Melon aphid yellowed virus infectious clone recombinant vector
阅读说明：本技术 甜瓜蚜传黄化病毒侵染性克隆重组载体 (Melon aphid yellowed virus infectious clone recombinant vector ) 是由 刘莉铭 古勤生 彭斌 康保珊 吴会杰 于 2020-12-01 设计创作，主要内容包括：本发明提供了甜瓜蚜传黄化病毒侵染性克隆重组载体,涉及生物领域。该重组载体中含有MABYV病毒的基因组全长序列,所述MABYV病毒可为能侵染瓠瓜、甜瓜、西瓜和/或黄瓜的病毒,如,所述基因组全长序列的克隆如SEQ ID NO.1所示。本发明克服了现有技术的不足,发现：MABYV基因组基因难以一步克隆至质粒,发明人设计采用同源重组和定点插入相结合的策略才成功；本发明克隆病毒基因组载体侵入植物后,通过观察植物数周,最终鉴定其能侵染植物、使其发病。本发明可用于甜瓜蚜传黄化病毒致病性相关研究,也可用于瓜类作物甜瓜蚜传黄化病毒抗性鉴定,推进了该病毒相关的致病机理和瓜类抗病机理的研究。(The invention provides a melon aphid yellowed virus infectious clone recombinant vector, and relates to the field of biology. The recombinant vector contains a full-length genome sequence of a MABYV virus, wherein the MABYV virus can be a virus capable of infecting bottle gourds, melons, watermelons and/or cucumbers, and the full-length genome sequence is cloned as shown in SEQ ID No. 1. The invention overcomes the defects of the prior art and discovers that: the MABYV genome gene is difficult to clone to a plasmid in one step, and the inventor designs a strategy of combining homologous recombination and site-specific insertion to succeed; after the cloned virus genome vector disclosed by the invention invades a plant, the plant is finally identified to be capable of infecting the plant and causing the disease of the plant by observing the plant for a plurality of weeks. The invention can be used for researching the pathogenicity of melon aphid-borne yellowed viruses and identifying the resistance of melon aphid-borne yellowed viruses, which promotes the research on the pathogenesis and the disease-resistant mechanism of melons related to the viruses.)
1. An infectious cloning recombinant vector of melon aphid-borne yellows virus MABYV contains the full-length cDNA of the MABYV virus, wherein the MABYV virus can be a virus capable of infecting bottle gourd, melon, watermelon and/or cucumber, and the full-length cDNA sequence of the virus is shown as SEQ ID NO. 1.
2. The recombinant vector according to claim 1, wherein the recombinant vector is constructed by using a plant expression vector as a backbone vector, the plant expression vector comprises CaMV35S promoter (the number can be 2), ribozyme and/or terminator, the ribozyme can be HDV-RZ or TRSV-RZ, and the terminator can be Nos terminator or CaMV35S terminator; for example, the plant expression vector is pXT1, pCASS-4Z or pJL 89.
3. The recombinant vector according to any preceding claim, wherein the recombinant vector is constructed by the steps of:
4) carrying out PCR amplification by taking cDNA of a MABYV virus genome full-length sequence as a template to obtain an amplified fragment, and carrying out seamless connection on the amplified fragment and pXT1 by utilizing a homologous recombination strategy to obtain a defective vector pXT1-MABYV (D); before seamless connection, carrying out double enzyme digestion treatment on pXT1 by using Stu I and Sma I;
5) according to sequence information of a region rich in a base 'C' in a full-length sequence of a MABYV virus genome, combining sequence recombination conditions of corresponding regions in a defective vector pXT1-MABYV (D), designing a site-directed repair primer to repair base mutation corresponding to the full-length sequence of the genome of MABYV in pXT1-MABYV (D);
6) carrying out PCR amplification by taking pXT1-MABYV (D) as a template and the fixed-point repair primer as a primer to obtain a repair product, transforming the repair product into bacteria (such as escherichia coli), and screening to obtain an infectious clone recombinant vector pXT1-MABYV containing a MABYV virus genome full-length sequence; before transformation, any residual pXT1-MABYV (D) may be removed, e.g., by digestion with Dpn I.
4. The recombinant vector as claimed in claim 3, wherein the region rich in base "C" is located at 4129-4162nt of the viral genome.
