Sweet potato Hubei leaf curl virus infectious clone and construction method thereof
阅读说明：本技术 一种甘薯湖北曲叶病毒侵染性克隆及其构建方法 (Sweet potato Hubei leaf curl virus infectious clone and construction method thereof ) 是由 王永江 张振臣 乔奇 张德胜 秦艳红 王爽 田雨婷 赵付枚 于 2019-10-16 设计创作，主要内容包括：本发明属于植物基因工程技术领域,具体涉及一种甘薯湖北曲叶病毒侵染性克隆及其构建方法。该方法以甘薯湖北曲叶病毒为材料对病毒基因组通过无缝克隆技术插入植物表达载体中,得到含有2倍甘薯湖北曲叶病毒全基因组重组载体；所述重组载体即为甘薯湖北曲叶病毒侵染性克隆；所述甘薯湖北曲叶病毒基因组DNA序列如SEQ ID NO:8所示。本发明首次制备得到能通过农杆菌浸润法接种侵染本氏烟的甘薯湖北曲叶病毒侵染性克隆,对进一步研究甘薯湖北曲叶病毒致病机制、甘薯病毒基因功能、病毒与寄主互作、弱毒株系创制等研究奠定基础,研究结果具有重要意义和参考价值。(The invention belongs to the technical field of plant genetic engineering, and particularly relates to sweet potato Hubei leaf curl virus infectious clone and a construction method thereof. The method takes sweet potato Hubei leaf curl virus as a material, inserts virus genome into a plant expression vector through a seamless cloning technology, and obtains a recombinant vector containing 2 times of sweet potato Hubei leaf curl virus whole genome; the recombinant vector is the infectious clone of the sweet potato Hubei leaf curl virus; the sweet potato Hubei leaf curl virus genome DNA sequence is shown in SEQ ID NO. 8. The sweet potato Hubei leaf curl virus infectious clone capable of infecting the Benzeng tobacco is prepared for the first time by the method of agrobacteria infiltration, and lays a foundation for further research on the pathogenic mechanism of the sweet potato Hubei leaf curl virus, gene function of the sweet potato virus, interaction between the virus and a host, creation of a weak toxic strain and the like, and the research result has important significance and reference value.)
1. A construction method of sweet potato Hubei leaf curl virus infectious clone is characterized by comprising the following steps: connecting the sweet potato Hubei leaf curl virus genome DNA fragment to a plant expression vector in a seamless cloning manner to obtain a recombinant expression vector containing 2 times of sweet potato Hubei leaf curl virus genome DNA; the recombinant expression vector is the infectious clone of the sweet potato Hubei leaf curl virus; the sweet potato Hubei leaf curl virus genome DNA sequence is shown in SEQ ID NO. 8.
2. The method for constructing the sweet potato Hubei leaf curl virus infectious clone according to claim 1, wherein the cloning method of the sweet potato Hubei leaf curl virus genome DNA is as follows: the total DNA of the sweet potato is extracted from the infected and diseased plant of the sweet potato Hubei leaf curl virus, and the circular DNA amplification is carried out by using the total DNA as a template and utilizing a rolling reduction amplification method to obtain the whole genome sequence of the virus.
3. The method for constructing the sweet potato Hubei leaf curl virus infectious clone as claimed in claim 1, wherein said plant expression vector is pCB301, said recombinant expression vector contains two segments of sweet potato Hubei leaf curl virus whole genome DNA fragments, respectively fragment A and fragment B, said fragment A is located at the multiple cloning site of pCB301XbaI /BamHI enzymesBetween the cleavage sites, the fragment B is located at the pCB301 multiple cloning siteBamHI/SmaI between the enzyme cutting sites.
4. The method for constructing the infectious clone of sweet potato Hubei leaf curl virus of claim 3, wherein the primers for seamless cloning used for amplifying the fragment A are In-301-V3F1(X-B) and In-301-V3R1(X-B), respectively, as shown In SEQ ID NO: 1-2, and the primers for seamless cloning used for amplifying the fragment B are In-301-V3F2(B-S) and In-301-V3R2(B-S), respectively, as shown In SEQ ID NO: 3-4.
5. The sweet potato Hubei leaf curl virus infectious clone prepared by the method of any one of claims 1 to 4.
6. The sweet potato Hubei leaf curl virus infectious clone of claim 5, wherein the nucleic acid sequence of the sweet potato Hubei leaf curl virus infectious clone is shown in SEQ ID.7.
7. A recombinant bacterium containing the sweet potato Hubei leaf curl virus infectious clone of claim 5.
8. The use of the sweet potato Hubei leaf curl virus infectious clone of claim 5 or the recombinant bacterium of claim 7 in the study of the biological characteristics and/or pathogenic mechanism of the sweet potato Hubei leaf curl virus and/or gene function of the sweet potato Hubei leaf curl virus.
9. The sweet potato Hubei leaf curl virus infectious clone of claim 5 or the recombinant bacterium of claim 7 is applied to research of interaction between the sweet potato Hubei leaf curl virus and host plants.
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to sweet potato Hubei leaf curl virus infectious clone and a construction method thereof.
