CN102690776A - Agrobacterium strain carrying Yunnan tomato leaf curl virus infectious clones and application thereof - Google Patents
Agrobacterium strain carrying Yunnan tomato leaf curl virus infectious clones and application thereof Download PDFInfo
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- CN102690776A CN102690776A CN2012101936645A CN201210193664A CN102690776A CN 102690776 A CN102690776 A CN 102690776A CN 2012101936645 A CN2012101936645 A CN 2012101936645A CN 201210193664 A CN201210193664 A CN 201210193664A CN 102690776 A CN102690776 A CN 102690776A
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Abstract
The invention discloses a agrobacterium strain carrying Yunnan tomato leaf curl virus infectious clones and application thereof. 1.4 forward repetitive Yunnan tomato leaf curl virus full-length genomes of DNAs (deoxyribonucleic acids) are constructed to an efficient plant expression vector pBinPLUS by a molecular biology method, and an agrobacterium strain EHA105 is introduced by an electric shock method to obtain a virus infectious clone agrobacterium strain ToLCYnV-Y194 capable of efficiently infecting plants, wherein the collection number of the agrobacterium strain is CGMCC No.5931. The strain can generate massive Yunnan tomato leaf curl virus infectious clone which can efficiently infect multiple host plants, and provides technical and material supports for pathogenicity analysis and genome function research of Yunnan tomato leaf curl virus as well as tomato breeding for disease resistance.
Description
Technical field
Field under the present invention is the genetically engineered field, especially relates to a kind of agrobacterium strains and application thereof of carrying Yunnan tomato curve leaf disease virus infectious clone.
Background technology
Since nineteen nineties; Increase along with global warming and international trade; There is the unprecedented expansion in zone in Bemisia tabaci; By also worldwide (Africa, Mediterranean basin, East Asia, South Asia, Middle and North America and area, the Caribbean Sea etc.) big area outburst of the louse-borne geminivirus infection of tobacco powder, and on multiple important crop (tomato, cassava and cotton etc.), caused serious harm, caused heavy losses to crop production.The crops such as tomato, cassava, cotton and tobacco of existing more than 40 countries are endangered by the destructiveness of this viroid at present.China has found more than 40 kind of geminivirus infection on weeds such as crop such as tobacco, tomato, papaya and the pumpkin of 17 provinces and cities such as Yunnan, Guangxi, Guangdong, Fujian, Hainan, Beijing, Shanghai and false horsewhip, Herba Agerati Conyzoidis, rare alkali, and is the trend that expansion spreads.
Geminivirus infection is one type of unique in plant virus strand ring-type dna virus with twin particle form, and virus particle is the doublet structure, and particle size is 18 nm * 30 nm, no coating.The virus that has Economic Importance mostly be geminivirus infection section (
Geminiviridae) in bean golden mosaic virus belong to (
Begomovirus) virus, under the state of nature, this belong to virus by Bemisia tabaci (
Bemisia tabaci) propagate with the persistence mode, thereby this genus virus also claim whitefly-transmitted geminivirus (whitefly-transmitted geminivirus, WTG).
Because having become China WTGs, Special geography position, Yunnan concentrate one of generation range of distribution.In Yunnan Province; The geminivirus infection disease mainly is distributed in the torrid zone, subtropical climate type area and the temperate zone installment agriculture growth district of height above sea level below 1300 meters, comprises basin, northern Jinsha jiang River, the western Lancang River-basin, Nujiang, middle and south Red River and basin, the southern Lancang River.At present, the WTGs that has identified in Yunnan has 21 kinds, accounts for nearly 50% of China's kind.
Tomato is the important vegetable crop in the whole world, and it is wide in variety, and wide adaptability is nutritious, is one of 30 kinds of the highest farm crop of whole world ultimate production.At present, existing nearly 20 kinds of the geminivirus infection kind of on tomato, identifying, geminivirus infection has become one of important restraining factors of tomato production.(Tomato yellow leaf curl virus is to endanger the heaviest geminivirus infection of tomato in the world TYLCV) to tomato yellow leaf curl virus.In recent years, on the tomato of Yunnan, found a geminivirus infection novel species---Yunnan tomato curve leaf disease virus, the harm of this disease on tomato increases the weight of day by day.
