CN105063035B - The molecular labeling of Xanthomonas oryzae pv oryzae and its application - Google Patents
The molecular labeling of Xanthomonas oryzae pv oryzae and its application Download PDFInfo
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Abstract
The present invention relates to plant aetiology and plant quarantine technical field, there is provided the molecular labeling of the unique identification Xanthomonas oryzae pv oryzae of a collection of energy more convenient and quicker, in particular, provide the dominant molecular labeling and codominant marker of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola identification, and apply the two kinds of applications of pathogenetic bacteria of these Markers for Detection, using these molecular labelings and the method for multiple molecular labeling joint identifications, more ensure that the reliability of result, disease can not only accurately be identified so as to the development for taking correct prophylactico-therapeutic measures to meet current trial quarantine is sent to, and can in time control the extension of disease to spread.
Description
The application is directed to the entitled " molecule of rice Xanthomonas filed in applicant 2014 year 04 month 25 days
Mark and its apply " (original applying number is：2014101695173) divisional application that patent of invention is done.
Technical field
The present invention relates to field of plant genetic, there is provided the molecular labeling of Xanthomonas oryzae pv oryzae and its should
With the dominant molecular labeling and codominance in particular, providing rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola identification divide
Son mark, and apply the two kinds of applications of pathogenetic bacteria of these Markers for Detection.
Background technology
Xanthomonas oryzae pv oryzae is the Gram-negative bacteria of the yellow of a quasi-representative, is main on paddy rice bacillary cause a disease
Bacterium, including rice leaf spot bacteria Xanthomonas oryzae pv.oryzae (Xoo) and xanthomonas oryzae pv. oryzicola
Xanthomonas oryzae pv.oryzicola (Xoc) (Zhao, Ardales et al.2004) .Xoo can cause paddy rice
Vascular bundle diseases be bacterial blight of rice, and Xoc is that paddy rice is infected by tissue space so as to cause paddy bacterial bar
Pinta.Due to bacterial blight of rice (BB) by temperature and region influenceed turn into paddy bacterial disease the most serious it
One, influenceed serious mainly Asia and portions of Africa area.But the rice varieties of high yield are that to be more vulnerable to paddy rice white
Leaf blight (BB's) infects, therefore bacterial blight of rice (BB) often results in serious economic loss, and output reduction is up to 50%.Compared to it
Under, the loss that xanthomonas oryzae pv. oryzicola (BLS) causes is relatively small, and output reduction is up to 30% when serious.But, with
The extensive plantation of hybrid rice, paddy bacterial disease has the trend for aggravating year by year.Therefore, the development of paddy rice bacteriosis is contained
It is most important for stablizing grain-production and improvement people's living standard.
With global trade spreading in agricultural, the inspection work that rice paddy seed is imported and exported is particularly important.And in Asia
Including including China, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola being defined as paddy rice quarantine disease.Its
Seed-borne fungi propagates serious, therefore, quick detection recognizes that pathogen is the key of quarantine with accurate, while can be effective
Controlling disease there is and reduce the loss that brings of disease.Although rice leaf spot bacteria and the side of infecting of paddy bacterial germ
Formula is different, but due to belonging to the leaf spot bacteria (Xanthomonas oryzae pv.oryzae, Xoo) of rice Xanthomonas together
Affiliation is very near, and genome sequence phase with Population of Xanthomonas Oryzae Pv (Xanthomonas oryzae pv.oryzicola, Xoc)
More than 90% is up to like property, identified differentiation is difficult in quarantine.Traditional screening separation means are for different causes of the same race
Lesion kind, the very much like bacterial strain of morphological feature, differentiation gets up to take time and effort.Phage technology paddy rice bacteriosis in the groove compared with
It is conventional, but the vacation sun that antiserum quality problems are also easy to produce in the false negative that easily causes of bacteriophage specialization and serological method
Property so that the popularization of this method is restricted.In current inspection and quarantine work, using convenient, economic PCR molecule marks
Note is carried out related work and has become main method, and various is the detection method relied in an endless stream with PCR, and such as TaqMan is real-time
Although fluorescence PCR method is accurate sensitive but costly, it is very difficult to apply in routine check quarantine.In two kinds of bacteriums of paddy rice
In the inspection and quarantine of venereal disease evil, forefathers utilize the difference and transmembrane protein base of electro transfer Huang protein alpha subunit in two kinds of pathogens
Dominant specific primer is designed because of the difference of (AY319937, AY319941) to carry out identification differentiation, obtain certain effect.By
There is the exchange of a certain degree of inhereditary material between bacterium, single molecular labeling easily causes the error in detection, and PCR
Experiment easily forms certain contamination probability in practical operation, there is certain false positive risk, therefore develop enrich, guarantor
Keeping property molecular labeling high carries out the accuracy that many site primers advantageously ensure that detection.
