CN109652503A - A kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup - Google Patents
A kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup Download PDFInfo
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Abstract
The present invention relates to a kind of PCR-RFLP discrimination methods of Sugarcane white leaf phytoplasma difference subgroup.Using Sugarcane white leaf phytoplasma tuf gene as object designs specific primer, tuf genetic fragment is obtained by PCR amplification, then digestion is carried out to the tuf genetic fragment that amplification obtains using restriction endonuclease Msp I, carries out the differentiation of different subgroups to Sugarcane white leaf phytoplasma by the RFLP restriction enzyme mapping of otherness.The present invention can quickly, accurately and efficiently identify Sugarcane white leaf phytoplasma 16SrXI-B subgroup and Sugarcane white leaf phytoplasma 16SrXI-D subgroup without sequencing, be of great significance to the Pathogen identification and prevention and control of Sugarcane white leaf.
Description
Technical field
The invention belongs to technical field of plant protection, and in particular to a kind of Sugarcane white leaf phytoplasma 16SrXI-B subgroup and
The PCR-RFLP discrimination method of 16SrXI-D subgroup.
Background technique
Sugarcane white leaf is the sugarcane destructive disease as caused by phytoplasma, can cause sugarcane and the huge warp of sugaring industry
Ji loss.The disease in 1954 for the first time Thailand find, betided extensively India, Pakistan, Sri Lanka, Laos,
Sugarcane country is planted by Burma, Vietnam, Philippine and Australia etc..It is white that Yunnan Province of China Baoshan sugarcane district in 2012 detects discovery sugarcane for the first time
Leaf disease phytoplasma, Sugarcane white leaf spreads rapidly sprawling in Yunnan Sugarcane Area later, Lincang, Pu'er sugarcane district has been expanded to, to cloud
Southern sugar industry, which produces, to be seriously threatened.Previously showed to cause the phytoplasma of Sugarcane white leaf can be with the Study on Diversity of cause of disease
It is divided into 2 subgroups, i.e. 16SrXI-B subgroup and 16SrXI-D subgroup, wherein 16SrXI-D subgroup is newfound subgroup.Yunnan
Lincang sugarcane district and the Sugarcane white leaf phytoplasma of Baoshan sugarcane district discovery are belonging respectively to 16SrXI-B subgroup and 16SrXI-D subgroup.
Currently, the differentiation of 16SrXI-B subgroup and 16SrXI-D subgroup is designed by using phytoplasma 16S rRNA gene
Universal primer (P1/P7 and R16F2n/R16R2) carry out nest-type PRC, virtual RFLP digestion then is carried out to nest-type PRC sequencing
To identify, this method needs to carry out product sequencing analysis for analysis, time-consuming and laborious, in addition, the mistake generated in sequencing procedure is right
The accuracy of virtual RFLP restriction analysis result can also have an impact.It not yet establishes at present and carries out phytoplasma using practical digestion
The method of 16SrXI-B subgroup and 16SrXI-D subgroup identification.Establish a kind of quick, sensitive, accurate differentiation Sugarcane white leaf plant
The method of substance difference subgroup is of great significance to the Pathogen identification of Sugarcane white leaf and prevention and control.
Summary of the invention
Lack the technical issues of simply and quickly identifying Sugarcane white leaf phytoplasma difference subgroup to solve the prior art,
The present invention provides a kind of identification Sugarcane white leaf phytoplasma 16SrXI-B subgroup and 16SrXI-D subgroup quickly, accurate, easy
Method.
The technical scheme adopted by the invention is as follows:
A kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup, includes the following steps:
(1) using the genomic DNA of Sugarcane smut to be identified as template, PCR expansion is carried out using tuf gene-specific primer
Increase, the tuf gene-specific primer is made of tuf-F primer and tuf-R primer, the nucleotide sequence of tuf-F primer such as SEQ
Shown in ID NO:1, the nucleotide sequence of tuf-R primer is as shown in SEQ ID NO:2；
(2) pcr amplification product digestion of the restriction endonuclease Msp I to step (1) is used；
(3) result differentiates, digestion products is taken to carry out agarose gel electrophoresis, and restriction enzyme digestion and electrophoresis map is shown by Msp I digestion
For two bands, one is 124bp, another be 344bp Sugarcane smut cause of disease to be identified be Sugarcane white leaf phytoplasma
16SrXI-B subgroup；It is not digested, i.e., the Sugarcane smut cause of disease to be identified of only 1 468bp size strip is that sugarcane is white
Leaf disease phytoplasma 16SrXI-D subgroup.