Background
Sweet potato geminivirus (sweet potato) is an important virus on sweet potatoes, belongs to Geminiviridae (Geminiviridae) bean golden mosaic virus (Begomovirus), is widely distributed in the world, and can cause yield reduction and seed nature degradation of sweet potatoes. Sweet potato Hubei leaf curl virus (SPLCHbV) is a new species of Begomovirus. The genome is about 2.8Kb in size, has 6 Open Reading Frames (ORF) in the genome, and respectively encodes AV1 and AV2 for virus chains and AC1, AC2, AC3 and AC4 for complementary chains. A non-coding region, also known as Intergenic Region (IR), is located between AV2 and
To study SPLCHbV gene function and pathogenesis, SPLCHbV infectious clones were constructed. The virus infectious clone refers to a recombinant vector which has the function of infecting a host and is obtained by inserting a gene sequence containing the full-length genome of a virus into a specific vector by using a gene recombination technology. The construction of the geminivirus infectious clone usually constructs a geminivirus genome recombinant plasmid (Trenado et al, 2011; Bi, et al,2014) with 1.3-2.0 copies of positive repeats. Since the IR (endogenous region) region contains the regulatory elements necessary for viral replication, 2 complete IR regions must be included in the forward repeat (Trenado et al, 2011; Bi et al, 2014). Since the Sweet potato geminivirus belongs to the old world virus, only the component of DNA-A is found at present, the infectious clone of the Sweet potato geminivirus is difficult to obtain, at least 2 cases of reports fail to obtain the infectious clone, such as the Sweet potato Georgia virus (SPLCGoV) genome obtained from circular plasmid by Lotrakul and the like and in vitro cyclization is carried out, and then 27 Sweet potato plants are infected by electric transformation, so that no one Sweet potato plant is infected, and the infectious clone can not be obtained (Lotrakul et al 2003). Paprotka et al amplified sweetpotato geminivirus genomic DNA multimers from sweetpotato infected with sweetpotato geminivirus using the RCA method, followed by inoculation of this tobacco and some plants of several species of Ipomoea with a gene gun, and as a result, demonstrated that the obtained genome was non-invasive (Paprotka et al, 2010).
The SPLCHbV is a newly discovered Sweet potato double-borne virus species, the highest similarity of a virus genome and the most similar nucleic acid (HQ393477) of the Sweet potato Saint Paulo leaf curl virus (SPLCPV) is 72.7%, the classification standard of the Sweet potato double-borne virus species is that the nucleic acid similarity is less than 91%, the nucleic acid similarity of the SPLCHbV and other virus nucleic acids in the species is low, the SPLCHbV is specific, the SPLCHbV has significance for establishing gene functions, pathogenic mechanisms, in-vitro protein interaction and attenuated strains, and an infectious clone vector of the SPLCHbV is constructed, so that a good foundation is laid for further researching the gene functions, the pathogenic mechanisms and the like.
Disclosure of Invention
The invention aims to provide an infectious clone of sweet potato Hubei leaf curl virus and a construction method thereof. The constructed sweet potato Hubei leaf curl virus infectious clone can stably infect host plants such as Nicotiana benthamiana and sweet potatoes.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a construction method of sweet potato Hubei leaf curl virus infectious clone, which comprises the following steps: connecting the sweet potato Hubei leaf curl virus genome DNA fragment to a plant expression vector in a seamless cloning manner to obtain a recombinant expression vector containing 2 times of the sweet potato Hubei leaf curl virus whole genome DNA; the recombinant expression vector is the infectious clone of the sweet potato Hubei leaf curl virus; the sweet potato Hubei leaf curl virus genome DNA sequence is shown in SEQ ID NO. 8.
Further, the cloning method of the sweet potato Hubei leaf curl virus genome DNA comprises the following steps: the total DNA of the sweet potato is extracted from the infected and diseased plant of the sweet potato Hubei leaf curl virus, and the circular DNA amplification is carried out by using the total DNA as a template and utilizing a rolling reduction amplification method to obtain the whole genome sequence of the virus.
Furthermore, the plant expression vector is pCB301, the recombinant expression vector contains two DNA fragments of sweet potato Hubei leaf curl virus whole genome, namely a fragment A and a fragment B, the fragment A is positioned between XbaI/BamHI enzyme cutting sites of the pCB301 multiple cloning site, and the fragment B is positioned between BamHI/SmaI enzyme cutting sites of the pCB301 multiple cloning site.
Furthermore, primers for seamless cloning used for amplifying the fragment A are In-301-V3F1(X-B) and In-301-V3R1(X-B), respectively, as shown In SEQ ID NO: 1-2, and primers for seamless cloning used for amplifying the fragment B are In-301-V3F2(B-S) and In-301-V3R2(B-S), respectively, as shown In SEQ ID NO: 3-4.
The construction method of the sweet potato Hubei leaf curl virus infectious clone specifically comprises the following steps:
(1) extracting total DNA of the sweet potato from the infected and diseased plant of the sweet potato Hubei leaf curl virus, and performing circular DNA amplification by using a Rolling Circle Amplification (RCA) method by taking the total DNA as a template to obtain a virus whole genome sequence;
(2) carrying out full enzyme digestion on the amplified product by using an endonuclease BamHI, and recovering a sweet potato Hubei leaf curl virus (SPLCHbV) genome fragment of about 2.8 kb;
(3) carrying out BamH I single enzyme digestion on the PUC19 vector, then carrying out dephosphorylation treatment on a plasmid enzyme digestion product, and purifying and recycling the dephosphorylated vector;
(4) connecting the products obtained in the step (2) and the step (3) to obtain a recombinant vector PUC19-SPLCHbV containing one time of sweet potato Hubei leaf curl virus genome;
(5) specific primers In-301-V3F1(X-B)/In-301-V3R1(X-B) and In-301-V3F2(B-S)/In-301-V3R2(B-S) for seamless cloning are designed by utilizing the enzyme cutting site on the vector pCB301 and combining with the viral genome sequence, so that the amplified sequence contains the viral whole genome sequence and also contains 16 bp-18 bp basic groups complementary to the vector. The cloning of the viral genome using this primer is not restricted by endonuclease.
And (3) respectively amplifying by using the recombinant plasmid PUC19-SPLCHbV as a template and using primers In-301-V3F1(X-B)/In-301-V3R1(X-B) and In-301-V3F2(B-S)/In-301-V3R2(B-S) to respectively obtain a product A and a product B for In-fusion ligation.
