CN106939311B - Cereal cyst nematode Ha-56573 gene and its application - Google Patents
Cereal cyst nematode Ha-56573 gene and its application Download PDFInfo
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Abstract
The present invention relates to cereal cyst nematode Ha-56573 gene and its applications.The Ha-56573 gene of cereal cyst nematode, sequence is as shown in SEQ ID NO:1.Ha-56573 gene provided by the invention is lethal phenotype gene.DsRNA processing is measured by the method interfered in vitro to have an impact to nematode activity, infection ability and the completion history of life, demonstrates the function of gene Ha-56573.Present invention demonstrates that the target gene Ha-56573 with lethal effect, in Ha-56573-dsRNA1, after Ha-56573-dsRNA2 and Ha-56573-dsRNA3 processing, female adult amount declines 38.5% respectively compared with the control, 65.81%, 53.8%, show that Ha-56573 can be used as target gene for nematode prevention and control.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of Ha-56573 gene from cereal cyst nematode and its answers
With.
Background technique
Cereal cyst nematode (Cereal cyst nematodes, abbreviation CCNs), which has Wheat Production, to be seriously threatened,
38 national occurrence injuries in world wide, China since 1991 for the first time since Hubei is found, less than 30 years when
In, it include Henan, Hebei, Tibet, the province ,city and area's discovery of 16, Xinjiang etc. in China.Wheat of the CCN in China 80%
There is generation in producing region, covers China Major Wheat producing region, endangers most serious especially with Yellow River-Huai River region, and hazard area is up to more than 6,000 ten thousand
Mu, annual output loss reach 73%-89% in 23%-50% or more, the serious plot underproduction, or even ruin kind of a total crop failure, and have constantly
Build up trend [Peng D L, Nicol J M, Li H, Hou S, Li H, Chen S, Ma P, Li H, and Riley I
T.Current knowledge of cereal cyst nematode(Heterodera avenae)on wheat in
China.In'Cereal cyst nematodes:status,research and outlook.'Editered by IT
Riley,JM Nicol and AA Dababat.2009:29-34；Peng Deliang, Subbotin S, Moens M. Singularity SCAR mark of wheat spore
Ribosomal gene (rDNA) restriction fragment length polymorphism research plant pathology of capsule nematode (Heterodera avenae)
Journal .2003,33:323-329；Distribution of Zhao Honghai, Yang Yuanyong, Peng Deliang, Liu Feng the wheat cearal cyst nematode in Shandong Province
New report and occurrence characteristic simply analyse Qingdao Agricultural University journal .2011,28:17-22].
RNAi (RNA interference) refers to endogenous or external source double-stranded RNA (double-strand RNA, dsRNA)
The phenomenon that specifically leading to its homologous gene silencing after importing cell.RNAi is in mode nematode Caenorthaditis elegans ws123 first
(Caenorhabditis elegans) discovery (Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE,
Mello CC.Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans.Nature.1998,391:806-811).RNAi points are in vivo interference (in vivo
RNAi) and in vitro (in vitro RNAi) is interfered.Currently, in vitro RNAi is main in the application of anchorage endoparasitism nematode
It is directed to second instar larvae, and second instar larvae is mainly in transition state in vivo, has no trophic behaviour, but in neural stimulator
Under the action of (mainly including octopamine, resorcinol and hydroxytryptamine etc.), the lancet of nematode can be caused frequently to twitch, favorably
In nematode to feeding (Bakhetia M, Charlton WL, Urwin PE, the McPherson MJ, Atkinson of allogenic material
HJ.RNA interference and plant parasitic nematodes.Trends in Plant
Science.2005,10:362-367).Urwin etc. (2002) has used octopamine as stimulation inducer, and first passage impregnates
DsRNA is directed into soy bean cyst roundworm H.glycines and G.pallida Globodera pallida second instar larvae by method
In vivo, and silencing effect has been caused, several nematode target genes of successful silencing.This study demonstrate plant nematode with
Mode Caenorthaditis elegans ws123 RNAi effect having the same, while also showing RNAi and can be used for plant nematode gene function
Research (Urwin PE, Lilley CJ, the Atkinson HJ.Ingestion of double-stranded RNA by of energy
preparasitic juvenile cyst nematodes leads to RNA interference.Molecular