Further, the reaction condition of PCR amplification described in step (1) are as follows: 25 μ L PCR reaction systems, wherein sterilizing
ddH215.3 μ L, 10 × PCR buffer of O, 2.5 μ L, 25mmol/L MgCl22.0 μ L, 10mmol/L dNTPs 2.0 μ L, 20 μ
Mol/L tuf-F primer 1.0 μ L, 20 μm of 1.0 μ L, 5U/ μ L Taq archaeal dna polymerase of ol/L tuf-R primer, 0.2 μ L, template DNA
1.0μL；Response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 15s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed
Ring；Last 72 DEG C of extensions 7min；
Further, the enzyme cutting reaction condition in step (2) are as follows: 25 μ L endonuclease reaction systems, wherein 15 μ L of PCR product goes out
Bacterium ddH27.5 μ L, 10 × buffer buffer of O, 1.5 μ L, 20000U restriction endonuclease Msp I, 1.0 μ L.Reaction interval
Sequence are as follows: 37 DEG C of 30min；65℃10min；
Further, taking digestion products to carry out agarose gel electrophoresis in step (3) is to take 10 μ L digestion products, 3% agar
Sugared gel carries out electrophoresis.
The present invention also provides a kind of based on PCR-RFLP method identify Sugarcane white leaf phytoplasma difference subgroup kit,
The kit contains tuf gene-specific primer, restriction endonuclease Msp I or its isoschizomers, the tuf gene specific
Property primer is made of tuf-F primer and tuf-R primer, and the nucleotide sequence of tuf-F primer is as shown in SEQ ID NO:1, tuf-R
The nucleotide sequence of primer is as shown in SEQ ID NO:2.
The present invention also provides a kind of above-mentioned examinations of based on PCR-RFLP method identification Sugarcane white leaf phytoplasma difference subgroup
Agent box answering in identification Sugarcane white leaf phytoplasma 16SrXI-B subgroup and/or Sugarcane white leaf phytoplasma 16SrXI-D subgroup
With.
Compared with prior art, beneficial effects of the present invention:
The basis of the tuf gene order for the Sugarcane white leaf phytoplasma that the present inventor of the invention obtains in early-stage study
On, specific primer is designed, amplification tuf genetic fragment size is 468bp, on this basis, is established for the first time a kind of easy, fast
Speed, the method for accurately and efficiently identifying Sugarcane white leaf phytoplasma difference subgroup.
The method that the prior art determines Sugarcane white leaf phytoplasma difference subgroup is the nido to 16S rRNA gene
PCR product carries out virtual RFLP restriction analysis to determine, actual RFLP digestion discrimination method is not yet established.It is compared with it, this
Invention establishes a kind of actual RFLP digestion discrimination method to distinguish Sugarcane white leaf based on different genes (tuf gene)
Phytoplasma difference subgroup, according to the endonuclease reaction banding pattern of pcr amplification product can it is easy, quickly, accurately and efficiently judge sugarcane
The type of hoja blanca phytoplasma subgroup does not need to carry out sequencing analysis, has saved the time, reduced costs, improved identification
Accuracy.
The method of the present invention is quick, easy, quasi- to Sugarcane white leaf phytoplasma 16SrXI-B subgroup and 16SrXI-D subgroup
True identification is of great significance to the Pathogen identification and prevention and control of Sugarcane white leaf, different for research Sugarcane white leaf phytoplasma
Pathogenicity, regularty of epidemic and its prevention and control of subgroup provide technical support.
It is the nucleotide sequence of tuf-F primer shown in SEQ ID NO:1 in sequence table.
It is the nucleotide sequence of tuf-R primer shown in SEQ ID NO:2 in sequence table.
Detailed description of the invention
Fig. 1 is Sugarcane white leaf phytoplasma tuf gene PCR electrophoretogram, wherein M is DNAMarker E, and 1 is sweet to have infected
The Sugarcane Leaves sample of sugarcane hoja blanca phytoplasma 16SrXI-B subgroup shows a band；2 plant original to have infected Sugarcane white leaf
The Sugarcane Leaves sample of body 16SrXI-D subgroup shows a band；3 be health cane leaf sample, and 4 be ddH2O。
Fig. 2 is the restriction enzyme digestion and electrophoresis figure using the method for the present invention, wherein M is DNAMarker E, and 1 is to have infected the white leaf of sugarcane
The Sugarcane Leaves sample of sick phytoplasma 16SrXI-B subgroup shows two band；2 be to have infected Sugarcane white leaf phytoplasma
The Sugarcane Leaves sample of 16SrXI-D subgroup shows a band.
