CN109627311A

CN109627311A - Cereal cyst nematode HaHSP4 albumen and its encoding gene and application

Info

Publication number: CN109627311A
Application number: CN201910097638.4A
Authority: CN
Inventors: 简恒; 杨姗姗; 刘倩
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2019-04-16

Abstract

The invention discloses the HaHSP4 albumen and its encoding gene of cereal cyst nematode and applications.The present invention provides a kind of protein, are obtained from cereal cyst nematode, are named as HaHSP4 albumen, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.The gene for encoding HaHSP4 albumen, is named as HaHSP4 gene, also belongs to protection scope of the present invention.The present invention also protects the application of HaHSP4 albumen: regulating and controlling host's taxis of cyst roundworm；Regulate and control the parasitic ability of cyst roundworm；Regulate and control the pathogenecity of cyst roundworm；Regulate and control the development of cyst roundworm.A kind of method that the present invention also protects plant cultivated and improved to cyst roundworm resistance includes the following steps: to import the substance for inhibiting HaHSP4 protein expression in purpose plant, obtains the plant improved to cyst roundworm resistance.The present invention has substantial worth for cereal cyst nematode pathogenesis and the preparation of anti-nematode plant.

Description

Cereal cyst nematode HaHSP4 albumen and its encoding gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to cereal cyst nematode HaHSP4 albumen and its encoding gene with answer With.

Background technique

Cereal cyst nematode (Heterodera avenae) is a kind of sessile form endoparasitism Plant nematode, mainly invades wheat Class plant root inhibits the normal growth of root, causes very big loss to crop yields such as wheat, barleys.Studies have shown that in China The area that cereal cyst nematode (H.avenae) endangers wheat reaches 4,000,000 hm²More than, and in trend is risen year by year, cause small The production loss of wheat 15.5%-55.0%, it has also become the significant problem in China's Wheat Production.

Due to insufficient to cereal cyst nematode pathogenesis, there are poor specificities, side effect for traditional control method Greatly, the outstanding problems such as preventive effect is limited.RNA interference is brought as a kind of new control strategy and technology for engineering plants for nematode resistance New breakthrough.RNA interferes the major programme of anti-nematode are as follows: building target is that the RNA of nematosis pathogenic related gene interferes load Rna interference vector is imported and expresses double-stranded RNA (dsRNAs) or siRNA (siRNAs) in plant by body, through lancet feeding into Enter in nematode body, causes systemic rnai response, the correlation functions such as nematosis, development, metabolism, movement is caused to hinder Hinder even death, so that render transgenic plant realizes the resistance to parasitic nematode.

Therefore, the new gene for excavating cereal cyst nematode studies the work of its development parasitic, pathogenic to cereal cyst nematode With mechanism, the plant of anti-nematode is cultivated as target, and there is great prospect and value.

Summary of the invention

The purpose of the present invention is improve wheat to the resistance of cyst roundworm.

The present invention provides a kind of protein, are obtained from cereal cyst nematode, are named as protein HaHSP4, are following (a1) Or (a2) or (a3) or (a4) or (a5) or (a6) or (a7):

(a1) amino acid sequence is protein shown in sequence 1 in sequence table；

(a2) contain the fused protein of (a1)；

(a3) fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；

(a4) by (a1) by one or several amino acid residues substitution and/or deletion and/or addition and with sporangiocyst line It the host's taxis and/or parasitism of worm and/or causes a disease and/or develops the relevant protein as derived from (a1)；

(a5) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into inhibition The protein as derived from (a1) for the function that active oxygen generates in plant；

(a6) from cereal cyst nematode and with (a1) have 98% or more homology and with the host of cereal cyst nematode It taxis and/or parasitism and/or causes a disease and/or develops the relevant protein as derived from (a1)；

(a7) with 98% or more homology and there is inhibition plant activity in vivo from cereal cyst nematode and with (a1) The protein as derived from (a1) for the function that oxygen generates.

It, can amino acid sequence shown in sequence 1 forms in by sequence table egg in order to which protein is convenient for purifying and detection The amino terminal or carboxyl terminal of white matter connect 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

Above-mentioned protein can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.

The encoding gene of above-mentioned protein can be by will lack one or several in DNA sequence dna shown in sequence 2 in sequence table The codon of a amino acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or in its end 5' and/or 3' The coded sequence that end connects label shown in table 1 obtains.

The nucleic acid molecules of code for said proteins HaHSP4 also belong to protection scope of the present invention.

The nucleic acid molecules of code for said proteins HaHSP4 can be following (b1) or (b2) or (b3) or (b4):

(b1) code area DNA molecular as shown in sequence 2 in sequence table；

(b2) nucleotides sequence is classified as DNA molecular shown in sequence 2 in sequence table；

(b3) hybridize under strict conditions with (b1) or (b2) described DNA molecular and code for said proteins HaHSP4 DNA molecular；

(b4) there is 90% or more homology and volume from cereal cyst nematode and with (b1) or (b2) DNA molecular The DNA molecular of the code protein HaHSP4.

Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA It hands over and hybridizes at 65 DEG C in experiment and wash film.

The gene of the nucleic acid molecules of code for said proteins HaHSP4 concretely code for said proteins HaHSP4, name For HaHSP4 gene.

Recombinant expression carrier, expression cassette, Transgenic plant tissue containing any of the above-described nucleic acid molecules or recombination are micro- Biology also belongs to protection scope of the present invention.

The recombinant expression carrier of HaHSP4 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 at it when recombinant expression carrier gene constructed using HaHSP4 Plus any enhanced, composing type, organizing specific type or inducible promoter before transcription initiation nucleotide, they can be independent It is used in combination using or with other promoters；In addition, also can be used and increase when recombinant expression carrier gene constructed using HaHSP4 Hadron, 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 present invention also protects the application of the protein HaHSP4, can be at least one of following (c1) to (c6):

(c1) regulate and control host's taxis of cyst roundworm；

(c2) regulate and control the parasitic ability of cyst roundworm；

(c3) regulate and control the pathogenecity of cyst roundworm；

(c4) regulate and control the development of cyst roundworm；

(c5) regulate and control active oxygen in plant to generate；

(c6) active oxygen in plant is inhibited to generate.

The present invention also protects a kind of method for cultivating genetically modified plants, it may include imports in the plant that sets out and inhibits the egg The substance of white matter HaHSP4 expression, the step of obtaining genetically modified plants；Compared with the plant that sets out, the genetically modified plants pair The resistance of cyst roundworm improves.Described improve to cyst roundworm resistance can improve for the resistance infected to cyst roundworm.

In the above method, " substance for inhibiting the protein HaHSP4 expression " concretely inhibits HaHSP4 gene The interference carrier of expression.