5. The recombinant vector according to claim 3 or 4, wherein the site-directed repair primers comprise the following primer pairs:
MABYV-S：5＇-GGGTTGGTGTTGGTGCTGGGGGGGGGGAGGTGCTT-3＇
MABYV-X：5＇-AGCACCAACACCAACCCCCCCCCCACCGGCC-3＇。
6. the recombinant vector according to claim 3, wherein the recombinant vector is constructed further comprising step 3): selecting three pairs of primers required by the PCR in the step 4, wherein the sequences of the primer pairs are as follows in sequence:
1F：5＇-gttcatttcatttggagaggACAAAAGATACAAGCGGGGGATGC-3＇
1R：5＇-GTCGAAGAATCGCTGGAAGTC-3＇；
2F：5＇-GACTTCCAGCGATTCTTCGAC-3＇
2R：5＇-GTTGTGGTTGGCCAGTGG-3＇；
3F：5＇-CCACTGGCCAACCACAAC-3＇
3R：5＇-tggagatgccatgccgacccACACCGAAATGCCAGGGGGAG-3＇。
7. the recombinant vector according to claim 3, wherein the cDNA used in step 4 is prepared by:
(1) extracting plant total RNA from tissues (such as leaves) of melon crops infected with MABYV virus, wherein the melon crops can be bottle gourd, melon, watermelon or cucumber;
(2) the total RNA is reverse transcribed to synthesize cDNA.
8. Recombinant containing a recombinant vector according to any preceding claim, e.g. a recombinant host cell is an agrobacterium capable of infecting a crop which is capable of being infected by MABYV, e.g. agrobacterium GV3101 or C58C 1.
9. A method of infecting a melon crop (such as bottle gourds, melons, watermelons and/or cucumbers) comprising the use of a recombinant vector or recombinant according to any preceding claim.
10. The method of claim 9, wherein said using comprises injecting said recombinant vector or recombinant into said crop plant.
Technical Field
The present invention relates to the field of biology.
Background
The melon aphid yellowed virus (MABYV) is mainly confined to the phloem cells of hosts, cannot be inoculated mechanically, and can only be transmitted in a persistent and non-proliferative manner by aphids, which greatly limits their research on pathogenicity and host disease resistance.
Disclosure of Invention
The invention overcomes the defects of the prior art and discovers that: the full-length sequence of the MABYV genome is difficult to clone to a plasmid in one step, and the inventor designs a strategy of combining homologous recombination and fixed-point insertion to succeed; after the virus genome vector is introduced into a plant, the experiment is continued for a plurality of weeks, and the virus genome vector can be identified to infect the plant and cause the plant to be attacked.
In a first aspect, the invention provides a MABYV infectious clone recombinant vector, which contains full-length cDNA (namely, the full-length sequence of a virus genome is cloned into cDNA) of a MABYV virus, wherein the MABYV virus can be a virus capable of infecting bottle gourd, melon, watermelon and/or cucumber (the infecting part can be a leaf), and the full-length sequence of the cDNA is shown as SEQ ID No. 1. The infectious cloning recombinant vector refers to a vector capable of infecting a natural host of the MABYV, such as melons (bottle gourd, melon, watermelon or cucumber). Specifically, the recombinant vector is constructed by taking a plant expression vector as a framework vector, wherein the plant expression vector contains CaMV35S promoters (the number can be 2), ribozyme genes and/or terminators, the ribozyme genes can be HDV-RZ or TRSV-RZ, and the terminators can be Nos terminators or CaMV35S terminators; for example, the plant expression vector is pXT1, pCASS-4Z or pJL 89.
In a second aspect, the present invention provides a method for constructing the infectious cloning recombinant vector, comprising: constructing a recombinant vector by taking a plant expression vector as a framework vector from cDNA of a MABYV genome full-length sequence, wherein the plant expression vector can contain CaMV35S promoters (the number can be 2), ribozyme genes and/or terminators, the ribozyme genes can be HDV-RZ or TRSV-RZ, and the terminators can be Nos terminators or CaMV35S terminators; for example, the plant expression vector is pXT1, pCASS-4Z or pJL 89.