Plant-Microbe Interactions.2002,15:747-752).Chen etc. (2005) impregnates thorn by further improving
Swash system, is knocked out using the protein gene Gr-ams-1 that globodera rostochiensis G.rostorchiensis side device is secreted as target
Silencing results in G.rostorchiensis second instar larvae and declines (Chen Q, Rehman S, Smant to the stationkeeping ability of host
G,Jones JT.Functional analysis of pathogenicity proteins of the potato cyst
nematode Globodera rostochiensis using RNAi.Molecular Plant-Microbe
Interactions.2005,18:621-625.).It is cereal by dsRNA silencing β-Isosorbide-5-Nitrae-endoglucanase parasitism gene
Cyst roundworm larva invades the ability decline of root, it was demonstrated that β-Isosorbide-5-Nitrae-endoglucanase is during nematode is nematosis
Important albumen (Long H, Peng H, Huang W, Wang G, Gao B, Moens M, Peng D.Identification and
molecular characterization of a newβ-1,4-endoglucanase gene(Ha-eng-1a)in the
cereal cyst nematode Heterodera avenae.Eur J Plant Pathol.2012,134:391-400)。
The screening of nematode lethal gene is of great significance to nematode prevention and control.It is directed to model organism Caenorthaditis elegans ws123 at present
The research of genome and lethal gene is more comprehensive, and Alkharouf etc. is existed using development mutation and RNAi lethal phenotype
RNAi lethal gene [Alkharouf NW, Klink VP, Matthews are identified in H.glycines
BF.Identification of Heterodera glycines(soybean cyst nematode)cDNA sequences
with high identity to those of Caenorhabditis elegans having lethal mutant or
RNAi phenotypes.Exp Parasitol.2007,115:247-258].Then, in parasitic nematode Pratylenchus
Multiple RNAi lethal phenotype genes (Nicol P, Gill R, Fosu-Nyarko J, Jones MGK.De is identified in thornei
novo analysis and functional classification of the transcriptome of the root
lesion nematode,Pratylenchus thornei,after 454 GS FLX
Sequencing.International Journal for Parasitology 2012,42:225-237), it is phytotrophy
Solid foundation is established in the prevention and control and resistance breeding of nematode.
Summary of the invention
The object of the present invention is to provide a kind of Ha-56573 genes from cereal cyst nematode, with cereal cyst nematode
Growth and development it is related, the prevention and control using the gene silencing, for cereal cyst nematode.
The Ha-56573 gene of cereal cyst nematode, nucleotide sequence is as shown in SEQ ID NO:1.
For Ha-56573-dsRNA1, Ha-56573-dsRNA2 and the Ha-56573-dsRNA3 piece of said gene synthesis
Section, nucleotide sequence are respectively SEQ ID NO:2, SEQ ID NO:3, shown in SEQ ID NO:4.
The dsRNA segment of the Ha-56573 gene design of the cereal cyst nematode is in prevention and treatment cereal cyst nematode
Using.
The application, for the Ha-56573 gene of cereal cyst nematode is designed the dsRNA segment feeding synthesized to standing grain
In paddy cyst roundworm body, inhibit cereal cyst nematode development, so that prevention and control cereal cyst nematode infects host.
The present invention is by having RNAi lethal phenotype in cereal cyst nematode RNA-seq data and C.elegans genome
Gene compares, and is screened out from it homologous gene, nucleotide sequence such as SEQ ID NO:1.
Recombinant expression carrier, interference carrier, recombinant virus, dsRNA, transgenic cell containing above-mentioned Ha-56573 gene
System, genetically modified plants or tissue, recombinant bacterium all belong to the scope of protection of the present invention.The recombinant expression of gene containing Ha-56573
Carrier includes double base agrobacterium vector, viral vectors, bacterial expression vector and Yeast expression carrier etc..Contain Ha-56573 gene
Vector construction during, can it is independent or it is multiple be applied in combination induction type, enhanced, composing type, organizing specific type starting
Son.Carrier may include the resistance screening label of antibiotic or anti-chemical reagent, can also contain the enzyme for generating color change, than
Such as GUS or luminescent marking albumen, such as red or green fluorescent protein, convenient for the screening of subsequent transformation.The carrier of building
Bacterium, fungi and single dicotyledon can be converted, is specifically as follows Escherichia coli, yeast, tobacco, arabidopsis and wheat, greatly
Wheat.
