CN109913492A - A kind of method that arabidopsis PEPR2 albumen and AtPep1 small peptide synergistic effect inhibit geminivirus infection to infect - Google Patents
A kind of method that arabidopsis PEPR2 albumen and AtPep1 small peptide synergistic effect inhibit geminivirus infection to infect Download PDFInfo
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Abstract
The invention discloses a kind of methods that arabidopsis PEPR2 albumen and AtPep1 small peptide synergistic effect inhibit geminivirus infection to infect, and in particular to the method field for inhibiting geminivirus infection to infect, specifically includes the following steps: Step 1: choosing the plant for suffering from geminivirus infection；Step 2: the overexpression PEPR2 in this plant, meanwhile, external source applies AtPep1 small peptide, the antiviral mechanism of synergistic activation.The method that the present invention is acted synergistically using the gene and small peptide of plant origin, plant is improved to the resistance of geminivirus infection, solve the problems, such as that geminivirus infection causes to seriously endanger to the production estimation in China, under the effect of AtPep1 small peptide, the regulating and controlling effect of PEPR2 C4 albumen encoded to BSCTV, develop a kind of special controllable plant disease-resistant method, since C4 is the virulence factor in a variety of geminivirus infections with conserved sequence, the technology can be applied to other geminivirus infection types, to improve the anti-virus ability of future crop plants.
Description
Technical field
The present invention relates to the method and technology fields for inhibiting geminivirus infection to infect, it is more particularly related to a kind of quasi-
The method that southern mustard PEPR2 albumen and AtPep1 small peptide synergistic effect inhibit geminivirus infection to infect.
Background technique
Geminivirus infection (Geminivirus) is a kind of plant DNA virus with extensive host, many important crops
All threatened by it.Geminivirus infection is successively found on the grain in the multiple provinces in China and industrial crops, agricultural production is caused to endanger
Evil.Therefore, the interaction for furtheing investigate geminivirus infection and plant improves crop yield to controlling disease, ensures that agricultural is raw
The development important in inhibiting of production.
Geminivirus infection has single stranded DNA genome, a few albumen is only encoded, by infecting host cell and and place
Chief cell carries out interaction, completes its life cycle.After insect vector enters plant host cell, viral DNA exists geminivirus infection
Massive duplication is carried out in core, and then mobile by intracellular and iuntercellular, migrated to other positions of plant, form systematicness and invade
Dye, to generate illness.The serious curly top of beet malicious (BSCTV, beet severe curly top virus) is geminivirus infection
One kind, can be with infection model plant Arabidopsis thaliana and Ben's tobacco, therefore frequently as plant and the research of geminivirus infection interaction
Representative species.
Most geminivirus infection all encodes conservative C4 albumen (being known as AC4, AL4 or L4 in certain kinds), Er Qie
Verified C4 is main illness factor of determination in a variety of Study on Geminiviruses Infecting.When the C4 gene delection of BSCTV, virus
Pathogenic significant decrease；And when the C4 of external source overexpression BSCTV, it can promote the cell division of plant, form callus, produce
The raw phenotype for being similar to virus infection.Existing research shows that C4 albumen may hormone signal transduction with plant and cell cycle
Regulation is related, and then influences the normal development of host.But also not about specific adjustment mechanism of the C4 albumen in plant cell
It is clear, more lack the method for inhibiting virus infection by interference C4 function.
The signal transduction that AtPeps small peptide relies on is the important access of activating plants immune response, but at present about the approach
Research concentrate on fungi and bacteriosis, and had not been reported about function of this access in plant and viral interaction.
AtPeps is the small peptide of a kind of plant code, is generated by the precursor protein of eight PROPEP gene codings, due to these small peptides
Sequence is close, therefore most research all utilizes AtPep1 as an example.AtPeps passes through in identification plant plasma membrane serves
Receptoroid kinases PEPR1 and PEPR2, start the signal transduction mechanism in downstream.Before research shows that AtPep1 and PEPR1 or
The combination of PEPR2 can cause these receptor kinases in the form of endocytosis, be transported by cytoplasma membrane intracellular.In the present invention, Shen
It asks someone to find that the PEPR2 albumen of arabidopsis can promote the C4 albumen endocytosis of BSCTV to position for the first time, to influence it in plant illness
Function in decision, and then geminivirus infection is inhibited to infect and (do not deliver).