Optionally, the construction of the recombinant vector comprises the following steps:
4) carrying out PCR amplification by taking cDNA of a MABYV virus genome full-length sequence as a template to obtain an amplified fragment, and carrying out seamless connection (also called seamless cloning technology) on the amplified fragment and pXT1 by utilizing a homologous recombination strategy to obtain pXT1-MABYV (D) (the inventor finds that the virus genome sequence has deletion mutation, and is particularly called a defective vector) through sequencing;
5) according to sequence information of a region rich in a base 'C' in a full-length sequence of a MABYV virus genome, combining sequence recombination conditions of corresponding regions in a defective vector pXT1-MABYV (D), designing a site-directed repair primer to repair base mutation corresponding to the full-length sequence of the genome of MABYV in pXT1-MABYV (D);
6) carrying out PCR amplification by taking pXT1-MABYV (D) as a template and the site-directed repair primer as a primer to obtain a repair product, transforming the repair product into bacteria (such as escherichia coli), and screening to obtain an infectious clone recombinant vector pXT1-MABYV containing the full-length sequence of the MABYV virus genome.
Optionally, before transformation into bacteria, any residual pXT1-MABYV (D) is removed, e.g., by digestion with Dpn I.
Optionally, before seamless connection, carrying out double enzyme digestion treatment on the pXT1 by using Stu I and Sma I;
alternatively, homologous recombination may use the reagent NEBuilderHiFi DNA Assembly Master Mix (NEBuilder high fidelity DNA Assembly premix) from New England Biolabs (NEB).
Alternatively, the region rich in base "C" is located at 4129-4162nt of the viral genome.
Optionally, the site-directed repair primers comprise the following primer pairs:
MABYV-S：5＇-GGGTTGGTGTTGGTGCTGGGGGGGGGGAGGTGCTT-3＇
MABYV-X：5＇-AGCACCAACACCAACCCCCCCCCCACCGGCC-3＇。
optionally, the construction of the recombinant vector further comprises step 3): selecting three pairs of primers required by the PCR in the step 4, wherein the sequences of the primer pairs are as follows in sequence:
1F：5＇-gttcatttcatttggagaggACAAAAGATACAAGCGGGGGATGC-3＇
1R：5＇-GTCGAAGAATCGCTGGAAGTC-3＇；
2F：5＇-GACTTCCAGCGATTCTTCGAC-3＇
2R：5＇-GTTGTGGTTGGCCAGTGG-3＇；
3F：5＇-CCACTGGCCAACCACAAC-3＇
3R：5＇-tggagatgccatgccgacccACACCGAAATGCCAGGGGGAG-3＇。
alternatively, the preparation of the cDNA used in step 4 comprises:
(1) extracting plant total RNA from tissues (such as leaves) of melon crops infected with MABYV virus, wherein the melon crops can be bottle gourd, melon, watermelon or cucumber;
(2) the total RNA is reverse transcribed to synthesize cDNA.
As a more specific example, the construction method of the melon aphid-borne yellow virus infectious clone recombinant vector of the present invention comprises:
(1) extracting plant total RNA from bottle gourd leaves infected with melon aphid yellow virus;
(2) reverse transcription of total RNA to synthesize cDNA;
(3) three pairs of primers are designed according to sequence information of a virus genome and a plant expression vector pXT1, wherein the primer sequences are as follows:
1F：5＇-gttcatttcatttggagaggACAAAAGATACAAGCGGGGGATGC-3＇
1R：5＇-GTCGAAGAATCGCTGGAAGTC-3＇
2F：5＇-GACTTCCAGCGATTCTTCGAC-3＇
2R：5＇-GTTGTGGTTGGCCAGTGG-3＇
3F：5＇-CCACTGGCCAACCACAAC-3＇
3R：5＇-tggagatgccatgccgacccACACCGAAATGCCAGGGGGAG-3＇
(4) carrying out PCR amplification by taking cDNA as a template and 3 pairs of primers in the step as primers to obtain three fragments with different lengths, and connecting the three fragments with a pUI and SmaI double-enzyme-digestion-treated pXT1 vector by utilizing a homologous recombination strategy to obtain a recombinant vector pXT1-MABYV (D);
(5) the inventor finds that the virus genome of the invention has a region (located at 4129-4162nt of the genome) rich in 'C', combines the recombination condition of the region sequence in a recombination vector pXT1-MABYV (D) according to the sequence information, and designs a fixed-point repair primer:
MABYV-S：5＇-GGGTTGGTGTTGGTGCTGGGGGGGGGGAGGTGCTT-3＇
MABYV-X：5＇-AGCACCAACACCAACCCCCCCCCCACCGGCC-3＇
(6) carrying out PCR amplification by taking a plasmid of the recombinant vector pXT1-MABYV (D) as a template and MABYV-S/MABYV-X as a primer to obtain a repair product, digesting the repair product by Dpn I, directly transferring the repair product into escherichia coli, and screening to obtain the infectious clone recombinant vector pXT1-MABYV containing the full-length sequence of the melon aphid-borne yellows virus.