The present invention is directed to Ha-56573 gene order, and screening specific region synthesizes dsRNA, is surveyed by the method interfered in vitro
DsRNA processing is determined to nematode activity, infection ability and the influence for completing the history of life, verifies the lethal function of gene.Present invention tool
There is the target gene Ha-56573 of lethal effect, after dsRNA processing, female adult amount decline 65.81%.It is anti-to can be applied to nematode
Control is specially carried using the interference of Ha-56573 gene order or genetic fragment building expression dsRNA or hairpin structure RNA
Body or recombinant virus, are used for wheat or Transgenic plants of barley, and silencing cereal cyst nematode Ha-56573 gene invades it
The decline of dye ability, cannot complete the history of life, reach nematode prevention and control purpose.
Detailed description of the invention
Fig. 1 is the synthesis of dsRNA.Wherein 1:eGFP compares dsRNA；2:Ha-56573-dsRNA1；3:Ha-56573-
dsRNA2；4:Ha-56573-dsRNA3
Fig. 2 fluorescence microscope wheat cearal cyst nematode second instar larvae imitates the feeding of fluorescein isothiocynate FITC
Fruit.
Wherein S: lancet；Pl: pharyngeal cavity；M: oesophageal bulb.
The nematode of Fig. 3 Ha-56573-dsRNA1, Ha-56573-dsRNA2 and Ha-56573-dsRNA3 processing is in inoculation 50
White female adult amount after it, wherein CK and eGFP is control
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.Applicant can also be to public affairs from granting.Quantitative test in following embodiment, is all provided with
Three repeated experiments are set, results are averaged.
The screening of embodiment 1, RNAi lethal phenotype gene
To cereal cyst nematode carry out transcript profile sequencing, with Wormbase database (http: //
Www.wormbase.org/) the lethal database of RNAi phenotype carries out homologous sequence alignment in the C.elegans collected.Identification
With C.elegans homologous (e-value of≤1.0e-40) and with the base of RNAi lethal phenotype in wheat cearal cyst nematode
Cause.Vegetable protein (Embryophyta protein is downloaded from ncbi database (http://www.ncbi.nlm.nih.gov/)
Database, NCBI txid3193), vertebrate albumen (Craniata protein database, NCBI
) and insect protein (Insect protein database, NCBI txid6960) database txid89593.According to being collected into
This protein sequence establish local nucleic acid database respectively.Singularity SCAR mark of wheat sporangiocyst line had into RNAi using Local BLAST
The lethal candidate gene of phenotype carries out tBLASTX or BLASTX analysis (e-value≤1.0e-5) respectively.Obtain Singularity SCAR mark of wheat spore
The gene lethal without the RNAi phenotype of homologous sequence with the gene of these species in capsule nematode transcript profile.5 tables are therefrom screened
Type is stablized, the candidate targets with embryonic death (EMB embryonic lethal) and larva lethal (larval lethal)
Sequence (table 1).Ha-56573 gene order is as shown in SEQ ID NO:1.Synthesis gene specific is designed for target-gene sequence to draw
Object, and held in special primer 5 ' and introduce T7 promoter sequence TAATACGACTCACTATAGGG, synthesis dsRNA template is simultaneously pure
Change.According to HiscribeTMThe specification of RNAi Transcription kit carries out, the specific steps are as follows: RNase-Free water
24 μ l, 10 × Transcription Buffer, 4 μ l, 20 × Ribonucleotide Solution Mix, 2 μ l, DNA profiling
6 μ l (the obtained PCR product of upper step, 1 μ g), 20 × HMV Mix, 2 μ l, T7 RNA polymerase 2 μ l, 40 μ l of total volume.
After each reactant is mixed, 42 DEG C of incubation reaction 4h in PCR instrument are placed in, synthesize dsRNA, dsRNA treatment process is in example 2
It is described in detail.DsRNA processing result shows that Ha-56573 gene female adult amount after dsRNA processing is remarkably decreased, other 4 genes do not have
It is remarkably decreased (table 2), therefore screens Ha-56573 gene and carry out further functional verification analysis, such as embodiment 2.
