CN105063035A - Molecular markers of xanthomonas oryzae pv oryzae and applications thereof - Google Patents
Molecular markers of xanthomonas oryzae pv oryzae and applications thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant aetiology and plant quarantine, provides a batch of molecular markers which can specifically identify xanthomonas oryzae pv oryzae more conveniently and rapidly, and particularly provides dominant molecular markers and codominant molecular markers which identify xanthomonas oryzae pv. oryzae and xanthomonas oryzae pv. oryzicola, and applications for detecting the two pathogenic bacteria by applying the molecular markers. By adopting a method for identification through the molecular markers and combination of the molecular markers, the reliability of results is guaranteed, and diseases can be accurately identified, so that correct prevention and control measures which are in accordance with the development direction of current inspection and quarantine, and extending and spreading of the diseases can be controlled in time.
Description
The application is that in the denomination of invention of application on 04 25th, 2014, for " molecule marker of rice Xanthomonas and application thereof ", (original applying number is the divisional application that patent of invention 2014101695173) is done for applicant.
Technical field
The present invention relates to field of plant genetic, provide molecule marker and the application thereof of Xanthomonas oryzae pv oryzae, in particular, provide dominant molecule marker and the codominant marker of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola qualification, and apply the application of these Markers for Detection two kinds of pathogenetic bacterias.
Background technology
Xanthomonas oryzae pv oryzae is the Gram-negative bacteria of the yellow of a quasi-representative, bacillary pathogenic bacterium main on paddy rice, comprise rice leaf spot bacteria Xanthomonasoryzaepv.oryzae (Xoo) and xanthomonas oryzae pv. oryzicola Xanthomonasoryzaepv.oryzicola (Xoc) (Zhao, Ardalesetal.2004) .Xoo can cause the vascular bundle diseases of paddy rice to be bacterial blight of rice, and Xoc infects paddy rice by tissue space thus causes bacterial leaf streak of rice.The impact being subject to temperature and region due to bacterial blight of rice (BB) becomes one of the most serious paddy bacterial disease, is affected serious mainly Asia and portions of Africa area.But the rice varieties of high yield is more vulnerable to infecting of bacterial blight of rice (BB), therefore bacterial blight of rice (BB) often causes serious financial loss, and output reduction reaches 50%.By contrast, the loss that xanthomonas oryzae pv. oryzicola (BLS) causes is relatively little, and time serious, output reduction reaches 30%.But along with the extensive plantation of hybrid rice, paddy bacterial disease has the trend increased the weight of year by year.Therefore, contain that the development of paddy rice Micobial Disease is for stable grain-production with to improve people's living standard most important.
Along with global trade spreading in agricultural, the inspection work that rice paddy seed is imported and exported is particularly important.And China is comprised in Asia, rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola are defined as paddy rice quarantine disease.Its seed-borne fungi is propagated serious, and therefore, rapid detection and identify that pathogen is the key of quarantine accurately, simultaneously can effectively preventing disease occurs and minimizing disease is brought loss.Although rice leaf spot bacteria is different with the mode of infection of paddy bacterial germ, but owing to belonging to the bacterial leaf spot pathogenic bacteria (Xanthomonasoryzaepv.oryzae of rice Xanthomonas together, and Population of Xanthomonas Oryzae Pv (Xanthomonasoryzaepv.oryzicola Xoo), Xoc) sibship is very near, and genome sequence similarity is up to more than 90%, not easily identified differentiation in quarantine.Traditional screening and separating means are for different pvs oryzae and oryzicola of the same race, and morphological specificity is similar strain very, distinguish to get up to take time and effort.Phage technology is comparatively conventional in the groove in paddy rice Micobial Disease, but the false positive that in the false negative that easily causes of phage specialization and serological method, antiserum(antisera) quality problems easily produce, the universal of this method is restricted.In current inspection and quarantine work, employing is convenient, economic PCR molecule marker carries out related work becomes main method, various with PCR be rely on detection method in an endless stream, although as accurately sensitive in TaqMan real time fluorescent PCR method but cost intensive, be difficult to be applied in routine check quarantine.In the inspection and quarantine of paddy rice two kinds of bacterial diseases, forefathers utilize the yellow difference of protein alpha subunit of transfer transport in two kinds of pathogenic bacterias and the difference of Genes Encoding Transmembrane Proteins (AY319937, AY319941) to design dominant specific primer to carry out qualification and distinguish, and obtain certain effect.Owing to there is the exchange of genetic material to a certain degree between bacterium, single molecule marker easily causes the error in detection, and PCR experiment easily forms certain contamination probability in actually operating, there is certain false positive risk, therefore exploitation abundant, molecule marker that conservative property is high carries out multidigit point and detects the accuracy being conducive to ensureing to detect.