And along with the development of biological order-checking technology, complete many genome sequencing work.Up to the present,
The bacterial strain for having four Xanthomonas campestris completes sequencing (including KACC10331, PXO99A, MAFF311018, and
), BLS256 and complete rice leaf spot bacteria strain AX01947 sketch.Based on acquired information, it is possible to use biological
The comparison algorithm of informatics studies the difference between different microspecies.It is how white to distinguish paddy rice using these existing achievements in research
Leaf spoting bacteria and paddy bacterial germ turn into our study frontier.
The content of the invention
The research conditions that the present inventor is just being directed to prior art are entered based on these issued species sequences
Row whole genome sequence is compared, there is provided the molecular labeling of Xanthomonas oryzae pv oryzae and its application, in particular, provides paddy rice white
Leaf spoting bacteria and the dominant molecular labeling and codominant marker of xanthomonas oryzae pv. oryzicola identification, and apply these points
Son mark two kinds of applications of pathogenetic bacteria of detection.
Inventor is first according to 3 bacterial leaf-blight bacterial strain MAFF311018, KACC10311, the PXO99A for having announced and 2
The genome sequence of cecospora spot bacterial strain BLS256 and the partial gene sequence of RS105, using the method for bioinformatics to Xoo bacterium
Analysis is compared in strain full-length genome and between Xoc full-length genomes, leaf spot bacteria MAFF311018 and Population of Xanthomonas Oryzae Pv is selected
BLS256 is analysis object, according to the diversity sequence of two strain gene groups, designs specific primer, right by Standard PCR technology
40 candidate strains comprising rice leaf spot bacteria and Population of Xanthomonas Oryzae Pv and other related strains carry out detection differentiation.Finally
The molecular labeling that can be used for distinguishing is determined as follows：The molecular labeling of rice leaf spot bacteria, its sequence such as SEQ ID
Shown in NO.1-58；The molecular labeling of xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQ ID NO.59-104；Paddy rice is white
The codominant marker of leaf spoting bacteria and xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQ ID NO.105-126；Profit
The two of Xanthomonas oryzae pv oryzae can also not only be caused a disease with unique identification Xanthomonas oryzae pv oryzae with these molecular labelings
Mutation rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola carry out further careful identification, so as to solve to reflect in the prior art
Determine the problem of accuracy difference, insecure problem in identification technology.
By the molecular labeling of above-mentioned determination, examined with reference to simple round pcr and common DNA detecting systems
Survey, be easy, quickly and accurately detect that two kinds of pathogenetic bacterias open effective way, can be for substantial amounts of sample using many
Mark cooperation detection, is conducive to improving detection reliability, meets the direction of detection technique development.
Brief description of the drawings
Fig. 1 is specific primer design principle schematic of the present invention；
Fig. 2 is rice leaf spot bacteria specific primer the result schematic diagram,
1-10 is xanthomonas oryzae pv. oryzicola in figure, is respectively RH3, JSB2-24, RS85, RS105, HNB8-47,
HCA1,HCA2,HCB1,HCC1,HGA1；
11-35:It is rice leaf spot bacteria, is respectively PXO99, PXO86, PX071, PXO61, JL691-1, PXO339a,
PXO339b,PXO339c,SC-yc-a,FuJian,GD414,HeN11,YN24,YN18,YN11,YN1,T3,T2,T1,JL691-
2,PXO349,PXO364,PXO347,Zhe173-1,Zhe173-3；
36-40:It is reference strain, is respectively Aac, Xcv, Xac, Xcc, Pst；
Fig. 3 is xanthomonas oryzae pv. oryzicola specific primer the result schematic diagram,
1-10 is xanthomonas oryzae pv. oryzicola in figure, is respectively RH3, JSB2-24, RS85, RS105, HNB8-47,
HCA1,HCA2,HCB1,HCC1,HGA1；
11-35:It is rice leaf spot bacteria, is respectively PXO99, PXO86, PX071, PXO61, JL691-1, PXO339a,
PXO339b,PXO339c,SC-yc-a,FuJian,GD414,HeN11,YN24,YN18,YN11,YN1,T3,T2,T1,JL691-
2,PXO349,PXO364,PXO347,Zhe173-1,Zhe173-3；
36-40:It is reference strain, is respectively Aac, Xcv, Xac, Xcc, Pst；
Fig. 4 be rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola codominance primer the result schematic diagram,
1-10 in figure:It is xanthomonas oryzae pv. oryzicola, is respectively RH3, JSB2-24, RS85, RS105, HNB8-47,
HCA1,HCA2,HCB1,HCC1,HGA1；
11-35:It is rice leaf spot bacteria, is respectively PXO99, PXO86, PX071, PXO61, JL691-1, PXO339a,
PXO339b,PXO339c,SC-yc-a,FuJian,GD414,HeN11,YN24,YN18,YN11,YN1,T3,T2,T1,JL691-
2,PXO349,PXO364,PXO347,Zhe173-1,Zhe173-3；
36-40:It is reference strain, is respectively Aac, Xcv, Xac, Xcc, Pst.
