CN102277335B - Chinese Baoding isolate of sugarcane mosaic virus and complete sequence of genome thereof - Google Patents
Chinese Baoding isolate of sugarcane mosaic virus and complete sequence of genome thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology. The inventor utilizes the nationally existed virus resource to obtain a Chinese Baoding isolate of sugarcane mosaic virus (SCMV) from infected plants and obtain a complete sequence of a genetic group. The biological collection number of the isolate is CGMCC No.4577; a detection primer can be designed by utilizing the sequence and the detection primer can be used for rapidly detecting SCMV; and a Ribonucleic Acid interference (RNAi) carrier also can be constructed by the sequence to convert corns to culture new disease-resistant species.
Description
Technical field
The present invention relates to microbial genome and plant protection field, particularly relate to a kind of genom sequence and application thereof of corn mosaic virus.
Background technology
Maize dwarf mosaic is a kind of important worldwide disease.This disease was at first found at Ohio, USA in 1963
10, the so far successively main maize area of more than 20 countries discovery in the world.China's maize dwarf mosaic is found in Xinxiang, Henan and Anyang early than nineteen sixty-eight, and each corn-growing regions that spread all over the country has at present caused loss in various degree for local Maize Production, and is wherein the heaviest with North China and northwest loss.
What cause having identified before the virales of maize dwarf mosaic has: maize dwarf mosaic virus (Maize dwarf mosaic virus, MDMV), corn mosaic virus (Sugarcane mosaic virus, SCMV), Johnson grass mosaic virus (Johnsongrass mosaic virus, JGMV), Zea mosaic virus (Zea mosaic virus, ZeMV), Pennisetum mosaic virus (Pennisetum mosaic virus, PenMV) and sorghum mosaic virus (Sorghum mosaic virus, SrMV), above-mentioned 6 kinds of viruses have formed corn mosaic virus (SCMV) subgroup that infects grass in the Potyvirus jointly.And the virus that causes at present China's maize dwarf mosaic is mainly corn mosaic virus and Pennisetum mosaic virus.
The classification of virus is mainly based on serology, biology and molecular biology method, wherein by molecular level virus is classified and become more and more important, continuous improvement along with sequencing technologies, the reduction of cost, coming virus is classified by the sequence that records virus is one of current most important method.Present classification for potyvirus is based on mostly analyzes and researches to Nucleotide or the aminoacid sequence of certain gene, but because these genes can only represent the complete genomic part of virus, therefore based on virus genome complete sequence virus being carried out sort research will be more reliable also more meaningful.In the 8th virus taxis report of up-to-date ICTV (ICTV), the criteria for classifying of Potyvirus inner virus kind is stipulated: CP consensus amino acid sequence rate＜80%, the genomic sequence concordance rate＜85%, and in an other research, show should be ORF and CP for potyvirus kind optimal dividing standard the nucleotide sequence concordance rate all＜76%.Just be based on molecular biology evidence just clear and definite, causing the virus of China's maize dwarf mosaic all to belong to corn mosaic virus, and be another strain that is different from SCMV-MDB.
SCMV belongs to Potyvirus, it can be propagated in non-persistent mode by aphid, also can carry out long-distance communications by the seed belt poison, therefore need to be by the determination and analysis to the SCMV complete sequence that causes maize dwarf mosaic, grasp its strain variation situation, make up rapid detection system, cultivate disease-resistant varieties, formulate positive prevention and control measure, thereby guarantee the safety of Maize Production.
Therefore how to know the complete sequence of this main Causative virus, and use the prevention and control that this complete sequence carries out disease, just become one of present problem demanding prompt solution.
Summary of the invention
Based on above-mentioned reason, the contriver utilizes domestic existing Virus Resource Virus Resource, from ill plant, obtained a kind of genom sequence of viral isolates, the contriver is with its called after corn mosaic virus (SCMV) Baoding isolate (BD8) (being called for short SCMV-BD8), by this sequence and existing SCMV genom sequence are analyzed existence and the distribution situation that can be used for clear and definite China SCMV strain, cultivate transgenosis disease-resistant maize new variety for design RT-PCR rapid detection primer with structure RNA silent carrier foundation is provided, also can be the corresponding Maize Production prevention and control measure of formulation theoretical foundation is provided.