In a third aspect, the present invention discloses a recombinant of the recombinant vector of any of the previous aspects, wherein the recombinant vector is introduced into a recipient using genetic engineering techniques.
Alternatively, the host cell for the recombinant is an Agrobacterium, which infects a crop plant which is infested by MABYV, such as Agrobacterium GV3101 or C58C 1.
Alternatively, the recombinant vector containing pXT1-MABYV was transferred into Agrobacterium strain GV3101 by electrotransformation or liquid nitrogen freeze-thaw method.
In a fourth aspect, the present invention discloses a recombinant of any of the recombinant vectors of the previous aspects, for use in infecting melon crops (such as bottle gourds, melons, watermelons and/or cucumbers). It can be inoculated into crops, such as plant leaves, by using a syringe.
It should be noted that, based on the simple modification of the present patent, it is also within the scope of the present patent, such as constructing the mutant, defect (e.g. pXT1-MABYV (D)) of the vector of the present patent with reference to the method of the present invention, or performing deletion, insertion, mutation, etc. on the vector of the present invention to obtain a series of different types of mutants.
Has the advantages that:
1. the inventor finds that the full-length sequence of the viral genome is difficult to clone to a plasmid in one step, and the design adopts a strategy of combining homologous recombination and site-specific insertion to succeed. Through repeated screening and verification, the MABYV genome is identified by observing the plant for several weeks, and the plant can be infected and attacked after the MABYV genome is cloned to a vector.
2. The infectious clone recombinant vector is inoculated by using an agrobacterium-mediated mode, a new inoculation mode or a plant virus obtaining mode is provided for melon aphid yellow virus, compared with the traditional mediator virus transmission, the operation is simple and efficient, and the infectious clone recombinant vector is particularly suitable for inoculation of phloem restricted viroid.
3. The acquisition of the infectious clone recombinant vector ensures that people can obtain the virus source of the melon aphid yellowed virus at any time by a way of preserving the bacteria source, thereby not only ensuring the stability of the genetic information of the virus, but also ensuring the uniqueness of the virus without the acquired virus source relating to the complex infection condition of various viruses, improving the reliability of virus inoculation related experiments, being used for the research on the pathogenicity of the melon aphid yellowed virus, being used for the resistance identification of the melon aphid yellowed virus of melon crops, and promoting the research on the pathogenic mechanism and the disease-resistant mechanism of melons related to the virus.
4. The method used by the invention can carry out deletion, insertion, mutation and other operations on the constructed vector to obtain a series of mutants of different types, is used for relevant research on pathogenicity of the virus, and also provides reference for other viruses with technical bottlenecks in the construction of infectious clones.
Drawings
FIG. 1 shows the results of sequencing the C-rich region and the corresponding region of pXT1-MABYV (D) of the full-length sequence of the MABYV genome;
FIG. 2 demonstrates that melon aphid-borne yellows virus infectious clones can induce symptoms after inoculation;
FIG. 3 shows RT-PCR detection results of systematic leaves of plants inoculated with bottle gourd, melon, watermelon and cucumber.
