CN106811466A - A kind of many slime mould partings of China's wheat root cereal and its method for identification - Google Patents
A kind of many slime mould partings of China's wheat root cereal and its method for identification Download PDFInfo
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Abstract
The present invention relates to a kind of many slime mould partings of China's wheat root cereal and its method for identification, methods described is to enter performing PCR to wheat root genomic DNA using specific forward primer PSP2 (f) and reverse primer ITS4 (r) of many slime moulds of cereal to expand, and judges that wheat root whether there is many slime moulds of cereal.Forward primer NS7 (f) and reverse primer ITS.CN (r), and forward primer Pg.F2 (f) and reverse primer Pg.R2 (r) is recycled to determine the parting of many slime moulds of cereal.
Description
Technical field
The present invention relates to the molecular detection technology of crop disease virus disease vector, belong to technical field of agriculture science.
Background technology
The many slime moulds of cereal (Polymyxa graminis) are a kind of low eucaryon lifes of obligatory parasitism of grass root
Thing, itself growth to plant will not produce obvious harm, but it is used as at least 11 kinds vectors of virus, propagation
Virus can cause various virus diseases on the crops such as wheat, barley, paddy rice.Because of its live body parasitic character, it is impossible to carry out room
Interior cultured in vitro, resting spore wall thickness, strong stress resistance can survive more than ten years in soil, cause the research to many slime moulds of cereal
It is very difficult, make slow progress.By worldwide a large amount of samplings and identification, the wheat class disease that many slime moulds of cereal are propagated is specify that
The malicious main Bymovirus category that is under the jurisdiction of is with Furovirus category.In the Winter Wheat Area of China, on many slime mould main propagation wheats of cereal
WYMV (WYMV) and Chinese wheat mosaic virus (CWMV), cause wheat plant tiller reduce, downgrade jaundice
Etc. symptom, underproduction 50-70% can be caused when falling ill serious, endangered huge.
There are two kinds, many slime moulds of cereal and many slime moulds of beet under many slime moulds.The two species initially mainly pass through form
And host distinguishes, many slime moulds of cereal mainly infect gramineous crop, and the host of many slime moulds of beet is then chenopod.Due to standing grain
The many slime mould genomic informations of paddy are unknown, and the classification under current its kind depends on the ITS1 and ITS2 of rDNA (rDNA)
Regional sequence.Ward in 1994 etc. utilizes RFLP (RFLP) technology, first passage ITS distinguished this two
Many slime moulds are planted, while finding that many slime moulds of cereal have two kinds of ribotypings by sequence analysis.Then, Ward and Adams exist
The third parting of many slime moulds of cereal is found that on India sorghum.With the announcement of many slime mould ITS of increasing cereal, Ward
Phyletic evolution point is carried out Deng by African Territories, Asia Japan Area sample and many slime mould ITS sequences of cereal known to remaining area
Find to have 6 kinds of many slime mould partings of cereal (I type-VI types) at present after analysis.Legreve in 2003 and the research that she works together then show
In addition to ITS sequence, many slime mould partings of cereal in different regions source are to host and temperature for the parting foundation of many slime moulds of cereal
There are different requirements, also need to pay attention in parting.The whole history of life is completed on sorghum from many slime moulds of the cereal of India
Optimum temperature be 27-30 DEG C, the sprouting of 23-26 DEG C of resting spore and the rate of development in each stage slow down, and 19-22 DEG C is infected
Not significantly, less than 19 DEG C nothings infect.And optimum temperature of many slime moulds of cereal in Temperate Region in China source on barley is 15-18 DEG C,
19-22 DEG C of infection rate is substantially reduced, more than 22 DEG C infect it is not notable.Therefore, Legreve is sticked more according to its result of study to cereal
When bacterium carries out parting in addition to ITS sequence is considered, host and temperature factor are also added into, many slime moulds of cereal have been divided into five kinds
Type (f.sp.temperate, f.sp.tepida, f.sp.tropicalis, f.sp.subtropicalis,
f.sp.colombiana).Although current the two categorizing systems are not unified, its corresponding relation is clearly.
