CN102816730A - Agrobacterium strain containing cotton leaf curl Multan virus (CLCuMV) infectious clones and applications thereof - Google Patents
Agrobacterium strain containing cotton leaf curl Multan virus (CLCuMV) infectious clones and applications thereof Download PDFInfo
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Abstract
The invention discloses an agrobacterium strain containing CLCuMV infectious clones and applications thereof. 1.9 direct repeat CLCuMV full-length genome DNAs and 1.8 direct repeat satellite DNA beta are constructed simultaneously in efficient plant expression vector pCambia2300 by a molecular biology method, an agrobacterium strain EHA105 is led by an electric shock method to obtain agrobacterium strain CLCuMV capable of efficiently infecting plant virus infectious clones, and the preservation number is CGMCCNo.5930. The strain can produce a large number of infectious clones of the CLCuMV, and the infectious clones can efficiently infect various host plants and can be used for the pathopoiesis analysis and virus gene function study of the CLCuMV. In addition, the infectious clones can improve virus expression vectors and provides technology and material supports for expressing foreign proteins and studying plant functional genomes after endogenous gene silencing.
Description
Technical field
Field under the present invention is a bioengineering field, especially relates to a kind of agrobacterium strains and application thereof that contains MULTAN cotton curve leaf disease virus infectious clone.
Background technology
(Cotton leaf curl disease CLCuD) is a kind of disease of the cotton production that seriously causes harm to cotton curve leaf disease.The host range of CLCuD is wider, the cotton of not only causing harm, hollyhock, sea island cotton, upland cotton, okra, Japan and the yellow rose of Sharon of the Malvaceae of also can causing harm, and the capsicum of Solanaceae, black nightshade and watermelon cucurbitaceous, sponge gourd.Its experiment host comprises cotton, tobacco, tomato, the rose of Sharon, okra and wrinkled giant hyssop etc.Since the Sudan finds CLCuD, CLCuD was the main disease of the Pakistani regional output of cotton of influence always from 1978.Cotton curve leaf disease is propagated by Bemisia tabaci; Main generation plant leaf curls, vein expands, the leaf back ear is uprushed gives birth to, plant is downgraded, 40%～60% of general unsoundness plant height of disease plant degree, and cotton boll is tied less or is not tied; The cotton fibre low yield, total crop failure waits classical symptom when serious.
So far the cause of disease of identifying that causes cotton curve leaf disease has 7 kinds, and these 7 kinds of viruses mainly are distributed in Asia and Africa, and wherein (Cotton leaf curl Multan virus CLCuMV) is the main pathogen that causes cotton curve leaf disease to MULTAN cotton curve leaf disease virus.CLCuMV be geminivirus infection section (
Geminiviridae) in Begomovirus (
Begomovirus) virus, have typical doublet particle form, about 18～20 nm of size * 30 nm, genome is the strand cyclic DNA.CLCuMV is one type of disease complex body that is formed by DNA A and satellite DNA β.
The plant virus genome is generally less, be easy to carry out genetic manipulation and also course of infection simple, can be widely used in the research of plant reverse genetics.Set up the complete reverse genetics means of a cover based on plant virus; At first to make up the infectious clone of virus; And then utilize sudden change, disappearance, insertion, displacement equimolecular biological means viral genome to be carried out the location of functional area, thereby can on the host plant phenotypic level, reflect and estimate the expression of gene function.Mainly contain the effect of following two aspects based on the infectious clone of plant virus.At first, utilize plant viral vector in the host plant body, to carry out proteinic overexpression, the second, utilize plant viral vector to pass through RNAi (RNA interference) technology, in the host plant body, carry out the research of autotelic gene silencing.The RNAi technology found in petunia early than 1998, was chosen as then ten big sciences in 2002 by " science " magazine and broke through.Vegetable cell can specific recognition exogenous RNA, through proteic effects such as RDR6, with the external source single stranded RNA duplicate produce double-stranded RNA (dsRNA) after, cause its degraded.The secondary little RNA (siRNA) that degraded produces can act on its homologous sequence again, makes reticent signal send out greatly.Utilize this principle, on plant viral vector, insert the native gene that one section plant endogenous gene just can special reticent plant, thereby Plant Genome is studied through the means of reverse genetics.