The prior art one related to the present invention
The technical solution of the prior art one:
The present invention is to apply the method combined using PEPR2 overexpression and AtPep1 small peptide, is determined by influencing illness
Determine positioning of the factor C4 in plant cell, inhibits infecting for geminivirus infection BSCTV.The visible national inventing patent of similar technology
" a kind of new application of GDU3 gene " (CN101775397B) (concrete scheme can be referring to the patent content), the technology also with
The gene in overexpression arabidopsis source resists infecting for BSCTV.The invention is mainly by by the gene LSB1/ in arabidopsis source
GDU3 overexpression enhances plant to the resistance of BSCTV, and the new application of the LSB1/GDU3 gene can be used for reducing BSCTV and make
At agricultural losses.
The shortcomings that prior art one:
The main problem of the technology is: overexpression LSB1/GDU3 gene composing type itself routinely has activated plant
The disease-resistant access of object, and the persistent accumulation of resistance signal causes plant growth and development under normal condition to be affected；Meanwhile the party
Method support Antiviral mechanism it is unknown, may cause other unknown side effects in application process.Compared with the technology, originally
The resistance virus access utilized is invented, need to express PEPR2 at the same time and is just activated when applying AtPep1 small peptide, thus generation pair
Therefore the resistance of BSCTV can add again AtPep1 small peptide under conditions of having virus infection risk；Meanwhile present invention finds
AtPep1 and PEPR2 influences the mechanism of its function and regulating and controlling the positioning of illness factor of determination C4 albumen jointly, be one more
Add special, controllable disease-resistant mode.
In addition, the existing technology for effectively preventing geminivirus infection is less, mainly using overexpression or strikes and subtract plant gene
Activation associated signal paths were infected to improve the resistance to virus, and by the gene editing for viral DNA with destroying
Journey；Wherein overexpression and striking, which subtracts plant gene and mainly passes through, constitutively activates the disease-resistant accesses of popularity such as salicylic acid, or
By influencing the duplication of plant cell cycle control virus, therefore, growth while improving disease resistance, to plant itself
Development may also have an impact；Although the gene editing technology for viral DNA specifically destroys the complete of this viral body structure
Whole property, but existing research shows that there may be miss the target the technology at present, it is possible to the genome of plant itself is produced
It is raw to influence.
Summary of the invention
In order to overcome the drawbacks described above of the prior art, the embodiment of the present invention provide a kind of arabidopsis PEPR2 albumen and
The method that AtPep1 small peptide synergistic effect inhibits geminivirus infection to infect passes through the gene and small peptide synergistic effect using plant origin
Method, improve plant to the resistance of geminivirus infection, be conducive to secure agricultural production, solve farming of the geminivirus infection to China
Object production causes the problem of seriously endangering, under the effect of AtPep1 small peptide, the regulation of PEPR2 C4 albumen encoded to BSCTV
Effect develops a kind of special controllable plant disease-resistant method, since C4 is the cause in a variety of geminivirus infections with conserved sequence
Cause of disease, which can be applied to other geminivirus infection types, to improve the anti-virus ability of future crop plants, solve existing
Have in technology using overexpression and strike and subtract the problem of plant gene method causes plant growth and development, and there may be de-
The phenomenon that target, the problem of being had an impact to the genome of plant itself.
To achieve the above object, the invention provides the following technical scheme: a kind of arabidopsis PEPR2 albumen and AtPep1 small peptide
The method that synergistic effect inhibits geminivirus infection to infect, specifically includes the following steps:
Step 1: the overexpression PEPR2 in plant, meanwhile, external source applies AtPep1 small peptide, and synergistic activation is antiviral
Mechanism.
In a preferred embodiment, the PEPR2 gene and PEPR2 albumen derive from arabidopsis.
In a preferred embodiment, the AtPep1 derives from the exciton small peptide of arabidopsis.
In a preferred embodiment, concretely the serious curly top of beet is malicious for geminivirus infection in the step 1
(BSCTV), the virulence factor of the serious curly top of the beet malicious (BSCTV) coding is C4, i.e. BSCTV C4.
