CN102379305A - Novel applications of corn ferredoxin - Google Patents

Abstract

The invention discloses a new application of corn ferredoxin. The new application provided by the present invention is the application of ferredoxin V in the preparation of products inhibiting the replication of mosaic virus, and the application of the gene encoding the protein shown in sequence 1 in the sequence listing in the preparation of products inhibiting the replication of mosaic virus , and the application of the recombinant expression vector containing the coding gene of the protein shown in Sequence 1 in the sequence listing in the preparation of products for inhibiting the replication of mosaic virus. The present invention co-transforms the plasmid capable of expressing Fd V with SCMV RNA in maize protoplasts, and overexpressing Fd V can reduce the replication level of SCMV virus by about 40%, indicating that overexpressing Fd V can effectively inhibit SCMV replication. This will play an important role in the prevention and treatment of maize virus diseases, and at the same time lay a good foundation for molecular breeding of maize.

Description

The new purposes of corn ferredoxin

Technical field

The present invention relates to the new purposes of corn ferredoxin.

Background technology

The corn short mosaic disease can be infected by the viral systematicness of one to multiple kind and cause, in worldwide, causes serious economy loss.The virus of the caused corn short mosaic disease of having reported in the world has 6 kinds; All belong to Potyvirus (Potyvirus); They be respectively corn mosaic virus (Sugar cane mosaic virus, SCMV), maize dwarf mosaic virus (Maize dwarf mosaic virus, MDMV), sorghum mosaic virus (Sorghum mosaic virus; SrMV), Johnson grass mosaic virus (Johnsongrass mosaic virus; JGMV), the Zea mosaic virus (Zea mosaic virus, ZeMV) with white showy flowers of herbaceous plants mosaic virus (Pennisetum mosaic virus, PenMV).In China; Corn mosaic virus Beijing separator (SCMV-BJ) is (the Fan Z F of dominant strain system that causes northern corn producing region corn short mosaic disease; Chen H, Liang X, and Li H F.Complete sequence of the genomic RNA of the prevalent strain of a potyvirus infecting maize in China.Arch Virol; 2003,148:773-782.).

As the plant virus of obligate parasite, the completion of its history of life must rely on host factor and participate in virus uncoating, genome duplication, virion assembling and viral movement process.Like (Wittmann S such as Wittmann; Chatel H; Fortin M G.et al.Interaction of the Viral Protein Genome Linked of Turnip Mosaic Potyvirus with the Translational Eukaryotic Initiation Factor (iso) 4E of Arabidopsis thaliana Using the Yeast Two-Hybrid System.Virology; 1997; 234 (1): 84-92.) with (L é onard S such as L é onard; Plante D, Wittmann S, et al.Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity.J.Virol.; 2000; 74 (17): experimental result 7730-7737.) shows, Brassica 2 et 4 (Turnip mosaic virus, (the virus genome-linked protein of genome linkage protein TuMV); VPg) possibly realize the synthetic of viral GAP-associated protein GAP through interaction, point out that this mutual work is that virus is made the key element of (virus production) with eukaryotic initiation factor (eukaryotic initiation factor 4E).(Dunoyer P such as Dunoyer; Thomas C; Harrison S; Et al.A cysteine-rich plant protein potentiates Potyvirus movement through an interaction with the virus genome-linked protein VPg.J.Virol.; 2004,78 (5): 2301-2309.) utilize called after PVIP protein-interacting in VPg that yeast two-hybrid finds Potyvirus and the plant, and the motion of PVIP this virus of support in plant corpus.NIb albumen is the rna replicon enzyme (RdRp) of the dependenc RNA of Potyvirus genome encoding; (Wang X such as Wang; Ullah Z, Grumet R.Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly (A) binding protein.Virology, 2000; 275 (2): 433-443.) with (Dufresne P J such as Dufresne; Thivierge K, Cotton S, etal.Heat shock ' 70 protein interaction with Turnip mosaic virus RNA-dependent RNApolymerase within virus-induced membrane vesicles.Virology; 2008; 374 (1): 217-227.) use methods such as tandem affinity purification method and yeast two-hybrid respectively, find that the poly (A) of cucumber and arabidopsis combines albumen and NIb to do mutually, show that poly (A) combines albumen in virus replication, to play an important role.HC-Pro is the 2nd albumen of Potyvirus viral gene group coding, is an important multifunctional protein.Research in recent years shows; This albumen has protease and auxiliary aphid passes cytotoxic activity, the participation viral genome is duplicated and virus moves, disturbs microRNA (miRNA) approach, participate in virus disease symptom expression etc.; Particularly this albumen has the plant gene silencing of inhibition function, is this viral virulence factor.

