CN103243092B - 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 ofly identify the method for fungal bacterial strain pathotype and a kind of test kit.
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 manifestation difference of the verticillium dahliae bacterial strain of different pathotype is very large, such as, can cause the serious plant disease of cotton after the strain infection cotton of some high pathotype and cause the decline of farmland output, and only causing the slight disease of cotton after the strain infection cotton of some low pathotype and less to farmland yield effect.In addition, the pathotype of verticillium dahliae also shows certain latent, and namely high pathotype bacterial strain then may be not pathogenic in infection, and seriously cause a disease on a large scale in next year.
If the main Strain type of verticillium dahliae in farmland can be detected, and its pathotype is made judge accurately, just the agricultural measure such as bound drug control and crop rotation can suppress the generation of disease, thus increase economic benefit and social benefit.
Summary of the invention
The object of the invention is the technical problem solving the pathotype how accurately judging verticillium dahliae bacterial strain, a kind of nucleic acid is provided, a kind ofly identifies the method for fungal bacterial strain pathotype and a kind of test kit.
Sequence shown in SEQ ID NO:1 be the present inventor find in the genome sequence of the verticillium dahliae bacterial strain of high pathotype occur and in the genome sequence of the verticillium dahliae of low pathotype absent variable sequence.Thus, for the verticillium dahliae bacterial strain that a certain pathotype is to be measured, as long as identify in the genomic sequence of this verticillium dahliae bacterial strain whether there is the sequence shown in SEQ ID NO:1, can know whether this verticillium dahliae bacterial strain is high pathotype verticillium dahliae.Particularly, there is not the sequence shown in SEQ ID NO:1 if identified in the genomic sequence of this verticillium dahliae bacterial strain, then 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 there is the sequence shown in SEQ ID NO:1, then 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 there is not the sequence shown in SEQ ID NO:1 in the genomic sequence of this verticillium dahliae bacterial strain of sufficient proof.Thus, the present inventor obtains 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 ID NO:1, or the sequence shown in specific sequence had 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, present invention also offers the nucleic acid with complementary nucleic acid as above.
On the other hand, present invention also offers a kind of method 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 of (1) preparation containing verticillium dahliae; (2) detect in described sample to be tested whether there is the first molecule marker, described first molecule marker is nucleic acid as above; (3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not described first molecule marker in described sample to be tested, then indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; Existence has nucleic acid or its complementary nucleic acid of the sequence shown in SEQ ID NO:1 in described sample to be tested, then the bacterial strain of this verticillium dahliae is indicated to belong to high pathotype.
On the other hand, present invention also offers a kind of test kit, described test kit contains nucleic acid as above, and PCR reagent and/or molecular hybridization reagent.
By technique scheme, the average disease index of cotton generation verticillium that causes of the verticillium dahliae of the high pathotype that the present invention identifies is 56.6; The average disease index of cotton generation verticillium that causes of the verticillium dahliae of the low pathotype that the present invention identifies is 12.0.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of 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 the specific sequence had 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) be published in public database, specifically refer to document (KlostermanSJ et.al, Comparative genomics yields insights into niche adaptation of plant vascular wiltpathogens.PLoS Pathog.2011Jul; 7 (7): e1002137.Epub 2011Jul 28.) in the whole genome sequence of verticillium dahliae (Verticillium dahliae) announced in the database (http://www.broadinstitute.org) mentioned.
Wherein, specific sequence in sequence shown in SEQ ID NO:1 based on the whole genome sequence of the sequence shown in SEQ ID NO:1 and verticillium dahliae (Verticillium dahliae), can be obtained by sequence alignment (blast) method of this area routine.Such as, those skilled in the art can rely on published computer program (business-like and free, such as NCBI-PRIMERBLAST), 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 carried out comparison 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 SEQ ID NO:1 described in the present invention exists for the identification of the sequence shown in SEQID NO:1, the not special requirement of length of the specific sequence therefore in the sequence shown in SEQ ID NO:1, those skilled in the art can carry out conventional selection according to selected detection method.Such as, the length of the specific sequence in the sequence shown in described SEQ ID NO:1 can be 15-5000bp.