Technical effect and advantage of the invention:
1, the present invention improves plant and resists to geminivirus infection using the method for gene and the small peptide synergistic effect of plant origin
Property, be conducive to secure agricultural production, solve the problems, such as that geminivirus infection causes to seriously endanger to the production estimation in China；
2, the present invention utilizes under the effect of AtPep1 small peptide, the regulating and controlling effect of PEPR2 C4 albumen encoded to BSCTV,
Develop a kind of special controllable plant disease-resistant method, due to C4 be in a variety of geminivirus infections with conserved sequence it is pathogenic because
Son, the technology can be applied to other geminivirus infection types, to improve the anti-virus ability of future crop plants, solve existing skill
It in art using overexpression and strikes and subtracts the problem of plant gene method causes plant growth and development, and there may be miss the target
Phenomenon, the problem of being had an impact to the genome of plant itself；
3, the present invention may be used using PEPR2 gene progress overexpression of the different types of plant expression vector to arabidopsis
There can be effect, meanwhile, it can use the arabidopsis of pCanG-PEPR2-MYC or the transgenic plant of other host plants, lead to
It crosses external source and applies AtPep1 small peptide, improve various plants to the resistance of BSCTV and other geminivirus infections；
4, the present invention proves that PEPR2 overexpression and being used in combination for AtPep1 can influence geminivirus infection from every aspect
The subcellular localization of the virulence factor C4 albumen of BSCTV, function of causing a disease and geminivirus infection infect；Due to individual PEPR2 mistake
Amount expression or AtPep1 application can not effectively inhibit virus infection, thus can by specific time and space to mistake
The plant of amount expression PEPR2 carries out AtPep1 processing, realizes while not sustained activation plant disease-resistant approach, small by external source
The shortcomings that peptide application promotes plant virus resistance ability, avoids other composing type sustained activations disease-resistant approach technology, together
When since the present invention is specifically to identify that mechanism is apparent to inhibit C4 protein active by AtPep1-PEPR2, favorably
In the influence for avoiding other side effects.
Detailed description of the invention
Fig. 1 is experimental result of the PEPR2 and AtPep1 synergistic effect in antiviral infect in the embodiment of the present invention 2
Figure.
Fig. 2 is that PEPR2 and AtPep1 influences C4 albumen in terms of plant callus induction in the embodiment of the present invention 2
Experimental result picture.
Fig. 3 is that PEPR2 and AtPep1 synergy geminivirus infection BSCTV infects plant in the embodiment of the present invention 2
Experimental result picture.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1:
A kind of arabidopsis PEPR2 albumen according to shown in Fig. 1-2 and AtPep1 small peptide synergistic effect inhibit geminivirus infection to invade