Table 1: 5 screened in wheat cearal cyst nematode transcript profile and Caenorthaditis elegans ws123 RNAi lethal phenotype are homologous
Sequence
White female adult amount of the wheat cearal cyst nematode of table 2:dsRNA processing after inoculation 50 days
Embodiment 2, external RNAi interference verifying Ha-56573 function
The synthesis of 1.dsRNA, the special primer of design amplification target fragment:
Ha-56573F1:5 '-GGAGCCATTCTTTGCTCAAG-3 '；
Ha-56573R1:5 '-TTCTGTACCGCTTCCGATTC-3 '
Ha-56573F2:5 '-ACCTCAACAGGGACAAATCG-3 '；
Ha-56573R2:5 '-TCATCTGTTCCTCCAACTCC-3 '
Ha-56573F3:5 '-GAATCGGAAGCGGTACAGAA-3 '；
Ha-56573R3:5 '-CGATTTGTCCCTGTTGAGGT-3 '
And it is held in special primer 5 ' and introduces T7 promoter sequence TAATACGACTCACTATAGGG.Synthesize dsRNA template
And it purifies for testing in next step.According to HiscribeTMThe specification of RNAi Transcription kit carries out, specific to walk
It is rapid as follows: 24 μ l, 10 × Transcription Buffer of RNase-Free water 4 μ l, 20 × Ribonucleotide
2 μ l of Solution Mix, 6 μ l of DNA profiling (the obtained PCR product of upper step, 1 μ g), 20 × HMV Mix, 2 μ l, T7 RNA
2 μ l of polymerase, 40 μ l of total volume.After each reactant is mixed, 42 DEG C of incubation reaction 4h in PCR instrument are placed in, synthesize Ha-
56573-dsRNA1 (is expanded, sequence is SEQ ID NO:2) using gene specific primer Ha-56573F1 and Ha-56573R1, or
Person Ha-56573-dsRNA2 (it is expanded using gene specific primer Ha-56573F2 and Ha-56573R2, sequence is SEQ ID NO:
3) or Ha-56573-dsRNA3 (is expanded, sequence SEQ using gene specific primer Ha-56573F3 and Ha-56573R3
ID NO:2).1 μ l product is taken to carry out electrophoresis detection (Fig. 1)
2. the wheat cearal cyst nematode second instar larvae of fresh hatching is collected, after cleaning 3 times with DEPC processing water, with 0.25
× M9buffer is cleaned 2 times and is stored in the solution, spare at any time.
3. using fluorescein isothiocynate (FITC) as tracer first to observe the effect of swallowing of second instar larvae
Matter prepares 0.25 × M9buffer solution containing FITC, and specific each ingredient and concentration include: 1mg/ml FITC；The sub- essence of 3mM
Amine；50mM octopamine；0.05% gelatin, each ingredient final concentration are adjusted using 0.25 × M9 solution.Nematode suspension is added (about
1000), until 200 μ l of final volume, 36h, Lycra fluorescence microscopy microscopic observation result (figure are impregnated in 16 DEG C of dark surrounds slight concussions
2)。
4. preparing the silencing solution containing dsRNA for disturb target gene, specific each ingredient and concentration include: to implement
The Ha-56573-dsRNA1 or Ha-56573-dsRNA2 or Ha-56573-dsRNA:3mg/ml of acquisition are expanded in example 2；
3mM spermidine；50mM octopamine；0.05% gelatin, each ingredient final concentration are adjusted using 0.25 × M9 solution.Nematode is added to suspend
Liquid (about 8000), until 300 μ l of final volume, 36h is impregnated in 16 DEG C of dark surrounds slight concussions.
5. control experiment uses the green fluorescence protein gene eGFP-dsRNA (CK1) containing non-nematode gene and does not contain
The solution (CK2) of dsRNA, other compositions and soaking conditions are identical as dsRNA processing experiment.
6. the wheat seed (warm wheat 19) of inoculation experiments surface sterilization the vernalization of dark surrounds room temperature two days later, kind is in containing
In the 50ml centrifuge tube of sterilizing sandy soil.3 centrifuge tubes of each processing, 3 wheat seedlings are connect after 6 days in each centrifuge tube
Kind experiment, separately sampled inoculation 19 wheat seedling of warm wheat, every pipe are inoculated with 300 two after 36h after soaking stimulation feeding dsRNA
Instar larvae, 16 DEG C of illumination box cultures.Experiment is divided into 3 groups, and inoculation experiments are in triplicate.
7. wheat root tissue interior lines worm is dyed
10th day wheat root after harvest inoculation, carries out root dyeing, microscopically observation line using acid fuchsin staining
Worm infect efficiency, the results showed that nematode infection rate is without significant difference.
8. counting each processing female adult quantity
After inoculation 50 days, each white female adult quantity of processing, shadow of the statistics dsRNA processing to elegans development are collected using 80 meshes
Ring, the results showed that Ha-56573-dsRNA1, Ha-56573-dsRNA2 and Ha-56573-dsRNA3 processing after, female adult amount with it is right
Photograph ratio declines 38.5%, 65.81%, 53.8% respectively, as shown in figure 3, showing that Ha-56573 can be used as target gene and be used for
Nematode prevention and control.