The plant virus infectious clone is the genomic prerequisite of research viral function.Nineteen eighty-two, the infectious clone of plant DNA virus is first tomato golden mosaic virus (Tomato golden mosaic virus, TGMV) the middle structure successfully.Though should technology produce lateer, development but be very rapid, particularly for virus that can not mechanical inoculation, be configured to further research viral genome structure, function and the expression and regulation mechanism of its infectious clone provide possible.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of agrobacterium strains and application thereof of carrying Yunnan tomato curve leaf disease virus infectious clone is provided.
Carry the agrobacterium strains of Yunnan tomato curve leaf disease virus infectious clone: preserving number is CGMCC No.5931, can produce the infectious clone of Yunnan tomato curve leaf disease virus.
The infectious clone of described Yunnan tomato curve leaf disease virus is: the molecular biology method that utilize that PCR, enzyme are cut, gene connects is structured in the full-length gene group DNA of 1.4 forward multiple Yunnan tomato curve leaf disease virus on the high-efficiency plant expression vector pBinPLUS.
The agrobacterium strains that carries Yunnan tomato curve leaf disease virus infectious clone is used for pathogenic analysis, the known viral gene function research of Yunnan tomato curve leaf disease virus.Be used for the resistant variety screening of anti-Yunnan tomato curve leaf disease virus, be the service of tomato breeding for disease resistance.
The present invention compared with prior art has beneficial effect: the agrobacterium strains that 1) provides can produce the infectious clone of Yunnan tomato curve leaf disease virus in a large number; 2) utilize infectious clone provided by the invention efficiently to infect various plants, need not expensive instrument, simple to operate, good reproducibility; 3) utilize infectious clone provided by the invention can be effective to pathogenic analysis, the known viral gene function research of Yunnan tomato curve leaf disease virus; 4) utilize infectious clone provided by the invention to be used for the resistant variety screening of Yunnan tomato leaf curl viral disease, be the service of tomato breeding for disease resistance.
Description of drawings
The pathogenic analysis of Fig. 1 Yunnan tomato curve leaf disease virus;
The symptom of Fig. 2 Yunnan tomato curve leaf disease virus on different tomato varieties;
Fig. 3 Yunnan tomato curve leaf disease virus is inoculated the PCR detected result of different tomato varieties.
Embodiment
Carry the agrobacterium strains of Yunnan tomato curve leaf disease virus infectious clone: be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 22nd, 2012; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; The classification name: the infectious clone of Yunnan tomato curve leaf disease virus, preserving number are CGMCC No.5931, and it can produce the infectious clone of Yunnan tomato curve leaf disease virus.
The infectious clone of described Yunnan tomato curve leaf disease virus is: the molecular biology method that utilize that PCR, enzyme are cut, gene connects is structured in the full-length gene group DNA of 1.4 forward multiple Yunnan tomato curve leaf disease virus on the high-efficiency plant expression vector pBinPLUS.
The agrobacterium strains that carries Yunnan tomato curve leaf disease virus infectious clone is used for pathogenic analysis, the known viral gene function research of Yunnan tomato curve leaf disease virus.Be used for the resistant variety screening of anti-Yunnan tomato curve leaf disease virus, be the service of tomato breeding for disease resistance.
Below in conjunction with embodiment and accompanying drawing the present invention is described further.
The structure of embodiment 1. Yunnan tomato curve leaf disease virus infectious clones
The agriculture bacillus mediated infectious clone of geminivirus infection needs 1.3-2.0 to repeat above full-length gene group, and must comprise 2 intergenic regions (IR district).With Yunnan tomato curve leaf disease virus genomic dna is template, to contain
EcoThe Y194EF (5 '-GCGGAATTCCTTAAAGTGCTTTAG-3 ') in R I single endonuclease digestion site and Y194ER (5 '-TAGGAATTCATGGGAGCCCAAAG-3 ') are primer, and pcr amplification also is cloned into pGEM-T Vector, obtain pGEM-T-Y194, use
BamH I with
EcoR I carries out enzyme to pGEM-T-Y194 and cuts, and obtains 0.4 full length DNA, inserts and uses
BamH I with
EcoThe binary vector pBinPLUS of R I double digestion obtains pBinPLUS-Y194-0.4A; Use
EcoR I cuts out the corresponding site of inserting pBinPLUS-Y194-0.4A behind the complete full length DNA from pGEM-T-Y194, obtains the infectious clone of pBinPLUS-Y194-1.4A.
