CN102234643A - Specific primer pair for assisting in identifying tomato yellow leaf curl virus and application thereof - Google Patents
Specific primer pair for assisting in identifying tomato yellow leaf curl virus and application thereof Download PDFInfo
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Abstract
The invention discloses a specific primer pair for assisting in identifying tomato yellow leaf curl virus (TYLCV) and application thereof. The primer pair consists of DNA shown as a sequence 1 in a sequence table and DNA shown as a sequence 2 in the sequence table. The primer pair has the advantages of high specificity and high sensitivity, and can accurately, quickly and efficiently detect the TYLCV in plants at the molecular level. The primer pair can be easily applied to detection of the toxicity of industrial concentrated seedlings to ensure nontoxic strong seedlings in vegetable production, can be easily applied to quick detection of plant disease conditions in large-scale vegetable production to guide the control of the viral disease, and can be easily applied to identification and screening of anti-virus breeding materials and culture of new anti-virus varieties. The technology can provide powerful technical support for comprehensive control of the TYLCV and safe and sustainable production of vegetables in Beijing and all over the country.
Description
Technical field
The special primer that the present invention relates to a kind of assistant identification tomato yellow leaf curl virus to and use.
Background technology
Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) be to belong to a kind of twin dna virus that bean golden mosaic virus belongs to (Begomoviruses), propagate by " super insect "-Bemisia tabaci, have sudden and unmanageable characteristics.This virus can infect various plants such as Solanaceae, pulse family, cash crop such as vegetables such as main harm tomato, capsicum, eggplant, wild cabbage and cucumber and tobacco, and particularly harm is heavier on tomato.Being injured, the plant symptom shows as poor growth, plant is obviously downgraded; Top vane shrinkage, leaf periphery upsweep, and the blade thickening diminishes, the leaf area yellow; It is few to bear fruit, and fruit is firmly little, and normally annesl is lost commodity; Endangering can not bloom when serious bears fruit, and causes total crop failure.
Tomato yellow leaf curl virus is the harmful organism of new incoming China in recent years.Took place as far back as Israel, and be diffused into numerous countries and regions such as the Middle East, Mediterranean Sea bank, East Asia, South Asia, Africa, Europe, the U.S., Central America, Australia afterwards rapidly in 1964.Imported province, China south in 2002 into, autumn in 2005 was broken out in tomato main producing region, Guangxi big area, import Jiangsu and Zhejiang Provinces one band the same year into, imported Henan and Shandong in 2007 into, 2008 should disease in the whole nation a plurality of provinces and cities developmenting spread, 2009 should disease in partial area, Hebei and the great outburst of Shouguang, Shandong, more than 10 ten thousand mu of areas take place, tomato loses more than 80,000 mu.Summer and autumn in 2009, TYLCV takes place successively in the Beijing suburb county, the big fruit tomato variety of the main pink colour of planting in Beijing area is mostly to this virus sensitivity, and this disease spreads all over districts such as Tongzhou, Daxing, Shunyi, Pinggu, Miyun, Yanqing, Changping, Fangshan, Huairou, Fengtai, Chaoyang, Haidian; The morbidity canopy general diseased plant rate 5-10% in chamber, grave illness canopy chamber diseased plant rate about 20%, indivedual canopy chambers sickness rate reaches more than 90%, cause the serious underproduction or total crop failure, and PD is spreading trend rapidly.From the course of countries in the world TYLCV virus harm, in case occur, as fail to take effective prevention and control measure, will become the developing major obstacle of Beijing industrialized agriculture, need pay close attention.
Being directed to the generation and the harm of TYLCV virus, how accurately detecting the existence and the generation of this virus quickly and efficiently, is pressing for of vegetables safety Sustainable Production.
Summary of the invention
The special primer that the purpose of this invention is to provide a kind of assistant identification tomato yellow leaf curl virus to and use.
It is right to the invention provides the primer that DNA forms shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table.
Described primer is to can be used for the assistant identification tomato yellow leaf curl virus.
The invention provides the method for assistant identification tomato yellow leaf curl virus, is that the genomic dna with virus to be measured is a template, with described primer to carrying out pcr amplification; If obtain the dna fragmentation of 543bp, virus to be measured is candidate's tomato yellow leaf curl virus; If do not obtain the dna fragmentation of 543bp, virus to be measured is candidate's non-tomato yellow leaf curl virus.Described virus to be measured specifically can be tomato yellow leaf curl virus (TYLCV), Tomato mosaic virus (ToMV), fern leaf of tomato poison (CMV) or tomato bushy stunt virus (ToBSV).