Although the communication media of the wheat yellow mosaic in clear and definite China winter wheat area is soil early in the eighties in last century
Not there is which parting at present also not in many slime moulds of cereal in earth, but few to the research of many slime moulds of cereal, many slime moulds of China cereal
Know, while not knowing whether its parting is relevant with the Wheat Virus species propagated.Therefore, China's wheat yellow mosaic outburst is specified
The parting of many slime moulds of region cereal contributes to the prevention and control for passing wheat yellow mosaic for soil to provide foundation.
The content of the invention
This research provides a kind of Chinese many slime mould parting Rapid identifications of cereal and the method distinguished.According to many slime moulds of cereal
Ribosomal dna sequence, is expanded using the Winter Wheat Area wheat root DNA of many slime mould primer pair China of known cereal, screening
Suitable for the primer of many slime mould partings of China cereal, while specific anti-for many slime mould parting designs of the distinctive cereal of China
To primer I TS.CN, and a large amount of optimizations of reaction system and amplification condition work to PCR, finally obtaining stably can expand and differentiate me
The method of many slime mould partings of two kinds of cereals of state.
Researcher of the invention is first with many slime mould rDNA (rDNA) the ITS2 regions of European Region detection cereal
Universal primer PSP2 (f) and ITS4 (r) performing PCR is entered to the wheat root genomic DNA for coming from wheat yellow mosaic area of China
Amplification (PCR primer about 250bp), amplified production is sequenced and carries out phylogenetic analysis according to ITS2 regions, preliminary clear and definite China
At least there are two kinds of partings in many slime moulds of Winter Wheat Area cereal, I types that are a kind of and having reported have certain homology, are distinctive China
Parting, is named as I-CN type bacterium, and another kind is II type bacterium.Further to obtain the complete ITS sequence (bag of many slime mould rDNA of cereal
Include ITS1 and ITS2) for distinguishing two kinds of bacterial types, the general forward primer NS5 of the ITS sequence reported using European Region
F (), NS7 (f), PSP1 (f), ITS5 (f) and reverse primer ITS4 (r) combination are expanded.But wheat is arrived in main amplification
RDNA sequences, fail stabilization specifically amplification to many slime mould ITS sequences of cereal.Therefore, researcher of the invention devises
Substantial amounts of primer, final screening obtains reverse primer ITS.CN (r), using forward primer NS7 pacts of the amplification comprising ITS1
800bp sequences, can be used for detecting the specific primer of I-CN type bacterium.In addition, using II types bacterium primer Pg.F2 (f) and Pg.R2
R () amplification obtains the special ITS sequence of II type bacterium, PCR primer length is 430bp.The specific present invention uses following technical side
Case：
1. the collection of sample
Collection field morbidity wheat samples, detect that blade is viral and micro- sem observation root cereal sticks more by RT-PCR
The resting spore situation of bacterium, specifies sample root and contains many slime moulds of cereal.
2. the determination of many slime mould partings of China cereal
PSP2 (f) and ITS4 (r) is combined first with primer and expand many slime mould ITS2 sequences of wheat root cereal, be sequenced bright
Really specific extension increasing sequence, chadogram is built using the known many slime mould partings of cereal on NCBI, specifies many slime moulds of China cereal
Parting is I-CN types and II types.
3. the specific primer design of parting
Sequence characteristic according to the distinctive parting I-CN types of China designs primer I TS.CN (r), with reference to forward primer NS7
(f) PCR specific amplification I-CN types.Using Pg.F2 (f) and Pg.R2 (r) PCR specific amplification II type bacterium.