TABLE 1 seamless cloning primers
Primer name
Primer base sequence (5'-3')
In-301-V3F1(X-B)
GCAGGTCGACTCTAGATTCACATTATTGTTGGCCCAGTC
In-301-V3R1(X-B)
GCCGACCCGGGGATCCGGATCCCGCTGCGCGGCC
In-301-V3F2(B-S)
CGACTCTAGAGGATCCGGATCCTTCACATTATTGTTGGCCCAGTC
In-301-V3R2(B-S)
ATGCCATGCCGACCCGGGCGCTGCGCGGCCGGAGAGGC
(6) The vector pCB301 was digested with XbaI/BamHI and BamHI/SmaI, respectively, and the purified double digestion products were recovered and ligated with the product A and product B, respectively, using the seamless Cloning technique (In-fusion Cloning) to construct recombinant plasmids pCB301-SPLCHbV-1.0A (X-B) and pCB301-SPLCHbV-1.0A (B-S).
(7) XbaI/BamHI double enzyme digestion is carried out on the recombinant plasmid pCB301-SPLCHbV-1.0A (X-B), and a fragment with the size of 2.8kb is recovered for later use; the plasmid pCB301-SPLCHbV-1.0A (B-S) is subjected to double enzyme digestion of BamHI and SmaI, and a 7.4kb fragment is recovered for later use.
(8) And (3) connecting the constructed 2.8kb fragment and the 7.4kb fragment obtained in the step (7) to construct a recombinant vector pCB301-SPLCHbV-2.0A (X-S) containing 2 times of the SPLCHbV whole genome.
In a second aspect of the invention, the sweet potato Hubei leaf curl virus infectious clone prepared by the method is provided.
Further, the nucleotide sequence of the sweet potato Hubei leaf curl virus infectious clone is shown as SEQ ID.7.
In a third aspect of the invention, a recombinant bacterium containing the sweet potato Hubei leaf curl virus infectious clone is provided.
The fourth aspect of the invention provides the application of the sweet potato Hubei leaf curl virus infectious clone or recombinant bacterium in the research of the biological characteristics and/or pathogenic mechanism of the sweet potato Hubei leaf curl virus and/or gene function of the sweet potato Hubei leaf curl virus.
The fifth aspect of the invention provides the application of the sweet potato Hubei leaf curl virus infectious clone or recombinant bacterium in researching the interaction between the sweet potato Hubei leaf curl virus and host plants.
The invention has the following beneficial effects:
the sweet potato Hubei leaf curl virus infectious clone which can infect the Hubei tobacco is prepared for the first time by the method of agrobacteria infiltration, lays a foundation for further researching the pathogenic mechanism of the sweet potato Hubei leaf curl virus, the virus gene function and the construction of a weak virus strain, and has important significance on the result.
The infectious clone constructed by the invention is the first infectious clone of sweet potato Hubei leaf curl virus. As described in the background art, the technical nature of virus infectious clone construction design is strong, and the problems of non-infectious recombinant plasmid vector constructed and unstable vector exist in the process. Such as a Wangbuie PCR method to obtain a Citrus leaf mottle virus (CLBV), then a virus genome is inserted into a binary expression vector pXT1 (homologous vector pCB301), the construction method and the vector are verified to be effective, but 4 positive clones are obtained by constructing a recombinant plasmid (vector) to transform agrobacterium, and after inoculation, infectious clones are not screened by molecular detection (Wangbuie, 2017). The infectious clone of the sweet potato geminivirus is more difficult to obtain, and at least 2 failed infectious clone research experiments are reported in the aspect of the sweet potato geminivirus. Sweet potato Georgia leaf curl virus (SPLCGoV) genomes are obtained from circular plasmids like Lotrakul and the like, and in vitro cyclization is carried out, and then 27 Ipomoea plants are infected by electric transformation, so that no one is infected and no infectious clone can be obtained (Lotrakul et al 2003). Paprotka et al amplified sweetpotato geminivirus genomic DNA multimers from sweetpotato infected with sweetpotato geminivirus using the RCA method, followed by inoculation of this tobacco and some plants of several species of Ipomoea with a gene gun, and as a result, demonstrated that the obtained genome was non-invasive (Paprotka et al, 2010).
Because the sweet potato Hubei leaf curl virus is a new species of sweet potato geminivirus and related research is not carried out, the infectious clone vector of the sweet potato Hubei leaf curl virus constructed for the first time provides an important tool for the deep research of the virus.
Drawings
FIG. 1 is a schematic diagram of the construction of pCB301-SPLCHbV-2.0A infectious cloning vector.
FIG. 2 shows the result of enzyme digestion identification of pCB301-SPLCHbV-2.0A infectious cloning vector. 1: DNA marker (15000bp), 2: plasmid Xba I cleavage product, 3: plasmid Xbal I and Sma I cleavage products, 4: plasmid Xba I/BamH I cleavage product, 5: plasmid BamHI/SmaI cleavage product.
FIG. 3 is the infectious identification of the pCB301-SPLCHbV-2.0A infectious clone.
FIG. 4 shows PCR detection results of the indigenous tobacco infected with pCB301-SPLCHbV-2.0A infectious clone. 1: DNAmarker (5000bp), 2-3: SPLCHbV infesting plants, 4: uninfected plants (-CK), 5: containing SPLCHbV plasmid (+ CK).
FIG. 5 shows the Southern Blot assay results. M DNA molecular-weight Marker II (125, 564, 2027, 2322, 4361, 6557, 9416, 23130bp) 1: DNA enzyme digestion products of tobacco leaves; and 2-5, treating the DNA enzyme digestion product of the tobacco leaves inoculated with the virus. OC stands for open circular double-stranded, SC for super-stranded double-stranded, and SS for Single-stranded.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the scope of the present invention is not limited to the following examples, and any technical solutions that can be conceived by those skilled in the art based on the present invention and the common general knowledge in the art are within the scope of the present invention.
