSAT 5, specific DNA molecular first (specific DNA is inserted between I site Nco I and Xho
Molecule first is double chain DNA molecule shown in the sequence 7 of sequence table), specific DNA molecular second is inserted between I site Pst I and Kpn
(specific DNA molecular second is double chain DNA molecule shown in the sequence 8 of sequence table).The 1st to 300 nucleosides of specific DNA molecular second
Acid and specific DNA molecular first reverse complemental.
2, construction recombination plasmid II
(1) recombinant plasmid I is taken, double digestion is carried out with restriction enzyme Nco I and BstE II, recycles small fragment.
(2) carrier pCAMBIA3301 is taken, double digestion is carried out with restriction enzyme Nco I and BstE II, recycles carrier bone
Frame.
(3) small fragment that step (1) obtains is connect with the carrier framework that step (2) obtains, obtains recombinant plasmid II.Weight
Group plasmid II is interference carrier.According to sequencing result, structure is carried out to interference carrier and is described as follows: with carrier pCAMBIA3301
For skeleton, there is the specific DNA molecular first between I site Nco I and Xho, there is the spy between II site Pst I and BstE
The 1st to 300 nucleotide of different DNA molecular second.
Two, the acquisition of genetically modified plants
1, interference carrier is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium.
2, Columbia ecotype arabidopsis is converted by dipping in the recombinational agrobacterium that colored method obtains step 1, using 0.5 ‰
Ppt herbicide sprays screening, collect T1 for seed.
T1 is T1 for plant for the plant that seed grows up to.T1 for plant selfing and is harvested into seed, as T2 is for seed.
T2 is T2 for plant for the plant that seed grows up to.T2 for plant selfing and is harvested into seed, as T3 is for plant.T2 generation kind
The plant that son grows up to is T3 for plant.
For a certain T1 is for plant, if the offspring (T2 is for plant and T3 for plant) of its sampling Detection is PCR
Identification is positive, which is a homozygous transgenic line for plant and its offspring.The method of PCR identification: genome is extracted
DNA carries out PCR amplification using the primer pair that Mesp1-qRT-F and Mesp1-qRT-R is formed, if the amplification for obtaining 178bp produces
Object, PCR are accredited as the positive.
3 homozygous transgenic line (Mesp1-1st-2 strains, Mesp1-1st-3 strain, Mesp1-1st-5 are taken at random
Strain) carry out step 4.
Three, turn the acquisition of empty carrier plant
It replaces interference carrier to carry out step 2 with carrier pCAMBIA3301, obtains turning empty carrier strain.
Four, the Study of Interference in nematosis pathogenecity body is carried out
Test plant: the T3 of Mesp1-1st-2 strain for plant, Mesp1-1st-3 strain T3 for plant, Mesp1-
The T3 of 1st-5 strain for plant, turn the T3 of empty carrier strain for plant, Columbia ecotype arabidopsis (Colombia's ecology
Type arabidopsis is indicated with Col or COL).25 plants of each strain.
Test method is as follows:
1, the test plant (sprouting one week) grown on MS culture medium flat plate is taken, is transferred to equipped with culture substrate (culture
What matrix i.e. 1 parts by volume Nutrition Soil and 1 parts by volume vermiculite were mixed to get) container in, each container transplants 1 plant.
2, after cultivating 3 weeks in culture substrate, 300 enterolobium cyclocarpum root knot lines are inoculated in the culture substrate in each container
Worm.
3, after being inoculated with Meloidogyne enterolobii 20 days, data statistics is carried out.
In each strain, the root knot number on average every plant of plant is shown in Fig. 4.Compared with Columbia ecotype arabidopsis,
The root knot number of three transgenic lines reduces 55.30%, 41.75% and 48.16% respectively.It is quasi- with Columbia ecotype
Southern mustard is compared, and the root knot number for turning empty carrier strain is not significantly different.
In each strain, the nematode count in the root of average every plant of plant is shown in Fig. 5.With Columbia ecotype arabidopsis phase
Than the nematode count in three transgenic line roots reduces 63.40%, 53.51% and 53.30% respectively.It is raw with Colombia
State type arabidopsis is compared, and the nematode count turned in empty carrier strain root is not significantly different.