Summary of the invention
The purpose of this invention is to provide a kind of agrobacterium strains and application thereof that contains MULTAN cotton curve leaf disease virus infectious clone.
The agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone, preserving number are CGMCC No.5930, can produce the infectious clone of MULTAN cotton curve leaf disease virus.
The described construction process that can produce the infectious clone of MULTAN cotton curve leaf disease virus is: utilize molecular biology method that 1.9 forward multiple MULTAN cotton curve leaf disease virus full-length gene group DNA and 1.8 forward multiple satellite DNA β are building up on the same high-efficiency plant expression vector pCambia2300, obtain MULTAN cotton curve leaf disease virus infectious clone.
The described agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone is used for pathogenic analysis, the known viral gene function research of MULTAN cotton curve leaf disease virus.
Described MULTAN cotton curve leaf disease virus infectious clone is used for expression of exogenous gene and reticent native gene is studied the Plant Genome function.
The invention has the beneficial effects as follows: the agrobacterium strains that 1) provides can produce the infectious clone of MULTAN cotton curve leaf disease virus in a large number; 2) utilize infectious clone provided by the invention can efficiently infect various modes plants such as Ben Shi cigarette, common cigarette and three lives cigarette, need not expensive instrument, simple to operate, good reproducibility; 3) utilize infectious clone provided by the invention can be effective to pathogenic analysis, the known viral gene function research of MULTAN cotton curve leaf disease virus; 4) utilize infectious clone provided by the invention can be transformed into virus expression carrier; 5) utilize infectious clone provided by the invention to be used for expression of exogenous gene and study the Plant Genome function through reticent native gene.
Description of drawings
Fig. 1. pCambiaA β-PDS induces Ben Shi cigarette PDS gene silencing phenotype;
Fig. 2. real-time quantitative PCR detects pCambiaA β-PDS and soaks into the reticent efficient of the endogenous PDS of Ben Shi cigarette;
Fig. 3. pCambiaA β-CP4EPSP soaks into Ben Shi cigarette resistance glyphosate phenotype.
Embodiment
The agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone: be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 22nd, 2012; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; The classification name: the infectious clone of MULTAN cotton curve leaf disease virus, preserving number are CGMCC No.5930, and it can produce the infectious clone of MULTAN cotton curve leaf disease virus.
The described construction process that can produce the infectious clone of MULTAN cotton curve leaf disease virus is: utilize molecular biology method that 1.9 forward multiple MULTAN cotton curve leaf disease virus full-length gene group DNA and 1.8 forward multiple satellite DNA β are building up on the same high-efficiency plant expression vector pCambia2300, obtain MULTAN cotton curve leaf disease virus infectious clone.
The described agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone is used for pathogenic analysis, the known viral gene function research of MULTAN cotton curve leaf disease virus.
Described MULTAN cotton curve leaf disease virus infectious clone is used for expression of exogenous gene and reticent native gene is studied the Plant Genome function.
Below in conjunction with embodiment and accompanying drawing the present invention is described further.
The structure of embodiment 1. MULTAN cotton curve leaf disease virus DNA A and DNA β infectious clone
The infectious clone of geminivirus infection needs 1.1-2.0 multiple full-length gene group, and must comprise 2 common districts of geminivirus infection.With cotton curve leaf disease virus Guangxi, MULTAN isolate gene group DNA (GenBank:JQ317603) is template, design
SalThe total length primers F of I single endonuclease digestion (5 '-GGTCGACGTCATCAATGACGTTGTA-3 ', SEQ ID NO:1) and R (5 '-ACGTCGACCCGCATTTCCTCAAA-3 ', SEQ ID NO:2) carry out pcr amplification, the PCR product is used
SalBe connected to after the I enzyme is cut on the pCambia2300 carrier, after the sequencing checking, obtain pCambia-A.With total length pCambia-A clone is template, design XbaI F (5 '-CCTGAAAGATCCTGTCTAGATTTGCAT-3 ', SEQ ID NO:3) and R (5 '-ACGTCGACCCGCATTTCCTCAAA-3 ', SEQ ID NO:2) for carrying out PCR, primer reacts.The PCR product is used
SalI with
XbaPCambia2300 (GenBank:AF234315) with same double digestion behind the I double digestion is connected, screens, and carries out sequence checking back acquisition pcambia-0.9A.Use
SalI is from being inserted into the corresponding site of pCambia-0.9A after pCambia-A cuts next complete DNA A total length, obtains pCambia-1.9A after utilizing PCR screening direction.
