CN111154731A - Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application - Google Patents
Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application Download PDFInfo
- Publication number
- CN111154731A CN111154731A CN202010014216.9A CN202010014216A CN111154731A CN 111154731 A CN111154731 A CN 111154731A CN 202010014216 A CN202010014216 A CN 202010014216A CN 111154731 A CN111154731 A CN 111154731A
- Authority
- CN
- China
- Prior art keywords
- tylcv
- dna
- tomato
- virus
- leaf curl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000702308 Tomato yellow leaf curl virus Species 0.000 title claims abstract description 27
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 27
- 230000002458 infectious effect Effects 0.000 title claims abstract description 26
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 241000700605 Viruses Species 0.000 claims abstract description 26
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims abstract description 20
- 238000012216 screening Methods 0.000 claims abstract description 12
- 239000013612 plasmid Substances 0.000 claims abstract description 11
- 230000000840 anti-viral effect Effects 0.000 claims abstract description 9
- 241000589158 Agrobacterium Species 0.000 claims abstract description 8
- 241000219194 Arabidopsis Species 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000002574 poison Substances 0.000 claims abstract description 3
- 231100000614 poison Toxicity 0.000 claims abstract description 3
- 241000227653 Lycopersicon Species 0.000 claims abstract 5
- 238000012258 culturing Methods 0.000 claims description 15
- 239000012634 fragment Substances 0.000 claims description 13
- 238000012795 verification Methods 0.000 claims description 9
- 238000001712 DNA sequencing Methods 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 6
- 230000007918 pathogenicity Effects 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 10
- 241000219195 Arabidopsis thaliana Species 0.000 abstract description 7
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 7
- 241000208125 Nicotiana Species 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 208000024891 symptom Diseases 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 31
- 240000003768 Solanum lycopersicum Species 0.000 description 19
- 238000011081 inoculation Methods 0.000 description 8
- 241000207746 Nicotiana benthamiana Species 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 4
- 241000702463 Geminiviridae Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 241000589159 Agrobacterium sp. Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241000219833 Phaseolus Species 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/12011—Geminiviridae
- C12N2750/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a tomato yellow leaf curl virus isolate TYLCV-BJ, a construction method of infectious clone and application thereof, wherein the whole genome sequence is shown as SEQ ID NO.2 in a sequence table, and the preservation number is CGMCC NO. 19119. 1.4 tandem repeats of TYLCV-BJ genome sequence to pCambia2300 vector, obtain pCambia2300-TYLCV-BJ-1.4DNA recombinant plasmid; introduced into the agrobacterium EHA105, the obtained agrobacterium can successfully infect tobacco and tomato plants. The recombinant vector pCambia2300-TYLCV-BJ-1.4DNA has the following characteristics: 1) the plant infection is achieved, and tobacco, tomato and Arabidopsis plants can be successfully infected; 2) can cause obvious virus symptoms on tobacco, tomato and arabidopsis plants and accumulate the DNA of the virus; 3) the mutant virus gene can be used for researching the functions of virus genes; 4) as a poison source, the method can be used for screening antiviral tomato varieties and antiviral arabidopsis thaliana mutants.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a tomato yellow leaf curl virus isolate and an infectious clone construction method and application thereof.
Background
Tomato Yellow Leaf Curl Virus (TYLCV) is a single-stranded, circular plant DNA virus belonging to the geminiviridae family, the genus phaseolus viridae. TYLCV can infect various vegetable crops and economic crops of solanaceae and is an important pathogen in agricultural production in China and the world. The pathogenicity and function of a plant virus is generally studied, requiring that an infectious clone of the virus be obtained first.
The infectious cloning of the virus is to construct the virus with infectivity in vitro by means of molecular biology and genetic engineering technology. The known tomato yellow leaf curl virus TYLCV-SH2 has weak infectivity, and is not beneficial to experimental development.
Disclosure of Invention
The invention aims to provide a tomato yellow leaf curl virus isolate TYLCV-BJ, and a construction method and application of an infectious clone.
