CN116445540A - Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method - Google Patents
Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method Download PDFInfo
- Publication number
- CN116445540A CN116445540A CN202310475108.5A CN202310475108A CN116445540A CN 116445540 A CN116445540 A CN 116445540A CN 202310475108 A CN202310475108 A CN 202310475108A CN 116445540 A CN116445540 A CN 116445540A
- Authority
- CN
- China
- Prior art keywords
- sweet potato
- leaf curl
- curl virus
- potato leaf
- infectious clone
- 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.)
- Granted
Links
- 240000002124 Sweetpotato leaf curl virus Species 0.000 title claims abstract description 68
- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 45
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 45
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 45
- 230000002458 infectious effect Effects 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 241000589158 Agrobacterium Species 0.000 claims description 38
- 238000002791 soaking Methods 0.000 claims description 17
- 241000700605 Viruses Species 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 230000029812 viral genome replication Effects 0.000 claims description 5
- 101150073246 AGL1 gene Proteins 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 239000004576 sand Substances 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 16
- 201000010099 disease Diseases 0.000 abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 6
- 208000024891 symptom Diseases 0.000 abstract description 5
- 230000003993 interaction Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 5
- 241000702463 Geminiviridae Species 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 240000006677 Vicia faba Species 0.000 description 3
- 235000010749 Vicia faba Nutrition 0.000 description 3
- 235000002098 Vicia faba var. major Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 3
- 229960001225 rifampicin Drugs 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000005723 virus inoculator Substances 0.000 description 2
- 101150057000 AV2 gene Proteins 0.000 description 1
- 241000702451 Begomovirus Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001506304 Kadsura japonica Species 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
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
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/40—Monitoring or fighting invasive species
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato. 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into a binary vector to obtain sweet potato leaf curl virus infectious clone. The invention provides a method for solving the problem of low infection rate of sweet potato by inoculating sweet potato leaf curl virus into a natural host. Through observation of disease symptoms and detection of PCR, the invention successfully improves the inoculation rate of the sweet potato leaf curl virus to different varieties to 100 percent. In addition, the method provided by the invention is not only efficient, but also easy to operate, can realize large-scale inoculation and screening, and can provide powerful support for subsequent screening of resistant varieties and research on interaction of sweet potato leaf curl virus and sweet potato.
Description
Technical Field
The invention relates to the fields of genetic engineering and plant virology, in particular to a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato.
Background
The sweet potato has the functions of grain and cash crops, has wide application and very important economic value. China is the largest sweet potato producing country in the world. In recent years, diseases and insect pests of sweet potatoes, particularly viral diseases, are increasingly occurring. The sweet potato belongs to asexual propagation crops, and the virus is easy to infect in the generation propagation. Among them, sweet potato leaf curl virus (Sweet potato leaf curl viru s, SPLCV) is the most predominant sweet potato DNA virus. To study the interaction between virus and host, the primary condition is to obtain efficient, stable, reproducible vaccination methods under laboratory conditions. Traditional grafting inoculation methods are difficult to control and infect single viruses, and symptoms are easily affected by various factors, so that the stability and the repeatability are poor. Sweet Potato Leaf Curl Virus (SPLCV) is a single-stranded DNA virus belonging to the family Geminiviridae (Geminiviridae), the genus Begomovirus. At present, most geminivirus infectious clones are obtained by connecting 1.2-2.0 virus genomes in tandem to an agrobacterium binary vector, and then performing virus inoculation by using an agrobacterium-mediated leaf penetration or stem injection method. Nevertheless, the research on the interaction relationship between SPLCV and sweet potato is still basically in a blank stage, and the main reason is that the construction of the SPLCV invasive clone is imperfect, the inoculation method is not mature, and the highest efficiency of the reported inoculation method can only reach about 20%.
Disclosure of Invention
The invention aims to provide a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato.
The construction method of the sweet potato leaf curl virus infectious clone comprises the step of inserting 1.01 repeated Sweet Potato Leaf Curl Virus (SPLCV) genome sequences into a binary vector to obtain the sweet potato leaf curl virus infectious clone.
Preferably, 1.01 repeated Sweet Potato Leaf Curl Virus (SPLCV) genome sequences are inserted into the multiple cloning site of a binary vector pCAMBIA1300 transformed by agrobacterium to prepare pCAMBIA1300-SPLCV-1.01 invasive clone.
