CN103243093B - Nucleic acid, method for identifying pathotype of fungus strain and kit - Google Patents
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Abstract
The invention provides a nucleic acid. The nucleic acid has the sequences shown in SEQ ID NO:1 or has specific sequences in the sequences shown in SEQ ID NO:1. The invention also provides a nucleic acid complementary with the above nucleic acid. The invention also provides a method for identifying the pathotype of a fungus strain. The fungus strain is verticillium dahliae. The method comprises the following steps of: (1) preparing a sample to be detected of complete genome DNA containing verticillium dahliae; (2) detecting whether a first molecular marker exists in the sample to be detected, wherein the first molecular marker is the above nucleic acid; and (3) judging the pathotype of verticillium dahliae according to the detection result in the step (2). The invention also provides a kit.
Description
Technical field
The present invention relates to crop disease control field, particularly, relate to a kind of nucleic acid, a kind of method and a kind of test kit of identifying fungal bacterial strain pathotype.
Background technology
Verticillium dahliae (Verticillium dahliae) is a kind of phytopathogenic fungi, and it can cause plant (particularly cotton) that verticillium occurs.But, the pathogenic performance difference of the verticillium dahliae bacterial strain of different pathotypes is very large, for example after the strain infection cotton of some high pathotype, can cause the serious plant disease of cotton and cause the decline of farmland output, and after the strain infection cotton of some low pathotype, only causing the slight disease of cotton and less to farmland yield effect.In addition, the pathotype of verticillium dahliae also shows certain latent, and high pathotype bacterial strain then may be not pathogenic in infection, and serious pathogenic on a large scale in next year.
If can detect the main Strain type of verticillium dahliae in farmland, and its pathotype is made accurately to judgement, just can bound drug control and the generation of the agricultural measure inhibition disease such as crop rotation, thus increase economic benefit and social benefit.
Summary of the invention
The object of the invention is to solve the technical problem that how accurately to judge the pathotype of verticillium dahliae bacterial strain, a kind of nucleic acid, a kind of method and a kind of test kit of identifying fungal bacterial strain pathotype are provided.
Sequence shown in SEQ ID NO:1 is appearance absent variable sequence in the genome sequence of the verticillium dahliae of low pathotype in the genome sequence of the verticillium dahliae bacterial strain at high pathotype of inventor's discovery.Thereby, for a certain pathotype verticillium dahliae bacterial strain to be measured, as long as whether there is the sequence shown in SEQ ID NO:1 in the genomic sequence of this verticillium dahliae bacterial strain of evaluation, can know whether this verticillium dahliae bacterial strain is high pathotype verticillium dahliae.Particularly, if identified in the genomic sequence of this verticillium dahliae bacterial strain, there is not the sequence shown in SEQ ID NO:1, judge that this verticillium dahliae bacterial strain does not belong to high pathotype; If identify in the genomic sequence of this verticillium dahliae bacterial strain and have the sequence shown in SEQ ID NO:1, judge that this verticillium dahliae bacterial strain belongs to high pathotype.And, if there is not the specific sequence in the sequence shown in SEQ ID NO:1 in the genomic sequence of this verticillium dahliae bacterial strain, just can fully prove in the genomic sequence of this verticillium dahliae bacterial strain and not have the sequence shown in SEQ ID NO:1.Thus, the present inventor has obtained technical scheme of the present invention.
To achieve these goals, on the one hand, the invention provides a kind of nucleic acid, this nucleic acid has the sequence shown in SEQ IDNO:1, or there is the sequence shown in the specific sequence in the sequence shown in SEQ ID NO:1, and the specific sequence in the sequence shown in described SEQ ID NO:1 exists only in the sequence shown in SEQ ID NO:1 within the scope of the whole genome sequence of verticillium dahliae (Verticillium dahliae).
On the other hand, the present invention also provides the nucleic acid with nucleic acid complementation as above.
On the other hand, the present invention also provides a kind of method of identifying fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae (Verticillium dahliae), and the method comprises the following steps: the sample to be tested of the complete genome DNA that (1) preparation contains verticillium dahliae; (2) detect in described sample to be tested whether have the first molecule marker, described the first molecule marker is nucleic acid as above; (3) according to the pathotype of the detected result judgement verticillium dahliae bacterial strain of step (2), the in the situation that of there is not described the first molecule marker in described sample to be tested, indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; In the situation that exist and there is the nucleic acid of the sequence shown in SEQ ID NO:1 or its complementary nucleic acid in described sample to be tested, indicate the bacterial strain of this verticillium dahliae to belong to high pathotype.