In the following examples of the invention, the experimental materials and reagent sources used are as follows:
the PUC19 vector was purchased from Dalibao, the dephosphorylation kit was purchased from Dalibao, the pCB301 vector was offered by Tao Xiaorong teacher of Nanjing agriculture university, the primers were synthesized by Shanghai Biotech, and the Agrobacterium strains LB4404 and GV3101 were purchased from Shanghai Yinjin.
The construction method of the sweet potato Hubei leaf curl virus infectious clone comprises the following specific steps:
1. whole genome extraction and enzyme digestion of sweet potato Hubei leaf curl virus
(1) Extracting total DNA of the sweet potato from the leaves of the infected and diseased plants of the sweet potato Hubei leaf curl virus by using a CTAB method, and performing circular DNA amplification by using the total DNA as a template and using a rolling reduction amplification (RCA) method to obtain the whole genome DNA of the sweet potato Hubei leaf curl virus.
(2) Subsequently, the amplified product was digested sufficiently with the endonuclease BamHI (overnight digestion at 16 ℃ C.), and 10. mu.L of the RCA product, 5. mu.L of the RCA product, and BamH I2. mu. L, DdH were contained in a 50. mu.L reaction system2O33. mu.L. The enzyme was cleaved at 37 ℃ for 16 hours. The products of endonuclease digestion were electrophoresed with 1% agarose, and the SPLCHbV genome fragment of about 2.8kb was recovered by cutting the gel.
2. Preparation of recombinant vector PUC19-SPLCHbV containing sweet potato Hubei leaf curl virus genome the product obtained in
3. Designing seamless cloning primer and amplifying recombinant plasmid PUC19-SPLCHbV
Designing seamless cloning primers (virus primers and vector primer complexes) according to the sequence of the polyclonal site of the vector pCB301 and the whole genome sequence and endonuclease site of the sweet potato Hubei leaf curl virus, wherein the primers comprise the corresponding enzyme cutting site of the polyclonal on the vector and 16-18 base sequences complementary to the vector, as shown In the table 1, In-301-V3F1(X-B)/In-301-V3R1(X-B) and In-301-V3F2(B-S)/In-301-V3R2(B-S), respectively amplifying by using the recombinant plasmid PUC19-SPLCHbV as a template and using the primers In-301-V3F1(X-B)/In-301-V3R1(X-B) and In-301-V3F2(B-S)/In-301-V3R2(B-S), the PCR amplification products were purified to obtain product A (inserted between Xba I and BamHI cleavage sites In the pCB301 multiple cloning site region) and product B (inserted between BamHI and SmaI cleavage sites In the pCB301 multiple cloning site region) for In-fusion ligation, respectively, each of which had a size of 2.8 kb. The seamless cloning PCR reaction system and the reaction program are operated according to the kit instructions. The seamless cloning kit was Clontech In-Fusion HD cloning kit from TAKARA.
4. Construction of recombinant plasmids pCB301-SPLCHbV-1.0A (X-B) and pCB301-SPLCHbV-1.0A (B-S) vector pCB301 was digested with XbaI/BamHI and BamHI/SmaI, respectively: to a 50. mu.L reaction system, 5. mu.L of 10 XT 4buffer, 2. mu.L (5U) of endonuclease 1(XbaI or BamHI), and 2. mu.L (5U) of endonuclease 2(BamHI/SmaI) were added, and the purified double-digested products were collected and ligated to the above-mentioned product A and product B, respectively, by using the seamless Cloning technique (In-fusion Cloning), to construct recombinant plasmids pCB301-SPLCHbV-1.0A (X-B) and pCB 301-SPLCV-1.0A (B-S).
In-fusion reaction procedure: after 20 minutes of reaction at 50 ℃, the mixture is placed on ice and used for transforming Escherichia coli competent cells TG1, and single spots are picked for detection, and the target plasmid is extracted.
5. Double restriction enzyme recombinant plasmid pCB301-SPLCHbV-1.0A (X-B)
The recombinant plasmid pCB301-SPLCHbV-1.0A (X-B) is subjected to XbaI/BamHI double enzyme digestion, the enzyme digestion system is shown in the following table, and the
TABLE 2 enzyme digestion System
Composition (I)
Volume (μ L)
10×
5
Xba Ⅰ(2U/μL)
3
BamH Ⅰ(2U/μL)
3
Recombinant plasmid (pCB301-SPLCHbV-1.0A (X-B))
30
ddH2O
9
Total volume
50
6. Double restriction enzyme pCB301-SPLCHbV-1.0A (B-S) plasmid
And carrying out double enzyme digestion on the pCB301-SPLCHbV-1.0B (B-S) plasmid by BamHI and SmaI, wherein the method is the same as the
7. Construction of recombinant vector pCB301-SPLCHbV-2.0A (X-S) containing 2 times of SPLCHbV whole genome links the enzyme digestion products of
9. The recombinant plasmid pCB301-SPLCHbV-2.0A (X-S) is subjected to XbaI single enzyme digestion, XbaI and SmaI, XbaI and BamHI, BamHI and SmaI and other double enzyme digestion, and the recombinant plasmid is identified, and is confirmed to be consistent with the expected result as shown in figure 2.