With cotton curve leaf disease virus Guangxi, MULTAN isolate DNA β (GenBank:JQ317604) is template; With GXbeta sal1 F (5 '-ACAGTCGACGTTCGCATCATGAC-3 '; SEQ ID NO:4) with GXbeta Hind III R (5 '-CTTGAAAGCTTTATACATGGGTTTGTACC-3 '; SEQ ID NO:5) carry out pcr amplification, the PCR reaction parameter is 94 ℃ of 3 min; 94 ℃ of sex change 45 sec, 52 ℃ of annealing 30 sec, 72 ℃ are extended 1.5 min, 35 circulations, last 72 ℃ are extended 10 min, PCR product utilization
SalI with
HinBehind the d III double digestion, with same process
SalI with
HinThe pCambia2300 that d III enzyme is cut connects, and obtains pcambia-0.8 β, and carries out the sequence checking.The total length copy of another one DNA β comes from pCambia-β.With its usefulness
SalThe I enzyme downcuts about 1.3Kb product and is connected on the pcambia-0.8 β, obtains pCambia-1.8 β.
With pCambia-1.8 β is template; Design primer GXbetaXmaF (cccgggAGCGGATAACAATTTCACACAGGA; SEQ ID NO:6) and GXbetaXmaR (cccgggCGCCAGGGTTTTCCCAGTCACGAC, SEQ ID NO:7) carry out pcr amplification, must arrive two ends and contain
XmaThe 1.8 β fragments that are about the 2.3Kb size of I restriction enzyme site, and carry out sequence verification.With this fragment warp
XmaBe inserted into the corresponding site of pCambia-1.9A after the I enzyme is cut, obtain the pCambia-1.9A-1.8 β that DNA A and satellite DNA β are positioned at identical carrier after the PCR screening direction.
Embodiment 2. recombinant vectors pCambia2300-1.9A-1.8 β electric shock transforms Agrobacterium
Method through electric shock imports Agrobacterium host cell EHA105 with recombinant plant expression vector pCambia2300-1.9A-1.8 β.The preparation of EHA105 competent cell: agrobacterium strains EHA105 inoculates 5 ml and contains Rif (50 mg/L) YEP liquid nutrient medium; 28 ℃ down 200 rpm be cultured to OD600 ≈ 0.6, abandon the supernatant nutrient solution after centrifugal, with the sterilized water thalline that suspends again; Centrifugal again; Abandon supernatant, repeat 3-4 time, obtain agrobacterium tumefaciens EHA105 competent cell.Get 2 μ l recombinant plasmid pCambia2300-1.9A-1.8 β and join in the 200 μ l agrobacterium tumefaciens competent cells, mixing changes in the electric shock cup gently, places 10 min on ice.With Bio-rad gene pluser xcell electric shock appearance, voltage 2400V, electric shock transforms Agrobacterium EHA105.Agrobacterium after transforming is added 1 ml YEP recovery media, and 28 ℃ of 220rpm shook bacterium after 3 hours, got 50ul bacterium liquid; Be uniformly coated on and contain kantlex (Km; 50 μ g/mL) and on the YEP solid medium of the two resistances of Rifampin (Rif, 50 μ g/mL), cultivate 48h for 28 ℃.The 3rd day, utilize PCR screening bacterium colony, obtain pCambia-1.9A-1.8 β (EHA105).