The tomato yellow leaf curl virus isolate TYLCV-BJ is separated from a tomato sample suspected of being infected by tomato yellow leaf curl virus collected from a Beijing greenhouse in a laboratory, and the whole genome sequence of the tomato yellow leaf curl virus isolate TYLCV-BJ is shown as SEQ ID NO.2 in a sequence table.
The tomato yellow leaf curl virus isolate TYLCV-BJ is introduced into agrobacterium, the strain is preserved in China general microbiological culture Collection center (CGMCC) in 12 and 9 months in 2019, and the registration number is CGMCC No.19119, and the classification and the name are as follows: agrobacterium sp. The preservation date is as follows: 12 and 9 months in 2019. China general microbiological culture Collection center address: xilu No.1 Hospital No.3, Beijing, Chaoyang, North.
The method for constructing the infectious clone of the tomato yellow leaf curl virus isolate TYLCV-BJ comprises the following steps: 1.4 tandem repeats of TYLCV-BJ genomic sequence was constructed into pCambia2300 vector.
The method specifically comprises the following steps:
(1) the T-TYLCV-BJ-DNA is cut by using restriction enzymes EcoRI and BamHI to obtain a DNA fragment of about 1.2kb, which is about 0.4 copy of the genome; connecting to a pCambia2300 vector after cutting EcoRI and BamHI; the recombinant vector pCambia2300-TYLCV-BJ-0.4 was transformed into E.coli DH5a using primers: screening positive clones by TYLCV-BJ-EcoRI-F and TYLCV-BJ-BamH-R; TYLCV-BJ-EcoRI-F and TYLCV-BJ-BamH-R are shown in SEQ ID: no. 5-6; selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing;
TYLCV-BJ-EcoRI-F：GAATTCCTTGAAGTGCTTTAAATAATG
TYLCV-BJ-BamH-R：AAGTGGATCCCACATAGTGCAAGAC
(2) using restriction enzyme BamHI to cut T-TYLCV-BJ-DNA, and recovering about 2.7kb DNA fragment which is 1 copy of genome; the BamHI-digested DNA fragment of about 2.7kb was ligated into the BamHI-digested pCambia2300-TYLCV-BJ-0.4DNA vector, and the recombinant vector pCambia2300-TYLCV-BJ-1.4DNA was transformed into E.coli DH5a using the following primers: screening positive clones by TYLCV-BJ-FW-F and TYLCV-BJ-FW-R; TYLCV-BJ-FW-F and TYLCV-BJ-FW-R are as shown in SEQ ID: no. 7-8; selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing; after successful verification, the strain is introduced into an agrobacterium EHA105 strain;
TYLCV-BJ-FW-F：ACTGCCTGAATTGAGTGCC
TYLCV-BJ-FW-R：GTACCGGAAGCCCAGAATATAC。
the recombinant vector pCambia2300-TYLCV-BJ-1.4DNA is applied to analyzing the pathogenicity of TYLCV-BJ virus, and is used as a toxicity source for screening antiviral tomato varieties and antiviral Arabidopsis thaliana mutants.
The recombinant vector pCambia2300-TYLCV-BJ-1.4DNA constructed by the above method contains the nucleotide sequence shown in SEQ ID: the sequence shown in No. 1.
The recombinant vector pCambia2300-TYLCV-BJ-1.4DNA constructed by the method of the invention has the following characteristics and advantages:
(1) has higher infectivity than the known tomato yellow leaf curl virus, can successfully infect tobacco, tomato and Arabidopsis plants;
(2) can cause obvious virus symptoms on tobacco, tomato and arabidopsis plants and accumulate the DNA of the virus;
(3) the mutant virus gene can be used for researching the functions of virus genes;
(4) as a poison source, the method can be used for screening antiviral tomato varieties and antiviral arabidopsis thaliana mutants.
The construction of the infectious clone provides a useful tool for researching the function of the TYLCV gene and screening plant varieties resisting TYLCV.