4. the specificity verification of primer
The wheat old complaint STb gene containing the different many slime moulds of parting cereal is extracted as masterplate PCR amplification checking primer specifics
Property, finally the clearly specific primer of the detection many slime moulds of cereal is PSP2 (f) and ITS4 (r), the detection many slime mould I type bacterium of cereal
Specific primer is Ns7 (f) and ITS.CN (r), detect the specific primer of many slime mould II type bacterium of cereal for Pg.F2 (f) and
Pg.R2(r)。
5. the optimization of reaction system and amplification condition
To each condition of influence PCR amplifications：Including annealing temperature, primer concentration, HiFi archaeal dna polymerases concentration, dNTP
The aspects such as concentration, extension of time and cycle-index are optimized, and the optimal PCR system that final determination is adapted to this research is：10×
1 μ L, dNTP 2mM of PCR Buffer 1 μ L, 10 μM of 0.25 μ L of sense primer, 10 μM of 0.25 μ of μ L, DNA 1.0 of anti-sense primer
The 6.25 μ L of μ L, ddH2O of L, HiFi DNA polymerase 5U/ μ L 0.25, final volume is 10 μ L；Optimal PCR amplification programs
For：94 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C of extension of time are respectively according to primer difference：PSP2
F () and ITS4 (r) extend 20s, Ns7 (f) and ITS.CN (r) and extend 60s, Pg.F2 (f) and Pg.R2 (r) extension 30s, carry out altogether
35 circulations；Last 72 DEG C of extensions 10min.
5. application under study for action
The many slime mould parting situations of cereal in winter wheat area of China are carried out with more comprehensive investigation using the technology, sample comes
Source includes that Shandong, Henan, Jiangsu, Anhui and five, Hubei province soil pass 66 wheat old complaint samples of wheat yellow mosaic lesion
Product, specify that situation of the various regions sample with many slime moulds of cereal and its parting.
Using technical scheme, following technique effect can be obtained：
1. the specific primer of the identification many slime mould I-CN types of cereal and II types is obtained；
2. the detection many slime mould I-CN types of cereal of stabilization and the PCR system of II type bacterium are obtained.
Brief description of the drawings
Fig. 1 is used for position view of the three pairs of primers of detection on many slime mould rDNAs of cereal, and (PSP2 and ITS4 are
The many slime mould specific primers of cereal；NS7 and ITS.CN is many slime mould I-CN type-special primers of cereal；Pg.F2 and Pg.R2 is standing grain
The many slime mould II type-special primers of paddy；→ represent forward primer；← represent reverse primer)
Sample detection effect (the M of many slime mould parting specific primers of Fig. 2 cereals：Marker；a：PSP2/ITS4 primer sets
Close many slime moulds of specific amplification cereal；NS7/ITS.CN primers combine the specific amplification I-CN many slime moulds of type cereal；Pg.F2/
Pg.R2 primers combine the specific amplification II many slime moulds of type cereal；Sample A：The wheat root that many slime moulds of I-CN type cereals are infected；Sample
Product B：The wheat root that I-CN types and many slime moulds of II type cereals are infected jointly；Sample C：Without the wheat root that many slime moulds of cereal are infected
Portion；Sample D：Blank (H2O))
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but be not limit of the invention
System, only illustrates.
Embodiment 1：The design of specific primer, synthesis and PCR Preliminary detections
It is general according to many slime moulds of cereal reported and parting specific primer sequence designs following forward primer：NS5,
NS7, PSP1, ITS5, PSP2, Pg.F2；And reverse primer：ITS4, Pg.R2.Preliminary PCR detections lesion wheat root sample
Afterwards, the sequence that band occurs in electrophoresis is carried out into rubber tapping to reclaim and connect carrier T sequencing.The pairing of primer, sequence and preliminary PCR
Result is as shown in table 1 below：
1 research institute's primer sequence of table and its PCR results
Note：Primer entrusts Beijing Qing Kexin industry Bioisystech Co., Ltd to synthesize.
Sequencing result shows that many slime moulds universal primer PSP2 (f) of cereal/ITS4 (r) can be expanded smoothly to many slime moulds of cereal
RDNA ITS2 sequences, the ITS2 sequencings to a large amount of wheat samples in China Winter Wheat Area (five provinces totally 66 parts of samples, detailed in Example 3)
At least there are two kinds of partings in the interpretation of result many slime moulds of preliminary clear and definite China Winter Wheat Area cereal, I types that are a kind of and having reported have one
Determine homology, be the distinctive parting of China, be named as I-CN type bacterium, another kind is II type bacterium.Pg.F2 (f)/Pg.R2 (r) is same
Specific can expand to many slime mould sequences of cereal (II types).