Corn mosaic virus provided by the present invention China Baoding isolate gene group complete sequence, its genom sequence shown in SEQ IDNO.1, its protein sequence namely the amino acid whose sequence of open reading frame coding shown in SEQ ID NO.2;
The measuring method of this virogene complete sequence is as follows: the total RNA of maize leaf that shows obvious flower leaf paresthesia from a strain extracts, according to the listed SCMV complete sequence of GenBank conservative region design primer (seeing Table 1), use the RT-PCR method to increase, the purified recovery of the gene fragment that obtains is connected to later in the pMD18/19-T carrier and forms recombinant plasmid, the method that transforms by heat shock again is transformed into it in competence bacillus coli DH 5 alpha, the screening of amoxicillin, utilize alkaline process to extract in a small amount plasmid after the picking list spot enlarged culturing, after plasmid PCR is defined as positive strain, it is delivered to order-checking company the goal gene fragment is checked order.(5 ' UTR) utilize the terminal rapid amplifying method of cDNA 5 ', and (5 ' RACE) obtain 5 ' terminal non-translational region.Each fragment sequence that obtains DNAMAN software package (Lynnon BioSoft, ver.5.2.2) and DNASTAR 5.00 software packages (DNASTAR Inc., USA) SeqMan in carries out sequence assembly, finally obtains complete SCMV-BD8 complete nucleotide sequence.
Table 1BD8 gene fragment amplification the primer
▲ utilize the degenerate primer of the conservative section design of SCMV isolate Nucleotide
◆ the position of primer sequence in the final SCMV-BD8 complete nucleotide sequence that obtains
★ annexs site Y=T/C, R=A/G, N=A/C/G/T, H=A/C/T
☆ is used for the anchor primer of 5 ' RACE
The gene complete sequence that the present invention obtains, shown in SEQ ID NO.1, its maximum characteristics are: this sequence total length is 9576nt (nucleotide), based composition is: A:33.38%, G:21.99%, T:24.74%, C:19.89%, basically identical with other members' of Potyvirus based composition.The long 148nt of its 5 ' UTR nucleotide sequence, 3 ' UTR is long to be 236nt.Its ORF is by 9192 based compositions, and will encode one and contain 3063 amino acid, and molecular weight is the polyprotein of 345.97kDa.By SCMV-BD8 is compared with the conservative motif in 13 each protease hydrolysis sites of SCMV isolate complete sequence coding region ORF having reported in addition, 9 protease cracking sites (table 2) have been determined
2, in the protease P 1 of encoding viral, under the hydrolytic action of HC-Pro and NI a-Pro polyprotein is cut into the albumen of 10 maturations
8
Table 2SCMV-BD8 genome structure and functional protein cracking site sequence thereof
Font-weight illustrates this amino acid to be conservative site
ORF Nucleotide and the consensus amino acid sequence rate scope of the complete nucleotide sequence of SCMV-BD8 disclosed in this invention and 13 SCMV isolate complete sequences having reported in addition and one MDMV isolate AJ 001691 complete sequence being carried out concordance rate comparison result shows itself and other 13 SCMV isolates are respectively 78.9%-80.7% and 89.7%-91.5%, and wherein Nucleotide and the consensus amino acid sequence rate with Guangdong isolate AJ 310105 is the highest; The genomic sequence concordance rate scope is 79.1%-80.8%; The Nucleotide of CP and consensus amino acid sequence rate scope are respectively 76.9%-82.6% and 82.8%-86.9%.ORF Nucleotide and the consensus amino acid sequence rate of itself and MDMV are respectively 69.5% and 74.3%, and the genomic sequence concordance rate is that 69.2%, CP Nucleotide and consensus amino acid sequence rate are respectively 71.0% and 75.6%.Therefore gained isolate SCMV-BD8 of the present invention and other 13 SCMV isolates should belong to virus of the same race.