The tomato yellow leaf curl virus isolate TYLCV-BJ and the construction method and application of the infectious clone of the invention are further described in the following description and specific examples with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the construction of a pCambia2300-TYLCV-BJ-1.4DNA invasive cloning (TYLCV-BJ-1.4 DNA for short) vector.
FIG. 2 shows the symptoms of viral infection of plants inoculated with TYLCV-BJ-1.4DNA infectious clones in Nicotiana benthamiana and tomato plants 8, 16 and 24 days later (dpi) and buffer inoculation as a control (Mock);
FIG. 3 shows the virus DNA accumulation after 8 days, 16 days and 24 days of TYLCV-BJ-1.4DNA infectious clone inoculation on Nicotiana benthamiana (A) and tomato (B) plants by real-time quantitative PCR detection, wherein 25sRNA of Nicotiana benthamiana and tomato is used as an internal reference;
FIG. 4(A) TYLCV-BJ-1.4DNA infectious clones after 20 days of inoculation (dpi) into Arabidopsis plants, the plants were infected with symptoms of virus, and buffer was inoculated as a control (Mock); FIG. 4 (B) PCR detection of (A) the presence of virus in systemic leaves of plants.
FIG. 5(A) real-time quantitative PCR detection of the accumulation of viral DNA after 10, 18 and 26 days of tomato inoculation of TYLCV-BJ-1.4DNA and TYLCV-SH2-1.4DNA infectious clones, and tomato 25sRNA was used as an internal control. FIG. 5 (B) Westernblot analysis of the amount of TYLCV virus Capsid Protein (CP) accumulation after 10, 18, and 26 days of tomato inoculation of TYLCV-BJ-1.4DNA and TYLCV-SH2-1.4DNA invasive clones, and the plant Rubisco stained with Coomassie Brilliant Blue (CBB) was used as an internal reference; FIG. 5(C) was performed to examine TYLCV-BJ-1.4DNA and TYLCV-SH2-1.4DNA infectious clones by PCR C1(TYLCV-C1) of TYLCV virus 20 days after inoculation into Arabidopsis thaliana, and it was confirmed that the constructed infectious clone TYLCV-BJ-1.4DNA, but not TYLCV-SH2-1.4DNA, could really and effectively infect Arabidopsis thaliana plants.
Detailed Description
The tomato yellow leaf curl virus isolate TYLCV-BJ and the infectious clone construction method comprise the following steps:
(1) taking a tomato sample suspected of being infected with geminivirus from a greenhouse;
(2) extracting DNA of a tomato sample by using CTAB;
(3) taking DNA of 100ng tomato sample, amplifying a DNA fragment of about 500bp by using a geminivirus degenerate primer PA/PB, connecting the DNA fragment with a gold holo-pEASY-Blunt Simple Cloning T vector (T vector for short), transforming a recombinant vector into escherichia coli DH5a, and screening positive clones by using universal primers M13-F and M13-R (shown as SEQ ID: No. 12-13); selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing; obtaining the sequence SEQ ID: no. 1;
M13-F：TGTAAAACGACGGCCAGT
M13-R：CAGGAAACAGCTATGACC
PA：TAATATTACCKGWKGVCCSC
PB：TGGACYTTRCAWGGBCCTTCACA
*B＝C,T or G，K＝G or T，R＝Aor G，S＝C or G，V＝A，C or G，W＝Aor T，Y＝Cor T