And along with the development of biological sequencing technologies, complete a lot of genome sequencing work.Up to the present, there is the bacterial strain of four Xanthomonas campestris to complete order-checking (comprising KACC10331, PXO99A, MAFF311018, andBLS256), and completed the sketch of rice leaf spot bacteria strain AX01947.Based on acquired information, the comparison algorithm of information biology can be utilized to study the difference between different microspecies.How to utilize these existing achievements in research to distinguish the study frontier that rice leaf spot bacteria and paddy bacterial germ become us.
Summary of the invention
The present inventor carries out whole genome sequence comparison for the research conditions of prior art based on these species sequence issued just, provide molecule marker and the application thereof of Xanthomonas oryzae pv oryzae, in particular, provide dominant molecule marker and the codominant marker of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola qualification, and apply the application of these Markers for Detection two kinds of pathogenetic bacterias.
Contriver is first according to 3 the bacterial leaf-blight bacterial strain MAFF311018 announced, KACC10311, the genome sequence of PXO99A and 2 cecospora spot bacterial strain BLS256 and the partial gene sequence of RS105, utilize the method for information biology in Xoo bacterial strain full-length genome and and Xoc full-length genome between compare of analysis, selected bacterial leaf spot pathogenic bacteria MAFF311018 and Population of Xanthomonas Oryzae Pv BLS256 is analytic target, according to the diversity sequence of two strain gene groups, design Auele Specific Primer, by Standard PCR technology, carry out detection to 40 candidate strain comprising rice leaf spot bacteria and Population of Xanthomonas Oryzae Pv and other related strain to distinguish.Finally determine the molecule marker that may be used for distinguishing as follows: the molecule marker of rice leaf spot bacteria, its sequence is as shown in SEQIDNO.1-58; The molecule marker of xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQIDNO.59-104; The codominant marker of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQIDNO.105-126; Utilize these molecule markers not only can unique identification Xanthomonas oryzae pv oryzae, further careful qualification can also be carried out to two of an Xanthomonas oryzae pv oryzae pvs oryzae and oryzicola rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola, thus identify the problem of poor accuracy in solution prior art, insecure problem in authenticate technology.
By the above-mentioned molecule marker determined, detect in conjunction with simple round pcr and common DNA detection system, for easy, detect two kinds of pathogenetic bacterias quickly and accurately and open effective way, multiple labeling cooperation detection can be adopted for a large amount of samples, be conducive to improving detecting reliability, meet the direction of detection technique development.