Meanwhile, NS5 (f)/ITS4 (r), NS7 (f)/ITS4 (r), PSP1 (f)/ITS4 (r), ITS5 (f)/ITS4 (r),
Although PSP2 (f)/ITS4 (r) primers combination can be expanded smoothly to correct size strip, sequencing result is wheat rDNA, rather than
The many slime mould rDNA of cereal.Further sequence alignment result shows, in reverse primer ITS4 sequences and wheat rDNA section sequence
Row are matched completely.Thus while the ITS4 sequences reported can be widely used in many slime mould detections of cereal in Europe, but in China
Do not applied on wheat, this is probably because Europe is made for crop predominantly barley, rye etc. of many slime mould detections of cereal at present
Thing, rather than wheat.
Based on result above, according to wheat and the homology of many slime mould I-CN types rDNA sequences of cereal, in both difference
One reverse primer ITS.CN of Position Design, using forward primer NS7 about 800bp sequences of the amplification comprising cereal many slime mould ITS1
Row, the specific primer (being shown in Table 1) for detecting I-CN type bacterium.Therefore, by the screening to many slime mould primers of cereal, obtain
For universal primer PSP2 (the f)/ITS4 (r) of many slime mould detections of China cereal, many slime moulds of newfound China I-CN type cereals
Specific primer NS7 (f)/ITS.CN (r), and II type bacterium specific primer Pg.F2 (f)/Pg.R2 (r).
Embodiment 2：Set up the PCR system and condition of the detection many slime moulds of cereal and its parting
Clip every part of 30mg of dry old complaint, is extracted using the DNAsecure Plant Kit kit methods of TIANGEN companies
The STb gene of wheat old complaint sample, is dissolved in 50 μ L ddH2O.
The PCR reactions of 50 μ L systems are carried out, system is：The μ L of 10 × PCR Buffer, 5 μ L, dNTP 2mM 5, sense primer
10 μM of 1.25 μ L, anti-sense primer 10 μM of 1.25 μ L, DNA5 μ L, HiFi DNA polymerase 5U/ μ L 0.5 μ L, ddH2O
32 μ L, final volume is 50 μ L；PCR amplification programs are：94 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C are prolonged
Stretch the time is respectively according to primer difference：PSP2 (f) and ITS4 (r) extends 20s, Ns7 (f) and ITS.CN (r) and extends 60s,
Pg.F2 (f) and Pg.R2 (r) extends 30s, and 35 circulations are carried out altogether；Last 72 DEG C of extensions 10min.PCR primer is carried out into electrophoresis
Analyze and observed under uviol lamp, it can be observed that the stripe size about 250bp that primer PSP2 (f) and ITS4 (r) are amplified,
What the stripe size about 800bp that primer Ns7 (f) and ITS.CN (r) are amplified, primer Pg.F2 (f) and Pg.R2 (r) were amplified
Stripe size is about 430bp.