10. Agrobacterium transformation
Transforming agrobacterium strains LB4404 and GV3101 by a liquid nitrogen freezing method, which comprises the steps of taking 200 mu L of competent cells, adding 1 mu g of plasmid DNA (2 mu L), uniformly mixing, placing on ice for 30 minutes, and quickly freezing for 1 minute by liquid nitrogen; quickly placing in a water bath at 37 ℃ for 5 minutes; then, the cells were placed on ice for 2 minutes, 1mL of LB liquid medium was added thereto, and after slow shaking culture at 28 ℃ for 4 to 6 hours (150rpm), the culture broth was spread on an LB solid medium plate containing 50mg/mL of kanamycin and 20mg/mL of rifampicin, and cultured at 28 ℃ for about 48 hours. Single plaque PCR detection is picked, and agrobacterium plaque containing plasmid pCB301-SPLCHbV-2.0A (X-S) is screened.
TABLE 3 primers for PCR detection of post-infection viral plaques
Primer name
Primer base sequence (5'-3')
V3F1
TTCACATTATTGTTGGCCCAGTC (shown as SEQ ID NO: 5)
V3R1
GGATCCCGCTGCGCGGCC (shown as SEQ ID NO: 6)
11. Agrobacterium inoculation Benshi tobacco
The selected single plaque containing the objective plasmid is inoculated into a liquid LB culture solution containing 50mL, wherein the culture solution contains 50mg/mL kanamycin and 20mg/mL rifampicin. The culture is carried out in a logarithmic growth phase (12-16 hours) under the condition of 220rpm at the temperature of 28 ℃. The cells were collected by centrifugation at 5000rpm and resuspended in Agrobacterium cell suspension (10mmol/L MgCl)210mmol/LMES and 0.15. mu. mol/L AS) so that the OD value of the solution is about 0.8. Then inoculating the burley tobacco by adopting an agrobacterium infiltration method, observing the growth condition of the plant after inoculating for 2 days, generating the symptoms of shrinkage mosaic and the like on the inoculated leaves after inoculating for 5 days, and generating the symptoms of shrinkage mosaic and the like on the inoculated new leaves of the plant after 2 weeks, as shown in figure 3.
12. PCR detection of inoculated plants
New leaf DNA from inoculated and uninoculated (-CK) plants was extracted and PCR detected using specific detection primers V3F1/V3R1 (see Table 4). And (3) confirming whether SPLCHbV exists in new leaves of inoculated plants and whether the recombinant plasmid has infectivity.
TABLE 4 plant PCR reaction System
Composition (I)
Volume (μ L)
2×Mix buffer
12.5
Upstream primer V3F1 (10. mu.M)
1
Downstream primer VR1 (10. mu.M)
1
Taq enzyme (5U/. mu.L)
0.5
DNA solution
0.5
ddH2O
9.5
Total volume
25
TABLE 5 PCR detection reaction procedure for plants
The results are shown in FIG. 4, the SPLCHbV infected plant has an amplification band at about 3000bp (lanes 2-3), and the non-infected plant does not have the amplification band, which indicates that SPLCHbV exists in the new leaves of the inoculated plant and the recombinant plasmid has infectivity.
13. Southern Blot assay
Hybridization was performed using a digoxin probe, and the specific method was performed with reference to a digoxin probe labeling kit (Roche digoxin kit). Hybridization was performed according to the instructions of the hybridization kit (Roche digoxigenin kit).
Wherein the probe is prepared by the following steps: firstly, designing a primer SF (TGGTACTGGTCTTACTCATCGTCTCTCTCTCTCTCTCCTCC)/SR (CTGATTCTCATACTTCGCCTCC) according to a recombinant plasmid nucleic acid sequence, and then preparing a probe by adopting PCR (polymerase chain reaction), wherein the result is shown in figure 5, and the pCB-SPLCHbV-2.0A constructed by the invention has the infectivity, can successfully infect the bursitia and can be copied and infected in a plant body.
Comparative examples
The construction of the recombinant vector pCB301-SPLCSCV-2.0a containing 2 times of the genomic DNA of the sweetpotato Sichuan leaf roll virus and the construction of the recombinant vector containing 1.5 times of the genomic DNA of the sweetpotato Sichuan leaf roll virus have no infectivity.
At least 2 failed infectious clone research experiments have been reported on the sweet potato geminivirus aspect. Sweet potato Georgia leaf curl virus (SPLCGoV) genomes are obtained from circular plasmids like Lotrakul and the like, and in vitro cyclization is carried out, and then 27 Ipomoea plants are infected by electric transformation, so that no one is infected and no infectious clone can be obtained (Lotrakul et al 2003). Paprotka et al amplified sweetpotato geminivirus genomic DNA multimers from sweetpotato infected with sweetpotato geminivirus using the RCA method, followed by inoculation of this tobacco and some plants of several species of Ipomoea with a gene gun, and as a result, demonstrated that the obtained genome was non-invasive (Paprotka et al, 2010).
The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.