Embodiment 2.pBinPLUS-Y194-1.4A recombinant vectors electric shock transforms Agrobacterium
Method through electric shock imports Agrobacterium host cell EHA105 with the recombinant plant expression vector.At first, the competent cell that needs preparation EHA105.Single colony inoculation of picking agrobacterium tumefaciens EHA105 contains in the YEP liquid nutrient medium of 40 μ g/mL Rifampins (Rif) in 5mL, and 28 ℃ of 200 rpm is cultured to OD600 ≈ 0.6; Collect enough bacterium with 1.5 mL centrifuge tubes; 4 ℃ of 8,000 centrifugal 1 min of rpm abandons most supernatant; Thalline is with 200 μ L ddH
2O is fully resuspended, and 4 ℃ of 8,000 centrifugal 1 min of rpm abandons most supernatant; Repeating step 3 three times is abandoned most supernatant; Last thalline is used 200 μ L ddH again
2O is fully resuspended, is the agrobacterium tumefaciens competent cell.Get 10 μ L recombinant plasmid pBinPLUS-Y194-1.4A and join in the 200 μ L agrobacterium tumefaciens competent cells, mixing changes in the electric shock cup gently, places 10 min on ice.Be ready to electric shock device (BIO-RAD) this moment, and voltage transfers to 2400 V, shocks by electricity.After having shocked by electricity, room temperature adds 800 μ L YEP recovery medias after leaving standstill 2 min, and 28 ℃ leave standstill 1 h, then at 28 ℃, and 200 rpm shaking culture, 2 h.Get 200 μ L at last and coat on the YEP solid medium that contains 100 μ g/mL kantlex (Kan) and 40 μ g/mL Rifampins (Rif), in incubator, be inverted cultivation 48 h for 28 ℃.
Embodiment 3. carries the Agrobacterium thorn inoculation plant of Yunnan tomato curve leaf disease virus infectious clone
PBinPLUS-Y194-1.4A cultivated 48 hours down for 28 ℃ in the YEP solid plate culture medium culturing that contains 100 μ g/mL kantlex and 40 μ g/mL Rifampins.The inoculation plant selects for use 4-6 leaf phase fully extended seedling to be advisable.Inoculation Ben Shi cigarette, tomato, Nicotiana glutinosa, three lives cigarette, common cigarette.Concrete inoculation method is following, gets a sterilization toothpick, and picking solid bacterium colony carries out phloem thorn inoculation getting at 3 apart from root 1-2cm place, and the inoculation plant places 25 ℃ of Isolation warm houses cultivations.
The pathogenic analysis of embodiment 4. Yunnan tomato curve leaf disease virus infectious clones
The inoculation plant carries out observation of symptoms, and all inoculation plant are carried out PCR and ELISA detection.Observation of symptoms shows, inoculates after 6 days, and the Ben Shi cigarette begins to appear the symptom that volume, vein thicken on the serious leaf; Inoculate after 10 days the symptom that tomato performance leafing last volume, vein thicken; The phenotype that Nicotiana glutinosa began to have vein to thicken in inoculation in back 8 days; Three lives cigarette then presents leaf last volume symptom in inoculation after 9 days; Petunia begins to produce Ye Shangjuan in inoculation after 7 days, vein thickens and the symptom of leaf surface nadel (Fig. 1).Y194 DNA infect on the different host to infect efficient all very high, more than 90%.