Specific fragment is carried out into gel recovery respectively, Cloning Transformation is carried out, transfers to Beijing to hold up the positive colony of acquisition
Ke Xin industry Bioisystech Co., Ltd carries out sequencing analysis.Sequencing result is respectively：
Extension increasing sequence (the SEQ ID NO of primer PSP2 (f) and ITS4 (r)：10)：
TTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCTATATCTCTCACATACTCAC
TGGTGGTGTGTGTGGTGGAGAATGGGAGTTGCGCACGTCTCTCTGCATCTCCTGAAGTCAGCGTCGGCCGAAGTCCA
TCGGGTTCTTGGAACGATAATCCGTCATGGCGCCCAAACCATACACAAAAGATCTCAGATGAGGTAAGACTACCCGC
CGAATTTAAGCATATCAATAAGCGGAGGA
Extension increasing sequence (the SEQ ID NO of primer Ns7 (f) and ITS.CN (r)：11)：
GAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACAATGCTAGGTTCAA
CGAGTCGATTTCGTACTTGGCCGAGAGGCCTGGTTAATCTTCTGAAATCCTAGCGTGCTTGGGCTTGCCTCTTGCAA
CTAGAGGCACCAACGAGGAATTCCTAGTAGACGCAGGTCATCAACCTGCATCGATTACGTCCCTGCCCTTTGTACAC
ACCGCCCGTCGCTTCTACCGATTGGTCGTCCCGGTGAACAATCGGGAGGCCGTGCTCATGGTCAGCAATGGCCGGAG
CGCGGTCGAACTTTTGTAAATTTGGACGACTAGAGGAAGAAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGG
AAGGATCATTAGCGTTGATTGGTCTTGGTGCCATGCGAAAAACATGTGGATCGTGGGCTATGTGACCCCTCTGTGGG
GGGTATTTTGTCGCGAACTTGGAGATTTGGCTGATGGATATACATGATAGATAAACAAAATACAACTCTTAGCAATG
GATATCTTGGTTCCCACAACGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGTGAATCA
TCGAATCTTTGAACGCAAGTTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCTATATCTCT
CACACACTCACTGGTGGTGTGTGTGGTGGAGAATGGGAGTTGCGCACGTCTCTCTGCATCTCCTGAAGTCAGCGTCG
GCCGAAGTCCATCGGGTTCTTGGAACGATAATCCGC
Extension increasing sequence (the SEQ ID NO of primer Pg.F2 (f) and Pg.R2 (r)：12)：
ATGTGGATCGTCTCTGTTGCTGGATACCGGGATGGAACGCCCTCGTGGTGGTTCTTGTCTTTCACGAAT
TGGATCAAACGGTGGCTAAAGTCGAATTGGTTTCATAACCTACAAACAATATATACAACTCTTAGCAATGGATATCT
TGGTTCCCACAACGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGTGAATCATCGAATC
TTTGAACGCAAGTTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCCCATCTCACACCCGGT
GTGGAGAATGAGGGTCGGGCGCCTCGGCGCCGGTCCCTTCAAATCAGGCCGGCCTGGTCAAGTCCATCGGATTGCTG
GTACGATAGTCCGTCATGGCGCAAGGAACCACTTGGCAAGATCTCAGATGAGG
Embodiment 3：
The application of detection method in the present invention in practical study
Included using the technology for detection from Shandong, Henan, Jiangsu, Anhui, five, Hubei province wheat yellowtop disease
62 wheat old complaint samples in area, specify that situation of the various regions sample with many slime moulds of cereal and its I types and II type bacterium.Can from table
To find out, the I many slime moulds of type cereal are all distributed with this five provinces, small part place is distributed with II type bacterium simultaneously, but simultaneously
Without the situation for finding individualism II type bacterium.