The present invention also protects the interference carrier or recombinant virus for inhibiting any of the above-described nucleic acid molecules expression." the suppression Make the interference carrier or recombinant virus of any of the above-described nucleic acid molecules expression " concretely inhibit the dry of HaHSP4 gene expression Disturb carrier or recombinant virus.

Any of the above-described interference carrier concretely recombinant plasmid BSMV:HaHSP4 for inhibiting HaHSP4 gene expression. Recombinant plasmid BSMV:HaHSP4 is following recombinant plasmid: in multiple cloning sites (such as ApaI digestion position of carrier pCa- γ bLIC Point) insert the sequence 2 of the sequence table DNA molecular shown in the nucleotide of 5 ' end 296-595.

Any of the above-described recombinant virus for inhibiting HaHSP4 gene expression.The recombinant virus concretely recombinates BSMV Virus.The preparation method of the recombinant virus is specific as follows: pCaBS- α, pCaBS- β and recombinant plasmid BSMV:HaHSP4 are total to With transfection tobacco plant, culture obtains recombinant virus.PCaBS- α transfects tobacco plant especially by recombinational agrobacterium first. PCaBS- β transfects tobacco plant especially by recombinational agrobacterium second.Recombinant plasmid BSMV:HaHSP4 is especially by recombinational agrobacterium Third transfection tobacco plant.Recombinational agrobacterium first is the recombinational agrobacterium for obtaining pCaBS- α importing Agrobacterium tumefaciems EHA105.Weight Group Agrobacterium second is the recombinational agrobacterium for obtaining pCaBS- β importing Agrobacterium tumefaciems EHA105.Recombinational agrobacterium third is will to weigh Group plasmid BSMV:HaHSP4 imports the recombinational agrobacterium that Agrobacterium tumefaciems EHA105 is obtained.The common transfection tobacco plant tool Body can grow to the blade of this life cigarette plant of 8-12 leaf phase for common transfection.The mode of the transfection, concretely injection contains There are recombinational agrobacterium first, recombinational agrobacterium second and the bacterium solution of recombinational agrobacterium third to vacuum side of blade.The time of the culture is specific It can be 3 days.Recombinant virus be specifically present in the 1st blade and/or blade of the blade being vaccinated and/or the blade or more with On the 2nd blade in.

The present invention also protects a kind of method for reducing cyst roundworm host taxis, can be to make the albumen in cyst roundworm body The expression quantity and/or activity of matter HaHSP4 reduces.

It is described " reducing the expression quantity of the protein HaHSP4 and/or activity in cyst roundworm body " in the above method It can be by feeding to cyst roundworm for inhibiting the substance of HaHSP4 gene expression to realize.

The present invention is also protected for inhibiting the protein HaHSP4 expression and/or active substance in preparing product Using.The product function is to inhibit cyst roundworm to host's taxis of plant and/or inhibit cyst roundworm to the parasitism of plant And/or inhibits cyst roundworm to the pathogenic of plant and/or inhibit cyst roundworm development.

It is described that for inhibiting, the protein HaHSP4 is expressed and/or active substance can be for for inhibiting HaHSP4 gene The substance of expression.

The inhibition of any of the above-described substance for inhibiting HaHSP4 gene expression concretely any description above The inhibition HaHSP4 base of the interference carrier of HaHSP4 gene expression, the RNA for inhibiting HaHSP4 gene expression or any description above Because of the recombinant virus of expression.

Any of the above-described " RNA for inhibiting HaHSP4 gene expression " can be double shown in the sequence 7 in sequence table Chain RNA.

The present invention also protects special RNA；The special RNA can be double-stranded RNA shown in the sequence 7 in sequence table.It is described Special RNA can inhibit HaHSP4 gene expression.

Any description above plant can be that monocotyledon or dicotyledon, concretely wheat, such as wheat are short anti- 58。

Any description above cyst roundworm concretely cereal cyst nematode.

Phasmid can secrete effect protein, but unlike can the puncturing and inject via lancet of esophagus glandular secretion is thin into plant It is intracellular, and the mode that may be by independently transporting enters plant cell, and weight is played in nematode and host's interaction pathogenic course It acts on.

HaHSP4 gene provided by the invention is expressed from the point of view of tissue specificity in phasmid, from the point of view of temporal In cereal cyst nematode after infecting three period in age expression quantity highests, therefore protein HaHSP4 may be important from infecting the later period Effect.Utilize gene silencing (BSMV-HIGS) system silencing of the hordeivirus BSMV host's induction mediated HaHSP4 gene, after discovery nematode infection wheat 7 days in wheat root nematode population significantly lower than control, and after 50 days sporangiocyst shape It is decreased obviously compared with the control at quantity.Protein HaHSP4 can significantly inhibit the generation of ROS in tobacco body simultaneously.Protein HaHSP4 plays a significant role in nematode parasitic processes, and HaHSP4 gene can be used as the target gene of Genes For Plant Tolerance line engineering.This Invention has substantial worth for the preparation of cereal cyst nematode pathogenesis and 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 the relative expression quantity of HaHSP4 gene in cereal cyst nematode in embodiment 4.

Fig. 4 is the quantity statistics result of cereal cyst nematode in average every plant of plant root in embodiment 4.

Fig. 5 is the sporangiocyst forming quantity statistical result of average every plant of plant in embodiment 4.

Fig. 6 is the result of embodiment 5.

Fig. 7 is the result of the relative expression quantity of HaHSP4 gene in cereal cyst nematode in embodiment 6.

Fig. 8 is the distribution situation that wheat periapex encloses cereal cyst nematode in embodiment 6.

Fig. 9 is the statistical result of the quantity of cereal cyst nematode within the scope of Helminthosporium sativum 1.5cm in embodiment 6.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

In following embodiments, cereal cyst nematode, this life cigarette and wheat short anti-58 are recorded in following document: Chen C, Liu S, Liu Q, et al.An ANNEXIN-Like Protein from the Cereal Cyst Nematode Heteroderaavenae Suppresses Plant Defense.PLOS ONE, 2015,10 (4): e122256..

In following embodiments, pGR107 carrier is recorded in following document: Changlong C, Yongpan C, Heng J, et al.Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana Benthamiana [J] .Frontiers in Plant Science, 2018,8:2062.

In following embodiments, " pCaBS- α ", " pCaBS- β " and " pCa- γ bLIC " are recorded in following document: barley item Application study of the gene silencing of line mosaic virus induction in plant gene function analysis.

100 × BSA, that is, 1g/100mL BSA aqueous solution.

The discovery of embodiment 1, HaHSP4 albumen and HaHSP4 gene

1, the sporangiocyst of the cereal cyst nematode of the fresh simple grain of picking, liquid nitrogen frozen, tissue grinder's disrupted sample use magnetic Pearl method (QIAGEN) extracts total serum IgE, and reverse transcription obtains cDNA.