SEQUENCE LISTING
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>cereal cyst nematode Ha-56573 gene and its application
<130>PP17067-ZWB
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 835
<212> DNA
<213>cereal cyst nematode Ha-56573 gene
<400> 1
acgggcagct gaacgcaccc gaatttgcgg attttgagcg aattgtgcgc gcacgcgcag 60
tcgaaacgtc gaaaaaggca ctgaaagtgg tggacacgga tgggaacaac gcactgacaa 120
tggacgaggc caagaaaatc gcctttgaac attacggatt tgatgaagtg acattggagc 180
cattctttgc tcaagcggac gaaaatgagg attcccaatt ggacccggtg gaatttgccg 240
gctttcgctc tgtcatccgc gggcgggcag tcaaaaacgc cggaggtctt tttgcagaaa 300
tggacgaaga tggggacggg atgatcagcg aatcggaagc ggtacagaag acacggcagg 360
aggatgacat gagcgcggca gaaacgcatg gattatttgt ggtggccgat caggacaaaa 420
acggactgtt ggacaaagtc gaattggccg atttcattcg cctcgtgcgt ctcactgcca 480
tcaaatatgt ggccgatcac ttccgcgact acgacctcaa cagggacaaa tcgatcactt 540
tggacgaagt ggagacactg gtcaaagagc ggtacaaatt ggggccggcg gtgacccgga 600
aatactatga caaggccgac ttggaccgca gtggcgattt gaacccaggg gaagtggtgg 660
actttcggca cgaaattaga cgctatacac aggacaaagc agcacaaaag gagttggagg 720
aacagatgag acgagagaca ggtgtcaaca aaaaggagag ggacgatgag gaaaaggagg 780
aaaagggaaa agacagacat aaacagggag aaagagaagg acaaacaagg acaga 835
<210> 2
<211> 174
<212> RNA
<213>Ha-56573-dsRNA1 sequence
<400> 2
ggagccauuc uuugcucaag cggacgaaaa ugaggauucc caauuggacc cgguggaauu 60
ugccggcuuu cgcucuguca uccgcgggcg ggcagucaaa aacgccggag gucuuuuugc 120
agaaauggac gaagaugggg acgggaugau cagcgaaucg gaagcgguac agaa 174
<210> 3
<211> 216
<212> RNA
<213>Ha-56573-dsRNA2 sequence
<400> 3
accucaacag ggacaaaucg aucacuuugg acgaagugga gacacugguc aaagagcggu 60
acaaauuggg gccggcggug acccggaaau acuaugacaa ggccgacuug gaccgcagug 120
gcgauuugaa cccaggggaa gugguggacu uucggcacga aauuagacgc uauacacagg 180
acaaagcagc acaaaaggag uuggaggaac agauga 216
<210> 4
<211> 204
<212> RNA
<213>Ha-56573-dsRNA3 sequence
<400> 4
gaaucggaag cgguacagaa gacacggcag gaggaugaca ugagcgcggc agaaacgcau 60
ggauuauuug ugguggccga ucaggacaaa aacggacugu uggacaaagu cgaauuggcc 120
gauuucauuc gccucgugcg ucucacugcc aucaaauaug uggccgauca cuuccgcgac 180
uacgaccuca acagggacaa aucg 204
Claims (4)
1. the Ha-56573 gene of cereal cyst nematode, nucleotide sequence is as shown in SEQ ID NO:1.
2. the dsRNA segment of the Ha-56573 gene design synthesis of cereal cyst nematode according to claim 1, nucleosides
Acid sequence is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4.
3. the dsRNA segment as claimed in claim 2 according to the design synthesis of the Ha-56573 gene of cereal cyst nematode is preventing and treating
Application in cereal cyst nematode.
4. application according to claim 3, for the dsRNA piece for synthesizing the design of the Ha-56573 gene of cereal cyst nematode
In section feeding to cereal cyst nematode body, inhibit cereal cyst nematode development, so that prevention and control cereal cyst nematode infects host.
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| US20070271630A1 (en) * | 2005-02-24 | 2007-11-22 | Boukharov Andrey A | Methods for genetic control of plant pest infestation and compositions thereof |
| CN102234651A (en) * | 2010-04-23 | 2011-11-09 | 中国科学院成都生物研究所 | Nucleotide sequence of gene for resistance to Cereal cyst nematode, Heterodera avenae and application thereof |
| CN104178490A (en) * | 2014-08-18 | 2014-12-03 | 中国科学院成都生物研究所 | Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and vector and application thereof |
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