Wherein, in order to amplified target (amplified target) sequence making the specific sequence in the sequence shown in the sequence shown in SEQ ID NO:1 can identify the sequence shown in SEQ ID NO:1 as polymerase chain reaction (PCR) method, under preferable case, the length of the specific sequence in the sequence shown in sequence shown in described SEQ ID NO:1 is 100-5000bp, be more preferably 100-1000bp, more preferably 100-500bp, is further preferably 150-250bp.
Wherein, in order to primer (primer) sequence making the specific sequence in the sequence shown in the sequence shown in SEQ ID NO:1 can identify the sequence shown in SEQ ID NO:1 as polymerase chain reaction (PCR) method, or as probe (probe) sequence of the sequence shown in nucleic acid hybridization (as Southern Blot) method qualification SEQ ID NO:1, under preferable case, the length of the specific sequence in the sequence shown in sequence shown in described SEQ ID NO:1 is 15-99bp, be more preferably 20-50bp, more preferably 20-40bp, further be preferably 22-35bp.
Present invention also offers the nucleic acid with complementary nucleic acid 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 modification to various nucleic acid as above, and the nucleic acid after modification also belongs to scope of the present invention.
Present invention also offers a kind of method identifying fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae, and the method comprises the following steps: the sample to be tested of the complete genome DNA of (1) preparation containing verticillium dahliae; (2) detect in described sample to be tested whether there is the first molecule marker, described first molecule marker is nucleic acid as above; (3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not described first molecule marker in described sample to be tested, then indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; Existence has nucleic acid or its complementary nucleic acid of the sequence shown in SEQ ID NO:1 in described sample to be tested, then the bacterial strain of this verticillium dahliae is indicated to belong to high pathotype.
Wherein, the step preparing sample to be tested can be carried out according to the method for the preparation of the sample detecting nucleic acid of this area routine, the not special requirement of the present invention, such as can gather tissue from cotton plants, and cultivate on the substratum being organized in containing resistance gathered, and screen, distinguish and be separated the bacterium colony of verticillium dahliae, then according to (" plant genetic engineering " (what light source work, Science Press, within 2007, publish)) described in method extract the genomic dna of verticillium dahliae, can sample to be tested be prepared.
Wherein, detecting the method that whether there is the first molecule marker in described sample to be tested can be the method for the detection nucleic acid of this area routine, the present invention does not have special requirement, such as, can be at least one in sequencing (as bi-deoxyribose Nucleotide stops 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 in described sample to be tested the nucleic acid that whether there is described first molecule marker or have the sequence shown in SEQ ID NO:1 and can carry out according to the method for this area routine and standard, such as by arranging outer ginseng and/or internal reference carries out Parallel testing, and can be judged further by Parallel testing result.
On the other hand, present invention also offers a kind of test kit, described test kit contains nucleic acid as above, and PCR reagent and/or molecular hybridization reagent.
Below will be described the present invention by embodiment.In following examples, the bacterial strain of various verticillium dahliae belongs to the genetic resources obtained through legitimate channels.
It should be noted that, in sequence shown in SEQ ID NO:1, occur that multiple (n is a by continuous print n, c, g, or the t) fragment that forms, this fragment represents 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 illustration of the preparation of sample to be tested.Particularly, be the preparation of genomic dna from 80 different verticillium dahliae bacterial strains (being numbered VDG1 to VDG80).
Cut cotton plants is about 10-35cm place stem from earth's surface, be cut into 1mm
3the fritter of left and right size, be seeded in containing microbiotic (penbritin, Rifampin and Streptomycin sulphate, concentration is 100 μ g/ml) PDA substratum (containing potato 200g/L, glucose 20g/L, agar 20g/L) on, cultivate 7 days at 25 DEG C, differentiate the bacterium colony of verticillium dahliae according to the Standard Plate form of verticillium dahliae.Obtain the bacterium colony of the verticillium dahliae differentiated namely as verticillium dahliae bacterial strain sample.
According to the method described above, 80 different verticillium dahliae bacterial strains are obtained from 80 different cotton plants, by above-mentioned 80 different verticillium dahliae bacterial strains according to " plant genetic engineering " (what light source work, Science Press, 2007 publish) described in method, extract genomic dna sample respectively, obtain sample to be tested 1-80.