2, using cDNA as template, PCR amplification is carried out using the primer pair that HaHSP4-F and HaHSP4-R is formed.

HaHSP4-F:5 '-ATGCATGTGCAAACTGTTTTCC-3 '；

HaHSP4-R:5 '-TCAGTTGTTGGCCAGCCC-3 '.

The reaction system (50 μ L) of PCR amplification: 5 × Q5Buffer, 10.00 μ L, Q5High-Fidelity DNA Polymerase 0.5 μ L, HaHSP4-F 2.5 μ L, HaHSP4-R2.5 μ L, 2.00 μ L of template are supplied with ddH2O.

PCR response procedures: 98 DEG C of 30s；98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃10min；4 DEG C of guarantors It deposits.

3, pGR107 carrier (Agricultural University Of Nanjing Wang Yuanchao professor laboratory present) is linearized into (37 DEG C of water-bath 3- 4h), linearization plasmid is recycled, is connect with the pcr amplification product of step 2, then converts bacillus coli DH 5 alpha, is sequenced.

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 HaHSP4 albumen, its encoding gene is named as HaHSP4 base Cause.In sequence 1, the 1st to 22 amino acids residue forms signal peptide.

The tissue positioning of embodiment 2, HaHSP4 gene

With DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) to standing grain Paddy cyst roundworm second instar larvae carries out hybridization in situ experiment.

1, using the cDNA of cereal cyst nematode as template, drawn using what HaHSP4-situ-F and HaHSP4-situ-R was formed Object recycles target fragment to amplification probe target fragment (263bp).

HaHSP4-situ-F:5 '-GAAGGAAGCCATTGAGGAG-3 '；

HaHSP4-situ-R:5 '-CCGTATCGGTGTAGTAGCG-3 '.

Reaction system (25 μ L): Phusion High-Fidelity DNA Polymerase 0.5 μ L, dNTP 1.0 2.5 2.5 μ L of μ L, HaHSP4-situ-R of μ L, HaHSP4-situ-F of Mixture (10mM each), template 2 μ L, 5 × Phusion HF Buffer 10 μ L, ddH2O are supplied.

Reaction condition: 94 DEG C of 4min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃10min；15℃ 5min。

2, Sense probes or antisense probe are prepared using asymmetric PCR.

Using HaHSP4-situ- or HaHSP4-situ-R as primer, carried out using the target fragment that step 1 recycles as template PCR amplification amplification, obtains antisense probe/Sense probes.

Reaction system (25 μ L): 10 × Taq Buffer 2.5 μ L, DIG/dNTP mix (1mM) 2.5 μ L, primer (10 μM) 5.0 μ L, 1.0 μ L, Taq DNA polymerase (5U/ μ L) of template 0.5 μ L, DEPC-H₂O is supplied.

3, in situ hybridization is carried out.

The result is shown in Figure 1.The results show that antisense probe has hybridization signal, hybridization signal is located at phasmid.The result shows that HaHSP4 gene may be expressed in phasmid cell.

The growth expression pattern of embodiment 3, HaHSP4 gene

Using real-time fluorescence quantitative PCR analysis HaHSP4 gene cereal cyst nematode each puberty (ovum, it is parasitic before Second instar larvae, parasitic stage second instar larvae, parasitic stage third-instar larvae, parasitic stage four-age larva and mature female adult) relative expression's water It is flat.Using GAPDH-1 gene as internal reference.UsingPremix Ex Taq^TMKit (Takara), in ABI PRISM Real time RT-PCR detection is carried out on 7500 fluorescence quantitative PCR instruments.For detecting the primer pair of HaHSP4 gene by qRT- HaHSP4-F and qRT-HaHSP4-R composition.For detecting the primer pair of GAPDH-1 gene by GAPDH-1-F and GAPDH-1-R Composition.Template is the cDNA that the total serum IgE reverse transcription of each budding cereal cyst nematode obtains.

QRT-HaHSP4-F (upstream primer): 5 '-ACCTGTTCGTTCCGTTGTT-3 '；

QRT-HaHSP4-R (downstream primer): 5 '-GCGTTATTGGCATGTTCTTG-3 '.

GAPDH-1-F (upstream primer): 5 '-AGCGGCACAGAACATCATCC-3 '；

GAPDH-1-R (downstream primer): 5 '-GGTCCTCCGTGTAGCCCAAA-3 '.

Reaction system (20 μ L):10 μ L, Rox Dye of Premix Ex TaqTM (2 ×), 0.4 μ L, upstream primer (10 μM) 0.4 μ L, 0.4 μ L of downstream primer (10 μM), template 1 μ L, ddH2O are supplied.

Response procedures: 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 31s, 40 circulations；95℃15s,60℃1min,95℃15s.

Biology three times is carried out respectively to repeat to test, and result is analyzed using 2- △ △ Ct method.

As a result see Fig. 2.Relative to the expression quantity in ovum period, expression quantity is most in parasitic stage third-instar larvae for HaHSP4 gene It is high.The results show that HaHSP4 gene mainly works in the Cereal cyst roundworm parasitic later period.

The parasitic ability of cereal cyst nematode weakens after embodiment 4, HaHSP4 gene silencing

PCass4-Rz-BSMV viral vectors totally three, be pCaBS- α, pCaBS- β and pCa- γ bLIC respectively.

One, interference carrier is constructed

1, using the cDNA of cereal cyst nematode as template, using the primer of HaHSP4-LIC-F and HaHSP4-LIC-R composition To progress PCR amplification.

HaHSP4-LIC-F:5 '-AAGGAAGTTTAAAGATGGAGACCAAAGCGGAC-3'；

HaHSP4-LIC-R:5 '-AACCACCACCACCGTTTTGCGTTGCTTCGTGCAAC-3’。

In HaHSP4-LIC-F and HaHSP4-LIC-R, underscore is labeled as joint sequence.

Through being sequenced, which is named as with connector sequence by pcr amplification product as shown in the sequence 3 of sequence table The HaHSP4 section of column.

2, with artificial synthesized eGFP gene (eGFP gene is as shown in sequence 4 of sequence table) for template, using eGFP- The primer pair of LIC-F and eGFP-LIC-R composition carries out PCR amplification.

EGFP-LIC-F:5 '-AAGGAAGTTTAAACCCTCGTGACCACCCTGAC-3'；

EGFP-LIC-R:5 '-AACCACCACCACCGTGTTCACCTTGATGCCGTTCT-3’。

In eGFP-LIC-F and eGFP-LIC-R, underscore is labeled as joint sequence.

Through being sequenced, which is named as with connector sequence by pcr amplification product as shown in the sequence 5 of sequence table The eGFP section of column.

