CN108315333A - Black streaked dwarf virus of rice disease-resistant gene RBSDV-6c and its coding albumen - Google Patents
Black streaked dwarf virus of rice disease-resistant gene RBSDV-6c and its coding albumen Download PDFInfo
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Abstract
The invention discloses a kind of black streaked dwarf virus of rice disease-resistant gene RBSDV 6c and its coding albumen and applications.The gene is SEQ ID NO in sequence table：CDS sequences shown in 1.It is SEQ ID NO in sequence table that it, which encodes albumen,：Amino acid sequence shown in 3.Black streaked dwarf virus of rice disease-resistant gene RBSDV 6c provided by the invention have important application value, can convert the gene and obtain the transgenic rice plant that resistance significantly improves.
Description
Technical field：
The invention belongs to gene orders and application field, and in particular to black streaked dwarf virus of rice disease-resistant gene RBSDV-6c and
It encodes albumen.
Background technology：
Rice is one of staple food crop, and black streaked dwarf virus of rice is by rice black-streaked dwarf virus (Rice black-
Streaked dwarf virus, RBSDV) caused by the important virus disease of rice, RBSDV by mediator small brown rice planthopper with persistently without
Ovum mode is propagated.After rice is infected, show typically to stunt, tiller it is few, leaf color is dark green, blade back portion, leaf sheath even stalk
On have wax white to the symptoms such as dun color strip-shaped arteries and veins is swollen.Since 21 century, due to promoting mainly rice varieties to black streaked dwarf virus of rice
Resistance is insufficient, the generation getting worse of the Jiangsu and Zhejiang Provinces area disease, especially the Single Late Rice rice seedling bed of early sowing.The disease Jiangsu,
The rice producing region in Zhejiang and Henan is in eruption and prevalence trend.Such as black streaked dwarf virus of rice in 2007 is universal on the japonica rice of Jiangsu for the first time
Occur, grave illness field has no harvest in flakes, and onset area in 2008 is up to 2.67 × 105hm2, 2009 then further up into 3.33 ×
105hm2, serious economic loss is caused to Jiangsu Rice Production.
Currently, the method for prevention black streaked dwarf virus of rice is mainly using pesticide control biography virus mediator small brown rice planthopper, however by
Big in mediator small brown rice planthopper population quantity, prevention is difficult, and as administration time is elongated, mediator insect develops immunity to drugs, and causes
Control effect is bad, and using pesticide there are problems that environmental pollution this, therefore excavate Resistance resource, cultivate disease-resistant variety be
Prevent disease means the most cost-effective.Although to some Resistance QTLs have been had now been found that, due to rice secret note
The Resistant germplasm of dwarf wilt is few, quantitative inheritance feature is presented in resistance, and resistance includes insect resistace and disease resistance, so so far
It there is no the report that resistant gene is cloned.
It is the effective way for excavating resistant gene that annotation of gene function, which obtains disease-resistant gene,.Pass through the base on antagonism QTL
Because annotation directly filters out possible candidate gene, again in F after the difference for comparing candidate gene in resisting, feeling parent2In group
Verification, and to structural and functional prediction, to screen and detach resistance candidate gene.Later candidate gene conversion is carried out turning base
Because of verification, so that it is determined that can disease-resistant gene improve black streaked dwarf virus of rice resistance.
Invention content：
One of the object of the invention is to provide a black streaked dwarf virus of rice disease-resistant gene RBSDV-6c.
The two of the object of the invention are to provide a pair for expanding above-mentioned black streaked dwarf virus of rice disease-resistant gene RBSDV-6c
Primer.
The three of the object of the invention are to provide the cDNA sequence of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c.
The four of the object of the invention are to provide the coding albumen of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c.
To achieve the goals above, the present invention has just used following technical scheme：
Black streaked dwarf virus of rice disease-resistant gene RBSDV-6c is SEQ ID NO in sequence table：Nucleotide sequence shown in 1,
SEQ ID NO：2 are and SEQ ID NO in sequence table：Sequence 90% shown in 1 or more homology and have and SEQ in sequence table
ID NO：The nucleotide sequence of sequence identical function shown in 1.
Black streaked dwarf virus of rice disease-resistant gene RBSDV-6c of the present invention is to carry out finely positioning using Kang Gan groups
What clone obtained.The present invention selects susceptible variety Huaihe River rice No. 5 and disease-resistant variety crow shell, builds F2And F2：3Genetic group, in conjunction with height
Density genetic linkage map and F2For phenotypic evaluation as a result, carrying out finely positioning to disease-resistant gene, acquisition black streaked dwarf virus of rice is anti-
Property QTL.By rice genome sequencing Gramene databases, bioinformatics and high-flux sequence report (http:// www.gramene.org/；http://blast.ncbi.nlm.nih.gov/；http://www.uniprot.org/), confrontation
Property QTLs sections existing for gene carry out gene annotation and electronics screening, emphasis screens R genes and defends relevant disease-resistant gene.