The method of dye, specifically includes the following steps:
Step 1: choosing the plant for suffering from geminivirus infection；
Step 2: the overexpression PEPR2 in this plant, meanwhile, external source applies AtPep1 small peptide, and synergistic activation is antiviral
Mechanism.
For the PEPR2 gene source in arabidopsis, gene order of the PEPR2 gene at the 3267bp of code area is specific
For TTTCAGTCTTGAGCTCTAATCTCAAATGAGGAATCTTGGGTTACTCGAAATTACTC TGCTTTGCTCTCTCTTTG
TCTATTTCCGTATAGATTCTGTCTCTAGTTTAAACTCAGATGGTTTGGCTTTACTCTCGCTTCTCAAGCACTTTGA
TAAAGTCCCACTTGAAGTAGCTTCGACGTGGAAGGAGAACACATCTGAAACCACTCCATGTAATAATAACTGGTTT
GGTGTCATTTGTGATCTTTCTGGTAATGTCGTCGAGACCCTTAATTTGTCTGCTTCTGGGCTTTCAGGCCAATTAG
GTTCTGAAATTGGGGAGCTTAAGAGCTT GGTCACATTGGATCTCAGTCTTAACAGTTTCTCTGGTTTATTGCCTT
CCACTTTAGGAAACTGTACTTCACTTGAGTATTTGGATTTGTCTAACAATGATTTTTCTGGAGAAGTTCCTGATAT
TTTTGGTAGCTTGCAGAATTTGACGTTTCTGTATCTTGATCGCAATAATCTTAGTGGTCTCATTCCTGCAAGTGTT
GGTGGGTTGATAGAGCTCGTAGATCTGAGGATGTCATATAATAACTTGTCTGGTACCATTCCAGAGTTGCTTGGGA
ACTGTAGTAAGCTGGAATATCTGGCTTTGAACAACAACAAGTTAAATGGTTCTTTGCCAGCAAGTCTCTATCTACT
CGAGAATCTTGGTGAGCTATTTGTCAGTAACAACAGCCTTGGAGGGAGGCTTCATTTTGGTTCTAGCAACTGCAAG
AAATTGGTTTCTTTAGATCTCTCGTTCAATGATTTCCAAGGCGGTGTTCCACCTGAGATAGGCAACTGCAGTAGCC
TTCACTCTTTAGTCATGGTGAAATGCAACTTGACAGGTACAATCCCATCATCAATGGGTATGTTGAGAAAGGTTTC
GGTTATTGACCTTTCCGATAATCGTCTCTCGGGGAATATCCCTCAAGAGCTTGGGAACTGCAGCAGCTTGGAAACC
TTGAAGCTGAACGACAACCAGCTCCAAGGCGAGATACCACCTGCATTGAGTAAGCTAAAGAAGCTACAAAGCCTGG
AGCTTTTTTTTAATAAGCTGTCCGGTGAGATTCCTATTGGCATATGGAAGATTCAGAGTCTGACACAGATGCTCGT
TTATAACAACACTCTCACCGGGGAACTACCAGTTGAAGTAACTCAGCTGAAGCACCTTAAGAAGCTTACACTGTTT
AACAACGGCTTTTATGGAGATATACCAATGAGTTTAGGCCTGAATCGAAGCTTAGAGGAGGTGGACCTTCTTGGTA
ACCGTTTTACAGGGGAGATACCACCCCATCTCTGCCATGGACAGAAGTTGAGATTGTTCATCTTGGGTTCTAATCA
GCTTCATGGTAAGATACCAGCGTCTATTCGTCAGTGTAAGACCCTTGAGCGAGTCAGACTTGAAGATAACAAACTT
TCAGGTGTTCTTCCGGAATTCCCTGAGAGTCTTAGTCTTTCCTATGTGAACCTCGGAAGCAATAGCTTTGAAGGAT
CCATCCCGCGCAGCTTGGGAAGCTGTAAAAATCTCTTGACTATTGACCTTTCTCAAAACAAACTCACGGGTCTGAT
ACCTCCAGAACTGGGAAATCTGCAAAGCCTTGGACTGTTGAACCTTTCACATAATTATCTGGAAGGTCCTCTGCCA
TCCCAGCTATCAGGCTGTGCGAGACTTCTGTATTTTGATGTTGGATCCAACTCATTGAACGGTTCTATTCCATCAA
GCTTCAGAAGCTGGAAAAGCTTGTCCACTTTAGTTCTCAGTGACAATAATTTTCTAGGAGCTATTCCACAGTTCTT
GGCAGAGCTTGACCGACTCTCAGATCTGCGGATAGCTCGAAATGCTTTTGGAGGTAAGATTCCTTCCTCGGTTGGC
TTGTTGAAGAGTCTACGCTATGGCTTAGACCTCAGTGCGAACGTATTTACGGGTGAGATTCCAACCACACTGGGGG
CTCTTAT CAATCTTGAACGTCTCAACATATCCAACAACAAGTTGACAGGGCCTTTATCGGTTCTTCAAAGTCTTA
AGTCATTGAATCAAGTTGACGTCTCGTATAATCAGTTCACGGGTCCAATACCCGTAAATCTGTTATCAAATTCTTC
AAAGTTTTCTGGAAATCCAGACCTCTGCATTCAAGCTTCTTACTCAGTGAGTGCCATAATCCGCAAAGAGTTTAAA
TCTTGCAAAGGTCAAGTCAAACTTAGCACGTGGAAGATCGCCCTTATAGCAGCTGGGTCCTCACTATCCGTATTGG
CTTTGCTTTTTGCTCTCTTTTTGGTTTTATGCCGGTGCAAAAGAGGAACCAAGACAGAAGATGCTAATATCCTCGC
AGAGGAAGGTCTGTCCTTGTTGCTGAACAAAGTTCTAGCAGCCACTGACAATCTAGATGACAAGTACATCATTGGA
AGAGGAGCTCATGGAGTTGTTTACAGAGCTTCTTTAGGATCAGGCGAAGAATACGCCGTGAAGAAACTCATCTTTG
CGGAACACATTCGCGCAAACCAAAATATGAAGCGGGAGATCGAAACAATCGGGCTAGTCAGGCACAGAAATCTCAT
TCGGTTAGAAAGATTTTGGATGAGGAAAGAAGATGGCTTAATGCTGTATCAGTACATGCCCAATGGAAGCCTACAC