3, carrier pCa- γ bLIC is taken to obtain linearization plasmid with restriction enzyme A paI digestion.

4, reaction system first (composition of reaction system first is shown in Table 2) is prepared, then 22-25 DEG C of incubation 30min, then 75 DEG C It is incubated for 15min, then rapid ice bath is cooling.The whole system for completing above-mentioned steps is named as product first.

Table 2

The linearization plasmid that step 3 obtains	200ng
		100mM dTTP	0.5μL
100×BSA	0.1μL
		10×T4DNA polymerase Buffer	1μL
T4DNA polymerase(NEB)	0.2μL
		ddH₂O	Supply 10 μ L

5, reaction system second (composition of reaction system second is shown in Table 3) is prepared, then 22-25 DEG C of incubation 30min, then 75 DEG C It is incubated for 15min, rapid ice bath is cooling.The whole system for completing above-mentioned steps is named as product second.

Table 3

In table 3, the section with connector refers to the HaHSP4 section or step 2 with joint sequence prepared by step 1 The eGFP section with joint sequence of preparation.When section with connector is the HaHSP4 section with joint sequence, obtain Product be product second-I.When section with connector is the eGFP section with joint sequence, obtained product is product second- Ⅱ。

6,2 μ L product first and 10 μ L product second-I are mixed, 66 DEG C of incubation 2min, then the cooling 2min of room temperature, obtains Connection product is interference carrier BSMV:HaHSP4 (also known as recombinant plasmid BSMV:HaHSP4).Through being sequenced, to recombinant plasmid BSMV:HaHSP4 carries out structure and is described as follows: inserting the sequence 2 of sequence table in the ApaI restriction enzyme site of carrier pCa- γ bLIC The DNA molecular shown in the nucleotide of 5 ' end 296-595.

7,2 μ L product first and 10 μ L product second-II are mixed, 66 DEG C of incubation 2min, then the cooling 2min of room temperature, obtains Connection product is interference carrier BSMV:eGFP (also known as recombinant plasmid BSMV:eGFP).Through being sequenced, to recombinant plasmid BSMV: EGFP carries out structure and is described as follows: last from 5 ' in the sequence 4 that the ApaI restriction enzyme site of carrier pCa- γ bLIC inserts sequence table Hold DNA molecular shown in 178-495 nucleotide.

Two, tobacco is transiently transfected

1, pCaBS- α is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, then suspended with buffer suspension liquid, Obtain OD_600nm=0.7 bacteria suspension.

2, pCaBS- β is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, then suspended with buffer suspension liquid, Obtain OD_600nm=0.7 bacteria suspension.

3, pCa- γ bLIC is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, it is then outstanding with buffer suspension liquid It is floating, obtain OD_600nm=0.7 bacteria suspension.

4, interference carrier BSMV:HaHSP4 is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, then with suspension Buffer suspends, and obtains OD_600nm=0.7 bacteria suspension.

5, interference carrier BSMV:eGFP is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, it is then slow with suspending Fliud flushing suspends, and obtains OD_600nm=0.7 bacteria suspension.

6, the bacteria suspension that bacteria suspension that step 1 obtains, step 2 obtain and the bacteria suspension that step 3 obtains are mixed in equal volume, 28 DEG C of standing 3h, obtain inoculation liquid γ.

7, the bacteria suspension that bacteria suspension that step 1 obtains, step 2 obtain and the bacteria suspension that step 4 obtains are mixed in equal volume, 28 DEG C of standing 3h, obtain inoculation liquid HaHSP4.

8, the bacteria suspension that bacteria suspension that step 1 obtains, step 2 obtain and the bacteria suspension that step 5 obtains are mixed in equal volume, 28 DEG C of standing 3h, obtain inoculation liquid eGFP.

9, (every using inoculation liquid γ, inoculation liquid HaHSP4 and inoculation liquid eGFP as following steps are carried out for examination bacterium solution respectively Kind bacterium solution operates 25 plants of plant):

(1) this life cigarette plant (vaccination ways: every 3 leaf inoculations 1 of 8-12 leaf phase will be grown to for examination bacterium solution inoculation Piece leaf carries out injection inoculation, and finger pressing is positive in this life Tobacco Leaves when injection, then from corresponding vacuum side of blade position Set and injected, bacterium solution is made to infiltrate entire blade in injection process as far as possible), normal culture 3 days.

(2) the 1st blade and the 2nd blade for taking the blade and the blade being vaccinated or more, according to 1g fresh weight blade ratio Phosphate buffer (20mM, pH7.0) is added in the ratio of 3mL phosphate buffer, is ground.

(3) it takes and grows to short anti-58 wheats of two leaf stage (every plant of plant plantation is equipped with the container of culture substrate, training in one What feeding matrix i.e. 1 parts by volume Nutrition Soil and 1 parts by volume vermiculite were mixed to get), diatomite is uniformly first sprayed on wheat leaf blade, so Latter hand holds wheat culm and blade intersection with fixation, and another hand dips the abrasive material that step (2) obtains, from bottom to top Gently smooth out with the fingers wheat leaf blade (inoculation dynamics can rub to make a sound with finger and wheat leaf blade to be advisable), wheat is then placed in 22 DEG C, 16h light/8h it is dark under conditions of normally cultivate 9 days.

(4) complete step (3) after, in the culture substrate in each container be inoculated with 300 cereal cyst nematodes, then after Continue and is normally cultivated under conditions of 22 DEG C, 16h light/8h dark.

(5) it after being co-cultured 7 days in step (4), is detected as follows: taking the entire root of wheat plant, extract total serum IgE simultaneously Reverse transcription is cDNA.Using cDNA as template, use the expression quantity of fluorescence quantitative PCR detection HaHSP4 gene (with GAPDH-1 gene For internal reference).Every kind of bacterium solution processing takes 5 plants of plant.

Primer for detecting HaHSP4 gene is as follows:

Q-HIG-HaHSP4-F:5 '-GCCAAGGCAGACACCCA-3 '；

Q-HIG-HaHSP4-R:5 '-TCGTGGTCCGGTCCAAC-3 '.

The nucleotide sequence such as 6 institute of sequence in sequence table of the amplification section of Q-HIG-HaHSP4-F and Q-HIG-HaHSP4-R Show.

Primer for detecting GAPDH-1 gene is as follows:

GAPDH-1-F (upstream primer): 5 '-AGCGGCACAGAACATCATCC-3 '；

GAPDH-1-R (downstream primer): 5 '-GGTCCTCCGTGTAGCCCAAA-3 '.