The gene screened is being resisted, feeling parent and F2Comparison is cloned and is sequenced in Kang Gan groups, and what is all had differences is selected as candidate mesh
Gene.
For the pair of primers of amplifying rice black streak dwarf disease-resistant gene RBSDV-6c, it is respectively provided with SEQ in sequence table
ID NO：4 and SEQ ID NO：Nucleotide sequence shown in 5.
The present invention is combined SMART on-line predictions (http according to the Rice Genome Sequence announced://
Smart.embl-heidelberg.de/) predicted gene structure, and according to the starting of prediction result and coded sequence is terminated, if
Counting full length gene amplimer a pair is respectively：SEQ ID NO in sequence table：4 and SEQ ID NO：Nucleotides sequence shown in 5
Row, the first chain carries out PCR amplification, anti-, the sense parent of acquisition as template after being utilized respectively anti-, sense parent total serum IgE reverse transcription
CDNA (mRNA) sequence of the cDNA of middle gene RBSDV-6c, the gene RBSDV-6c in anti-/ sense material are respectively sequence table
Middle SEQ ID NO：1 (disease-resistant parent) and SEQ ID NO：2 (Susceptible parents), confrontation, sense parent in gene cDNA sequence into
Row compares and analysis, and there are 4 Site discrepancies in anti-, sense parent for RBSDV-6c cDNA sequences.
Above-mentioned SEQ ID NO：Nucleotides sequence shown in 1 is classified as the cDNA of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c
Sequence.
The coding albumen of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c shown in above-mentioned SEQ is the SEQ ID in sequence table
NO：Amino acid sequence shown in 3.
Black streaked dwarf virus of rice disease-resistant gene amino acid sequence of the present invention is using RBSDV-6c genes in anti-/ sense
CDNA (mRNA) sequence in material, confrontation, sense parent in RBSDV-6c nucleotide sequences be compared, RBSDV-6c exists
There are the differences of 4 nucleotide in anti-, sense parent, are resisted/felt RBSDV-6c in material by Editseq software translations respectively
Amino acid (albumen) sequence SEQ ID NO：3 (disease-resistant parents) and SEQ ID NO：4 (Susceptible parents), and fight, feel in parent
RBSDV-6c amino acid sequences are compared, and there are the differences of 1aa in anti-, sense parent by RBSDV-6c, thus it is speculated that the difference
Cause disease-resistant structure to change, changes so as to cause the disease resistance of gene.
The present invention has the advantages that：
The present invention carries out essence using the method for annotation of gene function to the disease-resistant gene RBSDV-6c of black streaked dwarf virus of rice
Fine positioning and clone obtain black streaked dwarf virus of rice disease-resistant gene RBSDV-6c, and study the gene in anti-, sense parent and F2
Nucleotide sequence and amino acid sequence in Kang Gan groups are analyzed and are compared, and black streaked dwarf virus of rice disease-resistant gene is obtained
RBSDV-6c verifies black streaked dwarf virus of rice disease-resistant gene RBSDV-6c's in difference anti-, in sense parent by transgenosis
Function.Disease-resistant gene RBSDV-6c provided by the invention has important value.
Description of the drawings：
Fig. 1 is Huaihe River rice No. 5/crow shell F2RBSDV Resistance QTLs analysis chart in group.
Fig. 2 is that (figure A is nucleotide difference to nucleotide and amino acid of differences of the RBSDV-6c in anti-sense parent；Figure B is ammonia
Base acid difference).
For the gene expression amount and viral level of single plant, (figure A is RBSDV-6c to the resistant transgenic T1 that Fig. 3 is RBSDV-6c
The transcriptional level of gene；Figure B is viral level).
Specific implementation mode：
Below by specific implementation case, the present invention is described in detail, but the present invention is not limited thereto.