(4) according to SEQ ID: no.1DNA sequence, a pair of primer pairs TYLCV-BJ-BamHI-F/TYLCV-BJ-BamHI-R containing 15nt complementarity in between were designed as shown in SEQ ID: no. 3-4; amplifying DNA of a tomato sample to obtain a DNA fragment of about 3.0kb, connecting the DNA fragment with a T vector, transforming a recombinant vector into escherichia coli DH5a, and screening positive clones by using universal primers M13-F and M13-R; selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing; obtaining the full-length sequence of the virus, i.e. SEQ ID: no.2 shows
TYLCV-BJ-BamHI-F：atgtgggatccacttctaaatgaatttcct
TYLCV-BJ-BamHI-R：aagtggatcccacatagtgcaagac
(5) The T-TYLCV-BJ-DNA was digested with the restriction enzymes EcoRI and BamHI to obtain a DNA fragment of about 1.2kb (approximately 0.4 copy of the genome); connecting to a pCambia2300 vector after cutting EcoRI and BamHI; the recombinant vector pCambia2300-TYLCV-BJ-0.4 was transformed into E.coli DH5a using the following primers: screening positive clones by TYLCV-BJ-EcoRI-F and TYLCV-BJ-BamH-R; TYLCV-BJ-EcoRI-F and TYLCV-BJ-BamH-R are shown as SEQ ID: no. 5-6; selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing;
TYLCV-BJ-EcoRI-F：gaattccttgaagtgctttaaataatg
TYLCV-BJ-BamH-R：aagtggatcccacatagtgcaagac
(6) as shown in FIG. 1, T-TYLCV-BJ-DNA was digested with the restriction enzyme BamHI, and a DNA fragment of about 2.7kb (1 copy of the genome) was recovered; the BamHI-digested DNA fragment of about 2.7kb was ligated into the BamHI-digested pCambia2300-TYLCV-BJ-0.4DNA vector, and the recombinant vector pCambia2300-TYLCV-BJ-1.4DNA was transformed into E.coli DH5a using the following primers: screening positive clones by TYLCV-BJ-FW-F and TYLCV-BJ-FW-R; TYLCV-BJ-FW-F and TYLCV-BJ-FW-R are as shown in SEQ ID: no. 7-8; selecting positive clones, and culturing; extracting and culturing plasmids of target clones; verification by DNA sequencing; after successful verification, the strain is introduced into an agrobacterium EHA105 strain;
TYLCV-BJ-FW-F：actgcctgaattgagtgcc
TYLCV-BJ-FW-R：gtaccggaagcccagaatatac
(7) activating agrobacterium, inoculating agrobacterium liquid containing pCambia2300-TYLCV-BJ-1.4DNA plasmid to wild type Nicotiana benthamiana and tomato plants, observing plant symptoms as shown in figure 2, and determining that the constructed infectious clonal virus has infectivity;
as shown in fig. 3, total DNA on the nicotiana benthamiana plant and the new leaf of the tomato plant was extracted, and real-time quantitative PCR analysis was performed to determine that the constructed infectious clonal virus could be truly and effectively replicated and infected in the tomato and tobacco plants;
as shown in FIGS. 4 and 5, total DNA on new leaves of Arabidopsis plants was extracted and PCR analysis was performed to confirm that the constructed infectious clonal viruses could really and effectively infect Arabidopsis plants.
It is evident from FIG. 5(A) that the accumulation of viral DNA was much higher for TYLCV-BJ than for TYLCV-SH2 virus 10 days after inoculation; FIG. 5(C), detecting TYLCV-BJ-1.4DNA and TYLCV-SH2-1.4DNA infectious clone by PCR C1(TYLCV-C1) of TYLCV virus 20 days after inoculation into Arabidopsis thaliana, confirmed that the constructed infectious clone TYLCV-BJ-1.4DNA, but not TYLCV-SH2-1.4DNA, could really and effectively infect Arabidopsis thaliana plants. The primers used were TYLCV-C1-F and TYLCV-C1-R (SEQ ID: Nos. 10-11), and the sequence of TYLCV-BJ-C1 was amplified as SEQ ID: no. 9.
TYLCV-C1-F:atgcctcgtttatttaaac
TYLCV-C1-R:ttacgccttattggtttcttc
In conclusion, the constructed pCambia2300-TYLCV-BJ-1.4DNA infectious clone is beneficial to further researching the functions of virus proteins and the pathogenicity of viruses and is used as a toxic source for screening antiviral tobacco and tomato varieties.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> tomato yellow leaf curl virus isolate TYLCV-BJ, and construction method and application of infectious clone
<160>13
<170>SIPOSequenceListing 1.0
<210>1
<211>541
<212>DNA
<213> PA/PB amplification Sequence (Artificial Sequence)
<400>1
taatattacc ggaggcccgc gccttttctt tttatgtggt ccccacgagg gttccacaga 60
cgtcactgtc aaccaatcaa attgcatcct caaacgttag ataagtgttc atttgtcttt 120
atatacttgg tccccaagta gtttgtcttg cactatgtgg gatccacttc taaatgaatt 180
tcctgaatct gttcacggat ttcgttgtat gttagctatt aaatatttgc agtccgttga 240
ggaaacttac gagcccaata cattgggcca cgatttaatt agggatctta tatctgttgt 300
aagggcccgt gactatgtcg aagcgaccag gcgatataat catttccacg cccgtctcga 360
aggttcgccg aaggctgaac ttcgacagcc catacagcag ccgtgctgct gtccccattg 420
tccaaggcac aaacaagcga cgatcatgga cgtacaggcc catgtaccgg aagcccagaa 480
tatacagaat gtatcgaagc cctgatgttc ctcgtggatg tgaaggccca tgcaaagtcc 540
a 541
<210>2
<211>2781
<212>DNA
<213> TYLCV-BJ full length Sequence (Artificial Sequence)
<400>2
accggatggc cgcgcctttt ctttttatgt ggtccccacg agggttccac agacgtcact 60
gtcaaccaat caaattgcat cctcaaacgt tagataagtg ttcatttgtc tttatatact 120
tggtccccaa gtagtttgtc ttgcactatg tgggatccac ttctaaatga atttcctgaa 180
tctgttcacg gatttcgttg tatgttagct attaaatatt tgcagtccgt tgaggaaact 240
tacgagccca atacattggg ccacgattta attagggatc ttatatctgt tgtaagggcc 300
cgtgactatg tcgaagcgac caggcgatat aatcatttcc acgcccgtct cgaaggttcg 360
ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg 420
cacaaacaag cgacgatcat ggacgtacag gcccatgtac cggaagccca gaatatacag 480
aatgtatcga agccctgatg ttcctcgtgg atgtgaaggc ccatgtaaag tccagtctta 540
tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg 600
tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt 660
aggtaaagtc tggatggatg aaaatattaa gaagcagaat cacactaatc aggtcatgtt 720
cttcttggtt cgtgatagaa ggccttatgg aaacagccca atggattttg gacaggtttt 780
taatatgttc gataatgagc ccagtaccgc aaccgtgaag aatgatttgc gggataggtt 840
tcaagtgatg aggaaatttc atgctacagt tattggtggg ccctctggaa tgaaggaaca 900
ggcattagtt aagagatttt ttagaattaa cagtcatgta acttataatc atcaggaggc 960
agccaagtat gagaaccata ctgaaaacgc cttgttattg tatatggcat gtacccatgc 1020
ctctaatcca gtgtatgcaa ctatgaaaat acgcatctat ttctatgatt caatatcaaa 1080
ttaataaaat ttatatttta tatcatgagt ttctgttaca tttattgtgt tttcaagtac 1140
atcatacaat acatgatcaa ctgctctgat tacattgtta attgaaatta caccaagact 1200
atctaaatac ttaagaactt gatatctaaa tactcttaag aaacgaccag tctgaggctg 1260
taatgtcgtc caaattcgga agttgagaaa acatttgtga atccccaata tcttcctgat 1320
gttgtggttg aatcttatct gaatggaaat gatgtcgtgg ttcattagaa atggcctctg 1380
gctgtgttct gttatcttga aatagagggg attgtttatc tcccagataa aaacgccatt 1440
ctctgcttga ggagcagtga tgagttcccc tgtgcgtgaa tccatgattg ttgcagttga 1500
tgtggaggta gtatgagcag ccacagtcta ggtctacacg cttacgcctt attggtttct 1560
tcttggctat cttgtgttgg accttgattg atacttgcga acagtggctc gtagagggtg 1620