Using for the expression quantity of HaHSP4 gene as 1, use is various in cereal cyst nematode when being inoculation liquid γ for examination bacterium solution The result of the relative expression quantity of HaHSP4 gene is shown in Fig. 3 in cereal cyst nematode when for examination bacterium solution.With use inoculation liquid γ conduct It is compared for examination bacterium solution, using inoculation liquid HaHSP4 as the relative expression quantity for HaHSP4 gene in examination bacterium solution cereal cyst nematode It is remarkably decreased.Compared with being used as using inoculation liquid γ for examination bacterium solution, it is used as using inoculation liquid eGFP for trying bacterium solution cereal sporangiocyst line The relative expression quantity of HaHSP4 gene is without significant difference in worm.The result shows that can be successfully by nematode using inoculation liquid HaHSP4 The expression quantity of internal HaHSP4 gene reduces.

(6) it after being co-cultured 7 days in step (4), is detected as follows: taking the entire root of plant, every plant of statistical average plant The quantity of cereal cyst nematode in strain root.Every kind of bacterium solution processing takes 10 plants of plant.

The quantity statistics result of cereal cyst nematode is shown in Fig. 4 in average every plant of plant root.It is supplied with being used as using inoculation liquid γ Examination bacterium solution is compared, and is remarkably decreased using inoculation liquid HaHSP4 as the quantity for cereal cyst nematode in examination bacterium solution plant.With adopt Inoculation liquid γ is used to compare as examination bacterium solution, using inoculation liquid eGFP as the quantity for cereal cyst nematode in examination bacterium solution plant Without significant difference.

(7) it after being co-cultured 50 days in step (4), is detected as follows: taking the entire root of plant, sufficiently rinsed with water, Count all cereal cyst nematode sporangiocysts in flushing liquor；Collect the plant all culture substrate in a reservoir, count it In all cereal cyst nematode sporangiocysts；Two sporangiocyst quantity are added, obtained total quantity is that the sporangiocyst of the plant forms number Amount.Every kind of bacterium solution processing takes 10 plants of plant.

The sporangiocyst forming quantity statistical result of average every plant of plant is shown in Fig. 5.It is used as with using inoculation liquid γ for trying bacterium solution phase Than being remarkably decreased using inoculation liquid HaHSP4 as the sporangiocyst forming quantity for trying bacterium solution plant.With use inoculation liquid γ conduct It is compared for examination bacterium solution, using inoculation liquid eGFP as the sporangiocyst forming quantity for trying bacterium solution strain without significant difference.

The result shows that interfering to the HaHSP4 gene in cereal cyst nematode and cereal cyst nematode can be inhibited to small Wheat is infected.

Embodiment 5, HaHSP4 albumen can inhibit the generation of active oxygen in plant

Using luminol-HRP-based chemiluminescence testing inspection HaHSP4 albumen to flg22 polypeptide The influence of the active oxidative burst of mediation.

The nucleotide sequence (annular) of p3301 carrier is shown in sequence 9 in sequence table.

1, by the restriction enzyme of the insertion p3301 carrier of DNA fragmentation shown in the sequence of sequence table 2 67-975 The recognition site of BamHI obtains recombinant plasmid p3301-HaHSP4.

2, recombinant plasmid p3301-HaHSP4 is imported into agrobacterium strains GV3101, obtains recombinational agrobacterium.

3, the recombinational agrobacterium obtained using tobacco buffer resuspending step 2, obtains OD_600nm=0.4 bacterium solution, it is dark quiet Set 3h.

Tobacco buffer (pH5.2): MgCl containing 10mM₂, 10mM MES, 0.2mM acetosyringone, surplus is water.

4,3 main this life cigarette plant for sprouting 4-5 weeks are taken, 3 blades of every plant of plant pair are injected (non-plant top Blade；It is subject to the full blade of injection), after 36h, this blade of injection is taken, is punched, obtains the circle that 12 diameters are 40mm altogether Piece, as test group disk.

5, p3301 carrier is replaced into recombinant plasmid p3301-HaHSP4, successively carries out step 2,3 and 4, obtains 12 circles Piece, as a control group disk.

6, test group disk and control group disk proceed as follows respectively:

96 orifice plates are taken, a disk is placed in every hole, and every hole is added 100 μ L aqua sterilisas, is stored at room temperature 12h, then reject hole Interior liquid；Then 100 μ L elicitor master mix are added (containing 100 μM of luminols, 20 μ g/mL horseradish peroxidating in every hole Object enzyme and 100nM flg22 polypeptide, surplus are water)；Then 96 orifice plates are put into microplate reader (infinite 200) monitoring 40min The variation of interior ROS, directly output result are RLU (relative light units).

As a result see that Fig. 6: HaHSP4 albumen can significantly inhibit the outburst of ROS in tobacco body.The result shows that HaHSP4 egg The immune response of white ginseng and plant.

Embodiment 6, external interference HaHSP4 influence cereal cyst nematode to the taxis of wheat root

1 × M9Buffer: by Na₂HPO₄·12H₂O1.5g、KH₂PO₄0.3g and NaCl 0.5g is dissolved in DEPC processing water, Then it is settled to 100mL with DEPC processing water, finally adjusts pH value to 7.0.1 × M9Buffer dilute 4 times obtain 0.25 × M9Buffer。

1, double stranded rna molecule (being named as dsRNA-HaHSP4) and sequence table shown in sequence 7 in artificial synthesized sequence table Double stranded rna molecule (being named as dsRNA-GFP) shown in middle sequence 8.

2, the cereal cyst nematode second instar larvae for collecting fresh hatching, is cleaned three times with DEPC water, with 0.25 × M9Buffer is cleaned three times, spare.

3, after completing step 2,1mL solution 1 to be measured, solution to be measured 2 or solution to be measured 3 are taken, about 8000 cereal spores are added Capsule nematode second instar larvae, under dark surrounds, 36h are impregnated in 16 DEG C of slight concussions.

Solution 1 to be measured is dsRNA-HaHSP4 containing 2mg/mL, 3M spermidine, 50mM octopamine and 0.05% (m/v) gelatin 0.25 × M9Buffer.

Solution 2 to be measured is dsRNA-GFP containing 2mg/mL, 3M spermidine, 50mM octopamine and 0.05% (m/v) gelatin 0.25×M9Buffer。

Solution 3:0.25 × M9Buffer to be measured.

4, it after completing step 3, extracts the total serum IgE of cereal cyst nematode and reverse transcription is cDNA.Using cDNA as template, use The expression quantity of fluorescence quantitative PCR detection HaHSP4 gene (using GAPDH-1 gene as internal reference).

Primer for detecting HaHSP4 gene is as follows:

Q-HIG-HaHSP4-F:5 '-GCCAAGGCAGACACCCA-3 '；

Q-HIG-HaHSP4-R:5 '-TCGTGGTCCGGTCCAAC-3 '.

Primer for detecting GAPDH-1 gene is as follows:

GAPDH-1-F (upstream primer): 5 '-AGCGGCACAGAACATCATCC-3 '；

GAPDH-1-R (downstream primer): 5 '-GGTCCTCCGTGTAGCCCAAA-3 '.