The resistance screening of the anti-Yunnan of embodiment 5. different tomato varieties tomato curve leaf disease virus
Utilize the infectious clone of Yunnan tomato curve leaf disease virus, inoculate 3 tomato variety Zhejiang A, Zhejiang B, Zhejiang C, and it is carried out evaluation of resistance, make positive control, and the healthy plant that each kind is set respectively is as negative control with susceptible kind ruby.Handle inoculation 30 strain tomatoes for every kind.After inoculation, carry out observation of symptoms and write down disease time, disease symptom, statistics morbidity strain number, the sickness rate of calculating different varieties tomato.Yunnan tomato curve leaf disease virus inoculation susceptible kind ruby begins morbidity after 10 days, the last volume symptom appears in sick leaf, and 30 strain rubies are all fallen ill after 20 days, and sickness rate is 100%; Tomato curve leaf disease virus inoculating tomato Zhejiang, Yunnan A kind, 15 days begin to produce leaf rolling, yellow symptom, and the sequela rate was 20% in 40 days; Inoculation Zhejiang B kind begins to produce yellow, leaf-shrinkage symptom after 14 days, the sequela rate was 12% in 40 days; Inoculation Zhejiang C kind begins to produce blade edge yellow symptom after 18 days, the sequela rate was 7% in 40 days.Yunnan tomato curve leaf disease virus inoculation 35 days disease symptom of each tomato variety such as Fig. 2.Behind the virus inoculation 35 days; Get plant young leaves CTAB method extracting DNA of plants; With the Auele Specific Primer ToLCYnVF of Yunnan tomato curve leaf disease virus (5 '-GTCCCGCCGATATAGTCATTTCC-3 ') and ToLCYnVR (5 '-GAGAATCATAAAAATAGATACGG-3 ') every strain plant being carried out PCR detects; The band (Fig. 3) of special 750bp all can increase; Explain that Yunnan tomato curve leaf disease virus can both each tomato variety of successful systemic infection, and the symptom difference that shows behind the virus infection is caused by different tomato varieties.The result who detects the virus infection rate from disease symptom, sickness rate, PCR can draw, and 3 tomato variety Zhejiang A, Zhejiang B, Zhejiang C all have certain resistance to Yunnan tomato curve leaf disease virus, and wherein tomato variety Zhejiang C resistance is the strongest.
Y194EF（5’-?gcggaattccttaaagtgctttag?-3’）
Y194ER?(5’-?taggaattcatgggagcccaaag?-3’)
ToLCYnVF（5’-gtcccgccgatatagtcatttcc-3’）
ToLCYnVR?(5’-gagaatcataaaaatagatacgg-3’)
Claims (4)
1. agrobacterium strains that carries Yunnan tomato curve leaf disease virus infectious clone, it is characterized in that: preserving number is CGMCC No.5931, can produce the infectious clone of Yunnan tomato curve leaf disease virus.
2. a kind of agrobacterium strains that carries Yunnan tomato curve leaf disease virus infectious clone as claimed in claim 1 is characterized in that the infectious clone of described Yunnan tomato curve leaf disease virus is: the molecular biology method that utilize that PCR, enzyme are cut, gene connects is structured in the full-length gene group DNA of 1.4 forward multiple Yunnan tomato curve leaf disease virus on the high-efficiency plant expression vector pBinPLUS.
3. an application of carrying the agrobacterium strains of Yunnan tomato curve leaf disease virus infectious clone as claimed in claim 1 is characterized in that being used for pathogenic analysis, the known viral gene function research of Yunnan tomato curve leaf disease virus.
4. application of carrying the agrobacterium strains of Yunnan tomato curve leaf disease virus infectious clone as claimed in claim 1 is characterized in that being used for the resistant variety screening of anti-Yunnan tomato curve leaf disease virus, is the service of tomato breeding for disease resistance.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113999872A (en) * | 2021-11-17 | 2022-02-01 | 中国农业科学院植物保护研究所 | Application of tobacco DCP1/ATG8i gene in inhibiting tomato yellow leaf curl virus infection |
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| CN1424400A (en) * | 2002-12-24 | 2003-06-18 | 浙江大学 | Method for establishing carrier by plant DNA virus infestation |
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| CN101884281A (en) * | 2010-06-23 | 2010-11-17 | 江苏省农业科学院 | Method for determining resistance of tomatoes to tomato yellow leaf curl viruses by injection inoculation |
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| CN1424400A (en) * | 2002-12-24 | 2003-06-18 | 浙江大学 | Method for establishing carrier by plant DNA virus infestation |
| CN1425678A (en) * | 2002-12-24 | 2003-06-25 | 浙江大学 | Plant virus satellite DNA beta molecule, infectivity clone and method for preparing expression carrier |
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Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999872A (en) * | 2021-11-17 | 2022-02-01 | 中国农业科学院植物保护研究所 | Application of tobacco DCP1/ATG8i gene in inhibiting tomato yellow leaf curl virus infection |
| CN113999872B (en) * | 2021-11-17 | 2023-09-22 | 中国农业科学院植物保护研究所 | Application of tobacco DCP1/ATG8i gene in inhibiting tomato yellow leaf curl virus infection |
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Application publication date: 20120926 |