After inoculation Meloidogyne enterolobii 20 days, the nematode photo in plant root is shown in Fig. 6.With the quasi- south of Columbia ecotype
Mustard is compared, and nematode developmental state is obviously slack-off in three transgenic lines.
Refer to the result shows that carrying out interference to the Mesp1 gene in Meloidogyne enterolobii and will lead to nematode pathogenecity
Mark --- root knot number and root interior lines borer population are decreased obviously, and illustrate that Mesp1 gene causes a disease in Meloidogyne enterolobii and host's interaction
Critical function is played in the process.
Sequence table
SEQUENCE LISTING
<110>China Agricultural University
<120>from the Mesp1 albumen of Meloidogyne enterolobii and its encoding gene and application
<130> GNCYX162320
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 174
<212> PRT
<213>Meloidogyne enterolobii
<400> 1
Met Ser Ile Phe Leu Thr Ser Ala Leu Leu Ile Ile Ser Met Ile Ala
1 5 10 15
Met Thr Glu Gly Ala Gly Asp Arg Ser Ala Ser Thr Ser Thr Gly Cys
20 25 30
Thr Thr Tyr Phe Gly Met Leu Asp His Ala Asp Thr Lys Glu Asn Asn
35 40 45
Lys Arg Lys Thr Phe Lys Pro Asn Val Glu Asn Ile Ser Asn Thr Leu
50 55 60
Lys Val Thr Gly Gly Ala Thr Phe Ser Asn Thr Ser Val Ala Leu Val
65 70 75 80
Val Gly Asn Glu Val Leu Cys Met Ala Lys Thr Glu Gly Ser Gly Asp
85 90 95
Cys Gly Met Arg Asn Glu Ala Leu Thr Gly Thr Met Lys Phe Phe Ile
100 105 110
Ser Glu Asn Ile Ile Val Glu Val Pro Phe Lys Asp Val Phe Phe Phe
115 120 125
Thr Asp Asn Lys Cys Val Ile Gln Leu Val Ser Tyr Asn Val Gly Thr
130 135 140
His Glu Thr Leu Leu Lys Ile Asn Asp Val Asp Phe Lys Ile Thr Ala
145 150 155 160
Thr Asp Lys Lys Ile Ser Pro Lys Ala Cys Thr Met Lys Met
165 170
<210> 2
<211> 525
<212> DNA
<213>Meloidogyne enterolobii
<400> 2
atgtccatct tccttacttc tgctcttcta atcatttcaa tgattgctat gaccgaggga 60
gcaggcgatc gaagcgcttc aacctctact ggttgtacaa cctattttgg aatgcttgat 120
cacgcggata ccaaggaaaa taacaaaagg aaaactttca aacccaacgt tgaaaacata 180
tccaacacct tgaaagtgac tggtggggct acgtttagca atacctcggt ggctttggtt 240
gtcggtaatg aggtgttatg tatggctaag acagagggtt caggcgattg cggaatgcgc 300
aacgaagcgt tgactggaac tatgaaattt ttcatttctg agaatattat tgttgaggtt 360
ccattcaaag acgttttttt cttcaccgac aacaagtgtg tcatccagct tgtaagctac 420
aatgttggaa cgcatgaaac tcttctcaaa attaatgatg tcgacttcaa aattaccgct 480
actgacaaga aaatttcccc gaaggcttgt actatgaaaa tgtga 525
<210> 3
<211> 155
<212> PRT
<213>artificial sequence
<400> 3
Met Ala Gly Asp Arg Ser Ala Ser Thr Ser Thr Gly Cys Thr Thr Tyr
1 5 10 15
Phe Gly Met Leu Asp His Ala Asp Thr Lys Glu Asn Asn Lys Arg Lys
20 25 30
Thr Phe Lys Pro Asn Val Glu Asn Ile Ser Asn Thr Leu Lys Val Thr
35 40 45
Gly Gly Ala Thr Phe Ser Asn Thr Ser Val Ala Leu Val Val Gly Asn
50 55 60
Glu Val Leu Cys Met Ala Lys Thr Glu Gly Ser Gly Asp Cys Gly Met
65 70 75 80
Arg Asn Glu Ala Leu Thr Gly Thr Met Lys Phe Phe Ile Ser Glu Asn
85 90 95
Ile Ile Val Glu Val Pro Phe Lys Asp Val Phe Phe Phe Thr Asp Asn
100 105 110