Embodiment：The acquisition of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c
One, black streaked dwarf virus of rice disease-resistant gene RBSDV-6c is just positioned
Specific localization method includes the following steps：
(1) black streaked dwarf virus of rice localization of disease resistance genes research parent and genetic group：
It is anti-sense parent to select black shell (male parent) and Huaihe River rice No. 5 (female parent) respectively, builds the F more than 2000 plants2Group and
100 F2：3Family.
(2) black streaked dwarf virus of rice artificial infection idenfication
By Huaihe River rice No. 5, black shell and F more than 2000 plants2Group's seed is placed in culture dish, and presoaking and germinating 2d is broadcast respectively
In the disposable beaker of 500ml, every glass is broadcast 40, when seedling is grown to 2 leaf ages or so, rejects weak seedling, it is good to leave 35 plants of growing ways
For being inoculated with, each kind is repeated 3 times good seedling.According to the band poison rate of small brown rice planthopper, calculated by effective inoculation worm 4/seedling of amount
Practical inoculation worm amount, the malicious small brown rice planthopper of access band.Small brown rice planthopper is scanned out after meeting worm 3d, crop field is transplanted to after 1 day slow.Start after one month
Incidence is investigated, investigation is primary per 7d, and continuous observation 3 times accurately calculates incidence, analyzes the genetics of resistance pattern of black shell.
According to the method described above to 100 F2：3Family carries out black streaked dwarf virus of rice artificial infection idenfication, accurate to calculate morbidity
Rate.
(3) Resistance QTL finely positioning
Two parents and F2100 single plant high-flux sequences (Beijing Biomarker Technologies Co., Ltd.) of group, SLAF-
Seq technology rapid build high density genetic linkage maps, collection of illustrative plates SLAF marker numbers are 3835, and two adjacent marks are lost in linkage group
It is 99.51% to pass region of the distance less than 5cM and account for overall area number ratio, total figure away from for 1726.024cM, mean chart away from for
0.45cM.In conjunction with F2：3Family black streaked dwarf virus of rice artificial infection idenfication on No. 6 chromosomes as a result, navigate to rice secret note
Dwarf wilt Resistance QTL qRBSDV-6c.
The chromosome mapping and statistics feature of 1 Resistance QTL qRBSDV-6c of table
Two, the acquisition of candidate gene
(1) the electronics screening of QTL region candidates gene
Resistance QTL qRBSDV-6c is compared by genetic distance soap to genomic locations, and more than 400 candidate bases are obtained
Cause.In conjunction with high-flux sequence report in provide 5 database annotation of gene function information (COG, GO, KEGG, Swissprot,
Nr) primary dcreening operation disease-resistant related gene mainly checks various molecular functions and the bioprocess of participation, in Swiss- in GO databases
Prot databases check coding protein types, and the signal path of participation is checked in KEGG databases, are checked in COG databases
Its most like albumen.In Gramene (http://www.gramene.org/) obtain gene order in website, including introne,
Exon, cDNA, CDS, 5 ' UTR and 3 ' UTR；Blastp(http://blast.ncbi.nlm.nih.gov/) in NCBI albumen
Matter database carries out homology and conserved structure domain search；In SMART (http://smart.embl-heidelberg.de/)
The functional domain of predictive coding protein in website；In UniProt (http://www.uniprot.org/) it is integrated in website
Protein information, as protein function, enzymatic property, functional domain and site, gene family, posttranslational modification situation,
The induction and conclusion of subcellular localization and all relevant reports of same albumen.Screening goes out 78 and disease-resistant phase altogether in candidate section
The gene of pass.
(2) clone of candidate gene, sequencing
According to candidate gene cDNA or the CDS sequence obtained on the websites Gramene, designed in Primer primer 5
Primer, set primer include whole areas CDS of candidate gene.For convenience of next step construction of expression vector, when design primer, is distinguished
It is added to suitable restriction enzyme site in primer upstream and downstream.
