CN106701749A - Rice blast resistance gene Piz-t functional specific molecular marker and detection method and application thereof - Google Patents
Rice blast resistance gene Piz-t functional specific molecular marker and detection method and application thereof Download PDFInfo
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Abstract
The invention provides a rice blast resistance gene Piz-t functional specific molecular marker and a detection method and application thereof. The molecular marker shows a specific banding pattern of the rice blast resistance gene Piz-t amplified from a rice genome DNA through primer pairs SEQ ID NO.1 and the SEQ ID NO.2. The rice blast resistance gene Piz-t gene specific molecular maker provided by the invention has important application values, and the utilization efficiency of the gene in each of germplasm resource screening, molecular marker-assisted selective breeding, gene pyramiding breeding and transgenic breeding can be improved by use of the marker.
Description
Technical field
The invention belongs to agricultural biological technical field, more particularly to a kind of rice blast resistance gene Piz-t functional moleculars
Mark Piz-t-InDel and its detection method and application.
Background technology
Rice blast is one of hazard rice stable high yield disease the most serious.Though the chemical prevention commonly used in current production
Influence of the disease to Rice Production can be solved or mitigated to a certain extent, but farmland long-term prescription not only has pollution to make to environment
With, and pesticide residue also affects the edible quality of rice.Long-term production practices show, seed selection with utilize disease-resistant product
Kind it is preventing and treating rice blast the most safely and effectively one of method.There is frequently variation yet with physiological races of rice blast fungus is pathogenic
Characteristic, this can cause the single kind of resistance can gradually lose in the 3-5 years after planting resistance (abundant institute etc.,
2011)；Therefore, excavate and using resistant gene be obtain persistently, the important channel of broad-spectrum disease resistance kind.The resistant gene of early stage
Authentication method is mainly inoculated identification, but the inoculation identification method of routine can not exactly reflect disease-resistant gene type, it is difficult to full
The requirement of the disease-resistant molecular breeding of foot.With the development of paddy disease-resistant molecular genetics, many resistant genes by finely positioning or gram
Grand (Su et al.2015)；With the development and application of molecular labeling, it is gradually suitable for the identification of resistant gene genetic background
And development (the Hittalmani et al.2000 of many disease-resistant gene pyramiding breedings；Jena and Mackill,2008).
But in the resistant gene cloned, some important disease-resistant genes are located at same site, such as Pik and Pi2/Pi9
Sequence very high homology between multiple allele on site, between functional form sequence nand function type, general linked marker is still
So it is difficult to accurately screen function disease-resistant gene (the Qu et al.2006 of various materials；Zhou et al.2006；Ashikawa
et al.2008；Takahashi et al.2010；Yuan et al.2011；Zhai et al.2011；Yuan et
al.2011；Hua et al.2012；Ma et al.2015；Tian et al.2016).Therefore, Direct Analysis function equipotential base
Because itself sequence and develop the molecular labeling of its specific Function target gene selected, not only select reliability
Height, can also greatly accelerate breeding paces.
Up to the present, at least six rice blast resistance genes (Piz-t, Pi2, Pi9, Piz, Pigm, Pi50) have been
Be positioned in the Pi2/Pi9 gene clusters at paddy rice Short arm of chromosome 6 end (Jiang etal.2012), wherein Piz-t, Pi2,
Pi9 and Pi50 etc. is by successful clone (Qu et al.2006, Zhou et al.2006).Research shows, the work(in the site
Having wide spectrum anti-characteristic high gene, wherein Piz-t is related to leaf blast in rice resistance, to domestic and international multiple rice blast microspecies more
Show stronger resistance to leaf blast.Magnificent rosy clouds etc. (2015) have developed the dominant marker of Piz-t, and this is marked at resistance breeding work
The situation of wrong choosing or leakage choosing is susceptible in work.In order to be able to more accurately and effectively rice blast broad-spectrum resistance gene Piz-t is applied
To in paddy rice resistance breeding work, it is necessary to develop the specific function mark of the gene.In view of early stage we developed
Pi2 and Pi9 functional labels (Yang Liming etc., 2014；2015), this research will focus on parting Piz-t and Pi2/Pi9 sites other
The specific marker of functional gene and the non-functional gene of equipotential.