Isolate SCMV-BD8 of the present invention and other 13 SCMV isolate complete nucleotide sequences are passed through phylogenetic tree construction behind the Multiple Sequence Alignment take MDMV as the other nationality, the result shows that SCMV-BD8 all independently becomes one with Australian A strain isolate AJ278405, and Sino-Spanish, Mexican corn isolate is poly-for cluster is called as corn group (Maize group), and the sugarcane isolate of Zhejiang Province, China and corn isolate are poly-to be called as corn or sugarcane is organized (Sugarcane﹠amp for cluster; Maize)
1Corn mosaic virus isolate SCMV-BD8 involved in the present invention should belong to a new strain of this virus in sum, our called after MDC (causing maize dwarf in China) strain.
Take viral isolates involved in the present invention as toxogen, 12 commercially available corn varieties have been carried out the juice frictional inoculation in the greenhouse, each kind all shows as high sense to SCMV-BD8 as a result, illustrates that the new strain virulence of gained of the present invention is very strong, is a virulent strain department.Therefore this new strain virus probably becomes the Maize Production that next popular strain threatens China.In this case, the new strain isolate of corn mosaic virus involved in the present invention SCMV-BD8 complete sequence will provide important clue and theoretical foundation for effective prevention and control of detection, disease-resistant maize breed of variety and the corn disease viral disease of China's corn mosaic virus.Preservation information
The preservation time: on February 18th, 2011
Depositary institution's title: the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms
Deposit number: CGMCC NO.4577
Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Classification And Nomenclature: corn mosaic virus
Description of drawings
Fig. 1 SCMV-BD8 full length nucleotide sequence clone strategy schematic diagram;
The full-length gene group of BD8 obtains by 4 RT-PCR and 5 '-RACE, and the used primer of reverse transcription generally all is special, and forward primer is the degenerated primer according to the sequences Design of SCMV;
The systematic evolution tree that Fig. 2 utilizes SCMV-BD8 and other 13 SCMV isolate complete nucleotide sequences to make up;
Each isolate is listed with accession number/title/host/source place form among the figure, the value of bootstrapping of the numeral on the branch;
From evolutionary tree, begin to find out, SCMV-BD8 form one independently with A, corn group, the sugarcane/parallel branch of corn group, illustrate that BD8 is the new strain of SCMV.
Embodiment
Embodiment 1: the acquisition of viral isolates
From the corn field of Baoding, Hebei province, gather the blade that is obvious flower leaf paresthesia in July, 2009, obtain viral isolates, called after BD8.Sample retention is numbered No.4577 at China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).Take this virus strain as malicious source frictional inoculation on the healthy milpa so that virus under greenhouse experiment, copied.
The extraction of embodiment 2:RNA and RT-PCR
The amplification of BD8 genomic fragment is carried out according to strategy shown in Figure 1.Genome divides first A, B, C, D 4 sections and increases, and per two intersegmentally have the overlapping of 100-200 base.5 '-UTR increases by RACE.
1.RNA extract
According to the method that TransZol test kit (Beijing TransGen Biotech Co., Ltd) provides, from susceptible maize leaf, extract total RNA.
2. reverse transcription-PCR (RT-PCR) reaction
The viral RNA that contains in the total RNA of plant carries out reverse transcription as template.
Gene fragment A
The reverse transcription system
5 * reverse transcription damping fluid (TransGen), 4 μ l
Hatch 50min for 42 ℃ behind the above composition mixing, hatch the 15min stopped reaction for 70 ℃.The gained reverse transcription product is used for the PCR reaction.
The PCR system
Above component mixes to be placed in the PCR instrument and increases.Response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2.5min, totally 35 circulations; 72 ℃ are extended 10min.
Gene fragment B
The reverse transcription system
Hatch 50min for 42 ℃ behind the above composition mixing, hatch the 15min stopped reaction for 70 ℃.The gained reverse transcription product is used for the PCR reaction.
The PCR system is
Above component mixes to be placed in the PCR instrument and increases.Response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2.5min, totally 35 circulations; 72 ℃ are extended 10min.
Gene fragment C
Hatch 50min for 42 ℃ behind the mixing, hatch 15mi n stopped reaction for 70 ℃.The gained reverse transcription product is used for the PCR reaction.