Described primer is to can be used for preparing the test kit of assistant identification tomato yellow leaf curl virus.
The present invention also protects a kind of test kit of assistant identification tomato yellow leaf curl virus, comprises that described primer is right.
Whether described primer infects tomato yellow leaf curl virus to also can be used for the assistant identification tomato.
The invention provides the method whether the assistant identification tomato infects tomato yellow leaf curl virus, comprise the steps:
(1) total DNA of extraction tomato leaf to be measured;
(2) the total DNA that extracts with step (1) is a template, with described primer to carrying out pcr amplification; If obtain the dna fragmentation of 543bp, tomato to be measured has infected tomato yellow leaf curl virus; If do not obtain the dna fragmentation of 543bp, tomato to be measured does not infect tomato yellow leaf curl virus.
Described tomato specifically can be tomato variety M82 or tomato variety CM.
Described primer is to can be used for tomato breeding, the prevention and control of tomato yellow leaf curl virus in the also available tomato.
It is right to the invention provides based on the special section nucleotide sequence of tomato yellow leaf curl virus (TYLCV) primer, has good, the highly sensitive advantage of specificity, can detect TYLCV the plant accurately, fast and efficiently from molecular level.
Primer of the present invention concentrates the band toxicity of growing seedlings to detect to being applied to batch production easily with method, guarantees to use nontoxic healthy seedling in the vegetables production; Can be conveniently used in the rapid detection of plant incidence in the vegetables scale operation, instruct prevention and control this virus disease; Can be applied to the evaluation and screening of antiviral breeding material and the cultivation of disease-resistant drugs new variety easily.This technology will provide strong science and technology support for the vegetables safety Sustainable Production in the comprehensive prevention and control of TYLCV and the Beijing and the whole nation.
Description of drawings
Fig. 1 is the electrophorogram of the sample of embodiment 2; M:DL2000plus; 1:TYLCV; 2:ToMV; 3:CMV; 4:ToBSV; 5: blank.
Fig. 2 is the electrophorogram of the sample of embodiment 3.
Fig. 3 is the electrophorogram of the sample of embodiment 4.
Fig. 4 is the electrophorogram of the sample of embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Special section design one couple of PCR primers (TYLCV-F/R) according to the TYLCV sequence is as follows:
TYLCV-F (upstream primer): 5 '-ACG CAT GCC TCT AAT CCA GTG TA-3 ' (23bp; Sequence 1);
TYLCV-R (downstream primer): 5 '-CCA ATA AGG CGT AAG CGT GTA GAC-3 ' (24bp; Sequence 2).
Tomato yellow leaf curl virus (TYLCV), Tomato mosaic virus (ToMV), fern leaf of tomato poison (CMV) and tomato bushy stunt virus (ToBSV) all are to separate by the sick leaf of the tomato of gathering classical symptom in Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences's tomato booth respectively to obtain again.The separation of tomato yellow leaf curl virus and identifying: genomics and applied biology, 2009,28 (1): 115-118 referring to document.The separation of Tomato mosaic virus and identifying: Scientia Agricultura Sinica, 2004,37 (7): 982-986 referring to document.The separation of fern leaf of tomato poison and identifying: Hebei agricultural science and technology, 2006,3:18-18 referring to document.The separation of tomato bushy stunt virus (ToBSV) and identifying: Chinese virusology, 2007,22 (3): 199-206 referring to document.
Extract the genomic dna of four kinds of viruses respectively, with the TYLCV-F/R primer of embodiment 1 design to carrying out pcr amplification.The pcr amplification condition: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 30 circulations, 72 ℃ are extended 10min.The PCR product carries out 1% agarose gel electrophoresis (containing EB), detects on the gel automated imaging instrument and takes pictures.
The results are shown in Figure 1.The result shows: the TYLCV-F/R primer has obtained specific band (TYLCVSF543) to the amplification tomato yellow leaf curl virus, and other three kinds of viruses all do not show this band.Thereby proved that the TYLCV-F/R primer is to having specificity and high efficiency to tomato yellow leaf curl virus.
Reclaim specific band (TYLCVSF543), check order.The result shows: the nucleotide sequence of this specific band is shown in the sequence 3 of sequence table, and length is 543 Nucleotide (bp).Sequence 3 in the sequence table is made up of 543 Nucleotide, protein (C3 albumen), the 214-543 position Nucleotide shown in the sequence 5 in the protein (V1 albumen) shown in the sequence 4, the 69-473 position nucleotide coding sequence table in 5 ' end 1-72 position nucleotide coding sequence table, the protein (C2 albumen) shown in the sequence 6 in the code sequence tabulation.