Specific embodiment
Further definition is of the invention in following examples, description and these embodiments, people in the art according to more than
Member can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, can be to the present invention
Make various changes and modifications, so that its applicable various uses and condition.It is of the present invention to be this in addition to special indicating
Field prior art；
Embodiment one designs the molecular labeling that can distinguish identification rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
Three microspecies KACC10331 of rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae),
Two of PXO99A, MAFF 311018 and xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola)
Microspecies BLS256, RS105, the genome sequence and relevant information of five germs are from NCBI (http://
Www.ncbi.nlm.nih.gov/) download and obtain.(table 1)
1 five kinds of data statistics of bacterial gene group information of table
Inventor compares to the full-length genome of Xoo (MAFF311018, KACC10311, PXO99A) 3 microspecies, hair
Existing sequence similarity degree is higher, chooses pattern microspecies of the MAFF311018 as water Xanthomonas oryzae pv.oryzae.
Compared with RS105 and BLS256, it is found that BLS256 contains the full detail of RS105, similarity degree is very high, therefore
With BLS256 as X. c. pv. oryzicola bacterium pattern microspecies.
Whole genome sequence is carried out to two-mode microspecies MAFF311018 and BLS256 to compare, use perl programs with BLAST
Match information and mismatch (the containing gap) unpack in result, using non-matching information design specific marker.(table
2)
The comparison result of table 2 type strain MAFF311018 and BLS256
Using in MAFF3011018 genomes with the unmatched information of BLS256, interception fragment length more than 200bp piece
Duan Bingyong eprimer3 design primer.First detect designed primer respectively in five kinds of bacterium with ePCR, then screened with Perl
The dominant molecular labeling of MAFF3011018.Similarly, the dominant molecular labeling (I in Fig. 1) of BLS256 is developed.
Using Perl programs, the both sides for mismatching section in two strains respectively intercept the similar fragments of 60bp, separately design
Trip and anti-sense primer, the amplified production being achieved in that include three parts, a complete non-matching section, two parts Matching band
Section.And then the inconsistent primer of its amplification length in two strains is screened with ePCR, as there can be polymorphism in two kinds of bacterium
Codominant marker (II in Fig. 1).The information of the relevant primer for wherein designing is as shown in table 3.
The primer relevant information of the design of table 3
The bacterial strain that the present invention is detected is for 40 plants, and specifying information is as shown in table 4
Table 4 tests rice leaf spot bacteria used, xanthomonas oryzae pv. oryzicola and other related strains
Embodiment two is to the dominant molecular marker screening of rice leaf spot bacteria specialization and checking
We have chosen according to the ePCR the selection results of the designed specific primer to rice leaf spot bacteria specialization
40 pairs of primer pairs, 25 plants of rice leaf spot bacterias and 10 plants of xanthomonas oryzae pv. oryzicolas and other related strains are verified.
Strains tested is lined on TSA solid medium flat boards, after 28 DEG C of culture 2d, picking single bacterium colony is placed in TSA liquid
Body culture medium, in 180rmin-1, 28 DEG C of shaking table cultures two days.Bacterium solution 3mL is drawn, the genome of bacterial strain is extracted with kit
DNA.And as the template of PCR reactions.
The reaction system of optimization is：The μ L of Taq enzyme 0.2 (5u μ L-1), 10 × PCR Buffer (Mg2+Free) 2 μ L, 25mM
MgCl2The μ L of 1.2 μ L, 2.5mM dNTP Mixture 1.5, take bacterium solution for the μ L of template 1, primers F (10 μM) 0.6 μ L, primer R (10
μM) 0.6 μ L, it is supplemented to 20 μ L systems with sterilizing distilled water.Response procedures are 94 DEG C of predegeneration 3min；94 DEG C are denatured 15s, 58 DEG C
Annealing 15s, 72 DEG C of extension 30s, 30 reaction cycles；Last 72 DEG C of extensions 7min.Examined with 2% TBE agarose gel electrophoresis
Survey PCR primer.