Specific embodiment
The present invention is described in detail with reference to embodiments, without specified otherwise is conventional method in embodiment.
1. Genome DNA extraction
By document " Zhang R Y, Li W F, Huang Y K, et al.Group 16SrXI phytoplasma
strains,including subgroup 16SrXI-B and a new subgroup,16SrXI-D,are
associated with sugar cane white leaf.International journal of systematic and
Evolutionary microbiology, 2016,66 (1): 487-491. " method, which detects, has been infected Sugarcane white leaf plant
The Sugarcane Leaves of substance 16SrXI-B subgroup and 16SrXI-D subgroup take infected Sugarcane white leaf phytoplasma 16SrXI-B respectively
Each 0.2g of the Sugarcane Leaves of subgroup and 16SrXI-D subgroup, using plant genome DNA extracts kit (with the full Shi Jinsheng in Beijing
For the Easy Pure plant Genomic DNAKit plant DNA extraction kit of object technology company) blade is extracted respectively
Total DNA, specific steps are operated according to the specification.
2.PCR amplification
Use Sugarcane white leaf phytoplasma tuf gene-specific primer: tuf-F primer/tuf-R primer is respectively to extraction
Total DNA carries out PCR amplification, and sterilizing distilled water (ddH is added in 25 μ L PCR reaction systems2O) 15.3 μ L, 10 × PCR buffer
2.5 μ L, MgCl22.0 μ L, tuf-F primer (20 μm of ol/L) of (25mmol/L) 2.0 μ L, dNTPs (10mmol/L) 1.0 μ L, tuf-
R primer (20 μm of ol/L) 1.0 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.2 μ L, 1.0 μ L of template DNA.Response procedures are as follows: 94 DEG C pre-
It is denaturalized 3min；94 DEG C of denaturation 15s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 recycle；Last 72 DEG C of extensions 7min.Amplification
Product with 1.5% agarose gel electrophoresis detect, as a result as shown in Figure 1, Sugarcane white leaf phytoplasma 16SrXI-B subgroup and
16SrXI-D subgroup sample only amplifies the specific band of 1 treaty 468bp.Fig. 1 shows: in no progress digestion,
No matter infect the Sugarcane smut of Sugarcane white leaf phytoplasma 16SrXI-B subgroup or infects Sugarcane white leaf phytoplasma
The Sugarcane smut of 16SrXI-D subgroup, pcr amplification product band is all the same, only shows that a size is the band of 468bp
(Sugarcane white leaf phytoplasma tuf gene band).
3.RFLP analysis
PCR after reaction, takes 15 μ L PCR products to carry out endonuclease reaction.PCR is added in 25 μ L endonuclease reaction systems to produce
15 μ L of object, sterilize distilled water (ddH2O) 7.5 μ L, 10 × buffer buffer, 1.5 μ L, restriction endonuclease Msp I
(20000U)1.0μL.Response procedures are as follows: 37 DEG C of 30min；65℃10min.10 μ L digestion products are taken to carry out 3% after digestion
Agarose gel electrophoresis analysis, digestion result is as shown in Fig. 2, be two bands by Msp I digestion, a band is about 124bp, another
Band is about that 344bp is Sugarcane smut (the i.e. Sugarcane smut cause of disease for having infected Sugarcane white leaf phytoplasma 16SrXI-B subgroup
For Sugarcane white leaf phytoplasma 16SrXI-B subgroup), it is infection without being digested only 1 treaty 468bp size strip
(i.e. Sugarcane smut cause of disease is Sugarcane white leaf phytoplasma to the Sugarcane smut of Sugarcane white leaf phytoplasma 16SrXI-D subgroup
16SrXI-D subgroup).