(ferredoxin is the important component part of PSI in the plant chloroplast Fd) to ferredoxin, mediates photosynthetic electronics transmission.As other most of chloroplast proteins, Fd also is by nuclear gene encoding, synthetic precursor protein in cytoplasm, and leader peptide (transit peptide) the mediation amyloid protein precursor through its N end is transported in the chloroplast after translation then.Corn as the C4 plant has two kinds of photosynthetic cells, promptly vascular bundle sheath cell (the bundle-sheath cell, BSC) and mesophyll cell (mesophyll cell, MC).So far found that corn has six kinds of Fd to comprise three kinds of photosynthetic type Fd (Fd I, Fd II and Fd V) and 3 kinds of non-photosynthetic type Fd (Fd III, Fd IV and Fd VI).Photosynthetic type Fd II, Fd V mainly express in BSC, and (cyclic electron flow CEF), produces extra ATP and supplies Calvin cycle and CO the high activity circulating electron transmission among the participation PS I ₂Concentrating mechanism is required; Fd V transmission capacity is apparently higher than Fd II simultaneously.And CEF is for cold resistance (the Ducruet J M that strengthens corn plant; Roman M; Havaux M, et al.Cyclic electron flow around PSI monitored by afterglow luminescence in leaves ofmaize inbred lines (Zea mays L.): correlation with chilling tolerance.Planta, 2005; 221 (4): 567-579.), protection photosystem II (photosystem II; PS II) protected from light suppresses (Rumeau D, Peltier G, and Cournac L.Chlororespiration and cyclic electron flow around PSI duringphotosynthesis and plant stress response.Plant Cell Environ.; 2007,30 (9): the injury of 1041-1051) compeling with other association etc. are in close relations; SA CEF can cause that plant strain growth receives to press down, chloroplast content reduces, be vulnerable to (Kimata-Ariga Y such as photobleaching; Matsumura T; Kada S; Et al.Differential electron flow around photosystem I by two C (4)-photosynthetic-cell-specific ferredoxins.EMBO J., 2000,19 (19): 5041-5050.).

Summary of the invention

An object of the present invention is to provide the application of ferredoxin V in the product that preparation inhibition mosaic virus duplicates.

The amino acid sequence of said ferredoxin V is in the sequence table shown in the sequence 1.

Another object of the present invention provides the application of encoding gene in the product that preparation inhibition mosaic virus duplicates of the albumen shown in the sequence 1 in the sequence table; The encoding gene of said albumen is following 1)-3) in arbitrary described gene:

1) dna molecular shown in the sequence 2 in the sequence table;

2) under stringent condition with 1) shown in dna molecule hybridize and the gene of encoding said proteins;

3) with 1) or 2) gene have autoploidy and the gene of encoding said proteins more than 90%.

Contain the recombinant expression carrier of the encoding gene of albumen shown in the sequence 1 in the ordered list and also belong to protection scope of the present invention in the application that preparation suppresses in the product that mosaic virus duplicates.

Said recombinant expression carrier is the recombinant expression carrier that the encoding gene of albumen obtains shown in the sequence 1 in the MCS insetion sequence table of pGFP carrier.

Said mosaic virus is a corn mosaic virus.

Said inhibition mosaic virus copies as and suppresses mosaic virus duplicating in plant protoplast.

Said plant is a corn.

The present invention can express plasmid and the SCMV RNA of Fd V through cotransformation in the corn protoplast, and overexpression Fd V can reduce SCMV virus replication level about 40%, shows that overexpression Fd V can effectively suppress SCMV and duplicate.This will play a significant role in the control of maize virus disease, lay a good foundation for the molecular breeding of corn simultaneously.

Description of drawings

Fig. 1 cuts the checking result for plasmid pGFP-Fd V enzyme.

Fig. 2 is the influence of overexpression FdV to SCMV virus replication level.

Embodiment

Employed experimental technique is conventional method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Case study on implementation 1, overexpression ferredoxin V (FdV) suppress duplicating of corn mosaic virus (SCMV)

One, makes up FdV expression vector (pGFP-FdV)

According to the molecular cloning conventional method, make up pGFP-FdV.Concrete grammar is following: (public can obtain from China Agricultural University with carrier pGAD-FdV; The non-patent literature of putting down in writing this carrier is: Cheng YQ; Liu ZM, Xu J, Zhou T; Wang M; Chen YT, Li HF and Fan ZF (2008) .HC-Pro protein of Sugarcane mosaicvirus specifically interacts with maize ferredoxin-5in vitro and in planta.J.Gen.Virol.84 (7): 2046-2054) be template, use downstream primer:

Upstream primer: 5 '-ATA GAGCTCAATGGCCACCGTCCTGAGC-3 ' (underscore partly is a Sac I restriction enzyme site),

Downstream primer: 5 '-GGCGTCGACTTTAGGAGATAAGGTCATCCT-3 (underscore partly is a Sal I restriction enzyme site),

Amplification obtains the coding gene sequence of FdV albumen, and the nucleotide sequence of this encoding gene is shown in sequence in the sequence table 2, and the amino acid sequence of the FdV albumen that this sequence is coded is shown in sequence in the sequence table 1.(public can obtain from China Agricultural University to cut this coding gene sequence and carrier pGFP with Sal I and Sac I enzyme; The non-patent literature of putting down in writing this carrier is: Shi Y, Qin YH, Cao YY; Sun H; Zhou T, Hong YG and Fan ZF (2011), Influence of an m-type thioredoxin in maize on potyviral infection.Eur.J.Plant Pathol.131:317-326); According to operation instruction, reclaim kit (available from AXYGEN company) with DNA glue and reclaim genetic fragment and the big fragment of carrier after purifying enzyme is cut.Connect to reclaim big fragment of carrier and genetic fragment behind the purifying, and change attachment over to the bacillus coli DH 5 alpha competent cell, after bacterium liquid is coated on the LB flat board that contains ampicillin screening positive clone.Recombinant plasmid is carried out enzyme cut checking, testing result sees that (swimming lane M is a dna molecular amount standard to Fig. 1 among Fig. 1; Swimming lane 1 is cut product for pGFP-Fd V enzyme), visible from Fig. 1, enzyme is cut the fragment that has obtained about 420bp size, and is consistent with expected results, proves that plasmid construction is correct, will obtain recombinant plasmid called after pGFP-Fd V.

Two, the RNA of corn mosaic virus (SCMV) extracts

(public can obtain from China Agricultural University, and the non-patent literature of putting down in writing this carrier is: Cheng YQ, Liu ZM to combine 31 at corn variety; Xu J, Zhou T, Wang M; Chen YT, Li HF and Fan ZF (2008) .HC-Proprotein of Sugarcane mosaic virus specifically interacts with maize ferredoxin-5in vitro andin planta.J.Gen.Virol.84 (7): 2046-2054) on the 3rd leaf frictional inoculation corn mosaic virus (SCMV) (public can obtain from China Agricultural University, and the non-patent literature of putting down in writing this material is: Cheng YQ; Liu ZM; Xu J, Zhou T, Wang M; Chen YT; Li HF and Fan ZF (2008) .HC-Pro protein of Sugarcane mosaicvirus specifically interacts with maize ferredoxin-5in vitro and in planta.J.Gen.Virol.84 (7): 2046-2054), 24 ℃ of growths are after 10-12 days, get it is caused being flower leaf paresthesia (be inhomogeneous chlorisis and form floral leaf, striped etc.) by virus infection blade 200g; Be cut into segment, in liquid nitrogen, grind to form powdery.Add 400ml in the powder after grinding and extract buffer solution I (0.5M kaliumphosphate buffer, the DIECA of adding 0.01M before using (diethyl-dithio ammonia potassium acid sodium, DDTC) and 1%2-ME (pH 7.2 for 2 mercapto ethanol, V/V)), stir under the room temperature.Filter with the individual layer nylon yarn, filtrating adds 20% (V/V) CCl ₄, fully stir 20-30min.8, the centrifugal 25min of 500rpm.The NaCl (final concentration) that adds 0.25M in the supernatant, be stirred to dissolving fully after, add PEG 6000 again, making its final concentration is 6g/L,, be stirred to PEG in the ice bath and dissolve fully, leave standstill 2h in 4 ℃.8, the centrifugal 30min of 500rpm, deposition is spent the night in 4 ℃ of suspensions with the extraction buffer solution II (0.05M kaliumphosphate buffer, pH 7.2) that contains 1%Triton.7, the centrifugal 15min of 000rpm places supernatant on 20% the sucrose pad, and 38, the centrifugal 90min of 000rpm.Deposition is extracted buffer solution III (0.005M kaliumphosphate buffer, pH 7.2) with 1.0ml and is fully suspended 4 ℃ of refrigerator overnight.7, the centrifugal 10min of 000rpm goes deposition, and supernatant is the virus of thick purification, is the unit packing with 100 μ l then, in-80 ℃ of refrigerators, preserves subsequent use.