On the other hand, the present invention also provides a kind of test kit, and described test kit contains nucleic acid as above, and PCR reagent and/or molecular hybridization reagent.
By technique scheme, the average disease index that causes cotton generation verticillium of the verticillium dahliae of the high pathotype that the present invention identifies is 56.6; The average disease index that causes cotton generation verticillium of the verticillium dahliae of the low pathotype that the present invention identifies is 12.0.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of nucleic acid, this nucleic acid has the sequence shown in SEQ ID NO:1, or there is the specific sequence in the sequence shown in SEQ ID NO:1, and the specific sequence in the sequence shown in described SEQ ID NO:1 exists only in the sequence shown in SEQ ID NO:1 within the scope of the whole genome sequence of verticillium dahliae (Verticillium dahliae).
Wherein, term " whole genome sequence of verticillium dahliae (Verticillium dahliae) " refers to the whole genome sequence of the verticillium dahliae (Verticillium dahliae) being published in public database, specifically refer to document (Klosterman SJ et.al, Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.PLoS Pathog.2011Jul; 7 (7): the whole genome sequence of the verticillium dahliae (Verticillium dahliae) of announcing in the database (http://www.broadinstitute.org) of mentioning e1002137.Epub 2011 Jul 28.).
Wherein, specific sequence in sequence shown in SEQ ID NO:1 can be based on shown in SEQ ID NO:1 sequence and the whole genome sequence of verticillium dahliae (Verticillium dahliae), sequence alignment (blast) method by this area routine obtains.For example, those skilled in the art can rely on published computer program, and (business-like and free all can, NCBI-PRIMERBLAST for example), the all possible fragment of the sequence shown in exhaustive SEQ ID NO:1, then the whole genome sequence of itself and verticillium dahliae (Verticillium dahliae) is compared one by one, if this fragment only comes across in the sequence shown in SEQ ID NO:1, and do not come across in the sequence beyond the sequence shown in SEQ ID NO:1, this fragment is the specific sequence in the sequence shown in SEQ ID NO:1.
Wherein, whether the specific sequence in the sequence shown in the SEQ ID NO:1 described in the present invention is to exist for the identification of the sequence shown in SEQ ID NO:1, therefore the length of the specific sequence in the sequence shown in SEQ ID NO:1 does not have special requirement, and those skilled in the art can carry out conventional selection according to selected detection method.For example, the length of the specific sequence in the sequence shown in described SEQ ID NO:1 can be 15-5000bp.
Wherein, in order to make the specific sequence in the sequence shown in the sequence shown in SEQ ID NO:1 can be used as amplified target (amplified target) sequence that polymerase chain reaction (PCR) method is identified the sequence shown in SEQ ID NO:1, under preferable case, the length of the specific sequence in the sequence shown in the sequence shown in described SEQ ID NO:1 is 100-5000bp, 100-1000bp more preferably, more preferably 100-500bp, is further preferably 150-250bp.
Wherein, in order to make the specific sequence in the sequence shown in the sequence shown in SEQ ID NO:1 can be used as primer (primer) sequence that polymerase chain reaction (PCR) method is identified the sequence shown in SEQ ID NO:1, or as nucleic acid hybridization (as Southern Blot) method, identify probe (probe) sequence of the sequence as shown in SEQ ID NO:1, under preferable case, the length of the specific sequence in the sequence shown in the sequence shown in described SEQ ID NO:1 is 15-99bp, 18-50bp more preferably, 20-40bp more preferably, further be preferably 22-35bp.
The present invention also provides the nucleic acid with nucleic acid complementation as above.
It should be noted that, in the present invention's various nucleic acid as above, except base sequence, all, without special requirement, those skilled in the art can carry out various modifications to various nucleic acid as above, and the nucleic acid after modification also belongs to scope of the present invention.