GACGTTTTGCACAGAGGTAATCAAGGAGAAGCAGTTCTTGACTGGTCTGCACGGTTCAACATAGCCCTTGGGATTT
CACATGGACTGGCGTATTTACATCATGATTGTCATCCACCAATAATTCACCGCGACATCAAACCAGAGAACATACT
CATGGACTCGGATATGGAGCCTCACATTGGAGATTTCGGATTGGCTCGGATTCTAGATGACTCAACAGTTTCAACG
GCCACTGTTACTGGCACAACTGGGTACATTGCACCAGAAAATGCGTACAAGACGGTGAGGAGCAAGGAATCAGATG
TTTACAGTTATGGAGTTGTTTTGCTCGAGCTGGTAACAGGAAAGAGAGCACTGGACAGATCTTTCCCGGAAGATAT
CAACATTGTGAGCTGGGTCAGATCTGTATTAAGCAGCTACGAGGATGAAGACGATACTGCTGGTCCAATCGTTGAT
CCAAAACTTGTGGATGAGCTTCTGGATACGAAGCTCAGGGAACAAGCAATCCAAGTCACAGACTTGGCTCTTAGAT
GTACAGACAAGAGGCCGGAGAACAGACCATCGATGAGAGATGTGGTGAAAGATTTGACTGATTTGGAAAGTTTTGT
AAGAAGCACTTCGGGTTCAGTTCACTAG；
For the PEPR2 albumen source in arabidopsis, protein sequence of the PEPR2 albumen at the 1088aa of code area is specific
For MRNLGLLEITLLCSLFVYFRIDSVSSLNSDGLALLSLLKHFDKVPLEVASTWKENT SETTPCNNNWFGVICDLS
GNVVETLNLSASGLSGQLGSEIGELKSLVTLDLSLNSFSGLLPSTLGNCTSLEYLDLSNNDFSGEVPDIFGSLQNL
TFLYLDRNNLSGLIPASVGGLIELVDLRMSYNNLSGTIPELLGNCSKL EYLALNNNKLNGSLPASLYLLENLGEL
FVSNNSLGGRLHFGSSNCKKLVSLDLSFNDFQGGVPPEIGNCSSLHSLVMVKCNLTGTIPSSMGMLRKVSVIDLSD
NRLSGNIPQELGNCSSLETLKLNDNQLQGEIPPALSKLKKLQSLELFFNKLSGEIPIGIWKIQSLTQMLVYNNTLT
GELPVEVTQLKHLKKLTLFNNGFYGDIPMSLGLNRSLEEVDLLGNRFTGEIPPHLCHGQKLRLFILGSNQLHGKIP
ASIRQCKTLERVRLEDNKLSGVLPEFPESLSLSYVNLGSNSFEGSIPRSLGSCKNLLTIDLSQNKLTGLIPPELGN
LQSLGLLNLSHNYLEGPLPSQLSGCARLLYFDVGSNSLNGSIPSSFRSWKSLSTLVLSDNNFLGAIPQFLAELDRL
SDLRIARNAFGGKIPSSVGLLKSLRYGLDLSANVFTGEIPTTLGALINLERLNISNNKLTGPLSVLQSLKSLNQVD
VSYNQFTGPIPVNLLSNSSKFSGNPDLCIQASYSVSAIIRKEFKSCKGQVKLSTWKIALIAAGSSLSVLALLFALF
LVLCRCKRGTKTEDANILAEEGLSLLLNKVLAATDNLDDKYIIGRGAHGVVYRASLGSGEEYAVKKLIFAEHIRAN
QNMKREIETIGLVRHRNLIRLERFWMRKEDGLMLYQYMPNGSLHDVLHRGNQGEAVLDWSARFNIALGISHGLAYL
HHDCHPPIIHRDIKPENILMDSDMEPHIGDFGLARILDDSTVSTATVTGTTGYIAPENAYKTVRSKESDVYSYGVV
LLELVTGKRALDRSFPEDINIVSWVRSVLSSYEDEDDTAGPIVDPKLVDELLDTKLREQAIQVTDLALRCTDKRPE
NRPSMRDVVKDLTDLESFVRSTSGSVH；
The AtPep1 derives from the exciton small peptide of arabidopsis, albumen of the AtPep1 albumen at the 23aa of code area
Sequence is specially ATKVKAKQRGKEKVSSGRPGQHN；
Concretely the serious curly top of beet is malicious (BSCTV) for geminivirus infection in the step 1, the serious bent top of the beet
The virulence factor of viral (BSCTV) coding is C4, i.e. BSCTV C4；
Gene order of the BSCTV C4 gene at the 264bp of code area is specially ATGAAAATGGGGAACCACATC
TGCATGCCCTTATTCAATTCGAAGGAAAAGTCCAGATCCGTAATGCCCGTTACTTCGATCTGCAACATCGAAGTAC
CAGCAAACAATTCCACTGCAATATTCAGGGAGCTAAATCCAGTTCCGACGTCAAGTCCTACGTCTCAAAGGACGGA
GATCACATCGACTGGGGTGAATTTCAGGTCGATGGAAGATCTGCACGCGGAGGTCAACAGACGGCTAATGATGCTG
CAGCAGAGGCATTA；
Protein sequence of the BSCTV C4 albumen at the 87aa of code area is specially MKMGNHICMPLFNSKEKSRSVM
PVTSICNIEVPANNSTAIFRELNPVPTSSPTSQRTEITSTGVNF RSMEDLHAEVNRRLMMLQQRH。
It is demonstrated experimentally that under the treatment conditions of AtPep1 small peptide, overexpression PEPR2 can promote BSCTV it is pathogenic because
Sub- C4 albumen endocytosis infects by the way that the function inhibitio of influence C4 is viral, PEPR2 of the invention and AtPep1 collaboration is utilized to improve
Plant has great importance to the method for geminivirus infection resistance to agricultural production.