Preferably, the 1.01 repeated sweet potato leaf curl virus is a stem loop structure of a key sequence related to virus replication repeated at the tail end of the genome sequence of the sweet potato leaf curl virus.
Preferably, a key sequence stem loop structure related to virus replication is repeated at the tail end of a sweet potato leaf curl virus Guangzhou strain virus (SPLCV-GZ 02) gene and is connected to an agrobacterium-transformed binary vector pCAMBIA1300 through KpnI and XbaI double enzyme cutting sites to obtain pCAMBIA1300-SPLCV-1.01 invasive clone, wherein GenBank accession number of the sweet potato leaf curl virus Guangzhou strain virus is JX286654.
Preferably, the nucleotide sequence of the stem-loop structure is AAGGCGGGCACCGTATTAATATTACCGGTG CCCGCCGCGCC, which is 41bp.
The invention also provides a method for efficiently and simply infecting sweet potatoes by utilizing the sweet potato leaf curl virus infectious clone, which is to introduce the sweet potato leaf curl virus infectious clone into an agrobacterium AGL1 strain to obtain recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone; soaking sweet potato seedlings in 6-8 leaf period in recombinant agrobacterium liquid containing sweet potato leaf curl virus infectious clone, transferring to clear water for continuous soaking, and culturing in transplanting matrix.
The sweet potato leaf curl virus infectious clone is pCAMBIA1300-SPLCV-1.01 infectious clone.
Preferably, the soaking of the sweet potato seedling in the 6-8 leaf period in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is to soak the sweet potato seedling in the 6-8 leaf period and the length of 15cm in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone for 6 hours, and then transfer the sweet potato seedling to clear water for continuous soaking for 12 hours.
Preferably, the sweet potato seedling is cultured in a transplanting matrix, the redundant leaves are cut off, only 2-3 leaves are reserved, and the sweet potato seedling is transplanted into a matrix mixed with nutrient soil, perlite and sand in a volume ratio of 3:1:1 for continuous culture.
Preferably, the sweet potato seedling in the 6-8 leaf period is soaked in recombinant agrobacterium tumefaciens bacteria solution containing sweet potato leaf curl virus infectious clone, and the vine of the sweet potato seedling is cut into a fresh section with the angle of 45 degrees by a knife before the sweet potato seedling is soaked.
Preferably, the concentration of the recombinant agrobacterium solution containing sweet potato leaf curl virus infectious clone is OD 600 =1.0。
Preferably, the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is prepared by culturing recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone in LB culture medium with corresponding resistance overnight, re-suspending the agrobacterium in complete induction culture medium, and re-suspending the agrobacterium after overnight culture 1 / 4 And (3) soaking in a buffer solution for soaking and inoculating sweet potato seedlings.
The invention is characterized in that the stem-loop structure sequence in the genome of sweet potato leaf curl virus (sweet potato leaf curl virus, SPLCV) is repeated once at the tail end, and is constructed into pCAMBIA1300 vector, thus obtaining the minimum unit and efficient invasive clone of sweet potato leaf curl virus. The infectious clone can successfully infect different varieties of sweet potatoes by an agrobacterium tumefaciens AGL1 mediated efficient and simple agrobacterium tumefaciens soaking inoculation method. The invention provides a method for solving the problem of low infection rate of sweet potato by inoculating sweet potato leaf curl virus into a natural host. Through observation of disease symptoms and detection of PCR, the invention successfully improves the inoculation rate of the sweet potato leaf curl virus to different varieties to 100 percent. Moreover, the method provided by the invention is not only efficient, but also easy to operate, can realize large-scale inoculation and screening, and can provide powerful support for subsequent screening of resistant varieties and research on interaction of sweet potato leaf curl virus and sweet potato.
Drawings
Fig. 1: SPLCV-1.01 sequence diagram. The framed "TATTAATATTA" sequence is a conserved sequence of geminiviruses. Wherein the thickened "A" is the replication origin of the sweet potato leaf curl virus. The underlined 41bp sequence is the repetitive stem-loop structure necessary at both ends of the invasive clone.
Fig. 2: schematic of the invasive cloning of pCAMBIA 1300-SPLCV-1.01.
Fig. 3: disease symptoms of broad-potato 87 inoculated with sweet potato leaf curl virus. The control group was inoculated with Agrobacterium containing the empty vector of pCAMBIA1300 and the experimental group was inoculated with Agrobacterium containing the invasive clone pCAMBIA 1300-SPLCV-1.01.