Accompanying drawing explanation
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,
In figure, 1-10 is xanthomonas oryzae pv. oryzicola, is RH3, JSB2-24, RS85, RS105, HNB8-47, HCA1, HCA2, HCB1, HCC1, HGA1 respectively;
11-35: be rice leaf spot bacteria is PXO99, PXO86, PX071, PXO61 respectively, 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: be reference strain is Aac, Xcv, Xac, Xcc, Pst respectively;
Fig. 3 is xanthomonas oryzae pv. oryzicola specific primer the result schematic diagram,
In figure, 1-10 is xanthomonas oryzae pv. oryzicola, is RH3, JSB2-24, RS85, RS105, HNB8-47, HCA1, HCA2, HCB1, HCC1, HGA1 respectively;
11-35: be rice leaf spot bacteria is PXO99, PXO86, PX071, PXO61 respectively, 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: be reference strain is Aac, Xcv, Xac, Xcc, Pst respectively;
Fig. 4 is rice leaf spot bacteria and Xanthomonas campestris PV.oryzicola codominance primer the result schematic diagram,
1-10 in figure: be xanthomonas oryzae pv. oryzicola is RH3, JSB2-24, RS85, RS105, HNB8-47, HCA1, HCA2, HCB1, HCC1, HGA1 respectively;
11-35: be rice leaf spot bacteria is PXO99, PXO86, PX071, PXO61 respectively, 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: be reference strain is Aac, Xcv, Xac, Xcc, Pst respectively.
Embodiment
The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art;
Embodiment one design can distinguish the molecule marker of qualification rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola
Two microspecies BLS256, RS105 of three microspecies KACC10331 of rice leaf spot bacteria (Xanthomonasoryzaepv.oryzae), PXO99A, MAFF311018 and xanthomonas oryzae pv. oryzicola (Xanthomonasoryzaepv.oryzicola), the genome sequence of five germs and relevant information are downloaded from NCBI (http://www.ncbi.nlm.nih.gov/) and are obtained.(table 1)
The data statistics of table 1 five kinds of bacterial genomes information
The full-length genome of contriver to Xoo (MAFF311018, KACC10311, PXO99A) 3 microspecies is compared, and finds that sequence similarity degree is higher, chooses the pattern microspecies of MAFF311018 as water Xanthomonas oryzae pv.oryzae.
With RS105 and BLS256 comparison, find that BLS256 contains the full detail of RS105, similarity degree is very high, therefore uses BLS256 as the pattern microspecies of X. c. pv. oryzicola bacterium.
With BLAST, whole genome sequence comparison is carried out to two-mode microspecies MAFF311018 and BLS256, by perl program the match information in result and mismatch (containing gap) unpack, utilize non-matching information design specific marker.(table 2)
The comparison result of table 2 type strain MAFF311018 and BLS256
To utilize in MAFF3011018 genome the unmatched information with BLS256, intercept fragment length and be greater than the fragment of 200bp and design primer with eprimer3.First in five kinds of bacterium, detect designed primer with ePCR respectively, then screen the dominant molecule marker of MAFF3011018 with Perl.In like manner, the dominant molecule marker (in Fig. 1 I) of BLS256 is developed.
Utilize Perl program, respectively intercept the similar fragments of 60bp in the both sides that two bacterial classifications do not mate section, design upstream and downstream primer respectively, the amplified production obtained like this comprises three parts, and a complete non-matching section, two portions mate section.And then screen with ePCR the primer that its amplification length is inconsistent in two bacterial classifications, be the codominant marker (in Fig. 1 II) that can have polymorphism in two kinds of bacterium.The information of the relevant primer wherein designed is as shown in table 3.
The primer relevant information that table 3 designs
The bacterial strain that the present invention detects is for 40 strains, and specifying information is as shown in table 4
Table 4 tests rice leaf spot bacteria used, xanthomonas oryzae pv. oryzicola and other related strain
The dominant molecular marker screening that embodiment two pairs of rice leaf spot bacterias are specially changed and checking
We are according to the designed ePCR the selection result to the Auele Specific Primer that rice leaf spot bacteria is specially changed, and have chosen 40 pairs of primer pair 25 strain rice leaf spot bacterias and 10 strain xanthomonas oryzae pv. oryzicolas and other related strain and verify.
Lined by strains tested on TSA solid medium flat board, after 28 DEG C of cultivation 2d, picking list bacterium colony, is placed in TSA liquid nutrient medium, at 180rmin
-1, 28 DEG C of shaking tables cultivate two days.Draw bacterium liquid 3mL, extract the genomic dna of bacterial strain with test kit.And as the template that PCR reacts.