Lys Cys Val Ile Gln Leu Val Ser Tyr Asn Val Gly Thr His Glu Thr
115 120 125
Leu Leu Lys Ile Asn Asp Val Asp Phe Lys Ile Thr Ala Thr Asp Lys
130 135 140
Lys Ile Ser Pro Lys Ala Cys Thr Met Lys Met
145 150 155
<210> 4
<211> 468
<212> DNA
<213>artificial sequence
<400> 4
atggcaggcg atcgaagcgc ttcaacctct actggttgta caacctattt tggaatgctt 60
gatcacgcgg ataccaagga aaataacaaa aggaaaactt tcaaacccaa cgttgaaaac 120
atatccaaca ccttgaaagt gactggtggg gctacgttta gcaatacctc ggtggctttg 180
gttgtcggta atgaggtgtt atgtatggct aagacagagg gttcaggcga ttgcggaatg 240
cgcaacgaag cgttgactgg aactatgaaa tttttcattt ctgagaatat tattgttgag 300
gttccattca aagacgtttt tttcttcacc gacaacaagt gtgtcatcca gcttgtaagc 360
tacaatgttg gaacgcatga aactcttctc aaaattaatg atgtcgactt caaaattacc 420
gctactgaca agaaaatttc cccgaaggct tgtactatga aaatgtga 468
<210> 5
<211> 720
<212> DNA
<213>artificial sequence
<400> 5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 6
<211> 579
<212> DNA
<213>artificial sequence
<400> 6
atggacgggt ccggggagca gcttgggagc ggcgggccca ccagctctga acagatcatg 60
aagacagggg cctttttgct acagggtttc atccaggatc gagcagggag gatggctggg 120
gagacacctg agctgacctt ggagcagccg ccccaggatg cgtccaccaa gaagctgagc 180
gagtgtctcc ggcgaattgg agatgaactg gacagcaata tggagctgca gaggatgatt 240
gctgacgtgg acacggactc cccccgagag gtcttcttcc gggtggcagc tgacatgttt 300
gctgatggca acttcaactg gggccgcgtg gttgccctct tctactttgc tagcaaactg 360
gtgctcaagg ccctgtgcac taaagtgccc gagctgatca gaaccatcat gggctggaca 420
ctggacttcc tccgtgagcg gctgcttgtc tggatccaag accagggtgg ctgggaaggc 480
ctcctctcct acttcgggac ccccacatgg cagacagtga ccatctttgt ggctggagtc 540
ctcaccgcct cgctcaccat ctggaagaag atgggctga 579
<210> 7
<211> 300
<212> DNA
<213>artificial sequence
<400> 7
atggcaggcg atcgaagcgc ttcaacctct actggttgta caacctattt tggaatgctt 60
gatcacgcgg ataccaagga aaataacaaa aggaaaactt tcaaacccaa cgttgaaaac 120
atatccaaca ccttgaaagt gactggtggg gctacgttta gcaatacctc ggtggctttg 180
gttgtcggta atgaggtgtt atgtatggct aagacagagg gttcaggcga ttgcggaatg 240
cgcaacgaag cgttgactgg aactatgaaa tttttcattt ctgagaatat tattgttgag 300
<210> 8
<211> 307
<212> DNA
<213>artificial sequence
<400> 8
ctcaacaata atattctcag aaatgaaaaa tttcatagtt ccagtcaacg cttcgttgcg 60
cattccgcaa tcgcctgaac cctctgtctt agccatacat aacacctcat taccgacaac 120
caaagccacc gaggtattgc taaacgtagc cccaccagtc actttcaagg tgttggatat 180
gttttcaacg ttgggtttga aagttttcct tttgttattt tccttggtat ccgcgtgatc 240
aagcattcca aaataggttg tacaaccagt agaggttgaa gcgcttcgat cgcctgccat 300
ggtcacc 307
<210> 9
<211> 300
<212> RNA
<213>artificial sequence
<400> 9
auggcaggcg aucgaagcgc uucaaccucu acugguugua caaccuauuu uggaaugcuu 60
gaucacgcgg auaccaagga aaauaacaaa aggaaaacuu ucaaacccaa cguugaaaac 120
auauccaaca ccuugaaagu gacugguggg gcuacguuua gcaauaccuc gguggcuuug 180
guugucggua augagguguu auguauggcu aagacagagg guucaggcga uugcggaaug 240
cgcaacgaag cguugacugg aacuaugaaa uuuuucauuu cugagaauau uauuguugag 300
<210> 10
<211> 300
<212> RNA
<213>artificial sequence
<400> 10
cucaacaaua auauucucag aaaugaaaaa uuucauaguu ccagucaacg cuucguugcg 60