Detailed Description
A construction method of a melon aphid yellowed virus infectious clone recombinant vector comprises the following steps:
(1) extraction of plant Total RNA
Extracting plant total RNA from bottle gourd leaves infected with melon aphid yellow virus, and referring to the instruction of an RNAscope total RNA extraction kit (TIANGEN, DP 419);
(2) synthesis of cDNA
Synthesizing cDNA by taking the total plant RNA extracted in the step (1) as a template and taking Random Primer as a Primer, wherein the cDNA is specifically as follows: first, 1. mu.L of LRandom Primer (50. mu. mol/L), 1. mu.L of dNTP (10mmol/L each), 1. mu.L of RNA and 7. mu.L of RNase free dH were added2O, mixing uniformly, placing the system on ice immediately after preserving heat for 5min at 65 ℃; then, 4. mu.L of 5 XPrimeScriptII RT Buffer, 1. mu.L of the PrimeScriptII Reverse Transcriptase (200U/. mu.L), 0.5. mu.L of the RNase Inhibitor (40U/. mu.L) and 4.5. mu.L of the RNase free dH were added to the system2O, mixing uniformly, and placing the system at 30 ℃ for 10min, 42 ℃ for 1h and 70 ℃ for 15min to obtain a cDNA template;
(3) initial construction of infectious clone recombinant vectors
Firstly, three pairs of primers are designed according to sequence information of a virus genome and a plant expression vector pXT1, wherein the primer sequences are as follows:
1F：
5＇-gttcatttcatttggagaggACAAAAGATACAAGCGGGGGATGC-3＇
1R：5＇-GTCGAAGAATCGCTGGAAGTC-3＇
2F：5＇-GACTTCCAGCGATTCTTCGAC-3＇
2R：5＇-GTTGTGGTTGGCCAGTGG-3＇
23F：5＇-CCACTGGCCAACCACAAC-3＇
3R：5＇-tggagatgccatgccgacccACACCGAAATGCCAGGGGGAG-3＇
secondly, carrying out PCR amplification by using cDNA as a template and 3 pairs of primers in the above step as primers to obtain three fragments with different lengths. Wherein, the PCR amplification system is as follows: 2 XQ 5 Hot Start Mix, 10. mu.L, 1. mu.L of forward primer (10mM), 1. mu.L of reverse primer (10mM), cDNA, 1. mu.L; finally using ddH2Make up to 20. mu.l of O. The reaction procedure is as follows: 30s at 98 ℃; 10s at 98 ℃, 20s at 60 ℃, 2min at 72 ℃ and 35 cycles; 5min at 72 ℃.
And finally, carrying out seamless connection on the three fragments and a pUT 1 vector subjected to double enzyme digestion by Stu I and Sma I by utilizing a homologous recombination strategy to obtain a recombinant vector pXT1-MABYV (D). Wherein, the homologous recombinants are: NEBuilderHiFi DNA Assembly Master Mix (NEB), 5. mu.L, pXT1 cleavage product, 2. mu.L; recovering 1 mul of products 1, 2 and 3 respectively, the total volume is 10 mul, mixing the system evenly, and carrying out conversion and screening after heat preservation at 50 ℃ for 1 h.
(4) Site-directed repair of infectious clonal recombinant vectors
Firstly, designing a site-directed repair primer according to sequence information of a C-rich region (positioned at 4129-4162nt of a genome) of a virus genome and combining recombination conditions of the region sequence in a recombination vector pXT1-MABYV (D) (figure 1):
MABYV-S：5＇-GGGTTGGTGTTGGTGCTGGGGGGGGGGAGGTGCTT-3＇
MABYV-X：5＇-AGCACCAACACCAACCCCCCCCCCACCGGCC-3＇
secondly, carrying out PCR amplification by using a plasmid of the recombinant vector pXT1-MABYV (D) as a template and MABYV-S/MABYV-X as a primer to obtain a repair product, digesting the repair product by Dpn I, directly transferring the repair product into escherichia coli, and carrying out sequencing screening to obtain the infectious clone recombinant vector pXT1-MABYV containing the full-length sequence of the melon aphid-borne yellow virus. Wherein, the PCR amplification system is as follows: 2 XQ 5 Hot Start Mix 10. mu.L, forward primer (10mM) 1. mu.L, reverse primer (10mM) 1. mu.L, plasmid 1. mu.L; finally using ddH2Make up to 20. mu.l of O. The reaction procedure is as follows: 30s at 98 ℃; 10s at 98 ℃, 20s at 60 ℃, 4min at 72 ℃ and 35 cycles; 5min at 72 ℃. The digestion system is as follows: cutsmart 5. mu.l, Dpn I1. mu.l, PCR product 5. mu.l, with ddH2And supplementing O to 50 mu l, preserving the temperature at 37 ℃ for 6h, and then carrying out transformation and screening.
(5) Agrobacterium transformation of infectious cloning recombinant vectors
Adding 1 μ L of plasmid of LpXT1-MABYV into 50 μ L of melted GV3101 competent cells, standing in ice-water bath for 10min, quickly freezing with liquid nitrogen for 5min, warm-bathing at 37 deg.C for 5min, standing on ice for 2min, adding 500 μ L of LB liquid culture medium without antibiotic, resuscitating at 28 deg.C and 200rpm for 2h, and spreading on LB plate (50ng/μ L Kan, 50ng/μ L Rif). After the plate is cultured for 48h at 28 ℃, the grown single cells are screened after PCR detection to obtain positive clones.