Amino acid sequence table
<110>Zhejiang Academy of Agricultural Science
<120>A kind of many slime mould partings of China's wheat root cereal and its method for identification
<130> NKY001
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial
<220>
<223> NS5(f)
<400> 1
aacttaaagg aattgacgga ag 22
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
<223> ITS4(r)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 23
<212> DNA
<213> Artificial
<220>
<223> NS7(f)
<400> 3
gaggcaataa caggtctgtg atg 23
<210> 4
<211> 20
<212> DNA
<213> Artificial
<220>
<223> PSP1(f)
<400> 4
tagacgcagg tcatcaacct 20
<210> 5
<211> 22
<212> DNA
<213> Artificial
<220>
<223> ITS5(f)
<400> 5
ggaagtaaaa gtcgtaacaa gg 22
<210> 6
<211> 19
<212> DNA
<213> Artificial
<220>
<223> PSP2(f)
<400> 6
ttgcgctttc gagagccct 19
<210> 7
<211> 19
<212> DNA
<213> Artificial
<220>
<223> Pg.F2(f)
<400> 7
ttgcgctttc gagagccct 19
<210> 8
<211> 23
<212> DNA
<213> Artificial
<220>
<223> Pg.R2(r)
<400> 8
cctcatctga gatcttgcca agt 23
<210> 9
<211> 20
<212> DNA
<213> Artificial
<220>
<223> ITS.CN(r)
<400> 9
gcggattatc gttccaagaa 20
<210> 10
<211> 252
<212> DNA
<213>The many slime moulds of cereal（Polymyxa graminis）
<400> 10
ttgcgctttc gagagccctc ggaagcatgc ctctctgagt atcggtttct atatctctca 60
catactcact ggtggtgtgt gtggtggaga atgggagttg cgcacgtctc tctgcatctc 120
ctgaagtcag cgtcggccga agtccatcgg gttcttggaa cgataatccg tcatggcgcc 180
caaaccatac acaaaagatc tcagatgagg taagactacc cgccgaattt aagcatatca 240
ataagcggag ga 252
<210> 11
<211> 798
<212> DNA
<213>The many slime moulds of cereal（Polymyxa graminis）
<400> 11
gaggcaataa caggtctgtg atgcccttag atgttctggg ccgcacgcgc gctacaatgc 60
taggttcaac gagtcgattt cgtacttggc cgagaggcct ggttaatctt ctgaaatcct 120
agcgtgcttg ggcttgcctc ttgcaactag aggcaccaac gaggaattcc tagtagacgc 180
aggtcatcaa cctgcatcga ttacgtccct gccctttgta cacaccgccc gtcgcttcta 240
ccgattggtc gtcccggtga acaatcggga ggccgtgctc atggtcagca atggccggag 300
cgcggtcgaa cttttgtaaa tttggacgac tagaggaaga agaagtcgta acaaggtttc 360
cgtaggtgaa cctgcggaag gatcattagc gttgattggt cttggtgcca tgcgaaaaac 420
atgtggatcg tgggctatgt gacccctctg tggggggtat tttgtcgcga acttggagat 480
ttggctgatg gatatacatg atagataaac aaaatacaac tcttagcaat ggatatcttg 540
gttcccacaa cgatgaagaa cgcagcgaaa tgcgatacgt aatgcgaatt gcagaattca 600
gtgaatcatc gaatctttga acgcaagttg cgctttcgag agccctcgga agcatgcctc 660
tctgagtatc ggtttctata tctctcacac actcactggt ggtgtgtgtg gtggagaatg 720
ggagttgcgc acgtctctct gcatctcctg aagtcagcgt cggccgaagt ccatcgggtt 780
cttggaacga taatccgc 798
<210> 12
<211> 430
<212> DNA
<213>The many slime moulds of cereal（Polymyxa graminis）
<400> 12
atgtggatcg tctctgttgc tggataccgg gatggaacgc cctcgtggtg gttcttgtct 60
ttcacgaatt ggatcaaacg gtggctaaag tcgaattggt ttcataacct acaaacaata 120
tatacaactc ttagcaatgg atatcttggt tcccacaacg atgaagaacg cagcgaaatg 180
cgatacgtaa tgcgaattgc agaattcagt gaatcatcga atctttgaac gcaagttgcg 240
ctttcgagag ccctcggaag catgcctctc tgagtatcgg tttcccatct cacacccggt 300
gtggagaatg agggtcgggc gcctcggcgc cggtcccttc aaatcaggcc ggcctggtca 360
agtccatcgg attgctggta cgatagtccg tcatggcgca aggaaccact tggcaagatc 420
tcagatgagg 430
Claims (9)
1. many slime mould partings of a kind of China's wheat root cereal (I-CN types), it is characterised in that with SEQ ID NO：11 represent
Nucleotide sequence.
2. the specific reverse primers of many slime mould partings of cereal described in claim 1, it is characterised in that with ITS.CN (r)
The nucleotide sequence of expression.
3. a pair of specific primers of many slime mould partings of cereal described in claim 1, it is characterised in that with forward primer
NS7 (f) and reverse primer ITS.CN (r).