SEQUENCE LISTING
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tatctgtcac ttcatcgaaa ggacagtaga aaaggaagat ggcttctaca aatgccatca 540
ttgcgataaa ggaaaggcta tcgttcaaga atgcctctac cgacagtggt cccaaagatg 600
gacccccacc cacgaggaac atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 660
aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 720
cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggcct gacctgcagg 780
tcgactctag attcacatta ttgttggccc agtccaacaa cacacgaggt aaaacaaacg 840
aagaagaagg gaaaggatgt acatatgttt ctgggggagg agcaaaaatg cgttcataat 900
ttgcactcaa attatgaaat tgaagagtaa aatcccttgg tgctaactcc cgtataattc 960
tgagagcctc tgacttactg cctgcgttaa gagctgcggc gtaagcgtca ttggctgatt 1020
gttgtcctcc ccttgcagat cgtccatcaa tctggaattc gccccattca attgtgtctc 1080
catccttatc gatgtaggac ttgacgtcgg aactggattt agctccctga atgtttggat 1140
ggaaatgtgc tgacctggta ggggagacca gatcgaagaa tctgttattt ttgcattgga 1200
atttcccctc gaactgaaga agcacgtgga gatgagggct cccatcttcg tgaagctctc 1260
tacagatttt gatgaatttt ttattaactg gggtttggat gttttggatt tgctgtagtg 1320
cttcttcttt ggatagagaa cactgaggat atgtaagaaa ataattctta gcatttatgc 1380
gaaaacgatt aggtgtaggc attttgactt ggtcaatcgg tgtctctcgc tcacctttgc 1440
tctggcaatt ggtgtctggt gtcccattta tactcgttgt gtctaaaatt cgaaattcga 1500
atttgagctc caaaagcggc catccgtata atattaccgg atggccgcgc ccgcttttat 1560
gtgggcccca accacgtgaa aaaagaaaaa gaatgaataa ttgtggaccc cacaatttgg 1620
agataaggtt aaaagcatcc attcagattg caatccttgg tcatcaagga tggagcagtc 1680
attgtgggac cctctcactc atcctttacc agaaaccctt cacgggttta ggtgtatgct 1740
ttcgttaaag tatatgtcca atatacgtga caaatacgag ccgggtacgt tagggcacga 1800
attagccgtt ctgctaattc gatctcttag gggaaagaat tatgtcagat cgacttcgag 1860
ttacgaggag gtatgcgcct tactctcgga gaccgcaggc agctcgccgt ctaaacttca 1920
cgacagacat cgtcccgtat gtggggaatg ctgccccgct tgctgcagca acgtacgttc 1980
cagttcctgt gaaggcaagg aagcggacct ctcgaaagag aggcgattgg attcctaggg 2040
gctgtgttgg cccctgtaag gttcaggatt atgagttcaa gatggatgtt cctcacgggg 2100
ggacctttgt ttgtgtttct gattttactc gtggtactgg tcttactcat cgtctgggca 2160
agcgtgtttg tattaagtct atggggtttt ctggaaaggt ttggatggat gacaacatcg 2220
ctaagaagga tcatacgaat ataattacgt tttggttagt tagggatcgg cgtcctaaca 2280
aggacccgct cacgtttagt cagttgtttc acatgtttga taacgaaccc ttgacagcca 2340
aggttcgtac cgatcttcgt gatcggtttc aggtgttgcg tacgttttct gttacggtta 2400
gtggtggtcc ttatgcccat aaggagcagg ctcaggttag gcgtttcttc aagggtctta 2460
acaaccatgt gatttacaat cacaaggagg aggcgaagta tgagaatcag ttagagaatg 2520
ctatgcttgt gtactctgct agcagtcatg ctagcaatcc tgtgtatcaa tctttaagag 2580
ttcgagcata tttttatgac tcgcatatga attaatataa aagtgtttgt attgaatcct 2640
tacaatctat tacatcaatg tcatgtatcc agcttacatt tttcaattgc cagattgcca 2700
tgactaaagt actcaaactt aaaaaaccta tgctagctaa gttatcacaa attcgccatt 2760
taaggcgaga aatcaagctg tgaataacaa aattgggatg cccatgaata atccggaatt 2820
ggagaaagat cttctggtac cctatgctcc tccttgggct gtggttcagt tgaagctgaa 2880
ccttcatgat ctgtttcccg ctgcttgacg tgtgtatgta catcacacgc aggtggaatg 2940
gtgcagtccg acccgcactc atgggattgc tgtcgaattc gacagctctc ccagtctgta 3000
caagacttag taacgcccct gtgcgtgaat ccatggatga acttgcagtc tgggctaatg 3060
aaggctgtgc acccgcagcc gaaccaggtt atcctagtcc tcggttccct gtacttcctc 3120
ttctggggtg ctggtggagg ggacagactc ctttctgtac aggttctctt caatggagta 3180
gaatattgca ttcttatgtg tccactggta gagaccgaag ttctctgcct tctcaagata 3240
agacttataa gaagcgcctt ctcctggatt gcagagtacg attgctggga ttccgccttt 3300
aatttgaact ggctttccgt atttacagtt agattgccag tccttctggg cccctattag 3360
ctccttaaag tgctttaggt aatgcgggtt gacgtcatca atgacgttat accacgcatc 3420
attactgtac accttaggag aaagatccag atgaccgcat aagtaattat gcggtcccaa 3480
agagcgagcc caaactgttt tgcccaatct acttgggcct tctaatatca agctcatggg 3540
cctctccggc cgcgcagcgg gatccttcac attattgttg gcccagtcca acaacacacg 3600
aggtaaaaca aacgaagaag aagggaaagg atgtacatat gtttctgggg gaggagcaaa 3660
aatgcgttca taatttgcac tcaaattatg aaattgaaga gtaaaatccc ttggtgctaa 3720
ctcccgtata attctgagag cctctgactt actgcctgcg ttaagagctg cggcgtaagc 3780
gtcattggct gattgttgtc ctccccttgc agatcgtcca tcaatctgga attcgcccca 3840
ttcaattgtg tctccatcct tatcgatgta ggacttgacg tcggaactgg atttagctcc 3900
ctgaatgttt ggatggaaat gtgctgacct ggtaggggag accagatcga agaatctgtt 3960
atttttgcat tggaatttcc cctcgaactg aagaagcacg tggagatgag ggctcccatc 4020
ttcgtgaagc tctctacaga ttttgatgaa ttttttatta actggggttt ggatgttttg 4080
gatttgctgt agtgcttctt ctttggatag agaacactga ggatatgtaa gaaaataatt 4140
cttagcattt atgcgaaaac gattaggtgt aggcattttg acttggtcaa tcggtgtctc 4200
tcgctcacct ttgctctggc aattggtgtc tggtgtccca tttatactcg ttgtgtctaa 4260
aattcgaaat tcgaatttga gctccaaaag cggccatccg tataatatta ccggatggcc 4320
gcgcccgctt ttatgtgggc cccaaccacg tgaaaaaaga aaaagaatga ataattgtgg 4380
accccacaat ttggagataa ggttaaaagc atccattcag attgcaatcc ttggtcatca 4440
aggatggagc agtcattgtg ggaccctctc actcatcctt taccagaaac ccttcacggg 4500