Using the expression quantity of HaHSP4 gene in the cereal cyst nematode of feeding solution 3 to be measured as 1, feeding solution 1 to be measured The relative expression of HaHSP4 gene in the cereal cyst nematode of (i.e. dsRNA-HaHSP4) and solution to be measured 2 (i.e. dsRNA-GFP) Amount is shown in Fig. 7.The result shows that in the cereal cyst nematode of feeding solution 1 (i.e. dsRNA-HaHSP4) to be measured HaHSP4 gene table Be remarkably decreased up to amount, and in the cereal cyst nematode of feeding solution 2 (i.e. dsRNA-GFP) to be measured HaHSP4 gene expression quantity without It significantly affects.

5, after completing step 4, the cereal sporangiocyst line of the cereal cyst nematode of feeding solution 1 to be measured, feeding solution 2 to be measured 23%F127 glue is added in the cereal cyst nematode of worm or feeding solution 3 to be measured, mixes, and is subsequently placed in culture dish center.

6, after completing step 5, the culture dish is taken, places the Helminthosporium sativum part of growth 5d (about in culture dish center 1cm), it after 48h, observes the distribution of nematode around Helminthosporium sativum and counts the quantity of nematode within the scope of Helminthosporium sativum 1.5cm.

The distribution situation of nematode is shown in Fig. 8 around Helminthosporium sativum.The statistics knot of the quantity of nematode within the scope of Helminthosporium sativum 1.5cm Fruit sees Fig. 9.The result shows that distribution of the cereal cyst nematode of feeding solution 1 (i.e. dsRNA-HaHSP4) to be measured around the tip of a root It is substantially reduced, illustrates that HaHSP4 albumen is interfered to influence cereal cyst nematode to the taxis of wheat root in vitro.