The content of the invention
The technical problem of solution：In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the invention is to provide
A kind of rice blast resistance gene Piz-t gene function specific molecular markers Piz-t-InDel.
Another goal of the invention of the invention is the rice blast resistance gene Piz-t gene specific molecule marks described in offer
Remember the detection method of Piz-t-InDel.
Another goal of the invention of the invention is the rice blast resistance gene Piz-t gene functions specificity point described in offer
The application of son mark Piz-t-InDel.
Technical scheme：A kind of rice blast resistance gene Piz-t specific Function molecular labelings, the molecular labeling is to pass through
Primer pair SEQ ID NO.1 and SEQ ID NO.2 are amplified from oryza sativa genomic dna and are in rice blast resistance gene Piz-t
The molecular labeling of specific banding pattern, the primer pair sequence is：
SEQ ID NO.1Fl：5’-TTACTAGAATCGCTCCAT-3’；
SEQ ID NO.2Rl：5’-ACTGCGTGCGACTTGA-3’.
The sequence such as SEQ ID NO.3 institutes of the molecular labeling for rice blast resistance gene Piz-t being in specific banding pattern
Show.
The method for detecting the specific Function molecular labeling of the rice blast resistance gene Piz-t, by comparing multiple water
The allelic sequences of rice rice blast resistance gene Piz-t, comprise the steps of:(1) download what is obtained from public database
Piz-t and its homologous genome sequence, and the genome sequence in the corresponding region of kind Nipponbare is sequenced, for Piz-t
Site carries out sequence alignment, and examination Piz-t is special, can be different from the insertion of other rice blast resistance alleles of the site/
Missing (insertion-deletion, InDel) site；(2) the InDel information obtained using step (1), is marked according to InDel
The design principle of note, in the InDel sites upstream and downstreamPlace's design gene-specific primer, primer pair base
Sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；(3) carrying the rice blast resistance of rice blast resistance gene Piz-t
The STb gene of kind IRBLZT-T is template, enters performing PCR amplification, the PCR primer for being obtained as rice blast resistance gene Piz-t
Gene specific molecular labeling Piz-t-InDel.
Above-mentioned rice blast resistance gene Piz-t gene specifics molecular labeling is differentiating Rice Blast resistant gene
In application.
Above-mentioned rice blast resistance gene Piz-t gene specifics molecular labeling is differentiating Pi2/Pi9 gene cluster regions difference
Rice blast resistance gene in application.
A kind of kit for differentiating Rice Blast resistant gene, containing nucleotide sequence as shown in SEQ ID NO.3
Molecular labeling, and the primer pair as described in SEQ ID NO.1 and SEQ ID NO.2.
Principle of the invention:InDel refers to the same site of genome between sibling species or same species Different Individual
Sequence there occurs insertion or the missing of different size nucleotide fragments, i.e., another homologous is compared in a certain site in one sequence
One or more bases (Weber et al, 2002) are inserted or lacked to sequence.InDel marks PCR-based amplification technique, this
Belong to length polymorphism mark (Hyten et al, 2010) in matter.InDel marks good stability, polymorphism are high, classification system
Simply (Jander et al, 2002)；Compared with the complicated SNP marker of classification system, InDel detections are simpler convenient, right
Instrument and equipment and technical requirements are relatively low, can be carried out on electrophoretic techniques platform.Have started to be applied to animals and plants colony something lost at present
Pass the fields such as analysis, marker assisted selection and mankind's medicolegal genetics, medical diagnosis.The side that the present invention passes through Multiple Sequence Alignment
Method finds the InDel occurred in Piz-t gene orders, because this InDel is in other function/NOT functions of Piz-t and Pi2/Pi9
Energy gene has the difference of 27bp, therefore is easy to therefrom distinguish Piz-t.The present invention designs primer pair F1/R1 accordingly, leads to
Standard PCR amplification is crossed, when polyacrylamide gel electrophoresis is detected, segment in different sizes is presented.