Embodiment 3. contains the Agrobacterium infiltration inoculation plant of MULTAN cotton curve leaf disease virus infectious clone
PCambia-1.9A-1.8 β cultivates at the YEP liquid nutrient medium that contains Km (50 μ g/mL) and Rif (50 μ g/mL), cultivates 48 hours down for 28 ℃.The inoculation plant selects for use 4-6 leaf phase fully extended seedling to be advisable.Inoculation Ben Shi cigarette, three lives cigarette, common cigarette.Bacterium liquid when soaking into inoculation is handled with inoculation method following, and pCambia-1.9A-1.8 β (EHA105) 20 ml bacterium liquid are centrifugal through 12000 rpm 1 minute, abandons supernatant, with infiltration damping fluid (10 mM MgCl
2, 10 mM MES, 200 mM Syringylethanones) and the thalline that suspends again, adjustment OD600 is about 1.0, leaves standstill infiltration of plants after 2-3 hour.During infiltration, get one and do not carry out blade back with 1 ml disposable syringe of syringe needle at 5-6 leaf phase plant leaf and soak into, a strain plant generally needs 1ml bacterium liquid to soak into 3-4 sheet leaf, and the inoculation plant places 25 ℃ of Isolation warm houses to cultivate.
Embodiment 4. transform MULTAN cotton curve leaf disease virus as virus expression carrier
Design primer GXmcsF (GTCGACTTAATTAAGAGCTCGAGCTCACTAGTAATTACGGGAACGTAG; SEQ ID NO:8) and GXmcsR (GTCGACATGTCGAAGCGAGCTGCAGATATCGTCATTTC; SEQ ID NO:9); With MULTAN cotton curve leaf disease virus genomic dna is template, carries out pcr amplification, and response procedures is 94 ℃ of 3 min; 94 ℃ of sex change 45 sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 3 min, 35 circulations, last 72 ℃ are extended 10 min.The PCR product must arrive two ends and contain after Axygen PCR cleaning agents box is handled
SalI, CP disappearance place introduce
PacI with
SpeThe DNA-A fragment of the CP excalation of I cloning site, sequence checking back called after DNA-Amcs.DNA-Amcs is carried out
SalBe inserted on the pCambia-0.9A after the I enzyme is cut, utilize PCR screening direction after, obtain the CP excalation, and introduce
PacI with
SpeThe expression vector pCambia1.9Amcs of I cloning site.Because MULTAN cotton curve leaf disease virus contains DNA A and satellite DNA β; The accumulation volume of virus increases when DNA A and satellite DNA β infect jointly; For improving the expression amount of carrier; According to the method for embodiment 1, our structure obtains DNA A and satellite DNA β is positioned at identical carrier, viral CP excalation and the introducing of disappearance place
PacI with
SpeThe expression vector pCambiaA β mcs of I cloning site.
Embodiment 5. utilizes the reticent PDS gene of virus expression carrier
According to Ben Shi cigarette native gene phytoene desaturase (PDS) gene order (GenBank:EU165355); We design PDS F (GactagtCGGCACTTAACTTCATAAACCCTGA; SEQ ID NO:10) and PDS R (CCttaattaaGGAAAACATTTGACACTTCCATCCTC, SEQ ID NO:11) (small letter is respectively to primer for this
SpeI with
PacThe I site), be template with Ben Shi cigarette cDNA, pcr amplification obtains the about 200bp fragment of PDS, and (the PCR reaction parameter is 94 ℃ of 3 min; 94 ℃ of sex change 45 sec, 52 ℃ of annealing 30 sec, 72 ℃ are extended 15sec, 35 circulations, last 72 ℃ are extended 1min), sequencing after
PacI with
SpeThe I double digestion is inserted on the pCambiaA β msc, obtains pCambiaA β-PDS silent carrier.