Fig. 4: PCR detection of viral DNA content after 20 days (20 dpi) of inoculation of 10 Kadsura pepper 87. The viral AV2 gene, ibelF gene, was detected by PCR as an internal control. Water (H) 2 O) and the broad-potato 87 plants (Mock, M) inoculated with the empty vector are used as negative control, and 1-10 are broad-potato 87 plants inoculated with virus.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The specific examples are intended to be illustrative of the invention and are not intended to be limiting. Modifications of the present embodiments, which do not creatively contribute to the invention, can be made by a person skilled in the art after reading the present specification, for example, by enlarging the culture volume correspondingly to accommodate large-scale screening, but are protected by patent laws within the scope of the claims of the present invention.
Examples
A sweet potato leaf curl virus infectious clone and a sweet potato efficient and simple inoculation method comprise the following steps:
1. based on the published genome of the Guangzhou strain of sweet potato leaf curl virus (SPLCV-GZ 02) (GenBank accession number: JX286654, full length 2829 nt), a stem loop (stem loop) structure with a length of 41bp relevant for viral replication is repeated at the end, as shown in FIG. 1 and SEQ ID NO.1, the underlined part in FIG. 1 being the stem loop (stem loop) structure. The full length SPLCV-1.01 sequence of 2870bp is synthesized by gene synthesis technique (FIG. 1). The infectious clone pCAMBIA1300-SPLCV-1.01 of SPLCV is obtained by connecting the KpnI and XbaI double restriction sites to the binary vector pCAMBIA1300 transformed by agrobacterium.
2. The pCAMBIA1300-SPLCV-1.01 infectious clone is quickly frozen by liquid nitrogen, the agrobacterium AGL1 strain is introduced by a heat shock method of heat shock for 5 minutes at 37 ℃, and the recombinant agrobacterium monoclonal clone containing the sweet potato leaf curl virus infectious clone is obtained by screening by 50mg/L kanamycin and 10mg/L rifampicin resistant LB plates and inversely culturing for 2 days at 28 ℃.
3. Recombinant Agrobacterium was selected and subjected to shaking dark culture at 28℃for 10-12 hours in 5mL of LB liquid medium containing 50mg/L kanamycin and 10mg/L rifampicin resistance, to obtain an Agrobacterium culture.
4. 0.5mL of the above Agrobacterium culture was inoculated into 200mL of LB liquid medium containing 50mg/L kanamycin and 10mg/L rifampicin resistance, and shake-dark cultured overnight at 28℃and 200rpm to obtain an Agrobacterium solution.
5. The above overnight-cultured Agrobacterium solution was centrifuged at 3000rpm for 10min, the supernatant was discarded, and the Agrobacterium pellet was resuspended in 500mL of complete induction medium (Table 3) containing 50mg/L kanamycin resistance, and subjected to shaking dark culture at 28℃overnight at 200rpm to obtain an Agrobacterium solution.
6. Centrifuging the above overnight cultured Agrobacterium solution at 3000rpm for 10min, discarding supernatant, and re-suspending the Agrobacterium precipitate in 500mL 1 / 4 MS immersed in buffer (table 4). Detecting OD 600 By using 1 / 4 MS soaking buffer solution to dilute the bacterial solution to OD 600 =1.0, used for sweet potato inoculation.
7. Taking sweet potato seedling with length of 15cm at 6-8 leaf period, cutting vine of sweet potato seedling with knife to obtain 45 degree fresh section, soaking sweet potato seedling in OD 600 In the bacterial liquid of 1.0, after being soaked for 6 hours at room temperature in dark, the sweet potato seedlings are transferred into clear water, and the soaking is continued for 12 hours at room temperature.
8. Cutting off redundant leaves of the sweet potato seedlings, only keeping 2-3 leaves, and then transplanting the sweet potato seedlings into a matrix mixed by nutrient soil, perlite and sand in a volume ratio of 3:1:1 for continuous culture.
9. And respectively taking the new top leaves of the sweet potatoes as materials 10 days and 20 days after inoculation, extracting DNA, and detecting the content of the viral DNA by using PCR.