Sequence table
<110>Sugarcane Inst., Yunnan Prov. Agriculture Academy
<120>a kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaaacagaaa aaagacatta tgctca 26
<210> 2
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aacaccttca ataggcatta aaaatgg 27
Claims (4)
1. a kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup, which is characterized in that include the following steps:
(1) using the genomic DNA of Sugarcane smut to be identified as template, PCR amplification, institute are carried out using tuf gene-specific primer
It states tuf gene-specific primer to be made of tuf-F primer and tuf-R primer, the nucleotide sequence of tuf-F primer such as SEQ ID
Shown in NO:1, the nucleotide sequence of tuf-R primer is as shown in SEQ ID NO:2；
(2) pcr amplification product digestion of the restriction endonuclease Msp I to step (1) is used；
(3) result differentiates, digestion products is taken to carry out agarose gel electrophoresis, and restriction enzyme digestion and electrophoresis map shows that by Msp I digestion be two
Band, one be 124bp, another be 344bp Sugarcane smut cause of disease to be identified be Sugarcane white leaf phytoplasma 16SrXI-B
Subgroup；The Sugarcane smut cause of disease to be identified for the only 1 468bp size strip not being digested is Sugarcane white leaf phytoplasma
16SrXI-D subgroup.
2. the PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup according to claim 1, feature exist
In: the reaction condition of PCR amplification described in step (1) are as follows: 25 μ L PCR reaction systems, wherein sterilizing ddH2O 15.3 μ L, 10
2.5 μ L, 25mmol/L MgCl of × PCR buffer22.0 μ L, 10mmol/L dNTPs, 2.0 μ L, 20 μm of ol/L tuf-F primers
1.0 μ L, 20 μm of 1.0 μ L, 5U/ μ L Taq archaeal dna polymerases of ol/L tuf-R primer 0.2 μ L, 1.0 μ L of template DNA；Response procedures
Are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 15s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 recycle；Last 72 DEG C of extensions
7min；
Enzyme cutting reaction condition in step (2) are as follows: 25 μ L endonuclease reaction systems, wherein 15 μ L of PCR product, sterilize ddH2O 7.5μ
1.5 μ L, 20000U restriction endonuclease Msp I of L, 10 × buffer buffer, 1.0 μ L.Response procedures are as follows: 37 DEG C
30min；65℃10min；
Taking digestion products to carry out agarose gel electrophoresis in step (3) is that 10 μ L digestion products is taken to be carried out with 3% Ago-Gel
Electrophoresis.
3. a kind of based on PCR-RFLP method identifies the kit of Sugarcane white leaf phytoplasma difference subgroup, special containing tuf gene
Specific primer, restriction endonuclease Msp I or its isoschizomers, the tuf gene-specific primer by tuf-F primer and
Tuf-R primer composition, the nucleotide sequence of tuf-F primer is as shown in SEQ ID NO:1, and the nucleotide sequence of tuf-R primer is such as
Shown in SEQ ID NO:2.
4. the kit that based on PCR-RFLP method as claimed in claim 3 identifies Sugarcane white leaf phytoplasma difference subgroup is reflecting
Application in other Sugarcane white leaf phytoplasma 16SrXI-B subgroup and/or Sugarcane white leaf phytoplasma 16SrXI-D subgroup.
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| PCT/CN2019/116072 WO2020155730A1 (en) | 2019-02-02 | 2019-11-06 | Pcr-rflp identification method for different subgroups of sugarcane white leaf disease phytoplasma |
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| WO2020155730A1 (en) * | 2019-02-02 | 2020-08-06 | 云南省农业科学院甘蔗研究所 | Pcr-rflp identification method for different subgroups of sugarcane white leaf disease phytoplasma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3064592A1 (en) * | 2015-03-06 | 2016-09-07 | Brigitte König | Methods for the qualitative and quantitative detection of microbes in a sample |
| CN106399490A (en) * | 2016-09-04 | 2017-02-15 | 中国林业科学研究院森林生态环境与保护研究所 | LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit |
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| CN109652503B (en) * | 2019-02-02 | 2019-12-03 | 云南省农业科学院甘蔗研究所 | A kind of PCR-RFLP discrimination method of Sugarcane white leaf phytoplasma difference subgroup |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3064592A1 (en) * | 2015-03-06 | 2016-09-07 | Brigitte König | Methods for the qualitative and quantitative detection of microbes in a sample |
| CN106399490A (en) * | 2016-09-04 | 2017-02-15 | 中国林业科学研究院森林生态环境与保护研究所 | LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit |
Non-Patent Citations (2)
| Title |
|---|
| BERND SCHNEIDER等: "Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas", 《MICROBIOLOGY》 * |
| RONG-YUE ZHANG等: "Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D,are associated with sugar cane white leaf", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020155730A1 (en) * | 2019-02-02 | 2020-08-06 | 云南省农业科学院甘蔗研究所 | Pcr-rflp identification method for different subgroups of sugarcane white leaf disease phytoplasma |
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