Embodiment 2:
Application of the PEPR2 and AtPep1 synergistic effect in antiviral infect
1) positioning of the C4 albumen of PEPR2 and AtPep1 synergy BSCTV coding in plant cell
Early period finds that the PEPR2 albumen of arabidopsis Arabidopsis thaliana coding can by yeast two-hybrid screening
With the C4 protein-interacting (not delivering at present) with BSCTV, therefore PEPR2 and its corresponding small peptide A tPep1 is analyzed to C4 egg
The white influence positioned in plant cell, specific steps are as follows:
S101, using arabidopsis cDNA as template, by the method for PCR (the primer information is as follows: PEPR2-F primer:
5 '-ACTACTAGTTTTCAGTCTCTTGAGCTCTAATCT-3 ' are incited somebody to action since the 5 ' of the code area PEPR2 holds there are higher structure
The design of primers is in the 5 ' noncoding regions for closing on initiation codon；PEPR2-R primer: 5 '-AGTGTCGACAGTGAACTGAACC
CGAAGTGCTTC-3 '), amplification PEPR2 gene order is the gene order at the 3267bp of code area；
S102, then by PEPR2 gene cloning to pCanG-MYC (on the basis of pCambia1300 carrier plus MYC mark
Label, which is kalamycin resistance in plant；Can use other commercial plant over-express vectors and carry out similar building),
It is merged with MYC protein tag, constructs recombinant expression carrier pCanG-PEPR2-MYC；
S103, simultaneously, with BSCTV (ATCC PVMC-6；BCTV-CFH plants are formerly referred to as, this bacterial strain is by linearisation
The double-stranded DNA of BSCTV is stored on plasmid pCFH) it is template, using the method for PCR, (the primer information is as follows: C4-GFP-F
Primer: 5 '-AGTCCATGGTGATGAAAATGGGGAACCAC-3 '；C4-GFP-R primer: 5 '-AGTAAGCTTGATGCCTCTG
CTGCAGCATCATTAG-3 '), the C4 gene of BSCTV is expanded, is connected to pCambia1300-GFP (in pCambia1300 carrier
On the basis of increase GFP, CAMBIA, Canberra, Australia；It can be carried out using other commercial carriers with fluorescence labels
Similar building), obtain recombinant expression carrier pCambia1300-C4-GFP；
S104, above two recombinant plasmid is converted to Agrobacterium EHA105 respectively, obtains recombinant bacterium；
S105, the recombinant bacterium by culture, by the method for injection, in Ben's tobacco Nicotiana benthamiana leaf
Common overexpression C4-GFP and PEPR2-MYC in piece；Simultaneously with C4-GFP and pCanG-MYC empty carrier Agrobacterium co-injection work
For control sample；
S106, Agrobacterium inject 48 hours after, collect tobacco leaf, respectively inject final concentration 100nM AtPep1
Small peptide (amino acid sequence ATKVKAKQRGKEKVSSGRPGQHN)；
S107, GFP fluorescence signal is detected using laser confocal microscope, analyzes C4-GFP albumen in tobacco cell
Positioning；
In the case that S108, AtPep1 small peptide are handled 0 hour and 30 minutes, C4-GFP is in tobacco in empty vector control sample
Positioning on cytoplasma membrane simultaneously has not been changed (see attached drawing 1A)；And in the sample of overexpression PEPR2-MYC, do not handling
In the case where AtPep1, C4-GFP's is located a small amount of variation；When being handled 30 minutes with AtPep1, C4-GFP occurs apparent
Endocytosis phenomenon (see attached drawing 1B).
In Figure 1A, 1B, the upper left corner of each four figures is GFP fluorescence signal, and the upper right corner is the control of chlorophyll autofluorescence,
The lower left corner is light field cell outline, and the lower right corner is above-mentioned Signal averaging figure.