10. For example, the disease symptoms appear about 20 days after inoculation, using the broad-bean 87. Compared with the control group inoculated with empty vector, the new leaves of the broad-bean 87 inoculated with the virus are yellowing, curled, the growth of terminal buds is obviously inhibited, and lateral buds are increased (figure 3).
11. Taking the broad-bean 87 as an example, DNA of sweet potato leaf curl virus (SPLCV-AV 2) can be detected by PCR in 10 new-born leaves of sweet potato 20 days after virus inoculation (figure 4), and the control group has no virus DNA, which shows that the inoculation rate of the method reaches 100 percent and is far higher than 20 percent of the inoculation rate of the prior reported inoculation method.
TABLE 1
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
Claims (10)
1. A construction method of sweet potato leaf curl virus infectious clone is characterized in that 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into a binary vector to obtain sweet potato leaf curl virus infectious clone.
2. The method of claim 1, wherein the 1.01 repeat sweet potato leaf curl virus is a repeat of a viral replication-related key sequence stem loop structure at the end of the genomic sequence of the sweet potato leaf curl virus.
3. The construction method according to claim 1, wherein 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into the multiple cloning site of the binary vector pCAMBIA1300 transformed by agrobacterium to prepare pCAMBIA1300-SPLCV-1.01 invasive clone.
4. The construction method according to claim 1, wherein a key sequence stem loop structure related to virus replication is repeated at the tail end of a virus gene of a Guangzhou strain of sweet potato leaf curl virus, and the key sequence stem loop structure is connected to a binary vector pCAMBIA1300 transformed by agrobacterium through KpnI and XbaI double cleavage sites to obtain pCAMBIA1300-SPLCV-1.01 invasive clone, and GenBank accession number of the virus of the Guangzhou strain of sweet potato leaf curl virus is JX286654.
5. The method of claim 1, 2, 3 or 4, wherein the stem-loop structure has a nucleotide sequence of AAGGCGGGCACCGTATTAATATTACCGGTGCCCGCCGCGCC, which is 41bp.
6. A method for efficiently and simply infecting sweet potatoes by utilizing sweet potato leaf curl virus infectious clones, which comprises the steps of introducing the sweet potato leaf curl virus infectious clone according to any one of claims 1 to 5 into an agrobacterium AGL1 strain to obtain recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone; soaking sweet potato seedlings in 6-8 leaf period in recombinant agrobacterium liquid containing sweet potato leaf curl virus infectious clone, transferring to clear water for continuous soaking, and culturing in transplanting matrix.
7. The method of claim 6, wherein the sweet potato leaf curl virus infectious clone is pCAMBIA1300-SPLCV-1.01 infectious clone.
8. The method according to claim 6, wherein the soaking of the sweet potato seedling with 6-8 leaf period in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is carried out by soaking the sweet potato seedling with 6-8 leaf period and length of 15cm in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone for 6 hours, transferring to clear water and continuously soaking for 12 hours.
9. The method according to claim 6, wherein the cultivation in the transplanting matrix is that the sweet potato seedlings are cut off redundant leaves, only 2-3 leaves are reserved, and the sweet potato seedlings are transplanted into the matrix mixed with nutrient soil, perlite and sand in the volume ratio of 3:1:1 for continuous cultivation.