atgaaggttg cattcttgag agcccaattt ttcaaggata tgtttttttc ttcgtctaga 1680
tattccctat atgaggaggt aggtcctgga ttgcatagga agatagtggg aattccccct 1740
ttaatttgaa tgggcttccc gtactttgtg ttgctttgcc agtccctctg ggcccccatg 1800
aattccttga agtgctttaa ataatgcggg tctacgtcat caatgacgtt gtaccacgca 1860
tcattactgt acacctttgg gcttaagtct agatgtccac ataaataatt atgtgggcct 1920
agagacctgg cccacattgt cttgcctgtt ctgctatcac cctcaatgac aatacttatg 1980
ggtctccatg gccgcgcagc ggaagatacg acgttctcgg cgacccactc ttcaagttca 2040
tctggaactt gattaaaaga agaagaaaga aatggagaaa cataaacttc taaaggagga 2100
ctaaaaatcc tatctaaatt tgaacttaaa ttatgaaatt gtaaaatata gtcctttggg 2160
gccttctctt ttaatatatt gagggcctcg gatttactgc ctgaattgag tgcctcggca 2220
tatgcgtcgt tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact 2280
ccaaaatcaa tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta 2340
gctgcctgaa tgttcggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac 2400
cgttggttct tacattggta cttgccttcg aattggataa gcacatggag atgtggttcc 2460
ccattctcgt ggagttctct gcaaactttg atgtattttt tatttgttgg ggtttctagg 2520
ttttttaatt gggaaagtgc ttcctcttta gagagagaac aattgggata tgttaggaaa 2580
taatttttgg catatagttt aaataaacga ggcatgttga aatgaattgg tgtccctcaa 2640
agctctatgg caatcggtgt atcggtgtct tacttatacc tggacaccaa atggctattt 2700
ggtaattttg taaaagtaca ttgcaataca aaattcaaaa ttcaaaaatc aaatcattaa 2760
agcggccatc cgtataatat t 2781
<210>3
<211>30
<212>DNA
<213>TYLCV-BJ-BamHI-F(Artificial Sequence)
<400>3
atgtgggatc cacttctaaa tgaatttcct 30
<210>4
<211>25
<212>DNA
<213>TYLCV-BJ-BamHI-R(Artificial Sequence)
<400>4
aagtggatcc cacatagtgc aagac 25
<210>5
<211>27
<212>DNA
<213>TYLCV-BJ-EcoRI-F(Artificial Sequence)
<400>5
gaattccttg aagtgcttta aataatg 27
<210>6
<211>25
<212>DNA
<213>TYLCV-BJ-BamH-R(Artificial Sequence)
<400>6
aagtggatcc cacatagtgc aagac 25
<210>7
<211>19
<212>DNA
<213>TYLCV-BJ-FW-F(Artificial Sequence)
<400>7
actgcctgaa ttgagtgcc 19
<210>8
<211>22
<212>DNA
<213>TYLCV-BJ-FW-R(Artificial Sequence)
<400>8
gtaccggaag cccagaatat ac 22
<210>9
<211>1074
<212>DNA
<213>TYLCV-BJ-C1(Artificial Sequence)
<400>9
atgcctcgtt tatttaaact atatgccaaa aattatttcc taacatatcc caattgttct 60
ctctctaaag aggaagcact ttcccaatta aaaaacctag aaaccccaac aaataaaaaa 120
tacatcaaag tttgcagaga actccacgag aatggggaac cacatctcca tgtgcttatc 180
caattcgaag gcaagtacca atgtaagaac caacggttct tcgacctggt atccccaaac 240
aggtcagcac atttccatcc gaacattcag gcagctaaga gctcaacaga tgtcaagacc 300
tacgtggaga aagacggaga cttcattgat tttggagttt tccaaatcga tggcagatca 360
gctagaggag gtcagcaatc tgccaacgac gcatatgccg aggcactcaa ttcaggcagt 420
aaatccgagg ccctcaatat attaaaagag aaggccccaa aggactatat tttacaattt 480
cataatttaa gttcaaattt agataggatt tttagtcctc ctttagaagt ttatgtttct 540
ccatttcttt cttcttcttt taatcaagtt ccagatgaac ttgaagagtg ggtcgccgag 600
aacgtcgtat cttccgctgc gcggccatgg agacccataa gtattgtcat tgagggtgat 660
agcagaacag gcaagacaat gtgggccagg tctctaggcc cacataatta tttatgtgga 720
catctagact taagcccaaa ggtgtacagt aatgatgcgt ggtacaacgt cattgatgac 780
gtagacccgc attatttaaa gcacttcaag gaattcatgg gggcccagag ggactggcaa 840
agcaacacaa agtacgggaa gcccattcaa attaaagggg gaattcccac tatcttccta 900
tgcaatccag gacctacctc ctcatatagg gaatatctag acgaagaaaa aaacatatcc 960
ttgaaaaatt gggctctcaa gaatgcaacc ttcatcaccc tctacgagcc actgttcgca 1020