Mix to be placed in the PCR instrument and increase.Response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 2.5min, totally 35 circulations; 72 ℃ are extended 10min.
Gene fragment D
The reverse transcription system is:
Hatch 50min for 42 ℃ behind the mixing, hatch the 15min stopped reaction for 70 ℃.The gained reverse transcription product is used for the PCR reaction.
The PCR reaction system is:
Each component mixes to be placed in the PCR instrument and increases.Response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 2.5min, totally 35 circulations; 72 ℃ are extended 10min.
3.cDNA terminal rapid amplifying method (RACE) acquisition 5 '-the UTR sequence
Adopt the Methods For Purification SCMV virus of beam new talent.Get 100 μ l purified virus liquid and add 1ml Trizol, method is extracted with the total RNA of plant afterwards.The viral RNA that obtains is used for next step 5 ' RACE method, amplification 5 '-the UTR fragment.
(1) cDNA the first chain is synthetic
On ice following composition is joined in the Eppendorf pipe:
In the another one pipe, add 1 μ l reverse transcription special primer GSP456 (10 μ M) and 11 μ l RNA, totally 12 μ l, 80 ℃ of sex change 3min, place rapidly after the cooled on ice wink from.
Will be behind the mixed solution mixing in two pipes add 1 μ l (200U) SuperScript II RT, with rifle head mixing gently, hatch 1h in 42 ℃, hatch 10min for 50 ℃ afterwards.Form DNA-RNA heterozygote in the pipe this moment.Hatch 15min for 70 ℃ and make enzyme deactivation.
(2) degraded of RNA in the DNA-RNA heterozygote
The RNase H that uses TaKaRa company to produce, the RNA in degradation of dna-RNA heterozygote.
The following reaction solution of preparation in Eppendorf tube:
30 ℃ were reacted 1 hour.
(3) cDNA purifying
EasyPure rapid DNA gel with Beijing Quan Shi King Company reclaims test kit, by its supplying method purification cDNA, with 30 μ l H
2O returns molten.
(4) 5 ' add end reaction
The terminal enzyme (DNA) (TdT) that uses TaKaRa company to produce, the following reaction solution of preparation in Eppendorf tube, cumulative volume 50 μ l.
37 ℃ of reaction 15min, EDTA (pH 8.0) termination reaction that adds NaCl and the 1 μ l 0.5M of 5 μ l 5M, then after using phenol/chloroform/primary isoamyl alcohol (25: 24: 1) 50 μ l extractings, carry out 125 μ l (2.5 times of consumptions) dehydrated alcohol precipitation, place 30-60min for-20 ℃, the gained precipitation is dissolved in 10 μ l ddH after centrifugal
2Be used for pcr amplification (diluting 10 times uses afterwards) subsequently among the O.
(5) pcr amplification
The high-fidelity LA Taq archaeal dna polymerase that uses TaKaRa company to produce adds following reaction solution in Eppendorf tube:
Increase at the PCR instrument, program is: 94 ℃ of 3min, 48 ℃ of 2min, 72 ℃ of 40min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations; 72 ℃ are extended 10min.
Take turns nest-type PRC with carrying out second after 20 times of the first round PCR product dilutions, reaction solution is:
The pcr amplification program is 94 ℃ of 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations; 72 ℃ are extended 10min.
Embodiment 3:PCR product purification reclaims and the clone
Pcr amplification product downcuts the purpose band behind 1% agarose gel electrophoresis, reclaims test kit (centrifugal column type) (Beijing Bioteke Corporation) by the method purifying recovery DNA of its specification sheets with the multifunctional dna purifying.
Method on the by specification, the DNA that purifying is reclaimed is connected on pMD18/19-T (TaKaRa Biotechnology Dalian Co, the Ltd) carrier.The recombinant plasmid that connects heat shock method Transformed E .coli DH5 α, alkaline process extracts recombinant plasmid, PCR evaluation and screening positive colony.
Embodiment 4: determination and analysis of sequence
Each segment is got 3 positive colonies and is sent to Nanjing Genscript Biotechnology Co., Ltd. and checks order.At least to measure 6 clones' sequence to the positive colony of 5 ' RACE acquisition fragment.