Embodiment 2
The present embodiment is for illustration of 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 of bacterium liquid method qualification cotton variety (being). Cotton, 2010,37 (12): 15-17) vermiculite sandy soil bottomless bowl of paper in quantitatively dips in bacterium liquid method, and the verticillium dahliae bacterial strain different to 80 described in embodiment 1 carries out cotton in seedling stage Pathotypes.The height of characterizing pathogenetic type is carried out with the disease index of described 80 different verticillium dahliae bacterial strains after the healthy cotton plants of inoculation.
Particularly, verticillium dahliae is being looked into the Bick substratum (NaNO of 2g/L
3, the K of 1g/L
2hPO
4, the MgSO of the KCl of 0.5g/L, 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, obtain spore suspension with spore under aseptic washing, and the spore concentration in spore suspension be adjusted to 5 × 10
6individual/ml.Then, sow and inoculate when the cotton seedling of paper pot has at least a slice true leaf to launch, bottom paper an official document or note, inject 10mL spore suspension, paper pot is put into paper an official document or note, inoculate more than cotton seedling 30 strain.4 weeks incidences after inoculation, investigation employing 5 grades of stagings, grade scale: 0 grade, is good for 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, whole blade performance symptom, leaf abscission time serious, the top heart is withered.
According to investigation result, calculate disease index, formula is as follows:
The present embodiment detects the pathotype obtaining above-mentioned 80 different verticillium dahliae bacterial strains, by above-mentioned judging standard, identify the disease index of wherein 61 verticillium dahliae bacterial strains 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 belonging 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 not belonging 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 obtains the whole genome sequence of above-mentioned 2 different verticillium dahliae bacterial strains, pass through sequence alignment, identify containing the sequence shown in SEQ ID NO:1 in the whole genome sequence of VDG1, and not containing the sequence shown in SEQ ID NO:1 in the whole genome sequence of VDG2.
By above-mentioned identical method, 61 of identifying 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, further, there is not the sequence shown in described SEQ ID NO:1 in the genomic sequence of other 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).
Result according to embodiment 2 and embodiment 3 can be determined, there is not the sequence shown in SEQ ID NO:1 if identified in the genomic sequence of verticillium dahliae bacterial strain, then this verticillium dahliae bacterial strain does not belong to high pathotype; If identify in the genomic sequence of verticillium dahliae bacterial strain and there is the sequence shown in SEQ ID NO:1, then 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 IDNO: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 as the specific sequence in the sequence shown in SEQ IDNO:1 of the present invention:
Specific sequence in sequence shown in table 1SEQ ID NO:1
Embodiment 5
There is the primer of sequence shown in SEQ ID NO:26 and 27, with the specific sequence (position in SEQ ID NO:1 is for 97099-97612) of numbering 13 in the sequence shown in described SEQ ID NO:1 that increases from the customization synthesis of Beijing combining unit company of Sangon Biotech (Shanghai) Co., Ltd..
When 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 illustrates: 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 specific sequence (position in SEQ ID NO:1 is 97099-97612) 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 these verticillium dahliae bacterial strains do not belong to high pathotype accordingly.
After in use table 1, the primer pair of numbering 1-12 detects according to as above identical method respectively, judged result is completely the same, 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 the sequence shown in specific sequence do not existed in the sequence shown in described SEQ ID NO:1, then this verticillium dahliae bacterial strain does not belong to high pathotype.
Claims (4)
1. a nucleic acid, this nucleic acid is the nucleic acid shown in SEQ ID NO:1, or 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, the specific sequence in the sequence shown in described SEQ ID NO:1 for the position in SEQ ID NO:1 be the sequence shown in 97099-97612.
2. a nucleic acid, this nucleic acid and nucleic acid complete complementary according to 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 of preparation containing verticillium dahliae;
(2) detect in described sample to be tested whether there is the first molecule marker, described first molecule marker is nucleic acid according to claim 1 and 2;
(3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not described first molecule marker in described sample to be tested, then indicate the bacterial strain of described verticillium dahliae not belong to high pathotype; Existence has nucleic acid or its complete complementary nucleic acid of the sequence shown in SEQ ID NO:1 in described sample to be tested, then the bacterial strain of this verticillium dahliae is indicated 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 |
| 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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| CN102212121A (en) * | 2010-04-21 | 2011-10-12 | 中国科学院微生物研究所 | Pathogenic related protein VdGRP1 from verticillium dahliae kleb and coded gene thereof |
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