agtatcaatc aaggtccaac acaagatagc caagaagaaa ccaataaggc gtaa 1074
<210>10
<211>19
<212>DNA
<213>TYLCV-C1-F(Artificial Sequence)
<400>10
atgcctcgtt tatttaaac 19
<210>11
<211>21
<212>DNA
<213>TYLCV-C1-R(Artificial Sequence)
<400>11
ttacgcctta ttggtttctt c 21
<210>12
<211>18
<212>DNA
<213>M13-F(Artificial Sequence)
<400>12
<210>13
<211>18
<212>DNA
<213>M13-R(Artificial Sequence)
<400>13
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010014216.9A CN111154731A (en) | 2020-01-07 | 2020-01-07 | Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010014216.9A CN111154731A (en) | 2020-01-07 | 2020-01-07 | Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111154731A true CN111154731A (en) | 2020-05-15 |
Family
ID=70561881
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010014216.9A Pending CN111154731A (en) | 2020-01-07 | 2020-01-07 | Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111154731A (en) |
Cited By (4)
| 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 |
| CN115975950A (en) * | 2022-09-09 | 2023-04-18 | 广东省农业科学院植物保护研究所 | Tian Yang tomato leaf curl virus infectious clone and construction method thereof |
| CN116622739A (en) * | 2023-06-25 | 2023-08-22 | 中国农业科学院植物保护研究所 | Application of Tomato SlSUVH2 or SlSUVH4 Gene in Regulating Geminiviruses and Breeding Method of Transgenic Plants |
| CN118599864A (en) * | 2024-06-13 | 2024-09-06 | 中国农业科学院植物保护研究所 | Application of Nicotiana benthamiana NbRAD23 gene in regulating plant resistance to viruses and transgenic plant breeding methods |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392080A (en) * | 2011-11-17 | 2012-03-28 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance |
| CN102676564A (en) * | 2011-10-17 | 2012-09-19 | 湖南农业大学 | Method enabling scion variety to obtain virus resistance, RNA (ribonucleic acid) interference vector and transgenosis method |
-
2020
- 2020-01-07 CN CN202010014216.9A patent/CN111154731A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676564A (en) * | 2011-10-17 | 2012-09-19 | 湖南农业大学 | Method enabling scion variety to obtain virus resistance, RNA (ribonucleic acid) interference vector and transgenosis method |
| CN102392080A (en) * | 2011-11-17 | 2012-03-28 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance |
Non-Patent Citations (2)
| Title |
|---|
| EUI-JOON KIL等: "Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes", 《SCI REP.》 * |
| ZHOU,X.等: "登录号：MN432609", 《GENBANK》 * |
Cited By (7)
| 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 |
| CN115975950A (en) * | 2022-09-09 | 2023-04-18 | 广东省农业科学院植物保护研究所 | Tian Yang tomato leaf curl virus infectious clone and construction method thereof |
| CN116622739A (en) * | 2023-06-25 | 2023-08-22 | 中国农业科学院植物保护研究所 | Application of Tomato SlSUVH2 or SlSUVH4 Gene in Regulating Geminiviruses and Breeding Method of Transgenic Plants |
| CN116622739B (en) * | 2023-06-25 | 2023-11-28 | 中国农业科学院植物保护研究所 | Application of tomato SlSUVH2 or SlSUVH4 gene in regulating geminivirus and method of cultivating transgenic plants |
| CN118599864A (en) * | 2024-06-13 | 2024-09-06 | 中国农业科学院植物保护研究所 | Application of Nicotiana benthamiana NbRAD23 gene in regulating plant resistance to viruses and transgenic plant breeding methods |