With the SeqMan among DNAMAN (Lynnon BioSoft, ver.5.2.2) and the DNASTAR 5.00 (DNASTAR Inc., USA) each fragment sequence that records is spliced, carry out the comparison of sequence concordance rate with the MegAlign among the DNASTAR.Use the CLUSTAL W in MEGA 4.0 softwares to carry out Multiple Sequence Alignment, and then with adjacent method (neighbor-joining, NJ) constructing system evolutionary tree.
BD8 genome total length is 9576 Nucleotide (nt), with the genomic sequence concordance rate scope of other 13 SCMV isolates be 79.1%-80.8%, ORF Nucleotide and consensus amino acid sequence rate scope are respectively 78.9%-80.7% and 89.7%-91.5%, and wherein Nucleotide and the consensus amino acid sequence rate with Guangdong isolate (AJ310105) is the highest; The Nucleotide of CP and consensus amino acid sequence rate scope are respectively 76.9%-82.6% and 82.8%-86.9%.SCMV-BD8 and MDMV the genomic sequence concordance rate be 69.2%, ORF Nucleotide and consensus amino acid sequence rate be respectively 69.5% and 74.3%, CP Nucleotide and consensus amino acid sequence rate be respectively 71.0% and 75.6%.According to the criteria for classification of ICTV (ICTV), gained isolate BD8 of the present invention with should belong to corn mosaic virus (SCMV).
In systematic evolution tree, SCMV can be divided into four groups, and wherein Australian isolate (AJ278405) all independently becomes one, is called the A group; China, Spain and Mexican corn isolate are poly-for cluster, are called corn group (Maize group); The sugarcane isolate of Zhejiang Province, China and corn isolate are poly-to be cluster, is called corn or sugarcane group (Sugarcane﹠amp; Maize); Corn mosaic virus Baoding isolate SCMV-BD8 involved in the present invention states one of middle formation in evolution and independently organizes, and is a new strain (MDC) of this virus.
Embodiment 5: the biology test
12 kinds of common corn varieties on take greenhouse morbidity strain as frictional inoculation market, fresh malicious source: north, Tianjin 288, Five Sacred Mountins 97-1, Five Sacred Mountins 21, black beautiful No. 2, Luyuandan 22 is chatted jade 19, single 850, the Zheng Dan 958 in Shandong, Nongda108, single 981, the Lu Yu 15 in Shandong, No. 7, safe jade; 9-12 of every kind inoculation do not wait, and cultivates under 22～25 ℃ of conditions in greenhouse, observes and record incidence, result such as following table after 15 days:
The sickness rate of table 3 inoculation 12 corn varieties after SCMV-BD815 days
Experimental results show that, 12 corn varieties of test all show high sense to SCMV-BD8, illustrate that the new strain virus of gained of the present invention is virulent strain department, this new strain probably becomes the Maize Production that next popular strain threatens China, in this case, the new strain isolate of corn mosaic virus involved in the present invention SCMV-BD8 complete sequence will be the detection of China's corn mosaic virus, and the corn disease-resistant variety is cultivated and the formulation of Maize Production prevention and control measure provides important theoretical foundation.
Claims (1)
1. Chinese Baoding isolate SCMV-BD8 of corn mosaic virus (Sugarcane mosaic virus) that preserving number is CGMCC NO.4577, it is characterized in that its genom sequence shown in SEQ ID NO.1, the amino acid whose sequence of its open reading frame coding is shown in SEQ ID NO.2.
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| CN104846005B (en) * | 2015-03-02 | 2018-04-17 | 福建农林大学 | The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV3 sequences |
| CN105039355B (en) * | 2015-09-09 | 2019-07-12 | 福建农林大学 | The method for cultivating mosaic disease resisting sugar cane using RNAi silencing translation initiation factor gene |
| CN108728581B (en) * | 2018-06-26 | 2021-02-05 | 广西大学 | Multiple RT-PCR method for simultaneously detecting 5 sugarcane viruses, primers and kit thereof |
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