cauuccgcaa ucgccugaac ccucugucuu agccauacau aacaccucau uaccgacaac 120
caaagccacc gagguauugc uaaacguagc cccaccaguc acuuucaagg uguuggauau 180
guuuucaacg uuggguuuga aaguuuuccu uuuguuauuu uccuugguau ccgcgugauc 240
aagcauucca aaauagguug uacaaccagu agagguugaa gcgcuucgau cgccugccau 300
Claims (9)
1. a kind of protein is following (a1) or (a2):
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(a2) protein that the amino acid sequence shown in sequence 3 in sequence table forms.
2. encoding the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that: the gene is following (b1) or (b2):
(b1) code area DNA molecular as shown in sequence 2 in sequence table；
(b2) code area DNA molecular as shown in sequence 4 in sequence table.
4. recombinant expression carrier, expression cassette or recombinant bacterium containing gene described in Claims 2 or 3.
5. the application of protein described in claim 1 is at least one of following (c1) to (c3):
(c1) regulate and control the parasitic ability of root-knot nematode；
(c2) regulate and control the pathogenecity of root-knot nematode；
(c3) regulate and control the development of root-knot nematode.
6. inhibiting the interference carrier of gene expression described in Claims 2 or 3.
7. inhibiting the RNA of gene expression described in Claims 2 or 3.
8. a kind of method for cultivating genetically modified plants, includes the following steps: interference carrier described in claim 6 or claim
7 RNA import the plant that sets out, and obtain genetically modified plants；The genetically modified plants go out the resistance of root-knot nematode higher than described in
Send out plant.
9. application of the RNA described in interference carrier or claim 7 described in claim 6 in reagent preparation box；The kit
Function be to inhibit root-knot nematode to the parasitism of plant and/or inhibit root-knot nematode causing a disease and/or inhibit root knot line to plant
Worm development.
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|---|---|---|---|---|
| CN103524611A (en) * | 2013-10-11 | 2014-01-22 | 中国农业大学 | Meloidogyne incognita Chitwood esophageal gland specific gene Msp40, and coding protein and application thereof |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524611A (en) * | 2013-10-11 | 2014-01-22 | 中国农业大学 | Meloidogyne incognita Chitwood esophageal gland specific gene Msp40, and coding protein and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| AAQ10015.1;Huang，G.等;《GenBank》;20031017;ORIGIN * |
| De Novo Analysis of the Transcriptome of Meloidogyne enterolobii to Uncover Potential Target Genes for Biological Control;Xiangyang Li等;《International Journal of Molecular Sciences》;20160901;1442 * |
| RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes;Sagar Banerjee等;《Frontiers in Plant Science》;20170519;Article 834 * |
| 南方根结线虫蛋白Misp12的功能研究;谢家廉等;《中国植物病理学会2015年学术年会论文集》;20150721;第四部分线虫与线虫病害 * |
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