| CN118599864B (en) * | 2024-06-13 | 2024-11-15 | 中国农业科学院植物保护研究所 | Application of Nicotiana benthamiana NbRAD gene in regulation and control of plant virus resistance and transgenic plant cultivation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111154731A (en) | Tomato yellow leaf curl virus isolate TYLCV-BJ and infectious clone construction method and application | |
| EP0397687B1 (en) | Agrobacterium mediated transformation of germinating plant seeds | |
| Kumari et al. | Biological and molecular characterization of Cucumber mosaic virus subgroup II isolate causing severe mosaic in cucumber | |
| CN114317460B (en) | Snakegourd mottle mosaic virus and infectious cloning vector, construction method and application thereof | |
| CN112322652A (en) | Cucumber mosaic virus RNA2 attenuated mutant plasmid vector containing potato virus X fragment and application thereof | |
| Al-Musa et al. | Watermelon chlorotic stunt virus (WmCSV): A serious disease threatening watermelon production in Jordan | |
| CN115976040A (en) | Application of Bunyan NbTSG101 gene in regulation and control of plant virus resistance and transgenic plant cultivation method | |
| Xiong et al. | Ageratum yellow vein China virus is a distinct begomovirus species associated with a DNAβ molecule | |
| Wu et al. | A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification | |
| CN111635455A (en) | A class of plant disease resistance-related proteins and their applications | |
| CN115820681B (en) | A VIGS silencing system of Ilex dabieshanensis IdChlH gene and its construction method and application | |
| Nummert et al. | P1-independent replication and local movement of Rice yellow mottle virus in host and non-host plant species | |
| CN114015718B (en) | Soybean yellow leaf curl virus infectious cloning vector and its construction method and application | |
| CN1424400A (en) | Method for establishing carrier by plant DNA virus infestation | |
| CN111705075B (en) | Infectious clone of alfalfa mosaic virus, recombinant agrobacterium and construction method | |
| CN116837004A (en) | Chimeric structure IMC and its application in cultivating cucumber green mottle mosaic virus-resistant tobacco | |
| KR101090458B1 (en) | Gene and Recombinant Plasmids of DNA Viruses | |
| KR101925468B1 (en) | Infectious clone of Papaya leaf curl virus and uses thereof | |
| Hasanvand et al. | Genome sequence of new isolates of sesame curly top virus in Kerman Province and identification of new hosts of the virus. | |
| KR101962902B1 (en) | Infectious clone of Chilli leaf curl virus and uses thereof | |
| KR102351198B1 (en) | Infectious clone of Milk vetch dwarf virus DNA-R and DNA-S and uses thereof | |
| CN117126879B (en) | Application of tomato SlSUVH1 gene in regulating plant resistance to viruses and methods of breeding transgenic plants | |
| CN118685452B (en) | Infectious clone of Pinot Grigio grape virus variant and its construction method and application | |
| CN116836947A (en) | Cucurbit mosaic virus and its infective cloning vector, construction methods and applications | |
| CN115975950A (en) | Tian Yang tomato leaf curl virus infectious clone and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200515 |