In 100 μ l viruses, add the ddH that 160 μ l DEPC handle ₂O adds 200 μ l RNA extraction buffer (20mM Tris-HCl pH 8.0 again; 200mM NaCl; 5mM EDTA), add 40 μ l 10%SDS then, mixing, room temperature keeps 5min.Add 800 μ l PCI (phenol: chloroform: isoamyl alcohol=25: 24: 1), mixing, the centrifugal 5min of 12000 * g.Draw the upper strata water, add the PCI of 2 times of volumes, mixing, the centrifugal 5min of 12000g repeats extracting once again.Draw the upper strata water, confirm its volume after, add the NaAC (pH 5.2) of 1/10 (V/V) 3M, mixing; The absolute ethyl alcohol that adds 2.2 times of volumes, mixing ,-80 ℃ of deposition 1h or-20 ℃ of depositions are spent the night.The centrifugal 20min of 12000 * g is with 1ml 70% washing with alcohol deposition, drying at room temperature; Gained RNA is dissolved in the ddH that 30 μ l DEPC handle ₂Among the O ,-20 ℃ of preservations are subsequent use.

Three, the separation of corn protoplast and conversion

Corn variety Zheng 58 (available from Henan Academy of Agricultural Sciences) seed imbibition is placed in 25 ℃ the incubator germinate, the dark cultivation.When 2 leaves of corn etiolated seedling length to the are higher than the 1st 10-15cm; Cut the part of the middle 6-8cm of the 2nd leaf; Gently 20 blades of cutting are stacked, be cut into the filament of 0.5mm, put into and fill 30ml enzyme solutions (0.6M mannitol, 10mM MES pH5.7, ImM CaCl ₂, 5mM β-mercaptoethanol, 0.1%BSA, 0.3%Macerozyme R-10 and 1.5%Cellulase R-10) the flask of band sidewall in digest.The flask that fills enzyme solutions is vacuumized 30min, enzyme solutions fully is penetrated in the blade.In 25 ℃ of shaking tables (40rpm), continue digestion 2h then, and then digest 5min with 80rpm.The enzyme solutions that will contain protoplast filters the nylon wire of 35 μ m, transfers in the centrifuge tube that the bottom is a circle, and the centrifugal 2min of 150g precipitates protoplast.Wash 1-2 time with electric shock buffer solution (0.6M mannitol, 4mM MES, pH5.7, and 20mM KCl).The centrifugal 2min of 150g, the careful suction removed supernatant, with the resuspended protoplast of electric shock buffer solution, makes the protoplast concentration after resuspended reach 1 * 10 with the blood counting chamber counting ⁵/ ml-2 * 10 ⁵/ ml puts protoplast subsequent use on ice.In the 2ml round bottom centrifuge tube, add 25 μ g recombinant plasmid pGFP-Fd V (being prepared by step 1) and 300 μ l protoplasts, add 5 μ g SCMV RNA (being obtained by step 2) again, the rifle persorption is even; (5msec shocks by electricity 2 times; 200uF 400V), places 10min on ice.The centrifugal 2min of 150g inhales and removes supernatant, with the resuspended protoplast of 1ml incubation buffer (0.6M mannitol, 4mM MES pH5.7 and 4mM KCl), 25 ℃ of dark incubated overnight.Establish contrast protoplast (m), i.e. cotransformation empty carrier pGFP and SCMV RNA in protobiont simultaneously.

Four, the total RNA of corn protoplast and the Real-time RT-PCR that extract after transforming detect

The Trizol (the rich Deco skill Development Co., Ltd product that steps in Beijing) of 1ml is joined in the protoplast of step 3 thermal agitation 3min; Place 5min on ice.In 4 ℃, 12, centrifugal 10min changes supernatant in the one new 1.5ml centrifuge tube over to removing insoluble composition under the 000g condition with tabletop refrigerated centrifuge in homogenate.At room temperature leave standstill 5min, add the 0.2ml chloroform, concuss 15s at room temperature leaves standstill 2-5min then, more centrifugal 15min under 4 ℃, the condition of 12,000 * g.The upper water phase transfer in new 1.5ml centrifuge tube, is added the 0.5ml isopropyl alcohol, mix (turning upside down), make sample at room temperature leave standstill 15min, 4 ℃, the centrifugal 10min of 12,000 * g, then RNA can form deposition in the sidewall and the bottom of pipe.Abandon supernatant, add 75% washing with alcohol deposition, 4 ℃ then, the centrifugal 5min of 7,500 * g (suspend like the RNA deposition, then use 12,000 * g), abandon ethanol, obtain the total RNA of protoplast.Concentrate 5min with the at room temperature dry 10min of the total RNA that obtains or on the refrigerated centrifuge thickener, add the ultra-pure water dissolving that 30 μ l DEPC handle then ,-70 ℃ of preservations are subsequent use.