After testing, can be by 25 plants of rice leaf spot bacterias in all strains testeds we obtain 29 pairs of specific primers
In accurately identify come, the DNA fragmentation for being expanded is close with expected purpose clip size (clip size is shown in Table 5).It is such
Specific molecular marker can not amplify band (such as Fig. 2 institutes in xanthomonas oryzae pv. oryzicola and other reference strains
Show).
Sequence number of the such molecular labeling of table 5 in sequence table, the title of corresponding molecular labeling and institute's DNA amplification
The size of fragment
Embodiment three is to the dominant molecular marker screening of xanthomonas oryzae pv. oryzicola specialization and checking
EPCR the selection results according to the specific primer to xanthomonas oryzae pv. oryzicola specialization, using acquired confession
The genomic DNA of bacterial strain is tried, using PCR reaction systems and 2% TBE agaroses with the identical optimization of embodiment 2
Gel electrophoresis detection method, have chosen 40 pairs of primer pair strains testeds and verifies.
By detection, can be by 10 plants of xanthomonas oryzae pv. oryzicolas in all confessions we obtain 23 pairs of specific primers
Accurately expanded to the DNA fragmentation being consistent with target sizes in examination bacterial strain (clip size is shown in Table 6).Such specific primer is equal
Band (as shown in Figure 3) can not be amplified in rice leaf spot bacteria and other reference strains.
The title of sequence number and corresponding molecular labeling of the such molecular labeling of table 6 in sequence table
Example IV enters performing PCR examination to the codominant marker of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
Checking is bright
We are according to rice leaf spot bacteria and the ePCR of the codominant marker primer of xanthomonas oryzae pv. oryzicola
The selection result, equally, the 2 and 3 PCR detection architectures having built up and 2% TBE agarose gel electrophoresis in conjunction with the embodiments
Method, have chosen 40 pairs of primer pair strains testeds and verifies.
By detection, obtaining 11 pairs of specific primers can exactly by 10 plants of xanthomonas oryzae pv. oryzicolas and 25 plants
Rice leaf spot bacteria is distinguished and expanded to the DNA fragmentation being consistent with target sizes (clip size is shown in Table 7).Such is special
The DNA fragmentation that property molecular labeling can be amplified in rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola has significance difference
It is different, and identification (as shown in Figure 4) just can fast and accurately only be distinguished by a PCR and simple gel detection.
The title of sequence number and corresponding molecular labeling of the such molecular labeling of table 7 in sequence table
Claims (2)
1. the specific molecular marker of xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQ ID NO.59-104.
2. application of the molecular labeling described in claim 1 in xanthomonas oryzae pv. oryzicola identification and plant quarantine.
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| CN104316688B (en) * | 2014-10-16 | 2016-06-22 | 江南大学 | A kind of DASELISA immunization detecting xanthomonas oryzae pv. oryzicola |
| CN106011287B (en) * | 2016-07-27 | 2019-09-24 | 广西壮族自治区农业科学院水稻研究所 | The molecular labeling in the site rice bacterial leaf streak major gene resistance BLS1 and its application |
| CN107190096B (en) * | 2017-07-25 | 2021-05-14 | 山东农业大学 | General molecular marker primer for trees and combined development method and application thereof |
| CN112625961B (en) * | 2020-12-28 | 2022-06-28 | 上海交通大学 | Bacillus pumilus and application thereof |
| CN113186315B (en) * | 2021-05-20 | 2022-04-08 | 上海市农业技术推广服务中心 | Primer pair and detection method for detecting bacterial leaf streak germs of rice |
| CN114150077B (en) * | 2021-11-03 | 2023-05-12 | 江汉大学 | Molecular markers, primer compositions, kits and methods for identifying xanthomonas oryzae |
| CN114790484B (en) * | 2021-11-03 | 2023-12-22 | 江汉大学 | MNP (MNP) marking site of xanthomonas oryzae, primer composition, kit and application of MNP marking site |
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| CA2819547A1 (en) * | 2010-12-12 | 2012-06-21 | Pioneer Hi-Bred International, Inc. | Colonies of insects resistant to plants expressing insecticidal toxins |
| CN103333231A (en) * | 2013-05-24 | 2013-10-02 | 中国农业科学院作物科学研究所 | Plant disease-resistant protein Xa23 and its coding gene and use |
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| CN103993074B (en) | 2016-03-16 |
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| CN105063036B (en) | 2017-11-14 |
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