Inoculation and infectivity detection of infectious clone recombinant vectors:
agrobacterium containing pXT1-MABYV was transferred to 50mL LB liquid medium (50 ng/. mu.l Kan, 50 ng/. mu.l Rif) and cultured overnight, the cells were collected by centrifugation at 6,000g for 6min, the cells were induced to suspend in an inoculation buffer (10mM MgCl2, 10mM MES and 100. mu.MAS) for 2h, and then injected with a 1mL syringe to inoculate cotyledons of Lagenaria cucullata, Cucumis melo, Citrullus lanatus and Cucumis sativus plants, and the plants were placed in a greenhouse (25 ℃ to 28 ℃) for further culture.
After the culture for about three weeks, the lowest leaves of the bottle gourd plants inoculated with pXT1-MABYV begin to fade and yellow, then the yellow symptoms gradually become obvious, the adjacent leaves on the upper parts of the bottle gourd plants begin to show symptoms, and the bottle gourd plants which are not inoculated do not show the symptoms of fading, yellow and green (figure 2). Collecting bottle gourd systematic leaves, designing primers and carrying out RT-PCR detection, and finding that the infection of melon aphid-borne yellow viruses can be detected in bottle gourd obvious leaves (figure 3). Melon, watermelon and cucumber plants can show symptoms after being inoculated for 1-2 months, and the infection of melon aphid yellow-transmitted virus can be detected in the system leaves (figure 3). As shown above, the melon aphid-borne yellows virus infectious clone is successfully constructed, and can systematically infect melon crops such as bottle gourds, melons, watermelons, cucumbers and the like.
Sequence listing
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> melon aphid yellowed virus infectious clone recombinant vector
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5677
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acaaaagata caagcggggg atgcaaattg agactgtaaa acaacaactt gttttcagac 60
caacccgtcg agcaagcaca gacgatcgca gactatcaat atcttcattt ttagtcgatt 120
acgtctttct gtatcaattt acaaggcagt atggtgaaaa ttatctacgc ctttttcttg 180
ctcgcttgcc tctgctcatc agtgagcagc tatcagggga ctatgtttac acccctggag 240
tcaccagacg catcatggtg gctcgattcc accgccactg cggtgctccc ctcccctctt 300
caagcgcagt tgatttacga ctgcccgcca caaaagacgt tgcgcgattt ttcctcgcgc 360
gatattattc aagagttatg ggagaggggc tgcaacgacg ccagacagac ctctttagag 420
gttatgcaga atttcgaaaa tttcttactg tctggtgctc gtcgatttcg cgcaggctta 480
gggagcctac tccacacgct tttccagttg accgcatatt tgtggagctc tctaatctgg 540
gcatcggcct gcgcggtatg gcacttacta cgggagtaca ctatcgagat gctctcgctc 600
gcgtcgctct acatgtgcac cgtatatatg gtgaagacgg cggcttggat ttttggcgac 660
ttgccaattt tccttctaaa agctggcctt tcagcagcga gcggtatctc gcgggttctg 720
tggttcaaag ggaattacaa agctgagaaa tccgtggaag gatttctctc attcaaaatc 780
ccacagaccc ctcccggtaa aagcgtgttg caagtgcagc accaggatgg atcccacgca 840
ggttatgcaa cctgcgttgc acttttcaat ggatcaacag ggctcataac agcccaccat 900
gtgatcactc ctggtgctaa aatagtctcc actcggaatg gctctaaaat cccagcctcc 960
gagtttcaaa ttaaattaga aaactccaag agagacttga tactgatgac cggcccacca 1020