Beneficial effect:(1) the molecular labeling specificity that the present invention is provided is high:Genome area where Piz-t is existed
Candidate gene including the multiple resistant gene expression characteristicses including Piz-t, these candidate genes very high homology in sequence；This
Outward, the rice blast resistance gene cloned on Piz-t and the site is highly consistent on base sequence.What the present invention was provided divides
Son mark is that inventor is searched and obtained by experimental verification by constantly carrying out sequence polymorphism, can be significantly
Piz-t is made a distinction with the paralog gene being present in the genome area with Piz-t very high homologies.
(2) present invention provide molecular labeling in actual applications, low cost, high flux:It is at present mostly to utilize electrophoresis
Platform carries out parting, and the parting platform is fast economical, it is not necessary to complicated experimental facilities, workable.Electropherotyping platform
There are agarose gel electrophoresis, denaturation or native polyacrylamide gel electrophoresis and Capillary Electrophoresis.What the present invention was provided divides
Son mark only needs PCR combinations agarose gel electrophoresis or native polyacrylamide gel electrophoresis, and low cost, flux are high, add
Specific height (i.e. the degree of accuracy is high), is particularly well-suited in production practices.
(3) present invention is first Piz-t gene specifics InDel mark developed for Piz-t gene internal sequences
Note.The present invention can pass through the method for electrophoresis detection successfully by Piz-t and other the rice blast resistance bases being positioned on the site
Because distinguishing, up to the present, also without the report about this kind of mark.Piz-t specific moleculars mark provided by the present invention
Note Piz-t-InDel is a codominant marker, and its reliability and accuracy are better than dominant marker in actual application.This
Invention can be applied to the Screening of Germplasm of Piz-t, transgenosis identification and gene pyramiding and the paddy rice resistance based on MAS technologies
In breeding work.The mark is present in Piz-t gene internals, therefore to the screening capacity theoretical value of Piz-t up to 100%, its
Combination property is better than the molecular labeling and functional label chain with Piz-t reported.
(4) molecular labeling that the present invention is provided can be easily applied in the different colony of genetic background.It is existing with
What the most of sequence polymorphism both for 2 different parents of same colony of Piz-t chain molecular labeling was developed, this
The applicability being marked at a bit in other colonies is limited.The functional label of the exploitations such as magnificent rosy clouds etc. (2015), not only qualification process ratio
It is cumbersome, but also be dominant marker, it is not suitable in large-scale germplasm identification and MAS breedings.The present invention is applied to
Transgenic breeding under any genetic background, gene pyramiding and the resistance breeding based on MAS technologies, without repeating parent
The screening of polymorphism, substantially increases breeding efficiency.
Therefore, rice blast resistance gene Piz-t gene specific molecular labelings provided by the present invention have important answering
With value, can improve the gene using this mark and be educated in Screening of Germplasm, molecular marker assisted selection breeding, gene pyramiding
Kind, and the efficiency utilized in transgenic breeding.
Brief description of the drawings
Fig. 1 is Piz-t gene specific molecular labeling Piz-t-InDel in Pi2/Pi9 gene cluster regions difference rice blast
Resistant gene and Nipponbare, 9311 the result figure, wherein:Swimming lane DL500 is DNA ladder, swimming laneDNA moulds
Plate is followed successively by IRBLzt-T (Piz-t), IR65482-4-136-2-2, Gumei2, Gu Mei 4, Er Bazhan, Fukunishiki.
Fig. 2 is the result figures of the Piz-t gene specific molecular labeling Piz-t-InDel in other rice varieties,
Wherein:Swimming lane DL500 is DNA ladder, and the DNA profiling of swimming lane 1-13 is followed successively by IRBLzt-T (Piz-t), short foreign rice, distant round-grained rice
No. 5, TQ155, Masoli, BG1100, TRAT, 5011, the extensive 136-4 of Peiai 64,9311, poplar, Zhong Nongqing, 34 Chongqing it is extensive.