When using the total serum IgE of Trizol (Reagent) extraction Susceptible parents Huaihe River rice No. 5 and disease-resistant parent crow shell, examination is utilized
Agent box PrimeScriptTM RT reagent Kit with gDNA Eraser carry out reverse transcription, obtain the first chain cDNA.Point
Not using the cDNA of the Huaihe River rice of acquisition No. 5 and black shell as template, PCR is carried out with the upstream and downstream primer of each candidate gene, according to CDS's
Different enzymatic amplifications, product ＜ 2000bp general T aq archaeal dna polymerases or ExTaq archaeal dna polymerases is respectively adopted in clip size
Amplification, product ＞ 2000bp use LA Taq or PrimerSTAR GXL polymeric enzymatic amplifications.Respective reaction system is as follows：
1) general T aq archaeal dna polymerases PCR system：
2) ExTaq archaeal dna polymerases PCR system：
3) PrimerSTAR GXL archaeal dna polymerases PCR system：
4) LA Taq archaeal dna polymerases PCR system：
Its respective response procedures is as follows：
1) general T aq, ExTaq and LA Taq polymerases PCR response procedures：
2) PrimerSTAR GXL polymerases PCR response procedures：
After completion of the reaction, PrimerSTAR GXL enzymes additionally add A tails just to can connect on cloning vector PMD-18T to PCR, i.e.,
0.5 μ L Taq enzymes and 1.0 μ L (10mM each) dNTP, 72 DEG C of extensions are added into the thin wall centrifugal tube containing PCR product
15min.Finally, PCR product by 0.5 × TAE buffers at 1% Ago-Gel carry out electrophoresis detection, 150V
Electrophoresis 20min, ethidium bromide (EB) is middle to dye 10-15min, is watched under ultraviolet lamp after taking pictures, and cuts solidifying containing target fragment
Glue.Purify QIAquick Gel Extraction Kit with multifunctional dna and recycle target fragment, is connected to PMD-18T cloning vectors, thermal shock method will connection production
Object is converted to Escherichia coli, and cell suspension is spread evenly across on LB solid mediums (antibiotic containing Amp), 37 DEG C of culture 12-
15h.Picking several be of moderate size, full round and smooth bacterium colony, in the LB liquid medium containing 500 μ L added with Amp antibiotic
2mL centrifuge tubes in expand culture, 37 DEG C of shaken cultivation 3-5h of 150rpm shaking tables.Using the bacterium solution after culture as template, using each
The corresponding primer of candidate gene carries out PCR verifications, and PCR reaction systems are as follows：
Electrophoresis detection PCR amplification in 1% gel as a result, have correct target fragment and band it is bright be positive colony.It takes
The 200 fresh bacterium solutions of μ L send Nanjing Jin Sirui biotech companies to be sequenced.To ensure that result reliability, each sample at least send 3 pipes
Bacterium solution is sequenced respectively.
After sequencing, while gene order of the candidate gene in 3 samples is compared, i.e. Huaihe River rice No. 5, black shell and complete
At the Nipponbare of genome sequencing, the comparison of nucleotide sequence and amino acid sequence is carried out respectively.Sequence alignment is in software
It is carried out in ClustalX 2.0, comparison result is checked in analysis software Genedoc.Statistical analysis discovery has 21 gene cores
Thuja acid do not find with otherness, and 14 gene nucleotides are variant but amino acid not finding differences property, nucleotide and amino
Sour difference all exists but mutation does not have 18 in functional areas, and multiple design primer still expands having less than correct black shell gene
It 13, finally screens to 12 candidate genes.
Three, the structural and functional prediction of candidate gene
There are the candidate genes of amino acid of differences between confrontation, sense parent, further analyze difference to its structure or function domain
Influence, the possible influence of its antagonism of initial guess.Analysis includes：(1) whether difference changes albumen homology sequence：In NCBI
It is analyzed in the Blastp of website；(2) whether difference is happened at structure or function site：Pass through InterProScan databases
(http://www.ebi.ac.uk/interpro/scan.htmL) in functional site judged；(3) difference is to structural domain
Influence：In the websites SMART, the structural domain of the front and back gene order of comparing amino acid mutation judges its influence to structural domain.
Analysis finds that the anti-sense amino acid of differences of 12 candidate genes influence gene structure or are happened in functional site.
Further F of the verification candidate gene in Huaihe River rice No. 5/crow shell2Whether Dai Jikang and Ji Gan groups occur hereditary exchange,
The reliability of amino acid of differences is determined by double verification.From F2Respectively taken in 100 plants of mapping populations in generation 10 plants through artificial infection
Identification shows as that (incidence ＜ 33%) and pole is extremely resisted to feel the single-strain blade of black streak dwarf (incidence ＞ 80%), and CTAB methods carry
Take the total DNA of single-strain blade.According to the site of base or amino acid of differences, two flanks in gene DNA sequence difference site are set
Primer is counted, selects target fragment size suitable and includes the primer in difference site.Respectively with parents' sheet and F2It is chosen in generation each
Anti-, sense single plant DNA is template, using the DNA fragmentation comprising mutational site as upstream and downstream primer, using common DNA Taq enzymes body
System carries out PCR, and T-A clone and be sequenced, sequence alignment and interpretation of result respectively in software ClutalX 2.0 and GeneDoc into
Row judges the generation that heredity exchanges according to sequence alignment result.Further demonstrate the steady of 12 candidate gene amino acid mutations
Qualitative and consistency.
Four, the screening of candidate gene rice transformation and transfer-gen plant
Based on double verification and function prediction as a result, 12 candidate genes are inserted into respectively modified with maize ubiquitin
Ubi is promoter, using hygromycin gene as the plant binary expression vector pCAMBIA1301 of selected marker, builds Plant Transformation
Candidate gene is converted to Susceptible parent Huaihe River rice No. 5 through agriculture bacillus mediated method, has successfully received 8 transgenic lines by carrier.