4. a kind of detection method to many slime moulds of wheat root cereal, it is characterised in that using forward primer NS7 (f) and reversely
The parting (I-CN types) of many slime moulds of the clear and definite cereal of primer I TS.CN (r).
5. a kind of detection method to many slime moulds of wheat root cereal, it is characterised in that the specificity using many slime moulds of cereal is drawn
Thing PSP2 (f) and ITS4 (r) enter performing PCR amplification to primer pair wheat root genomic DNA, judge that wheat root whether there is standing grain
The many slime moulds of paddy.Recycle the parting of forward primer NS7 (f) and reverse primer ITS.CN (r) many slime moulds of clear and definite cereal.
6. a kind of authentication method to many slime moulds of wheat root cereal, it is characterised in that the specificity using many slime moulds of cereal is drawn
Thing PSP2 (f) and ITS4 (r) enter performing PCR amplification to primer pair wheat root genomic DNA, judge that wheat root whether there is standing grain
The many slime moulds of paddy.Recycle forward primer NS7 (f) and reverse primer ITS.CN (r), and forward primer Pg.F2 (f) and reverse primer
The parting of many slime moulds of the clear and definite cereals of Pg.R2 (r).
7. the authentication method according to claim 4-6 any claims, it is characterised in that PCR reaction systems are：10×
1 μ L, dNTP 2mM of PCR Buffer 1 μ L, 10 μM of 0.25 μ L of sense primer, 10 μM of 0.25 μ of μ L, DNA 1.0 of anti-sense primer
The 6.25 μ L of μ L, ddH2O of L, HiFi DNA polymerase 5U/ μ L 0.25, final volume is 10 μ L；PCR amplification programs are：94
DEG C predegeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C of extension of time are respectively according to primer difference：PSP2 (f) and
ITS4 (r) extends 20s, Ns7 (f) and ITS.CN (r) and extends 60s, Pg.F2 (f) and Pg.R2 (r) extension 30s, and 35 are carried out altogether
Circulation；Last 72 DEG C of extensions 10min.
8. the authentication method according to claim 5 or 6, it is characterised in that the judgement wheat root whether there is cereal
Many slime moulds refer to：Amplified production according to primer PSP2 (f) and ITS4 (r) for size whether be 250bp or so DNA bands,
Thus judge whether wheat root contains many slime moulds of cereal；Amplified production according to primer Ns7 (f) and ITS4.CN (r) is size
Whether be 800bp or so DNA bands, thus judge wheat root whether containing the I-CN many slime moulds of type cereal.
9. authentication method according to claim 6, it is characterised in that the judgement wheat root is sticked more with the presence or absence of cereal
Bacterium refers to：Amplified production according to primer Pg.F2 (f) and Pg.R2 (r) for size whether be 430bp or so DNA bands, by
This judges whether wheat root contains the II many slime moulds of type cereal.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD268256A1 (en) * | 1987-12-04 | 1989-05-24 | Adl D Ddr Inst F Phytopatholog | METHOD FOR DETECTING PILZ-TRANSFERABLE VIRUSES FROM GROUND SAMPLES |
| CN102424835A (en) * | 2011-11-10 | 2012-04-25 | 江苏省农业科学院 | Method for detecting Polymyxa graminis in soil |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD268256A1 (en) * | 1987-12-04 | 1989-05-24 | Adl D Ddr Inst F Phytopatholog | METHOD FOR DETECTING PILZ-TRANSFERABLE VIRUSES FROM GROUND SAMPLES |
| CN102424835A (en) * | 2011-11-10 | 2012-04-25 | 江苏省农业科学院 | Method for detecting Polymyxa graminis in soil |
Non-Patent Citations (3)
| Title |
|---|
| WARD等: "Analysis of ribosomal DNA sequences of Polymyxa species", 《MYCOL.RES.》 * |
| XU Y等: "Ribotypes of Polymyxa graminis in Wheat Samples Infected with Soilborne Wheat Viruses in China", 《PLANT DIS》 * |
| 陈剑平: "大麦黄花叶病及其真菌介体禾谷多粘菌的研究进展", 《中国病毒学》 * |
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