tttaggtgta tgctttcgtt aaagtatatg tccaatatac gtgacaaata cgagccgggt 4560
acgttagggc acgaattagc cgttctgcta attcgatctc ttaggggaaa gaattatgtc 4620
agatcgactt cgagttacga ggaggtatgc gccttactct cggagaccgc aggcagctcg 4680
ccgtctaaac ttcacgacag acatcgtccc gtatgtgggg aatgctgccc cgcttgctgc 4740
agcaacgtac gttccagttc ctgtgaaggc aaggaagcgg acctctcgaa agagaggcga 4800
ttggattcct aggggctgtg ttggcccctg taaggttcag gattatgagt tcaagatgga 4860
tgttcctcac ggggggacct ttgtttgtgt ttctgatttt actcgtggta ctggtcttac 4920
tcatcgtctg ggcaagcgtg tttgtattaa gtctatgggg ttttctggaa aggtttggat 4980
ggatgacaac atcgctaaga aggatcatac gaatataatt acgttttggt tagttaggga 5040
tcggcgtcct aacaaggacc cgctcacgtt tagtcagttg tttcacatgt ttgataacga 5100
acccttgaca gccaaggttc gtaccgatct tcgtgatcgg tttcaggtgt tgcgtacgtt 5160
ttctgttacg gttagtggtg gtccttatgc ccataaggag caggctcagg ttaggcgttt 5220
cttcaagggt cttaacaacc atgtgattta caatcacaag gaggaggcga agtatgagaa 5280
tcagttagag aatgctatgc ttgtgtactc tgctagcagt catgctagca atcctgtgta 5340
tcaatcttta agagttcgag catattttta tgactcgcat atgaattaat ataaaagtgt 5400
ttgtattgaa tccttacaat ctattacatc aatgtcatgt atccagctta catttttcaa 5460
ttgccagatt gccatgacta aagtactcaa acttaaaaaa cctatgctag ctaagttatc 5520
acaaattcgc catttaaggc gagaaatcaa gctgtgaata acaaaattgg gatgcccatg 5580
aataatccgg aattggagaa agatcttctg gtaccctatg ctcctccttg ggctgtggtt 5640
cagttgaagc tgaaccttca tgatctgttt cccgctgctt gacgtgtgta tgtacatcac 5700
acgcaggtgg aatggtgcag tccgacccgc actcatggga ttgctgtcga attcgacagc 5760
tctcccagtc tgtacaagac ttagtaacgc ccctgtgcgt gaatccatgg atgaacttgc 5820
agtctgggct aatgaaggct gtgcacccgc agccgaacca ggttatccta gtcctcggtt 5880
ccctgtactt cctcttctgg ggtgctggtg gaggggacag actcctttct gtacaggttc 5940
tcttcaatgg agtagaatat tgcattctta tgtgtccact ggtagagacc gaagttctct 6000
gccttctcaa gataagactt ataagaagcg ccttctcctg gattgcagag tacgattgct 6060
gggattccgc ctttaatttg aactggcttt ccgtatttac agttagattg ccagtccttc 6120
tgggccccta ttagctcctt aaagtgcttt aggtaatgcg ggttgacgtc atcaatgacg 6180
ttataccacg catcattact gtacacctta ggagaaagat ccagatgacc gcataagtaa 6240
ttatgcggtc ccaaagagcg agcccaaact gttttgccca atctacttgg gccttctaat 6300
atcaagctca tgggcctctc cggccgcgca gcgggatccc cgggtcggca tggcatctcc 6360
acctcctcgc ggtccgacct gggcatccga aggaggacgt cgtccactcg gatggctaag 6420
ggagagctcg aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa 6480
tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 6540
aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 6600
gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt 6660
atcgcgcgcg gtgtcatcta tgttactaga tcggaattca gattgtcgtt tcccgccttc 6720
agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 6780
tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 6840
tatgtgcatg ccaaccacag gagatctcag taaagcgctg gctgaacccc cagccggaac 6900
tgaccccaca aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc 6960
caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt 7020
cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg 7080
ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta 7140
cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc 7200
gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag 7260
cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg 7320
taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag 7380
gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc 7440
gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt 7500
gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg 7560
cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc 7620
tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg 7680
ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc 7740
ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga 7800
cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg 7860
cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg 7920
ctggaccatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct 7980
tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc 8040
cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg 8100
ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac 8160
cttatcggca atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt 8220
ccgaatcttg ccctgcacga ataccagcga ccccttgccc aaatacttgc cgtgggcctc 8280
ggcctgagag ccaaaacact tgatgcggaa gaagtcggtg cgctcctgct tgtcgccggc 8340
atcgttgcgc cacatctagg tactaaaaca attcatccag taaaatataa tattttattt 8400
tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc 8460
cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc 8520
cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt 8580
ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat 8640
catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat 8700
cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg 8760
tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct 8820
cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg 8880
cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa 8940
caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg 9000
tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct 9060
tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca 9120
gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct 9180
acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt 9240
ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt 9300
ggcgtataac atagtatcga cggagccgat tttgaaacca caattatggg tgatgctgcc 9360
aactcgagag cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg 9420
tcacgcgcac agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc 9480
aggttaaaag acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga 9540
ttttctgcct gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact 9600
ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg 9660
tcaataccgc agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta 9720
aaatcaggcg ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg 9780
agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc 9840
cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc 9900
gcggtgtctc gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc 9960
cgccagccca gcggcgaggg caaccagccc ggtgagcgtc tagtggactg atgggctgcc 10020
tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg 10080
caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac 10140
gtttttaatg tactggggtg gtttt 10165
<210>8
<211>2774
<212>DNA
<213> Sweet Potato Hubei leaf curl Virus (Sweet potato potatoleaf cur Hubei virus, SPLCHbV)
<400>8
accggatggc cgcgcccgct tttatgtggg ccccaaccac gtgaaaaaag aaaaagaatg 60
aataattgtg gaccccacaa tttggagata aggttaaaag catccattca gattgcaatc 120
cttggtcatc aaggatggag cagtcattgt gggaccctct cactcatcct ttaccagaaa 180
cccttcacgg gtttaggtgt atgctttcgt taaagtatat gtccaatata cgtgacaaat 240
acgagccggg tacgttaggg cacgaattag ccgttctgct aattcgatct cttaggggaa 300
agaattatgt cagatcgacttcgagttacg aggaggtatg cgccttactc tcggagaccg 360
caggcagctc gccgtctaaa cttcacgaca gacatcgtcc cgtatgtggg gaatgctgcc 420
ccgcttgctg cagcaacgta cgttccagtt cctgtgaagg caaggaagcg gacctctcga 480
aagagaggcg attggattcc taggggctgt gttggcccct gtaaggttca ggattatgag 540
ttcaagatgg atgttcctca cggggggacc tttgtttgtg tttctgattt tactcgtggt 600
actggtctta ctcatcgtct gggcaagcgt gtttgtatta agtctatggg gttttctgga 660
aaggtttgga tggatgacaa catcgctaag aaggatcata cgaatataat tacgttttgg 720
ttagttaggg atcggcgtcc taacaaggac ccgctcacgt ttagtcagtt gtttcacatg 780
tttgataacg aacccttgac agccaaggtt cgtaccgatc ttcgtgatcg gtttcaggtg 840
ttgcgtacgt tttctgttac ggttagtggt ggtccttatg cccataagga gcaggctcag 900
gttaggcgtt tcttcaaggg tcttaacaac catgtgattt acaatcacaa ggaggaggcg 960
aagtatgaga atcagttaga gaatgctatg cttgtgtact ctgctagcag tcatgctagc 1020
aatcctgtgt atcaatcttt aagagttcga gcatattttt atgactcgca tatgaattaa 1080
tataaaagtg tttgtattga atccttacaa tctattacat caatgtcatg tatccagctt 1140
acatttttca attgccagat tgccatgact aaagtactca aacttaaaaa acctatgcta 1200
gctaagttat cacaaattcg ccatttaagg cgagaaatca agctgtgaat aacaaaattg 1260
ggatgcccat gaataatccg gaattggaga aagatcttct ggtaccctat gctcctcctt 1320
gggctgtggt tcagttgaag ctgaaccttc atgatctgtt tcccgctgct tgacgtgtgt 1380
atgtacatca cacgcaggtg gaatggtgca gtccgacccg cactcatggg attgctgtcg 1440
aattcgacag ctctcccagt ctgtacaaga cttagtaacg cccctgtgcg tgaatccatg 1500
gatgaacttg cagtctgggc taatgaaggc tgtgcacccg cagccgaacc aggttatcct 1560
agtcctcggt tccctgtact tcctcttctg gggtgctggt ggaggggaca gactcctttc 1620
tgtacaggtt ctcttcaatg gagtagaata ttgcattctt atgtgtccac tggtagagac 1680
cgaagttctc tgccttctca agataagact tataagaagc gccttctcct ggattgcaga 1740
gtacgattgc tgggattccg cctttaattt gaactggctt tccgtattta cagttagatt 1800
gccagtcctt ctgggcccct attagctcct taaagtgctt taggtaatgc gggttgacgt 1860
catcaatgac gttataccac gcatcattac tgtacacctt aggagaaaga tccagatgac 1920
cgcataagta attatgcggt cccaaagagc gagcccaaac tgttttgccc aatctacttg 1980
ggccttctaa tatcaagctc atgggcctct ccggccgcgc agcgggatcc ttcacattat 2040
tgttggccca gtccaacaac acacgaggta aaacaaacga agaagaaggg aaaggatgta 2100
catatgtttc tgggggagga gcaaaaatgc gttcataatt tgcactcaaa ttatgaaatt 2160
gaagagtaaa atcccttggt gctaactccc gtataattct gagagcctct gacttactgc 2220
ctgcgttaag agctgcggcg taagcgtcat tggctgattg ttgtcctccc cttgcagatc 2280
gtccatcaat ctggaattcg ccccattcaa ttgtgtctcc atccttatcg atgtaggact 2340
tgacgtcgga actggattta gctccctgaa tgtttggatg gaaatgtgct gacctggtag 2400
gggagaccag atcgaagaat ctgttatttt tgcattggaa tttcccctcg aactgaagaa 2460
gcacgtggag atgagggctc ccatcttcgt gaagctctct acagattttg atgaattttt 2520
tattaactgg ggtttggatg ttttggattt gctgtagtgc ttcttctttg gatagagaac 2580
actgaggata tgtaagaaaa taattcttag catttatgcg aaaacgatta ggtgtaggca 2640
ttttgacttg gtcaatcggt gtctctcgct cacctttgct ctggcaattg gtgtctggtg 2700
tcccatttat actcgttgtg tctaaaattc gaaattcgaa tttgagctcc aaaagcggcc 2760
atccgtataa tatt 2774
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