The reaction system optimized is: Taq enzyme 0.2 μ L (5u μ L
-1), 10 × PCRBuffer (Mg
2+free) 2 μ L, 25mMMgCl
21.2 μ L, 2.5mMdNTPMixture1.5 μ L, getting bacterium liquid is template 1 μ L, and primers F (10 μMs) 0.6 μ L, primer R (10 μMs) 0.6 μ L, is supplemented to 20 μ L systems with sterilizing distilled water.Response procedures is 94 DEG C of denaturation 3min; 94 DEG C of sex change 15s, 58 DEG C of annealing 15s, 72 DEG C extend 30s, 30 reaction cycle; Last 72 DEG C extend 7min.TBE agarose gel electrophoresis with 2% detects PCR primer.
After testing, we obtain 29 pairs of Auele Specific Primers can identify out accurately by 25 strain rice leaf spot bacterias in all strains testeds, the DNA fragmentation increased all with expection object clip size close (clip size is in table 5).This type of specific molecular marker all can not amplify band (as shown in Figure 2) in xanthomonas oryzae pv. oryzicola and other reference strains.
The title of the sequence number of this type of molecule marker of table 5 in sequence table, corresponding molecule marker and the size of institute's amplification of DNA fragments
The dominant molecular marker screening that embodiment three pairs of xanthomonas oryzae pv. oryzicolas are specially changed and checking
According to the ePCR the selection result of the Auele Specific Primer specially changed xanthomonas oryzae pv. oryzicola, adopt the genomic dna of acquired strains tested, utilize and the PCR reaction system of the identical optimization of embodiment 2 and the TBE agarose gel electrophoresis detection method of 2%, have chosen 40 pairs of primer pair strains testeds and verify.
Through detecting, we obtain 23 pairs of Auele Specific Primers can increase to the DNA fragmentation be consistent with target sizes (clip size is in table 6) in all strains testeds accurately by 10 strain xanthomonas oryzae pv. oryzicolas.Such Auele Specific Primer all can not amplify band (as shown in Figure 3) in rice leaf spot bacteria and other reference strains.
The sequence number of this type of molecule marker of table 6 in sequence table and the title of corresponding molecule marker
The codominant marker of embodiment four pairs of rice leaf spot bacterias and xanthomonas oryzae pv. oryzicola carries out PCR test to be proved
We are according to the ePCR the selection result of the codominant marker primer of rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, equally, in conjunction with the embodiments the 2 and 3 PCR detection system set up and 2% TBE agarose gel electrophoresis method for detecting, have chosen 40 pairs of primer pair strains testeds and verify.
By detecting, obtaining 11 pairs of Auele Specific Primers can distinguish 10 strain xanthomonas oryzae pv. oryzicolas and 25 strain rice leaf spot bacterias exactly and increase to the DNA fragmentation be consistent with target sizes (clip size is in table 7).There were significant differences for the DNA fragmentation that such specific molecular marker can amplify in rice leaf spot bacteria and xanthomonas oryzae pv. oryzicola, and just can distinguish qualification (as shown in Figure 4) fast and accurately by means of only a PCR and simple gel detection.
The sequence number of this type of molecule marker of table 7 in sequence table and the title of corresponding molecule marker
Claims (2)
1. the specific molecular marker of xanthomonas oryzae pv. oryzicola, its sequence is as shown in SEQIDNO.59-104.
2. the application of molecule marker according to claim 1 in xanthomonas oryzae pv. oryzicola qualification 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 |
| 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 |
| 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 |
| CN114150077B (en) * | 2021-11-03 | 2023-05-12 | 江汉大学 | Molecular markers, primer compositions, kits and methods for identifying xanthomonas oryzae |
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| CN103993074B (en) | 2016-03-16 |
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| CN105063036A (en) | 2015-11-18 |
| CN105063035B (en) | 2017-06-27 |
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