Specific embodiment
A kind of rice blast resistance gene Piz-t gene functions specific molecular marker Piz-t-InDel, is by primer pair
SEQ ID NO.1 and SEQ ID NO.2 are amplified with rice blast resistance gene Piz-t in specificity from oryza sativa genomic dna
The molecular labeling of banding pattern；SEQ ID NO.1(5’-3’):TTACTAGAATCGCTCCAT；SEQ ID NO.2(5’-3’)：
ACTGCGTGCGACTTGA；
Described rice varieties are IRBLzt-T (Piz-t donor parents)；
Described rice blast resistance gene Piz-t includes the genetic fragment I of coding NBS-LRR albuminoids, the following institute of sequence
Show
SEQ ID NO.3:
TTACTGGAATCGCTCCATTTTCGTATTTGAAAATATTTGATTATGTTTTTTATGTGGGGTTTCTGATTC
CAATTAAAAAAA-TGAAAATAAAAATGGTATGATGGTTTCCGTTCGTTATGCATGCGCGGAACAATGGATCTCACTA
ATCAAGTAGCACGCAGT
The detection method of the specific Function molecular labeling Piz-t-InDel of described rice blast resistance gene Piz-t, leads to
The allelic sequences for comparing multiple resistance gene of rice blast Piz-t are crossed, is comprised the steps of:(1) from public database
It is middle to download the Piz-t that obtains and its homologous genome sequence Pi50 and sequencing kind Nipponbare (Nipponbare) is corresponding
The genome sequence in region, for NBS1~9- Piz-t sites carry out sequence alignment, and examination Piz-t is special, can be different from the position
Put insertion/deletion (insertion-deletion, InDel) site of other rice blast resistance alleles.(2) step is utilized
(1) the InDel information for obtaining, according to the design principle that InDel is marked, at the InDel sites upstream and downstream 100-200bp
Design gene-specific primer, (Pi50 is accounted for using primer pair amplifies IRBLz-FU (Piz), Gu Mei 4 (Pigm (t)), sixteen
(t)), Gu Mei 2 (P26 (t)), IR65482-4-136-2-2 (Pi40 (t)), Nipponbare and 9311, sequencing compare, until
NBS2-Piz-t is screened can substantially distinguish Piz-t primer pairs, and base sequence is as follows:Fl:5’-
TTACTAGAATCGCTCCAT-3’；Rl:5’-ACTGCGTGCGACTTGA-3’；(3) carrying rice blast resistance gene Piz-
The STb gene of the rice blast resistance kind IRBLzt-T of t is template, enters performing PCR amplification, the PCR primer for being obtained as rice blast
Resistant gene Piz-t gene specific molecular labelings Piz-t-InDel.
Rice blast resistance gene Piz-t gene specific molecular labeling Piz-t-InDel are differentiating Rice Blast
The application of resistant gene, is particularly suitable for differentiating the application of the different rice blast resistance gene in Pi2/Pi9 gene cluster regions；It is excellent
Choosing is comprised the following steps:(1) expand:Performing PCR is entered to the genome of rice varieties to be detected using primers F l and Rl；(2) examine
Survey:Detected using polyacrylamide gel electrophoresis, if detecting the nucleotide fragments that molecular size range is 163bp, carried
Rice blast resistance gene Piz-t；If detecting molecular size range for 190bp, rice varieties to be detected do not carry Piz-t
The functional gene of point.