Five, the verification of transfer-gen plant
After obtaining transfer-gen plant, plant total serum IgE is extracted using Trizol one-step method, using removing genome Reverse Transcription
Box reverse transcription obtains the first chain cDNA, positive plant is obtained with hygromycin specific primer and target gene primer screening, with reality
When quantitative fluorescent PCR measure transgenosis T0 for candidate gene transcriptional level.According to the transcriptional level of candidate gene height, select
Go out that expression quantity is higher and the lower T of expression quantity1In generation, carries out artificial infection Resistance Identification.Qualification result shows transformed gene
There were significant differences compared with transgene receptor Huaihe River rice No. 5 for the incidence of 1 line of qRBSDV-6c, and resistance significantly improves.Simultaneously
The candidate target gene transcriptional level and viral level for measuring disease-resistant single plant in this line, as a result show the change of gene expression amount
Change related to viral level variation.
Contain in the reaction system (20 μ l) of above-mentioned positive plant screening amplified reaction used：2 μ l the first chains of template, 10 μ l
2 × PCR Mix, 0.5 μM of sense primer, 0.5 μM of downstream primer, 7 μ l ddH2O。
PCR response procedures are：Stage 1：94 DEG C of pre-degeneration 5min；Stage 2：94 DEG C of 45s, 56 DEG C of 45s, 72 DEG C of 90s, totally 35
A cycle；Stage 3：72 DEG C of extension 10min；Stage 4：4 DEG C of preservations.
Contain in the reaction system (20 μ l) of real-time fluorescence quantitative PCR reaction：2 μ l the first chains of template, 10 μ l2 × SYBR,
0.5 μM of sense primer, 0.5 μM of downstream primer, 7 μ l ddH2O。
Real-time fluorescence quantitative PCR response procedures are：Stage 1：95 DEG C of pre-degeneration 15min；Stage 2：94 DEG C of 15s, 58 DEG C
30s, 79 DEG C of 30s, totally 45 recycle；Stage 3：65 DEG C of 30s, totally 61 recycle.
The part T of 3 qRBSDV-6c of table1For artificial Resistance Identification result
Note：Above-mentioned subscript lowercase letter indication difference conspicuousness (P ＜ 0.05), above-mentioned subscript capitalization indicate difference pole
Conspicuousness (P ＜ 0.01).
Claims (4)
1. black streaked dwarf virus of rice disease-resistant gene RBSDV-6c, (a) is SEQ ID NO in sequence table：CDS sequences shown in 1,
(b)SEQ ID NO：2 are and SEQ ID NO in sequence table：Sequence 90% shown in 1 or more homology and with in sequence table
SEQ ID NO：The CDS sequences of sequence identical function shown in 1.
2. the SEQ ID NO of the black streaked dwarf virus of rice disease-resistant gene RBSDV-6c described in claim 1 (a)：Nucleosides shown in 1
Acid sequence base occurs to be mutated at 4, and amino acid occurs to be mutated at 1.
3. the pair of primers for expanding the black streaked dwarf virus of rice disease-resistant gene RBSDV-6c described in claim 1 (a), respectively
To be SEQ ID NO in sequence table：4 and SEQ ID NO：Nucleotide sequence shown in 5.
4. the coding albumen of black streaked dwarf virus of rice disease-resistant gene RBSDV-6c described in claim 1 is SEQ in sequence table
ID NO：Amino acid sequence shown in 3.
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| CN105624170A (en) * | 2014-10-28 | 2016-06-01 | 北京大学 | Application of OsAGO18 protein or encoding gene of OsAGO18 protein to regulation and control on resistance of plants on RDV (Rice Dwarf Virus) or virus in same family as RDV |
| CN106834425A (en) * | 2015-12-04 | 2017-06-13 | 无锡南理工科技发展有限公司 | The anti-black streak dwarf site qRBSDV17 of rice varieties Zhejiang round-grained rice 5 and its molecule labelling method |
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- 2017-10-19 CN CN201711005609.8A patent/CN108315333B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624170A (en) * | 2014-10-28 | 2016-06-01 | 北京大学 | Application of OsAGO18 protein or encoding gene of OsAGO18 protein to regulation and control on resistance of plants on RDV (Rice Dwarf Virus) or virus in same family as RDV |
| CN106834425A (en) * | 2015-12-04 | 2017-06-13 | 无锡南理工科技发展有限公司 | The anti-black streak dwarf site qRBSDV17 of rice varieties Zhejiang round-grained rice 5 and its molecule labelling method |
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