aattgggaag gggccttggc ttgcaaagcg tcacaaatcc agactgctaa caatttatgc 1080
aagtccaagg cgaccttttt cgcctggaac ggtgaagatt gggagtcatc aaatgctgaa 1140
attgtcgggg tttcagcatg tagaaattat gtatcagtgc ttagcaacac gaatcccggc 1200
cacagcggca ccccgtactt caatggaaaa acattgttgg gtgtccatat tgggggcgca 1260
aacgatgaaa atgccaatta cttagccccc atccctgccg tgcccggtct cacgtcacca 1320
aaatacgttt tcgagaccac agcaccccaa ggccgcttgt ttaacgatga agaaatcgcg 1380
gcgttagtag aggagttcag catgtccgag gtcgcctcaa ttatgagggc ccggaaaggt 1440
aaacaagtat atgttgaaga ggctgctcct aagcagggaa acgacgacgc ggcggcgacc 1500
gcgcaaacaa ccggcccaca cttagccacc cagggcaacg gaggaagcag tacaaaagag 1560
ccatcaacaa ttgctgctcc ttcttcaccg aaggaaccct tgccccaaga accatcgcga 1620
gccacgagca cgaaccatgg aatgatgaaa aaccgcgatg cttctgtgca atccccggac 1680
acaactgctt cttcagaaac tttatcagag atcaagaatg caatactgga gagaataaac 1740
ctccagtcaa tagagaagca ggttgtggaa gccttgatga agaaggccac gaggaatcga 1800
ggaaagaggc aacaaaaaga caagccgaag acttccagcg attcttcgac tcccaataca 1860
catgggagcc gggaagtgga aaagaagccc ctggattcaa acaggtcggc cgcctccccg 1920
agttttacca ccccaagcaa aagacggggt cgaaatgggg ggccaagata tgccgacagc 1980
acccagagat ggatgcctac acccagggct tcgggtggcc acagttcggt gcccaggccg 2040
aactgaaatc cttgcggcta caagccgcca ggtggctgga gcgcgcccag ttcgttaaaa 2100
tcccctcatc tgaggagcgg gagcgcgtaa tcaggaaatg ctgtgaggcc tacaggaatg 2160
ccaaaactat agggcccaac gcaacaagag gagatagctt atcctgggaa ggattccttg 2220
aagattttaa gcaagctgtc ttctcgctcg aatttgatgc aggcatcggc gtaccataca 2280
ttgcttatgg aaggccaacc caccgggggt gggttgaaga tccaaaatta ttgccgatct 2340
tagctcgcct gacctttaac cggctacaga agatgttaga ggttaggttt gagcatctaa 2400
gccctgcgga gcttgtgcag gcgggtctct gtgacccgat acgtgtgttt gttaaaggtg 2460
aaccgcacaa gcagagcaaa ttagatgaag gccgctaccg cctcatcatg agcgtttcct 2520
tgatagatca attggtagcc cgggttttat tccagaacca gaataaaaga gaaatcactc 2580
tgtggagggc agtaccctct aaacccggtt ttggcttgtc tacggatgag caagtcgtgg 2640
agttcatgga aatactctcc gcgcaggtgg gggttgcacc caaagaatta ataggcaatt 2700
ggcaacacca ccttattgcg accgactgct ccggctttga ctggagcgtt tcggactggc 2760
tccttgaaga tgatatggaa gtccgaaacc gcctgacgct ggacattaat aagaccacca 2820
agcgcttaag atcagcttgg ttgaagtgca tctcgaatag cgtcctcagc ctctccgatg 2880
ggaccctcct gtcacaacag gtccccggag tacagaaatc tggcagctac aatacctcat 2940
cctccaattc taggattcga gtgatggctg cttatcattg cggggctgag tgggcaatgg 3000
cgatgggcga tgacgccctc gagtcagtcg gatcaaacct agcgaaatac gctaagctgg 3060
gttttaaagt cgaggtttcc tcaaaactgg aattttgctc acatatcttt gaacgtgagg 3120
acctcgccat tccggtcaac aaagccaaaa tgatatacaa gttgatacat ggctatgaac 3180
cggaatgtgg caatgctgag gtcttgatta actatctgac cgcctgtttc gcagttctca 3240
acgaattgcg gtcggatcct cagcttgtct cgactctcca tcagtggctg gttcttccag 3300
tgcagccaca aaagatataa ggggagtata aagaacacta gccggaaata cgttgttgca 3360
attgccggaa gtttaagcca actacaaaat aggccaagcg gacttcagat tcgtagcagg 3420
attcacatcg ggcttcctgg cagctatacc actttgcgca ctaggcctat acctaatcta 3480
cctaaagatt tcagcccacg ttcgatcaat tgttaatgaa tacggtcgcg gctaggaatc 3540
aaaatggcat gggaaggagg agacggaacc gtcgacgcac ttcaaagagt aaccgcgtgg 3600
ttgtggtcca aaccactggc caaccacaac gcggaagacg acgacgacga aatccaagac 3660