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
Rice blast resistance gene Piz-t gene specifics molecular labeling and its design of primers and detection:
(I) analysis in Piz-t insertion/deletions (insertion-deletion, InDel) site:
The partial genome sequence for obtaining the paddy rice donor kind such as Piz-t is downloaded from public database, for NBS1~9-
Piz-t sites carry out sequence alignment, and examination Piz-t is special, can be different from other rice blast resistance alleles of the site
Design primer, amplification IRBLz-FU (Piz), Gu Mei 2 (P26 (t)), Gu Mei 4 in specific insertion/deletion (InDel) difference site
(Pigm (t)), sixteen account for (Pi50 (t)), IR65482-4-136-2-2 (Pi40 (t)), Nipponbare, sequencing, comparison result following table
It is shown:
Wherein, ----it is the Piz-t gene specific deletion sequences for identifying；
(2) primer is designed:
According to the design principle that InDel is marked, primer is designed at the InDel sites upstream and downstream 100-200bp, drawn
Thing is as follows to base sequence:
Fl:5′-TTACTAGAATCGCTCCAT-3′；
Rl:5′-ACTGCGTGCGACTTGA-3′。
(3) paddy rice Representative Cultivars are selected:Selection carries the generation of Piz-t genes and Pi2/Pi9 gene cluster allele
Table kind is as follows:
IRBLzt-T (Piz-t), IR65482-4-136-2-2 (Pi40 (t)), Gu Mei 2 (P26 (t)), (Pigm of Gu Mei 4
(t)), sixteen account for (Pi50 (t)), Fukunishiki.
(4) PCR amplifications, obtain the fragment containing Piz-t gene specifics InDel
Using above-mentioned primer pair Fl and R1, the STb gene with above-mentioned rice varieties carries out standard PCR amplification laggard as template
Row polyacrylamide gel electrophoresis detects that the result for obtaining is as shown in Figure 1.
Amplification reaction system is as follows:
2x Reaction Mix:12.5μL
Primers F l (10 μ Μ):1μL
Primer Rl (10 μ Μ):1μL
Golden DNA Polymerase:0.2μL
DNA profiling (20-50ng/ μ L):1μL
ddH2O:Complement to 25 μ L.
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 63 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72
DEG C 7 minutes；10 DEG C of preservations.
After PCR reactions terminate, take appropriate amount of sample carries out electrophoresis detection, deposition condition on 8% polyacrylamide gel
It is 90V, 1 hour.
Wherein, the swimming lane I of Fig. 1 is obtained with Piz-t donor kind rice varieties IRBLzt-T genomes as template PCR
Fragment, is in that specific banding pattern, i.e. swimming lane 1 are Piz-t gene specific molecular labelings Piz-t- with rice blast resistance gene Piz-t
InDel.The result of Fig. 1 shows that Piz-t gene specifics molecular labeling can distinguish anti-sense allele, and Pik can be distinguished again
Other resistant genes identified on point.That is, the rice varieties of Piz-t are carried, the electrophoresis of its pcr amplification product
Test strip is presented with 163bp, and other Pi2/Pi9 gene cluster allele rice blast resistance genes and non-functional gene are with electrophoresis
Test strip 190bp is presented.
Embodiment 2
Detection application of the resistant gene Piz-t gene specifics molecular labeling in other rice varieties:
The representative rice varieties of selection 13, are followed successively by:IRBLzt-T (Piz-t), short foreign rice, distant round-grained rice 5, TQ155,
Masoli, BG1100, TRAT, 5011, the extensive 136-4 of Peiai 64,9311, poplar, Zhong Nongqing, 34 Chongqing it is extensive.