The present invention also provides a kind of method of identifying fungal bacterial strain pathotype, and described fungal bacterial strain is verticillium dahliae, and the method comprises the following steps: the sample to be tested of the complete genome DNA that (1) preparation contains verticillium dahliae; (2) detect in described sample to be tested whether have the first molecule marker, described the first molecule marker is nucleic acid as above; (3) according to the pathotype of the detected result judgement verticillium dahliae bacterial strain of step (2), the in the situation that of there is not described the first molecule marker in described sample to be tested, indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; In the situation that exist and there is the nucleic acid of the sequence shown in SEQ ID NO:1 or its complementary nucleic acid in described sample to be tested, indicate the bacterial strain of this verticillium dahliae to belong to high pathotype.
Wherein, preparing the step of sample to be tested can carry out according to the method for the preparation of detecting the sample of nucleic acid of this area routine, the present invention does not have special requirement, for example can gather tissue from cotton plants, and being organized on the substratum that contains resistance of gathering cultivated, and screening, distinguish the bacterium colony with separated verticillium dahliae, then according to (< < plant genetic engineering > > (what light source work, Science Press, publication in 2007)) method described in is extracted the genomic dna of verticillium dahliae, can prepare sample to be tested.
Wherein, detecting and in described sample to be tested, whether have the method for the first molecule marker can be the method for the detection nucleic acid of this area routine, the present invention does not have special requirement, for example, can be at least one in sequencing (as bi-deoxyribose Nucleotide is ended sequencing and chip sequencing), PCR method (as real-time quantitative PCR method) and nucleic acid hybridization (as Southern Blot method).
Wherein, judge that described detected result shows that the nucleic acid that whether has described the first molecule marker in described sample to be tested or have the sequence shown in SEQ ID NO:1 can carry out according to the method for this area routine and standard, for example can carry out Parallel testing by outer ginseng and/or internal reference are set, and further judge by Parallel testing result.
On the other hand, the present invention also provides a kind of test kit, and described test kit contains nucleic acid as above, and PCR reagent and/or molecular hybridization reagent.
Below will describe the present invention by embodiment.In following examples, the bacterial strain of various verticillium dahliaes belongs to the genetic resources obtaining through legitimate channels.
It should be noted that, in sequence shown in SEQ ID NO:1, occur that a plurality of (n is a by continuous n, c, g, or t) fragment forming, this fragment is illustrated in the base sequence that can freely change in different verticillium dahliae bacterial strains, and it does not affect the judgement of the pathotype of verticillium dahliae bacterial strain.
Embodiment 1
The present embodiment is for illustrating the preparation of sample to be tested.Particularly, be the preparation from the genomic dna of 80 different verticillium dahliae bacterial strains (being numbered VDG1 to VDG80).
The stem that cuts cotton plants about 10-35cm place from earth's surface, is cut into 1mm
3the fritter of left and right size, be seeded in and containing microbiotic (penbritin, Rifampin and Streptomycin sulphate, concentration is 100 μ g/ml) PDA substratum (contain potato 200g/L, glucose 20g/L, agar 20g/L) on, at 25 ℃, cultivate 7 days, according to the standard colonial morphology of verticillium dahliae, differentiate the bacterium colony of verticillium dahliae.Obtain the bacterium colony of the verticillium dahliae differentiated as verticillium dahliae bacterial strain sample.
According to the method described above, from 80 different cotton plants, obtain 80 different verticillium dahliae bacterial strains, by above-mentioned 80 different verticillium dahliae bacterial strains, according to < < plant genetic engineering > >, (what light source is outstanding, Science Press, publication in 2007) method described in, extract respectively genomic dna sample, obtain sample to be tested 1-80.
Embodiment 2
The present embodiment is for illustrating the detected result of the pathotype of above-mentioned 80 different verticillium dahliae bacterial strains.
Reference literature (Zhu Heqin, Feng Zili, Li Zhifang, Zhao Lihong, Shi Yongqiang. vermiculite sandy soil bottomless bowl of paper quantitatively dips in the greensickness-resistance that bacterium liquid method is identified cotton variety (being). Cotton, 2010,37 (12): the vermiculite sandy soil bottomless bowl of paper 15-17) quantitatively dips in bacterium liquid method, the verticillium dahliae bacterial strains different to 80 described in embodiment 1 carry out cotton in seedling stage Pathotypes.Disease index with described 80 different verticillium dahliae bacterial strains after the healthy cotton plants of inoculation characterizes the height of pathotype.