Tomato conventional cultivation kind M82 and conventional variety CM (Castlemart is abbreviated as CM) are all available from U.S. tomato genetic resources center (The C.M.Rick Tomato Genetics Resource Center), and the germplasm numbering is respectively LA3475 and LA2400.
With tomato yellow leaf curl virus (TYLCV) difference inoculating tomato conventional cultivation kind M82 and CM (every kind of tomato inoculates 20 strains), as experimental group; The not tomato of these two kinds of virus inoculation (every kind 20 strain) is set, in contrast group; Observe phenotype after 20 days, calculate its sickness rate respectively.
Experimental group, the sickness rate of M82 are 100%, and the sickness rate of CM is 100%; Control group, the sickness rate of M82 are 0%, and the sickness rate of CM is 0%.
Get the blade (getting 3 blades at random for every kind) of experimental group M82, experimental group CM, control group M82 and control group CM respectively, extract genomic dna.With the genomic dna is template, with the TYLCV-F/R primer of embodiment 1 design to carrying out pcr amplification.The pcr amplification condition: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 30 circulations, 72 ℃ are extended 10min.The PCR product carries out 1% agarose gel electrophoresis (containing EB), detects on the gel automated imaging instrument and takes pictures.With the genomic dna of TYLCV virus as template, as positive control.Do not add template, as blank.
The results are shown in Figure 2.Control group among M82 and the CM, does not all show specific band (543bp) because of it infects TYLCV; Experimental group among M82 and the CM, all shows specific band (543bp); Positive control shows specific band (543bp); Blank does not have specific band (543bp).The result shows: it is right to use TYLCV-F/R primer of the present invention, and behind pcr amplification, TYLCVSF543 segmental having or not can detect whether infected TYLCV virus in the tomato plant quickly and easily.
Tomato yellow leaf curl virus (TYLCV) incidence to the Beijing suburb county is investigated.337 portions of tomatoes of random acquisition sample.Utilize TYLCV-F/R primer provided by the invention to 337 parts of tomato samples are carried out Molecular Detection (method is with embodiment 3).The result shows: in 337 parts of tomato samples, 163 parts have TYLCV virus, account for 48% of total sample number; 171 parts do not detect TYLCV.Part infects the electrophoresis result of TYLCV Virus Sample and sees Fig. 3.Among Fig. 3, positive control is for determining to infect the TYLCV Virus Sample; Negative control is for determining not infect the TYLCV Virus Sample, and all the other swimming lanes are the part sample to be tested.
Carrying out disease symptom for all samples confirms.The result shows: the sample (showing the 543bp band) that detects virus all has the TYLCV classical symptom; The sample (not showing the 543bp band) that does not detect virus does not have the TYLCV symptom, and presents symptoms such as Tomato mosaic virus, bushy stunt virus or ferm-leaf poison.Present embodiment has further confirmed to use TYLCV-F/R primer of the present invention and whether has infected TYLCV virus to detecting tomato, and the real result that obtains is effective.
A situation arises carries out on the-spot investigation respectively to the TYLCV of (MAIND) (English is denoted as TORCH) of 7 mu of tomato variety U.S.s in (MAIND) (English is denoted as YARDEN827) of 30 mu of tomato variety U.S.s being planted in Zhu Keng village, Putian City Licheng District Huangshi town, Fujian Province and Kingsoft village.(MAIND) of the U.S. in Zhu Keng village (YARDEN827) and (MAIND) of the U.S. in Kingsoft village (TORCH) tomato field plant upper blade typical TYLCV symptom all appears, sickness rate is respectively 100% and 61%.
To morbidity sample and the sample of not falling ill sampling, utilize TYLCV-F/R primer provided by the invention respectively to carrying out Molecular Detection (method is with embodiment 3).Part infects the electrophoresis result of TYLCV Virus Sample and sees Fig. 4.The result shows, all detects TYLCV virus (showing the 543bp band) in the morbidity sample, does not all detect TYLCV virus (not showing the 543bp band) in the sample of not falling ill.