The above results show that the synergistic effect of PEPR2 and AtPep1 promotes the subcellular localization variation of C4 albumen, due to
Before studies have shown that positioning of the C4 on cytoplasma membrane is pathogenic most important to its, therefore, which may influence C4 and exist
Geminivirus infection infect in function.
2) PEPR2 and AtPep1 influences ability of the C4 albumen in terms of plant callus induction
Due to before research shows that the constitutive expression of C4 gene cause arabidopsis formed callus, can not be turned
Gene plant, therefore early-stage study passes through genetic transformation by the way that C4 to be implemented in estrogen-inducible expression vector pER8
Obtaining pER8-C4 Arabidopsis plant, (seed source is in Inst. of Genetics and Development Biology, CAS Xie Qi researcher
Laboratory can be used for measuring technology used in the present invention to the inhibitory effect of BSCTV illness factor of determination C4)；
Under regular culture conditions, which can be with normal growth；When applying estrogen induction C4 gene expression, the plant
Form callus；Therefore, the capability analysis C4 protein active of callus can be generated according to induction；
In order to study the influence of PEPR2 and AtPep1 to C4 function, specific research step is as follows:
S101, by above-mentioned pCanG-PEPR2-MYC and pCanG-MYC empty carrier (control) plasmid, be transformed into Agrobacterium
EHA105 obtains recombinant bacterium；
S102, the recombinant bacterium by culture are distinguished using the homozygote plant of flower drop dye method conversion pER8-C4 by selfing
The homozygote offspring for obtaining pCanG-PEPR2-MYC/pER8-C4 and pCanG-MYC/pER8-C4 plant, passes through the table of PEPR2
It is identified up to amount, chooses 2 independent PEPR2 and be overexpressed arabidopsis strain, be used for subsequent analysis；
S103, it the seed of above-mentioned plant is layered on the MS culture medium containing 2 μM of estrogen (is not added or is added final concentration
The above-mentioned AtPep1 small peptide of 100nM) on sprouted, plant phenotype was analyzed in 1 week after sprouting；
S104, be free of AtPep1 small peptide under conditions of, compared with pCanG-MYC empty carrier, overexpression PEPR2-MYC
Influence to C4 function is little；Under conditions of the above-mentioned AtPep1 small peptide of 100nM is added, the ability for the callus that C4 is generated
It is suppressed (see attached drawing 2A) completely, Fig. 2A claps the plant photo taken when being 1 week after sprouting；
S105, when above-mentioned plant grows 3 weeks on corresponding culture medium, be compared under Stereo microscope: not
Under conditions of AtPep1, pCanG-PEPR2-MYC slightly inhibits C4 calli induction (callus small volume)；
Under conditions of the above-mentioned AtPep1 small peptide of 100nM is added, the function that C4 evoked callus is formed is completely disappeared (see attached drawing
2B), the plant photo taken is clapped when Fig. 2 B is 3 weeks after sprouting.
The above results show that in the case where PEPR2 overexpression, the application of AtPep1 small peptide inhibits C4 protein induced
The ability of callus (illness decision).
3) PEPR2 and AtPep1 synergy geminivirus infection BSCTV infects plant
Since the above results show while overexpression PEPR2 and applying AtPep1 and can inhibit the function of C4, and C4 is
Virulence factor in BSCTV infection processs, therefore, the influence that analysis PEPR2 and AtPep1 infects BSCTV, concrete operations step
It is rapid as follows:
S101, the infectivity plasmid (pCambia1300-BSCTV1.8copy by BSCTV；It is lost from the Chinese Academy of Sciences
Pass and Developmental Biology research institute Xie Qi researcher laboratory) it converts into Agrobacterium EHA105 bacterial strain, obtain recombination agriculture
Bacillus EHA105-BSCTV；
S102, above-mentioned pCanG-PEPR2-MYC and pCanG-MYC (control) plasmid also converted into Agrobacterium respectively simultaneously
EHA105；
S103, the above Agrobacterium is suspended in respectively in 10mM MgCl2 solution, by pCanG-MYC and pCanG-PEPR2-
MYC bacterium solution is mixed with EHA105-BSCTV bacterium solution respectively, obtains two kinds of bacterium solutions, and (concentration is bacterium solution 1:pCanG-PEPR2-MYC
OD600=1)/BSCTV (concentration OD600=0.01) and bacterium solution 2:pCanG-MYC (concentration OD600=1)/BSCTV are (dense
Degree is OD600=0.01)；
S104, after placing 3 hours, in the method that Agrobacterium is injected, above-mentioned bacterium solution is injected into Ben's tobacco leaf；
S105, in the processing of addition AtPep1, AtPep1 is diluted in the MgCl2 solution of 10mM, with same side
Method is injected into tobacco leaf；
S106, after tobacco continued growth two weeks, the leaf roll and distortion illness at stem end are observed；
There is serious bent top symptom in S107, check plant (AtPep1 is not added in pCanG-MYC)；And independent overexpression
PEPR2 or independent applies AtPep1 and also is not sufficient to inhibit BSCTV's to infect illness；Simultaneously in blade overexpression PEPR2 and
There is not obvious illness in the tobacco for applying AtPep1 (see attached drawing 3A, 3B)；
S108, it is organized by the stem end for collecting above-mentioned processing plant, extracts DNA using CTAB method, drawn using above-mentioned C4
Object, by PCR amplification C4 gene (using C4 gene as the representative segment of detection BSCTV genome), to detect plant tip
The accumulation of BSCTV, the ACTIN gene of tobacco is as internal reference, compared with other samples, PEPR2 overexpression and AtPep1
Collaboration processing sample in, the DNA of BSCTV accumulation is substantially reduced (see attached drawing 3C).