Particularly, verticillium dahliae is being looked into the Bick substratum (NaNO of 2g/L
3, the K of 1g/L
2hPO
4, the KCl of 0.5g/L, the MgSO of 0.5g/L
4, the FeSO of 0.01g/L
4, the sucrose of 30g/L, the agar of 20g/L) on be cultured to generation spore, with spore under aseptic washing, obtain spore suspension, and the spore concentration in spore suspension be adjusted to 5 * 10
6individual/ml.Then, sowing inoculation when the cotton seedling of paper pot has at least a slice true leaf to launch, injects 10mL spore suspension to paper an official document or note bottom, paper pot is put into paper an official document or note, more than inoculating cotton seedling 30 strains.4 weeks incidences after inoculation, investigation adopts 5 grades of stagings, grade scale: 0 grade, strong seedling, without symptom; 1 grade, 1-2 sheet cotyledon performance symptom, cotyledons turn yellow, the not aobvious symptom of true leaf; 2 grades, cotyledon and 1 true leaf performance symptom; 3 grades, 2 true leaf performance symptom; 4 grades, all blade shows symptom, leaf abscission when serious, and the top heart is withered.
According to investigation result, calculate disease index, formula is as follows:
The present embodiment detects the pathotype that has obtained above-mentioned 80 different verticillium dahliae bacterial strains, by above-mentioned judging standard, identify the disease index of 61 verticillium dahliae bacterial strains wherein all higher than 35, belong to high pathotype, and the disease index of other 19 verticillium dahliae bacterial strains is all lower than 20, do not belong to high pathotype.
Above-mentioned 61 verticillium dahliae bacterial strains that belong to high pathotype are respectively: VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG68, VDG6, VDG52, VDG34, VDG7, VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, VDG17, VDG54, VDG39, VDG75, VDG20, VDG13, VDG22, VDG38, VDG19, VDG28, VDG14, VDG9, VDG53, VDG15, VDG42, VDG40 and VDG1.Above-mentioned 19 verticillium dahliae bacterial strains that do not belong to high pathotype are respectively: VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51.
Embodiment 3
According to the method for regulation in the specification sheets (Solexa technology) of the sequenator Genome Analyzer of Illumina company, measure the whole genome sequence of verticillium dahliae bacterial strain (being numbered VDG1 and VDG2).
The present embodiment has obtained the whole genome sequence of above-mentioned 2 different verticillium dahliae bacterial strains, pass through sequence alignment, identify in the whole genome sequence of VDG1 and contain the sequence shown in SEQ ID NO:1, and in the whole genome sequence of VDG2, do not contain the sequence shown in SEQ ID NO:1.
By above-mentioned identical method, 61 that identify in above-mentioned 80 different verticillium dahliae bacterial strains (are respectively VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG68, VDG6, VDG52, VDG34, VDG7, VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, VDG17, VDG54, VDG39, VDG75, VDG20, VDG13, VDG22, VDG38, VDG19, VDG28, VDG14, VDG9, VDG53, VDG15, VDG42, VDG40 and VDG1) genomic sequence in all there is the sequence shown in SEQ ID NO:1, and there is not the sequence shown in described SEQ ID NO:1 in 19 verticillium dahliae bacterial strains (being respectively VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51) in addition.
According to the result of embodiment 2 and embodiment 3, can determine, if identified in the genomic sequence of verticillium dahliae bacterial strain, do not have the sequence shown in SEQ ID NO:1, this verticillium dahliae bacterial strain does not belong to high pathotype; If identify in the genomic sequence of verticillium dahliae bacterial strain and have the sequence shown in SEQ ID NO:1, this verticillium dahliae bacterial strain belongs to high pathotype.
Embodiment 4
The present embodiment illustrates the specific sequence in the sequence shown in SEQ ID NO:1, and the specific sequence in the sequence shown in described SEQ ID NO:1 exists only in the sequence shown in SEQ ID NO:1 within the scope of the whole genome sequence of verticillium dahliae (Verticillium dahliae).
Through PCR and molecular hybridization experimental verification, fragment as shown in table 1 all can be used as the specific sequence in the sequence shown in SEQ ID NO:1 of the present invention:
Specific sequence in sequence shown in table 1SEQ ID NO:1
Embodiment 5
The synthetic primer with sequence shown in SEQ ID NO:12 and 13 of company of synthetic portion customization from Sangon Biotech (Shanghai) Co., Ltd. Beijing, take increases numbers 6 specific sequence (position in SEQ ID NO:1 is 53297-54736) in the sequence shown in described SEQ ID NO:1.