Sequence table
＜110〉Beijing City Agriculture and Forestry Institute
＜120〉special primer of assistant identification tomato yellow leaf curl virus to and use
<130>CGGNARY102280
<160>6
<210>1
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
acgcatgcct?ctaatccagt?gta 23
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
ccaataaggc?gtaagcgtgt?agac 24
<210>3
<211>543
<212>DNA
＜213〉tomato yellow leaf curl virus (Tomato yellow leaf curl virus)
<400>3
acgcatgcct?ctaatccagt?gtatgtaact?atgaaaatac?gcatctattt?ctatgattca 60
atatcaaatt?aataaaattt?atattttata?tcatgagttt?ctgttacatt?tattgtgttt 120
tcaagtacat?catacaatac?atgatcaact?gctctgatta?cattgttaat?ggaaattaca 180
ccaagactat?ctaaatactt?aagaacttca?tatctaaata?ctcttaagaa?atgaccagtc 240
tgaggctgta?atgtcgtcca?aattcggaag?ttgagaaaac?atttgtgaat?ccccattacc 300
ttcctgatgt?tgtggttgaa?tcttatctga?atggaaatga?tgtcgtggtt?cattagaaat 360
ggccgctggc?tgtgttctgt?tatcttgaaa?tagaggggat?tgtttatctc?ccaaataaaa 420
acgccattct?ctgcctgagg?agcagtgatg?agttcccctg?tgcgtgaatc?catgattgtt 480
gcagttgagg?tggaggtagt?atgagcagcc?acagtctagg?tctacacgct?tacgccttat 540
tgg 543
<210>4
<211>23
<212>PRT
＜213〉tomato yellow leaf curl virus (Tomato yellow leaf curl virus)
<400>4
Thr?His?Ala?Ser?Asn?Pro?Val?Tyr?Val?Thr?Met?Lys?Ile?Arg?Ile?Tyr
1 5 10 15
Phe?Tyr?Asp?Ser?Ile?Ser?Asn
20
<210>5
<211>134
<212>PRT
＜213〉tomato yellow leaf curl virus (Tomato yellow leaf curl virus)
<400>5
Met?Asp?Ser?Arg?Thr?Gly?Glu?Leu?Ile?Thr?Ala?Pro?Gln?Ala?Glu?Asn
1 5 10 15
Gly?Val?Phe?Ile?Trp?Glu?Ile?Asn?Asn?Pro?Leu?Tyr?Phe?Lys?Ile?Thr
20 25 30
Glu?His?Ser?Gln?Arg?Pro?Phe?Leu?Met?Asn?His?Asp?Ile?Ile?Ser?Ile
35 40 45
Gln?Ile?Arg?Phe?Asn?His?Asn?Ile?Arg?Lys?Val?Met?Gly?Ile?His?Lys
50 55 60
Cys?Phe?Leu?Asn?Phe?Arg?Ile?Trp?Thr?Thr?Leu?Gln?Pro?Gln?Thr?Gly
65 70 75 80
His?Phe?Leu?Arg?Val?Phe?Arg?Tyr?Glu?Val?Leu?Lys?Tyr?Leu?Asp?Ser
85 90 95
Leu?Gly?Val?Ile?Ser?Ile?Asn?Asn?Val?Ile?Arg?Ala?Val?Asp?His?Val
100 105 110
Leu?Tyr?Asp?Val?Leu?Glu?Asn?Thr?Ile?Asn?Val?Thr?Glu?Thr?His?Asp
115 120 125
Ile?Lys?Tyr?Lys?Phe?Tyr
130
<210>6
<211>109
<212>PRT
＜213〉tomato yellow leaf curl virus (Tomato yellow leaf curl virus)
<400>6
Pro?Ile?Arg?Arg?Lys?Arg?Val?Asp?Leu?Asp?Cys?Gly?Cys?Ser?Tyr?Tyr
1 5 10 15
Leu?His?Leu?Asn?Cys?Asn?Asn?His?Gly?Phe?Thr?His?Arg?Gly?Thr?His
20 25 30
His?Cys?Ser?Ser?Gly?Arg?Glu?Trp?Arg?Phe?Tyr?Leu?Gly?Asp?Lys?Gln
35 40 45
Ser?Pro?Leu?Phe?Gln?Asp?Asn?Arg?Thr?Gln?Pro?Ala?Ala?Ile?Ser?Asn
50 55 60
Glu?Pro?Arg?His?His?Phe?His?Ser?Asp?Lys?Ile?Gln?Pro?Gln?His?Gln
65 70 75 80
Glu?Gly?Asn?Gly?Asp?Ser?Gln?Met?Phe?Ser?Gln?Leu?Pro?Asn?Leu?Asp
85 90 95
Asp?Ile?Thr?Ala?Ser?Asp?Trp?Ser?Phe?Leu?Lys?Ser?Ile
100 105
Claims (10)
1. the primer of the composition of DNA shown in the sequence 2 of DNA and sequence table shown in the sequence 1 of sequence table is right.
2. the described primer of claim 1 is to the application in the assistant identification tomato yellow leaf curl virus.