It is above-mentioned the experimental results showed that this method enhances plant to the resistivity of geminivirus infection.
The technical program proves that PEPR2 overexpression and being used in combination for AtPep1 can influence twin disease from every aspect
The subcellular localization of the virulence factor C4 albumen of malicious BSCTV, function of causing a disease and geminivirus infection infect；
It, can be with since individual PEPR2 overexpression or AtPep1 application can not effectively inhibit virus infection
By carrying out AtPep1 processing to the plant of overexpression PEPR2 in specific time and space, realizes and planted in not sustained activation
While object disease-resistant approach, plant virus resistance ability is promoted by the application of outer source small peptide, other composing types is avoided and holds
Continuous the shortcomings that activating disease-resistant approach technology；
Simultaneously as the technical program is specifically identified by AtPep1-PEPR2 to inhibit C4 protein active, machine
It manages apparent, is conducive to the influence for avoiding other side effects；
Since C4 protein sequence has conservative between a variety of geminivirus infections, the technical program may be twin to other types
Virus disease prevention and treatment also has effect.
In the technical program, scale was carried out using PEPR2 gene of the different types of plant expression vector to arabidopsis
Up to may all have effect, meanwhile, the transgenosis of the arabidopsis or other host plants that can use pCanG-PEPR2-MYC is planted
Strain applies AtPep1 small peptide by external source, improves various plants to the resistance of BSCTV and other geminivirus infections；
Prevent in addition, the above method also can be used for the plant of non-illness.
Last: the foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, all in the present invention
Spirit and principle within, any modification, equivalent replacement, improvement and so on, should be included in protection scope of the present invention it
It is interior.
Claims (4)
1. a kind of method that arabidopsis PEPR2 albumen and AtPep1 small peptide synergistic effect inhibit geminivirus infection to infect, feature exist
In: specifically includes the following steps:
Step 1: choosing the plant for suffering from geminivirus infection；
Step 2: the overexpression PEPR2 in this plant, meanwhile, external source applies AtPep1 small peptide, the antiviral machine of synergistic activation
System.
2. a kind of arabidopsis PEPR2 albumen according to claim 1 and AtPep1 small peptide synergistic effect inhibit geminivirus infection
The method infected, it is characterised in that: the PEPR2 gene and PEPR2 albumen derive from arabidopsis.
3. a kind of arabidopsis PEPR2 albumen according to claim 1 and AtPep1 small peptide synergistic effect inhibit geminivirus infection
The method infected, it is characterised in that: the AtPep1 derives from the exciton small peptide of arabidopsis.
4. a kind of arabidopsis PEPR2 albumen according to claim 1 and AtPep1 small peptide synergistic effect inhibit geminivirus infection
The method infected, it is characterised in that: concretely the serious curly top of beet is malicious (BSCTV) for geminivirus infection in the step 1, institute
The virulence factor for stating the serious curly top of beet malicious (BSCTV) coding is C4, i.e. BSCTV C4.
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| CN112359066A (en) * | 2020-10-09 | 2021-02-12 | 华南师范大学 | Application of lipid-binding small peptide SSIP1 in mediating fusion protein of lipid-binding small peptide to enter cells |
| CN114107321A (en) * | 2021-12-08 | 2022-03-01 | 华南师范大学 | Method for inhibiting geminivirus infection by utilizing arabidopsis ABI5 protein overexpression |
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