After the above-mentioned total RNA that obtains cleared up with DNase I, on ultraviolet specrophotometer, measure respectively its 230,260 and the light absorption value of 280nm to confirm its purity and concentration.With kit Takaka SYBR premix Ex.Taq ^Tm(Takaka Company products) carried out Real-time RT-PCR and analyzed the influence that overexpression FdV duplicates SCMV.As template, carry Oligo-d (T) and Random 6 mers as reverse primer with the total RNA of 500ng, carry out the RT reaction with kit.Each reaction tube adds 4.0 μ l, 5 * Reaction Buffer successively; 1.0 μ l dNTPs (10mM), 0.5 μ l RNase inhibitor, 0.5 μ l Oligo-d (T) Primer (50 μ M), 0.5 μ l Random 6mers (100 μ M), the total RNA of 500ng, 0.5 μ l M-MLV Reverse Transcriptase use DEPC H at last ₂O is supplemented to 20 μ l.Reactant liquor is at 42 ℃ of water-bath 1h.

Add in each PCR reaction tube 1.0 μ l with the RT product of 10 times of dilution buffer liquid (EASY Dilution) dilutions, 10 μ l SYBR Green, 0.4 μ l DyeII, forward and reverse primer respectively 0.2 μ l, add 8.2 μ l dd H at last ₂O is supplemented to 20 μ l.Quantitative fluorescent PCR-ABI7500 amplification appearance of producing in ABI company reacts.Reaction condition is 95 ℃, 30s; 95 ℃, 5s; 58 ℃, 20s; 72 ℃, 35s; 40 circulations.After Real time PCR finishes, data are analyzed according to the software that the amplification appearance carries.

The primer that detects Fd VmRNA is following:

Forward primer: 5 '-ACTACCGTGGCCATGACCCGTG-3 ';

Reverse primer: 5 '-CCTCGGCGTAGTCCAGGATGTAG-3 '.

The primer that detects SCMV mRNA is following:

Forward primer: 5 '-GGCGAGACTCAGGAGAATACA-3 ';

Reverse primer: 5 '-ACACGCTACACCAGAAGACACT-3 '.

Experimental result is seen Fig. 2, and wherein A figure shows Fd V mRNA level; Wherein Fd and m represent the Fd V mRNA level in the protoplast of protoplast, cotransformation empty carrier pGFP and SCMV RNA of cotransformation pGFP-Fd V and SCMV RNA respectively; B figure shows the SCMV rna level; Wherein Fd and m are expressed as the SCMV rna level in the protoplast of protoplast, cotransformation empty carrier pGFP and SCMV RNA of cotransformation pGFP-Fd V and SCMV RNA respectively.Visible from A figure, compare with contrast, change that Fd V mRNA level significantly increases in the protoplast of pGFP-Fd V and SCMV RNA over to, show that Fd V has obtained overexpression; Visible from B figure, compare with contrast, change that the SCMV rna level significantly descends in the protoplast of pGFP-Fd V and SCMV RNA over to, the range of decrease is 40%, this explains that overexpression Fd V can effectively suppress duplicating of SCMV.

Claims (8)

1. the application of ferredoxin V in the product that preparation inhibition mosaic virus duplicates.

2. application according to claim 1 is characterized in that: the amino acid sequence of said ferredoxin V is in the sequence table shown in the sequence 1.

3. the application of the encoding gene of the albumen shown in the sequence 1 in the product that preparation inhibition mosaic virus duplicates in the sequence table; The encoding gene of said albumen is following 1)-3) in arbitrary described gene:

1) dna molecular shown in the sequence 2 in the sequence table;

2) under stringent condition with 1) shown in dna molecule hybridize and the gene of encoding said proteins;

3) with 1) or 2) gene have autoploidy and the gene of encoding said proteins more than 90%.

4. contain the application of the recombinant expression carrier of the encoding gene of albumen shown in the sequence 1 in the product that preparation inhibition mosaic virus duplicates in the ordered list.

5. application according to claim 4 is characterized in that: said recombinant expression carrier is the recombinant expression carrier that the encoding gene of albumen obtains shown in the sequence 1 in the MCS insetion sequence table of pGFP carrier.

6. according to arbitrary described application among the claim 1-5, it is characterized in that: said mosaic virus is a corn mosaic virus.

7. according to arbitrary described application among the claim 1-6, it is characterized in that: said inhibition mosaic virus copies as and suppresses mosaic virus duplicating in plant protoplast.

8. application according to claim 7 is characterized in that: said plant is a corn.

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