3. the method for assistant identification tomato yellow leaf curl virus is that the genomic dna with virus to be measured is a template, with the described primer of claim 1 to carrying out pcr amplification; If obtain the dna fragmentation of 543bp, virus to be measured is candidate's tomato yellow leaf curl virus; If do not obtain the dna fragmentation of 543bp, virus to be measured is candidate's non-tomato yellow leaf curl virus.
4. method as claimed in claim 3 is characterized in that: described virus to be measured is tomato yellow leaf curl virus, Tomato mosaic virus, fern leaf of tomato poison or tomato bushy stunt virus.
5. the described primer of claim 1 is to the application in the test kit of preparation assistant identification tomato yellow leaf curl virus.
6. the test kit of an assistant identification tomato yellow leaf curl virus comprises that the described primer of claim 1 is right.
7. whether the described primer of claim 1 is to infecting the application in the tomato yellow leaf curl virus the assistant identification tomato.
8. whether the assistant identification tomato infects the method for tomato yellow leaf curl virus, comprises the steps:
(1) total DNA of extraction tomato leaf to be measured;
(2) the total DNA that extracts with step (1) is a template, with the described primer of claim 1 to carrying out pcr amplification; If obtain the dna fragmentation of 543bp, tomato to be measured has infected tomato yellow leaf curl virus; If do not obtain the dna fragmentation of 543bp, tomato to be measured does not infect tomato yellow leaf curl virus.
9. method as claimed in claim 8 is characterized in that: described tomato is tomato variety M82 or tomato variety CM.
10. right following (a) and/or the application (b) of the described primer of claim 1:
(a) application in tomato breeding;
(b) application in the tomato yellow leaf curl virus prevention and control in tomato.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643805A (en) * | 2012-04-27 | 2012-08-22 | 北京市农林科学院 | Specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of specific primer pair |
| CN103014179A (en) * | 2012-12-19 | 2013-04-03 | 广东省农业科学院植物保护研究所 | Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease |
| CN104164483A (en) * | 2014-05-30 | 2014-11-26 | 天津市农业生物技术研究中心 | Method for detecting ToMV resistant gene Tm2<2> by using allele specific PCR technology |
| CN107058626A (en) * | 2017-04-19 | 2017-08-18 | 北京市植物保护站 | Special primer and its application for quantitatively detecting tomato yellow leaf curl China virus |
| CN110628947A (en) * | 2019-09-27 | 2019-12-31 | 江苏省农业科学院 | Method for rapidly identifying tomato yellow leaf curl virus and tomato chlorosis virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560566A (en) * | 2009-04-13 | 2009-10-21 | 浙江省农业科学院 | Molecular marker auxiliary selection method for TYLCV breeding |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560566A (en) * | 2009-04-13 | 2009-10-21 | 浙江省农业科学院 | Molecular marker auxiliary selection method for TYLCV breeding |
Non-Patent Citations (2)
| Title |
|---|
| 《中国果菜》 20091031 王富 李文丽 番茄黄化曲叶病毒病综合防控技术 31-33 1-10 , 第7期 * |
| 《中国蔬菜》 20100101 李常保 等 北京番茄黄化曲叶病毒病得发生及分子检测 第29页，图1 1-10 , 第1期 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643805A (en) * | 2012-04-27 | 2012-08-22 | 北京市农林科学院 | Specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of specific primer pair |
| CN102643805B (en) * | 2012-04-27 | 2013-07-10 | 北京市农林科学院 | Specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of specific primer pair |
| CN103014179A (en) * | 2012-12-19 | 2013-04-03 | 广东省农业科学院植物保护研究所 | Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease |
| CN103014179B (en) * | 2012-12-19 | 2014-04-16 | 广东省农业科学院植物保护研究所 | Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease |
| CN104164483A (en) * | 2014-05-30 | 2014-11-26 | 天津市农业生物技术研究中心 | Method for detecting ToMV resistant gene Tm2<2> by using allele specific PCR technology |
| CN104164483B (en) * | 2014-05-30 | 2016-06-08 | 天津市农业生物技术研究中心 | Utilize ApoE gene technology for detection ToMV resistant gene Tm22Method |
| CN107058626A (en) * | 2017-04-19 | 2017-08-18 | 北京市植物保护站 | Special primer and its application for quantitatively detecting tomato yellow leaf curl China virus |
| CN110628947A (en) * | 2019-09-27 | 2019-12-31 | 江苏省农业科学院 | Method for rapidly identifying tomato yellow leaf curl virus and tomato chlorosis virus |
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| Publication number | Publication date |
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| CN102234643B (en) | 2013-01-23 |
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