In the situation that experimental implementation person does not know each self-corresponding pathotype of sample to be tested 1-80 that embodiment 1 obtains, use above-mentioned primer pair, using sample to be tested 1-80 as template, carry out the detection of PCR method.
Detected result explanation: in above-mentioned 80 different verticillium dahliae bacterial strains, 19 verticillium dahliae bacterial strains (are respectively VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51) there is not the sequence shown in the specific sequence (position in SEQ ID NO:1 is 53297-54736) in the sequence shown in described SEQ ID NO:1, prove that these verticillium dahliae bacterial strains all do not exist the nucleic acid with the sequence shown in SEQ ID NO:1, judge that accordingly these verticillium dahliae bacterial strains do not belong to high pathotype.
After the primer pair of numbering 1-5 in use table 1 detects according to as above identical method respectively, judged result is in full accord, does not repeat them here.
According to the result of embodiment 2, the judged result of known embodiment 5 is entirely true, can determine thus, if identify in the genomic sequence of verticillium dahliae bacterial strain and do not have the sequence shown in the specific sequence in the sequence shown in described SEQ ID NO:1, this verticillium dahliae bacterial strain does not belong to high pathotype.
Claims (4)
1. a nucleic acid, the nucleotide sequence of this nucleic acid is as shown in SEQ ID NO:1, or the nucleotides sequence of this nucleic acid is classified the specific sequence in the sequence shown in SEQ ID NO:1 as, and the specific sequence in the sequence shown in described SEQ ID NO:1 exists only in the sequence shown in SEQ ID NO:1 within the scope of the whole genome sequence of verticillium dahliae (Verticillium dahliae);
Wherein, the specific sequence in the sequence shown in described SEQ ID NO:1 is that the position in SEQ ID NO:1 is the sequence shown in 53297-54736.
2. a nucleic acid, this nucleic acid and nucleic acid complete complementary claimed in claim 1.
3. identify a method for fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae (Verticillium dahliae), and the method comprises the following steps:
(1) sample to be tested of the complete genome DNA that preparation contains verticillium dahliae;
(2) detect in described sample to be tested whether have the first molecule marker, described the first molecule marker is nucleic acid according to claim 1 and 2;
(3) according to the pathotype of the detected result judgement verticillium dahliae bacterial strain of step (2), the in the situation that of there is not described the first molecule marker in described sample to be tested, indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; In the situation that there is the nucleic acid of nucleotide sequence as shown in SEQ ID NO:1 or its complete complementary nucleic acid in described sample to be tested, indicate the bacterial strain of this verticillium dahliae to belong to high pathotype.
4. a test kit, described test kit contains nucleic acid according to claim 1 and 2, and PCR reagent and/or molecular hybridization reagent.
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| CN103525833B (en) * | 2013-10-30 | 2015-07-15 | 中国农业科学院农产品加工研究所 | Gene, method used for identifying pathogenicity of Verticillium dahliae and kit |
| CN103525832B (en) * | 2013-10-30 | 2016-01-20 | 中国农业科学院农产品加工研究所 | The test kit of qualification verticillium dahliae pathotype and gene and method thereof |
| CN103525831B (en) * | 2013-10-30 | 2016-01-13 | 中国农业科学院农产品加工研究所 | Specific gene is utilized to identify method and the application thereof of high pathotype verticillium dahliae |
| ES2535577B1 (en) * | 2013-11-08 | 2016-02-26 | Universidad De Jaén | METHOD FOR THE DIAGNOSIS OF VERTICILOSIS IN THE OLIVE TREE |
| CN103602676B (en) * | 2013-11-20 | 2015-08-05 | 中国农业科学院农产品加工研究所 | The method of nucleic acid and primer pair and qualification verticillium dahliae pathotype and test kit |
| CN103571834B (en) * | 2013-11-20 | 2016-04-27 | 中国农业科学院农产品加工研究所 | The method of qualification verticillium dahliae pathotype and test kit and the nucleic acid contained and primer pair |
| CN103602677B (en) * | 2013-11-20 | 2015-08-05 | 中国农业科学院农产品加工研究所 | A kind of identify the weak pathotype of verticillium dahliae nucleic acid and primer pair and test kit |
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