gttctcctcg aggaggcaga gctggaggac gcccaggtga aacatttgta ttcagcaaag 3720
acaatctcac gggcagttcc tccggagcaa tcactttcgg gccgtctcta tcagagagcc 3780
cagcattcag ctctggaata ctcaaggcct accatgagta taaaatctca atggtcaagt 3840
tggagttcat ctccgaggcc tcttccacct cctcaggttc catctcttat gagctggacc 3900
cccactgcaa gcttaacgcc ctccaatcca cggttaataa attcggaatc acgaagagtg 3960
gatctagaac atggagcgcg aagctcatca acgggctgga atggcacgac gccacggaag 4020
atcaattccg catcctatac aaaggaaacg ggagctcttc aacggcggga tcgttcaggg 4080
tcaccatcaa gtgccaggtc cagaacccga aataggtaga aagcacctcc ccccccccag 4140
caccaacacc aacccccccc ccaccggccc cctcacccga gcccacacct tgtaaaggcg 4200
ctcgtttctg gggctatgag ggcaatccac aaagcaagat acaaacggca gaaaacaata 4260
gaaacattga ttcaaggccg ttgaattatg tatccatgta ccggtgggaa gatgagaaat 4320
gggatcaagt taatctgcaa gctggctact caagaaatga tagacgatgt atggaaacgt 4380
acttcgtgat accggcaaac aagggaaagt ttcacgtgta ccttgaagcc gatggagaat 4440
tcgtcgtaaa acacataggc ggcgatctcg atggtagctg gcttggaaac atcgcgtatg 4500
atgtatccca gcgaggttgg aatattggaa attacaaagg ttgcagcatt aagaattacc 4560
agtcgaaaac gacctttgta gctggacacc ctgacgcatc catgaatgga aagaactttg 4620
atgcggctag ggcagtggaa gttgactggt tcgcctcatt tgaactagaa tgtgatgatg 4680
acgaaggcag ttggaggatc tatcctcccc ccatccagaa ggacagttca tataattaca 4740
ctgtctcgta cggaaactac acagagaaat actgtgaatg gggagccgtt tccatctcta 4800
ttgatgagga caacgccaca gggagggtgc tacagagaat taaaccacgt aagggggtaa 4860
tgacgtggtc aacgcccgag ccggaaagac aaccggttga acaaactccg gttcaagaac 4920
cgtccgagac atctgggctc gatgccccgc ctacgacaaa acaagaagac gaaacaaccg 4980
acgacctagg cgggacgttt aaagaacccc aaatccctga gttttcgacg ccaatgcata 5040
tgctagcgcg agatttgggc gggctcacgg aatcttcttc cagagcgccc gagggagtgg 5100
ctagctgggt ccagctagaa gcggatccag tgaatgaaag tgaatattcc tcagataatg 5160
agacgatcgc gagccctccc accacaaccg tttcctctcc cttcacttcg attccgaaca 5220
cggaaagggt gcttcaaatg cccggagtgt accaaggtga ccgacagatc gcttcaagcg 5280
tgttggatga acaccgtaaa aggtccttcg caaagcgcct cctcccgagt ttgggaggat 5340
ctagagcttc agctctcagt ggagggactc ttcgccaaaa gcattcagat ttgattaagc 5400
aatatatgac ggcggcagag catgcggaag cgcagaggat ccggaaccaa ctcggcaaag 5460
gtgctcaaac tcgatacatc gagtctttaa acttgcacga ccgagtataa agaaccgctt 5520
tgtggagacg cgcgtgactc cacccggcta caggtgcccg accaaagacc tgatgacatc 5580
aagccaaaga tgtaaaattg gaacgactcc gaaaggatag gcaacgaatg ttctcactct 5640
tgtgagtaca gggggactcc ccctggcatt tcggtgt 5677
<210> 2
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gggttggtgt tggtgctggg gggggggagg tgctt 35
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agcaccaaca ccaacccccc ccccaccggc c 31
<210> 4
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gttcatttca tttggagagg acaaaagata caagcggggg atgc 44
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtcgaagaat cgctggaagt c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gacttccagc gattcttcga c 21
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttgtggttg gccagtgg 18
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccactggcca accacaac 18
<210> 9
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tggagatgcc atgccgaccc acaccgaaat gccaggggga g 41