<110>China Agricultural University

<120>cereal cyst nematode HaHSP4 albumen and its encoding gene and application

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 978

<212> DNA

<213> Heterodera avenae

<400> 1

atgcatgtgc aaactgtttt cctccacctg ttcgttccgt tgtttgttgc cagcattgtg 60

tgggctgaca ttttaaaaga tgaaccacca cgtgacacca agaacatgcc aataacgcgc 120

actcgtcgtt cactgtacaa agcgatgatg gaagacatcg gcatcaccaa ggaggccacc 180

cgctttgcat gccgggtgtt gcgcacgaaa ttctgcacca aatttgggac gaccttaggc 240

atggagcgaa cccgacgcga caccaaagcc gacacaaaca ccaaagctga caccaagatg 300

gagaccaaag cggacacaaa gatggacacg aaggcggaca cgaggaccga catggtggca 360

gacaccaagg ctgctgctac caagatggac accaaggcgg acacaaagac agacatggtc 420

tttgacacca aggctgctac caagatggac accagagctg aagccaaggc agacacccaa 480

atggacacca aggcggacac caacaatgta caagcaatga tggacagcat tgtgccatcc 540

aatgttgcca cactgttgga ccggaccacg aaggagttgc acgaagcaac gcaaagacaa 600

ggcttccacg agctgagcca ggcggaacag gaacgcgtga tgctcgaggc agccggcaaa 660

cagggcgaca tcgtcaaaac caagctgaag gaagccattg aggaggcccg aaaaatggtg 720

gagcgcgcaa ttggcacaat ggtttcctac tcggaaatgc cgattgagtc caaactggcc 780

atggagcggg tgttcatgac actgacggac gtgacgaaga cgcccacgca aaaggtggag 840

cgtatcaggg aaattgagcg tgactggacg ccggagattc gcgaggcatt gaacccgtgc 900

acggaggaca agcagattct ggaggcagcc cgctactaca ccgatacggc ccaacaaatt 960

gggctggcca acaactga 978

<210> 2

<211> 325

<212> PRT

<213> Heterodera avenae

<400> 2

Met His Val Gln Thr Val Phe Leu His Leu Phe Val Pro Leu Phe Val

1 5 10 15

Ala Ser Ile Val Trp Ala Asp Ile Leu Lys Asp Glu Pro Pro Arg Asp

20 25 30

Thr Lys Asn Met Pro Ile Thr Arg Thr Arg Arg Ser Leu Tyr Lys Ala

35 40 45

Met Met Glu Asp Ile Gly Ile Thr Lys Glu Ala Thr Arg Phe Ala Cys

50 55 60

Arg Val Leu Arg Thr Lys Phe Cys Thr Lys Phe Gly Thr Thr Leu Gly

65 70 75 80

Met Glu Arg Thr Arg Arg Asp Thr Lys Ala Asp Thr Asn Thr Lys Ala

85 90 95

Asp Thr Lys Met Glu Thr Lys Ala Asp Thr Lys Met Asp Thr Lys Ala

100 105 110

Asp Thr Arg Thr Asp Met Val Ala Asp Thr Lys Ala Ala Ala Thr Lys

115 120 125

Met Asp Thr Lys Ala Asp Thr Lys Thr Asp Met Val Phe Asp Thr Lys

130 135 140

Ala Ala Thr Lys Met Asp Thr Arg Ala Glu Ala Lys Ala Asp Thr Gln

145 150 155 160

Met Asp Thr Lys Ala Asp Thr Asn Asn Val Gln Ala Met Met Asp Ser

165 170 175

Ile Val Pro Ser Asn Val Ala Thr Leu Leu Asp Arg Thr Thr Lys Glu

180 185 190

Leu His Glu Ala Thr Gln Arg Gln Gly Phe His Glu Leu Ser Gln Ala

195 200 205

Glu Gln Glu Arg Val Met Leu Glu Ala Ala Gly Lys Gln Gly Asp Ile

210 215 220

Val Lys Thr Lys Leu Lys Glu Ala Ile Glu Glu Ala Arg Lys Met Val

225 230 235 240

Glu Arg Ala Ile Gly Thr Met Val Ser Tyr Ser Glu Met Pro Ile Glu

245 250 255

Ser Lys Leu Ala Met Glu Arg Val Phe Met Thr Leu Thr Asp Val Thr

260 265 270

Lys Thr Pro Thr Gln Lys Val Glu Arg Ile Arg Glu Ile Glu Arg Asp

275 280 285

Trp Thr Pro Glu Ile Arg Glu Ala Leu Asn Pro Cys Thr Glu Asp Lys

290 295 300

Gln Ile Leu Glu Ala Ala Arg Tyr Tyr Thr Asp Thr Ala Gln Gln Ile

305 310 315 320

Gly Leu Ala Asn Asn

325

<210> 3

<211> 327

<212> DNA

<213> Artificial sequence

<400> 3

aaggaagttt aaagatggag accaaagcgg acacaaagat ggacacgaag gcggacacga 60

ggaccgacat ggtggcagac accaaggctg ctgctaccaa gatggacacc aaggcggaca 120

caaagacaga catggtcttt gacaccaagg ctgctaccaa gatggacacc agagctgaag 180

ccaaggcaga cacccaaatg gacaccaagg cggacaccaa caatgtacaa gcaatgatgg 240

acagcattgt gccatccaat gttgccacac tgttggaccg gaccacgaag gagttgcacg 300

aagcaacgca aaacggtggt ggtggtt 327

<210> 4

<211> 720

<212> DNA

<213> Artificial sequence

<400> 4

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> 5

<211> 345

<212> DNA

<213> Artificial sequence

<400> 5

aaggaagttt aaaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 60

cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 120

gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 180

gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 240

aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 300

gacaagcaga agaacggcat caaggtgaac acggtggtgg tggtt 345

<210> 6

<211> 109

<212> DNA

<213> Artificial sequence

<400> 6

gccaaggcag acacccaaat ggacaccaag gcggacacca acaatgtaca agcaatgatg 60

gacagcattg tgccatccaa tgttgccaca ctgttggacc ggaccacga 109

<210> 7

<211> 300

<212> RNA

<213> Artificial sequence

<400> 7

agauggagac caaagcggac acaaagaugg acacgaaggc ggacacgagg accgacaugg 60

uggcagacac caaggcugcu gcuaccaaga uggacaccaa ggcggacaca aagacagaca 120

uggucuuuga caccaaggcu gcuaccaaga uggacaccag agcugaagcc aaggcagaca 180

cccaaaugga caccaaggcg gacaccaaca auguacaagc aaugauggac agcauugugc 240

cauccaaugu ugccacacug uuggaccgga ccacgaagga guugcacgaa gcaacgcaaa 300

<210> 8

<211> 318

<212> RNA

<213> Artificial sequence

<400> 8

acccucguga ccacccugac cuacggcgug cagugcuuca gccgcuaccc cgaccacaug 60

aagcagcacg acuucuucaa guccgccaug cccgaaggcu acguccagga gcgcaccauc 120

uucuucaagg acgacggcaa cuacaagacc cgcgccgagg ugaaguucga gggcgacacc 180

cuggugaacc gcaucgagcu gaagggcauc gacuucaagg aggacggcaa cauccugggg 240

cacaagcugg aguacaacua caacagccac aacgucuaua ucauggccga caagcagaag 300

aacggcauca aggugaac 318

<210> 9

<211> 10872

<212> DNA

<213> Artificial sequence

<400> 9

aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt 60

agaataacgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc 120

atgccaacca cagggttccc ctcgggatca aagtactttg atccaacccc tccgctgcta 180

tagtgcagtc ggcttctgac gttcagtgca gccgtcttct gaaaacgaca tgtcgcacaa 240

gtcctaagtt acgcgacagg ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg 300

ttttagtcgc ataaagtaga atacttgcga ctagaaccgg agacattacg ccatgaacaa 360

gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc accgacgacc aggacttgac 420

caaccaacgg gccgaactgc acgcggccgg ctgcaccaag ctgttttccg agaagatcac 480

cggcaccagg cgcgaccgcc cggagctggc caggatgctt gaccacctac gccctggcga 540

cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc tactggacat 600

tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga 660

caccaccacg ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga 720

gcgttcccta atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt 780

gaagtttggc ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat 840

cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac 900

cctgtaccgc gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg 960

tgccttccgt gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg 1020

ccaagaggaa caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc 1080

gaagagatcg aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc 1140

tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg 1200

ccggccagct tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt 1260

gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa 1320

atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca 1380

agacgaccat cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt 1440

tagtcgattc cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac 1500

cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc 1560

ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga 1620

tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca 1680

ccgccgacct ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag 1740

cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg 1800

cgctggccgg gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc 1860

caggcactgc cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc 1920

gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa 1980

agagaaaatg agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag 2040

caaggctgca acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca 2100

gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat 2160

taccgagctg ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa 2220

tgagtagatg aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca 2280

ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct 2340

gggttgtctg ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgac 2400

ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag 2460

aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt 2520

gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc 2580

ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg 2640

atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt 2700

ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac 2760

gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg 2820

atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag 2880

cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat 2940

ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt 3000

gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa 3060

gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag 3120

atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg 3180

acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg 3240

gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc 3300

agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca 3360

aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc 3420

atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag 3480

atgctagggc aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat 3540

agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac 3600

ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa 3660

ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc 3720

tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg 3780

tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca 3840

aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca 3900

ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga 3960

aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 4020

gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 4080

gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag 4140

attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 4200

taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 4260

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 4320

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 4380

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 4440

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4500

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4560

tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 4620

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4680

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4740

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4800

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4860

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4920

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4980

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5040

cgttaaggga ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa 5100

tattttattt tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac 5160

tgttcttccc cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg 5220

tccgccctgc cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag 5280

atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc 5340

tttaaaaaat catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg 5400

caatccacat cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct 5460

aagctattcg tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc 5520

gcatacagct cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg 5580

acgccatcgg cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg 5640

acctttggaa caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca 5700

tcataggtgg tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct 5760

cccaccagct tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat 5820

ttttcgatca gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt 5880

cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca 5940

attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt 6000

tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac 6060

aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg 6120

tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt 6180

ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc 6240

gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat 6300

atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt 6360

aatgtactga attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg 6420

ttttaggaat tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg 6480

gtttcttata tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg 6540

gaactactca cacattatta tggagaaact cgagtcaaat ctcggtgacg ggcaggaccg 6600

gacggggcgg taccggcagg ctgaagtcca gctgccagaa acccacgtca tgccagttcc 6660

cgtgcttgaa gccggccgcc cgcagcatgc cgcggggggc atatccgagc gcctcgtgca 6720

tgcgcacgct cgggtcgttg ggcagcccga tgacagcgac cacgctcttg aagccctgtg 6780

cctccaggga cttcagcagg tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc 6840

ggggggagac gtacacggtc gactcggccg tccagtcgta ggcgttgcgt gccttccagg 6900

ggcccgcgta ggcgatgccg gcgacctcgc cgtccacctc ggcgacgagc cagggatagc 6960

gctcccgcag acggacgagg tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga 7020

agttgaccgt gcttgtctcg atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg 7080