PCambiaA β-PDS electric shock is transformed among the Agrobacterium EHA105, obtain pCambiaA β-PDS (EHA105), and soak into the Ben Shi cigarette respectively with contrast pCambia-1.9A-1.8 β (EHA105).In infiltration back 15 days, the Ben Shi cigarette system blade table that pCambiaA β-PDS soaks into revealed the albefaction phenotype (Fig. 1) of PDS gene silencing.After this shows that pCambiaA β-PDS soaks into the Ben Shi cigarette, endogenous PDS expression of gene in the special reticent Ben Shi cigarette of ability.Find that through the real-time quantitative PCR detection pCambiaA β-PDS soaks into the Ben Shi cigarette that the Ben Shi cigarette soaks into than contrast pCambia-1.9A-1.8 β, its endogenous PDS expression amount decline 80% (Fig. 2).
Embodiment 6. utilizes MULTAN cotton curve leaf disease virus infectious clone expression alien gene
According to glyphosate gene group sequence (GenBank:AF464188); We design primer CP4EPSP F (ACTAGTCACATAAAACCCCAAGTTCCTAAA; SEQ ID NO:12) and CP4EPSP R (TTAATTAACATCAGGCAGCCTTCGTATCG; SEQ ID NO:13), be that template is carried out pcr amplification with transgenic resistance glyphosate cotton gene group, response procedures is 94 ℃ of 3 min; 94 ℃ of sex change 45 sec, 60 ℃ of annealing 30 sec, 72 ℃ are extended 2 min, 35 circulations, last 72 ℃ are extended 10 min.Reaction solution obtains two ends and has respectively after handling through Axygen PCR cleaning agents box
SpeI with
PacThe about 1600bp fragment of the Antiglyphosate gene CP4EPSP of I.Behind the sequencing, with this fragment warp
PacI with
SpeBe connected into pCambiaA β mcs behind the I double digestion, obtain pCambiaA β CP4EPSP.Again pCambiaA β-CP4EPSP is imported among the Agrobacterium EHA105, obtain pCambiaA β-CP4EPSP (EHA105).
PCambiaA β-CP4EPSP (EHA105) and contrast pCambia-1.9A-1.8 β (EHA105) are soaked into the Ben Shi cigarette respectively.Soak into after 20 days, after 100 times of 41% Glyphosate 62 IPA Salt aqueous pesticide solution dilutions, spray whole plant and be placed on 25 ℃ of hot-house cultures and spend the night.Can observe next day; Than the plant of having soaked into pCambiaA β-CP4EPSP (EHA105); Obvious wilting phenotype has appearred in the plant that pCambia-1.9A-1.8 β soaks into, and the tobacco that pCambiaA β-CP4EPSP soaks into does not then have obvious phenotypes (Fig. 3).We think, in the Ben Shi cigarette that pCambiaA β-CP4EPSP soaks into, have expressed the gene C P4EPSP of resistance glyphosate, thereby have shown the phenotype of resistance glyphosate.
SEQUENCE?LISTING
< 110>Zhejiang University
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Claims (4)
1. an agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone is characterized in that preserving number is CGMCC No.5930, can produce the infectious clone of MULTAN cotton curve leaf disease virus.
2. a kind of agrobacterium strains that contains MULTAN cotton curve leaf disease virus infectious clone as claimed in claim 1; It is characterized in that the described construction process that can produce the infectious clone of MULTAN cotton curve leaf disease virus is: utilize molecular biology method that 1.9 forward multiple MULTAN cotton curve leaf disease virus full-length gene group DNA and 1.8 forward multiple satellite DNA β are building up on the same high-efficiency plant expression vector pCambia2300, obtain MULTAN cotton curve leaf disease virus infectious clone.
3. an application that contains the agrobacterium strains of MULTAN cotton curve leaf disease virus infectious clone as claimed in claim 1 is characterized in that being used for pathogenic analysis, the known viral gene function research of MULTAN cotton curve leaf disease virus.
4. application that contains the agrobacterium strains of MULTAN cotton curve leaf disease virus infectious clone as claimed in claim 2 is characterized in that described MULTAN cotton curve leaf disease virus infectious clone is used for expression of exogenous gene and reticent native gene is studied the Plant Genome function.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024108742A1 (en) * | 2022-11-24 | 2024-05-30 | 江苏省农业科学院 | Bean common mosaic virus isolate, method for constructing full-length infectious cdna clone of bean common mosaic virus isolate and use of bean common mosaic virus isolate |
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