Extract its genomic DNA respectively, and as template, according to the method for embodiment 1, enter performing PCR amplification, digestion and
Electrophoresis detection.According to the size of digestion products (band), resistant gene Piz-t and other rice blast resistance genes can be distinguished
Come.As shown in Fig. 2 in anti-stave type for the individuality of Piz-t types (carries the disease-resistant variety of rice blast resistance gene Piz-t
IRBLZT-T, the sample of swimming lane 1) in can detect the presence of Piz-t-InDel gene specific molecular labelings, and other anti-spectrums
The individual of phenotype is then presented with another banding pattern.It can be seen that, result of the test matches with design analysis, illustrates resistant gene
Piz-t gene specifics molecular labeling can be obtained the aspects such as Piz-t genes and other Pi2/Pi9 gene cluster allele are differentiated
Using.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Yang Haijun
<120>A kind of rice blast resistance gene Piz-t specific Functions molecular labeling and its application
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
ttactagaat cgctccat 18
<210> 2
<211> 16
<212> DNA
<213>Artificial sequence
<400> 2
actgcgtgcg acttga 16
<210> 3
<211> 162
<212> DNA
<213>Artificial sequence
<400> 3
ttactggaat cgctccattt tcgtatttga aaatatttga ttatgttttt tatgtggggt 60
ttctgattcc aattaaaaaa atgaaaataa aaatggtatg atggtttccg ttcgttatgc 120
atgcgcggaa caatggatct cactaatcaa gtagcacgca gt 162
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence
<400> 4
taaaaatggt atgatggttt ccgttcgtta tgcat 35
<210> 5
<211> 62
<212> DNA
<213>Artificial sequence
<400> 5
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
<210> 6
<211> 62
<212> DNA
<213>Artificial sequence
<400> 6
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
<210> 7
<211> 62
<212> DNA
<213>Artificial sequence
<400> 7
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
<210> 8
<211> 62
<212> DNA
<213>Artificial sequence
<400> 8
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
<210> 9
<211> 62
<212> DNA
<213>Artificial sequence
<400> 9
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
<210> 10
<211> 62
<212> DNA
<213>Artificial sequence
<400> 10
taaaaatggt atgatggttt ccgttcgttt tcgagccgtt tcatccctaa ttgcacatgc 60
at 62
Claims (6)
1. a kind of rice blast resistance gene Piz-t specific Function molecular labelings, it is characterised in that the molecular labeling is to pass through
Primer pair SEQ ID NO.1 and SEQ ID NO.2 are amplified from oryza sativa genomic dna and are in rice blast resistance gene Piz-t
The molecular labeling of specific banding pattern, the primer pair sequence is：
SEQ ID NO.1Fl：5’-TTACTAGAATCGCTCCAT-3’；
SEQ ID NO.2Rl：5’-ACTGCGTGCGACTTGA-3’.
2. molecular labeling according to claim 1, it is characterised in that it is described with rice blast resistance gene Piz-t in specificity
The sequence of the molecular labeling of banding pattern is as shown in SEQ ID NO.3.
3. the method that test right requires the specific Function molecular labeling of rice blast resistance gene Piz-t described in 1 or 2, it is special
Levy and be：By comparing the allelic sequences of multiple resistance gene of rice blast Piz-t, comprise the steps of:
(1) Piz-t and its homologous genome sequence for obtaining, and sequencing kind Nipponbare phase are downloaded from public database
The genome sequence of corresponding region, sequence alignment is carried out for Piz-t sites, and examination Piz-t is special, can be different from the site
Insertion/deletion (insertion-deletion, InDel) site of other rice blast resistance alleles；
(2) the InDel information obtained using step (1), according to the design principle that InDel is marked, in the InDel sites
DownstreamPlace's design gene-specific primer, primer pair base sequence such as SEQ ID NO.1 and SEQ ID NO.2 institutes
Show；
(3) it is template to carry the STb gene of the rice blast resistance kind IRBLZT-T of rice blast resistance gene Piz-t, carries out
PCR is expanded, the PCR primer for being obtained as rice blast resistance gene Piz-t gene specifics molecular labeling Piz-t-InDel.
4. the rice blast resistance gene Piz-t gene specifics molecular labeling described in claim 1 or 2 is differentiating rice varieties rice
Application in seasonal febrile diseases resistant gene.
5. the rice blast resistance gene Piz-t gene specifics molecular labeling described in claim 1 or 2 is differentiating Pi2/Pi9 bases
Because of the application in the different rice blast resistance gene in cluster region.
6. it is a kind of differentiate Rice Blast resistant gene kit, it is characterised in that contain nucleotide sequence such as SEQ ID
Molecular labeling shown in NO.3, and the primer pair as described in SEQ ID NO.1 and SEQ ID NO.2.
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