cctcggtggc acggcggatg tcggccgggc gtcgttctgg gctcatggta gactcgagag 7140

agatagattt gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact 7200

tccttatata gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca 7260

gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc 7320

acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga 7380

acgatagcct ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact 7440

gtccttttga tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt 7500

accctttgtt gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc 7560

ttggagtaga cgagagtgtc gtgctccacc atgttcacat caatccactt gctttgaaga 7620

cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg 7680

accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt 7740

gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg 7800

gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc 7860

ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt 7920

ggcaagctgc tctagccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7980

tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 8040

gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 8100

ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga caatgattac 8160

gaattcgagc tcggtaccga caaaatttag aacgaactta attatgatct caaatacatt 8220

gatacatatc tcatctagat ctaggttatc attatgtaag aaagttttga cgaatatggc 8280

acgacaaaat ggctagactc gatgtaattg gtatctcaac tcaacattat acttatacca 8340

aacattagtt agacaaaatt taaacaacta ttttttatgt atgcaagagt cagcatatgt 8400

ataattgatt cagaatcgtt ttgacgagtt cggatgtagt agtagccatt atttaatgta 8460

catactaatc gtgaatagtg aatatgatga aacattgtat cttattgtat aaatatccat 8520

aaacacatca tgaaagacac tttctttcac ggtctgaatt aattatgata caattctaat 8580

agaaaacgaa ttaaattacg ttgaattgta tgaaatctaa ttgaacaagc caaccacgac 8640

gacgactaac gttgcctgga ttgactcggt ttaagttaac cactaaaaaa acggagctgt 8700

catgtaacac gcggatcgag caggtcacag tcatgaagcc atcaaagcaa aagaactaat 8760

ccaagggctg agatgattaa ttagtttaaa aattagttaa cacgagggaa aaggctgtct 8820

gacagccagg tcacgttatc tttacctgtg gtcgaaatga ttcgtgtctg tcgattttaa 8880

ttattttttt gaaaggccga aaataaagtt gtaagagata aacccgccta tataaattca 8940

tatattttcc tctccgcttt gaattgtctc gttgtcctcc tcactttcat cagccgtttt 9000

gaatctccgg cgacttgaca gagaagaaca aggaagaaga ctaagagaga aagtaagaga 9060

taatccagga gattcattct ccgttttgaa tcttcctcaa tctcatcttc ttccgctctt 9120

tctttccaag gtaataggaa ctttctggat ctactttatt tgctggatct cgatcttgtt 9180

ttctcaattt ccttgagatc tggaattcgt ttaatttgga tctgtgaacc tccactaaat 9240

cttttggttt tactagaatc gatctaagtt gaccgatcag ttagctcgat tatagctacc 9300

agaatttggc ttgaccttga tggagagatc catgttcatg ttacctggga aatgatttgt 9360

atatgtgaat tgaaatctga actgttgaag ttagattgaa tctgaacact gtcaatgtta 9420

gattgaatct gaacactgtt taaggttaga tgaagtttgt gtatagattc ttcgaaactt 9480

taggatttgt agtgtcgtac gttgaacaga aagctatttc tgattcaatc agggtttatt 9540

tgactgtatt gaactctttt tgtgtgtttg cagctcataa aaaatggctg aggctgatga 9600

tattcaacca atcgtgtgtg acctcgagac tagtatggac tacaaggacg acgatgacaa 9660

ggactacaag gacgacgatg acaagacgcg tcccggggga cctccacctg gtatggtgag 9720

caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 9780

aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 9840

gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 9900

caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 9960

cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 10020

cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 10080

catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 10140

gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 10200

ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 10260

ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 10320

cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 10380

gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggaccagc 10440

tccagctcca ggatccaagc tttctccata ataatgtgtg agtagttccc agataaggga 10500

attagggttc ctatagggtt tcgctcatgt gttgagcata taagaaaccc ttagtatgta 10560

tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc aaaatccagt 10620

actaaaatcc agatccccgt cgacctgcag gcatgtcaag cttggcactg gccgtcgttt 10680

tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 10740

cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 10800

tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt gtcgtttccc 10860

gccttcagtt ta 10872

Claims

1. protein HaHSP4 is following (a1) or (a2) or (a3) or (a4) or (a5) or (a6) or (a7):

(a1) amino acid sequence is protein shown in sequence 1 in sequence table；

(a2) contain the fused protein of (a1)；

(a4) by (a1) by one or several amino acid residues substitution and/or deletion and/or addition and with cyst roundworm It host's taxis and/or parasitism and/or causes a disease and/or develops the relevant protein as derived from (a1)；

(a5) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into inhibition plant The protein as derived from (a1) for the function that activity in vivo oxygen generates；

(a6) from cereal cyst nematode and with (a1) have 98% or more homology and with host's taxis of cereal cyst nematode And/or the parasitic and/or pathogenic and/or relevant protein as derived from (a1) of development；

(a7) with 98% or more homology and there is active oxygen in inhibition plant to produce from cereal cyst nematode and with (a1) The protein as derived from (a1) of raw function.

2. encoding the nucleic acid molecules of protein HaHSP4 described in claim 1.

3. nucleic acid molecules as claimed in claim 2, it is characterised in that: the nucleic acid molecules are following (b1) or (b2) or (b3) Or (b4):

(b1) code area DNA molecular as shown in sequence 2 in sequence table；

(b3) hybridize under strict conditions with (b1) or (b2) described DNA molecular and encode protein described in claim 1 The DNA molecular of HaHSP4；

(b4) there is 90% or more homology and coding power from cereal cyst nematode and with (b1) or (b2) DNA molecular Benefit requires the DNA molecular of the 1 protein HaHSP4.

4. recombinant expression carrier, expression cassette, Transgenic plant tissue or recombination containing nucleic acid molecules described in Claims 2 or 3 Microorganism.

5. the application of protein HaHSP4 described in claim 1 is at least one of following (c1) to (c6):

(c1) regulate and control host's taxis of cyst roundworm；

(c2) regulate and control the parasitic ability of cyst roundworm；

(c3) regulate and control the pathogenecity of cyst roundworm；

(c4) regulate and control the development of cyst roundworm；

(c5) regulate and control active oxygen in plant to generate；

(c6) active oxygen in plant is inhibited to generate.

6. a kind of method for cultivating genetically modified plants is included in the plant that sets out and imports protein described in inhibition claim 1 The substance of HaHSP4 expression, the step of obtaining genetically modified plants；Compared with the plant that sets out, the genetically modified plants are to sporangiocyst The resistance of nematode improves.

7. a kind of method for reducing cyst roundworm host taxis, to make protein described in claim 1 in cyst roundworm body The expression quantity and/or activity of HaHSP4 reduces.

8. inhibiting the interference carrier or recombinant virus of the expression of nucleic acid molecules described in Claims 2 or 3.

9. for inhibiting, protein HaHSP4 described in claim 1 is expressed and/or active substance is preparing the application in product.

10. special RNA；The special RNA is double-stranded RNA shown in the sequence 7 in sequence table.