10. The method according to claim 6, wherein the recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone is prepared by culturing the recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone overnight in LB medium with corresponding resistance, re-suspending the agrobacterium in complete induction medium, and re-suspending the agrobacterium in 1/4MS soaking buffer solution for soaking and inoculating sweet potato seedlings after overnight culture.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310475108.5A CN116445540B (en) | 2023-04-27 | 2023-04-27 | Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310475108.5A CN116445540B (en) | 2023-04-27 | 2023-04-27 | Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116445540A true CN116445540A (en) | 2023-07-18 |
CN116445540B CN116445540B (en) | 2024-05-10 |
Family
ID=87132023
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310475108.5A Active CN116445540B (en) | 2023-04-27 | 2023-04-27 | Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116445540B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060206959A1 (en) * | 2003-05-19 | 2006-09-14 | Mario Tavazza | Method for the preparation of transgenic plants characterised by geminivirus lasting resistance |
KR20090112492A (en) * | 2008-04-24 | 2009-10-28 | 성균관대학교산학협력단 | The bidirectional promoter using Sweet potato leaf curl virus Korean isolate |
CN102703435A (en) * | 2012-06-13 | 2012-10-03 | 浙江大学 | Isolation and identification of Yunnan tomato leaf curl viral genome and agrobacterium tumefaciens-mediated infective clone construction |
CN110669776A (en) * | 2019-10-16 | 2020-01-10 | 河南省农业科学院植物保护研究所 | Sweet potato Hubei leaf curl virus infectious clone and construction method thereof |
-
2023
- 2023-04-27 CN CN202310475108.5A patent/CN116445540B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060206959A1 (en) * | 2003-05-19 | 2006-09-14 | Mario Tavazza | Method for the preparation of transgenic plants characterised by geminivirus lasting resistance |
KR20090112492A (en) * | 2008-04-24 | 2009-10-28 | 성균관대학교산학협력단 | The bidirectional promoter using Sweet potato leaf curl virus Korean isolate |
CN102703435A (en) * | 2012-06-13 | 2012-10-03 | 浙江大学 | Isolation and identification of Yunnan tomato leaf curl viral genome and agrobacterium tumefaciens-mediated infective clone construction |
CN110669776A (en) * | 2019-10-16 | 2020-01-10 | 河南省农业科学院植物保护研究所 | Sweet potato Hubei leaf curl virus infectious clone and construction method thereof |
Non-Patent Citations (2)
Title |
---|
MOHD FAIZ MAT SAAD: "Construction of Infectious Clones of Begomoviruses: Strategies, Techniques and Applications", BIOLOGY, vol. 2021, no. 10, 29 June 2021 (2021-06-29) * |
黄艳岚: "湖南甘薯DNA病毒分子鉴定与甘薯曲叶病毒病理特征研究", 中国博士学位论文全文数据库—农业科技辑, 15 February 2023 (2023-02-15) * |
Also Published As
Publication number | Publication date |
---|---|
CN116445540B (en) | 2024-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109679993B (en) | Construction method of agrobacterium rhizogenes-mediated transgenic plant | |
CN102154364A (en) | Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane | |
CN111424022B (en) | Verticillium dahliae VdEG target gene fragment for pathogen-resistant bacteria, interference vector and application thereof | |
CN110295183A (en) | A method of citrus is improved to canker resistance based on CsPrx25 overexpression | |
CN102676510B (en) | Method for enhancing black streaked dwarf resistance of paddy rice by using artificial microRNA (micro Ribonucleic Acid) and special double chain RNA thereof | |
CN112195186A (en) | Application of SlBBX20 gene in regulation and control of tomato gray mold resistance | |
CN115927757A (en) | Method for efficiently screening antiviral germplasm resources based on PEMV-1 and PEMV-2 infectious clones | |
CN107828816A (en) | One primary yeast Agrobacterium shuttle vector and construction method and application | |
CN101121942B (en) | Corn genetic transferring method conducted by agrobacterium rhizogenes | |
CN100491535C (en) | Chuancao-II Laomangmai wheat pest-resisting gene transferring technology | |
CN116445540B (en) | Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method | |
CN114150003B (en) | Application of cucumber green mottle mosaic virus gene interval region in resistant tobacco cultivation | |
CN103667322B (en) | Polygene mosaic AVCTC of a kind of anti-tomato yellow leaf curl virus and its preparation method and application | |
CN108901844A (en) | A method of building Lycoris genetic conversion system | |
CN110004176B (en) | Construction method of hybrid larch genetic transformation system | |
CN114107371B (en) | Cucumber green mottle mosaic virus gene mediated transgenic tobacco method | |
CN115725644B (en) | Genetic transformation method and application of Polygonatum cyrtonema Fabricius | |
CN104313051A (en) | Method for cultivating high-TYLCV (tomato yellow leaf curl virus)-resistant tomatoes | |
CN109022479A (en) | A kind of screening technique of callus phase | |
CN109022468A (en) | The preparation method of plant callus | |
CN117126879B (en) | Application of tomato SlSUVH1 gene in regulation and control of plant virus resistance and transgenic plant cultivation method | |
CN103627725A (en) | Plant chloroplast poly-gene transformation vector, and construction method and application thereof | |
CN115094082B (en) | VIGS silencing system and method for identifying MsPDS gene | |
CN116926088B (en) | Verticillium dahliae VdNRPS6 gene antipathogenic target gene fragment, interference vector and application thereof | |
KR102342405B1 (en) | Cucurbit Aphid-Borne Yellows Virus Infectious Clone And Their Uses |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |