CN110484557A - A kind of instantaneous silent carrier of Atg5 - Google Patents
A kind of instantaneous silent carrier of Atg5 Download PDFInfo
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Abstract
The present invention utilizes the principle of RNA silencing approach silencing of target genes, by the way that two specificity silencing segment SL and SS of Atg5 gene are connected to binary expression vector pCV1300, Agrobacterium transient expression constructs a kind of instantaneous silent carrier of Atg5, i.e. siatg5 silent carrier.By it is quantitative and using GFP-Atg8f analysis shows, siatg5 can lead to Atg5 gene silencing, effectively inhibit autophagy approach, alleviate the degradation of the albumen of different organelles positioning, to promote the accumulation of organelle foreign protein.Present invention demonstrates that the autophagy approach of siatg5 silencing is related to most cells such as endoplasmic reticulum, plastid, mitochondria, peroxisome place, siatg5 silent carrier may have adjustment effect to the autophagy approach in a variety of places, organelle in plant cell.
Description
Technical field
The present invention relates to plant genetic engineering fields, and in particular to a kind of instantaneous silencing of autophagy pathway key gene Atg5
Carrier.The silent carrier can result in Atg5 gene silencing, inhibit autophagy approach, alleviate the drop of the albumen of different organelle positioning
Solution, to promote the accumulation of foreign protein.
Background technique
It is current by organism being expressed and being produced as bioreactor foreign protein using gene recombination technology
The hot issue of biotechnology research.Common exogenous protein expression system mainly has prokaryotic expression, yeast expression system, insect
Expression system, mammalian expression systems and plant expression system etc., different expression systems have different expression characteristics.With
Other expression systems are compared, and plant expression system has the advantage that (1) plant as eucaryote, has eukaryotic protein
Modified mechanism is able to carry out protein folding, posttranslational modification, to express the destination protein of high bioactivity；(2) there is no lactation
The pollution of animal pathogenic, hardly by pathogenic bacterial infection, product safety with higher；(3) low production cost of plant,
Its overall manufacturing cost is about the 2%-10% of microorganism, is the 0.1% of animal；(4) plant expresses isolating and purifying for product
Advantage of lower cost is suitable for large-scale production；(5) it is stored convenient for foreign protein, foreign protein can be stored in seed, fruit
Equal positions.Therefore, increasingly cause domestic and international researcher using plant expression system as bioreactor to express foreign protein
Attention.Plant expression system is usually used in the preparation of the pharmaceutical protein that there is Important Economic to be worth or vaccine, has utilized at present general
Lead to the cells such as cigarette, Ben Shi cigarette, rice cell or plant successful expression mouse IgG1 antibody, hGM-CSF, mankind VEGF etc., generates
Higher economic value.However plant expression system still has the low problem of target protein expression amount, which is system
About one of the principal element of plant expression system industrialization.
Autophagy approach (Autophagy) is to be prevalent in the intracorporal important protein degradation approach of biology, main by thin
After duplicature concave shape intracellular is at imitated vesicle structure, merging with lysosome (lysosome) becomes autophagy lysosome
(autolyosomes) degradation function is executed.Under stress conditions, can include by some cytoplasmic contents by autophagy approach
Extra or impaired protein and organelle, which are sent into vacuole, degrades, and is recycled, to remain intracellular
Stable state, and life entity is helped to resist stress from outside environment.Autophagy can be divided into three classes according to the mode difference for forming autophagosome:
Autophagy (the Chaperone- that huge autophagy (Macroautophagy), micro- autophagy (Microautophagy) and companion participate in
Mediated autophagy), and huge autophagy and micro- autophagy are only found in plant.
In plant, a variety of autophagy related genes (autophagy-related genes, ATG) participate in autophagy approach, plant
Middle autophagy approach is related to a albumen more than 30, wherein Atg5 is the important gene for participating in autophagy approach, and one is only found in plant
A, it is the important adjusting gene of autophagy approach that it, which participates in the ubiquitination system that Atg8 is mediated,.Autophagy approach detection studies have shown that
After Atg8 is modified by cuttings such as Atg4, Atg3, Atg7, assisted after forming mature Atg8 with Atg5-Atg12-Atg16 complex
Make, ultimately forms autophagy vesica.In this process, Atg5 is attached to by forming complex with Atg12, Atg16L1 interaction
The maturation of autophagosome is instructed on immature autophagosome outer membrane, it can be seen that, silencing Atg5 can inhibit autophagy approach.
Currently, being directed to some known gene, design can induce the siRNA of its silencing, be imported by suitable means thin
Born of the same parents or body make gene expression dose decline or complete silencing, are that RNA perturbation technique is most widely applied.
Summary of the invention
To solve the deficiencies in the prior art, an object of the present invention is to provide a kind of instantaneous silent carrier of Atg5, i.e.,
Siatg5 silent carrier, the carrier can special silencing Atg5 and cannot other genes be caused with silencing, to further investigate the base
Because individual function provides possibility.In addition, the segment for being selected in different location carries out silencing, discovery may be implemented to be accurately positioned thin
The silencing segment of born of the same parents' device provides possibility to disclose the mechanism of action of the Atg5 gene in organelle level.
The silent carrier includes the SL segment and SS segment of first dual-expression vector and the insertion binary expression vector.By
Single-stranded hair-pin structure is generated after two fragment length differences, Agrobacterium transient expression, which is cut by RNA silencing approach
It generates siRNAs and obtains siatg5 silent carrier so as to cause Atg5 silencing.
The building of carrier mainly includes the following steps:
(1) SL segment and SS fragment amplification primer are designed according to Nb-Atg5 gene order；
(2) pcr amplification product constructs T-SL, T-SS carrier respectively；
(3) double digestion T-SS and binary expression vector, connection obtain recombinant plasmid；
(4) recombinant plasmid and T-SL that double digestion step (3) obtains, are attached；
(5) connection product that step (4) obtains is converted to Agrobacterium competent cell, screening obtains positive colony, mentions
Plasmid is taken, silent carrier is obtained；
In some preferred embodiments, the nucleotide sequence of the SL segment as shown in SEQ ID No.1, the SS segment
Nucleotide sequence is as shown in SEQ ID No.2.
In some preferred embodiments, the SL segment is by being encoded to KX369397.1, the Atg5 gene sequence of overall length 1116bp
The 63-552 nucleotide composition of column, including 490bp base；The SS segment is by being encoded to KX369397.1, overall length
63-476 nucleotide of the Atg5 gene order of 1116bp form, including 414bp base.
In some preferred embodiments, the connection of the SS segment and binary expression vector is Opposite direction connection；The SL segment
It is that forward direction is connect with the connection of binary expression vector.
In some preferred embodiments, the base sequence for obtaining SL fragment primer is as follows:
Forward primer sequence is 5 '-GGGATCCACTACAGATTCACCTTCATAAA-3 '；
Reverse primer sequences are 5 '-GGGTACCATCCACTAATAGGCCAAGTTTAA-3 '；
The base sequence for obtaining SS fragment primer is as follows:
Forward primer sequence is 5 '-GGGTACCCAGAGTTCTAATTGGTCAGGTT-3 '；
Reverse primer sequences are 5 '-GGAGCTCACTACAGATTCACCTTCATAAA-3 '.
On the other hand, present aspect provides a kind of use of protein degradation that the instantaneous silent carrier of Atg5 is positioned in alleviation organelle
On the way, which can act on organelle, promote the expression of organelle foreign protein.
Mainly use following technical step:
1, the instantaneous silent carrier of Atg5 is constructed；
2, green fluorescent protein (Green Fluorescence Protein, GFP) is melted with organelle localization signal peptide
Close building organelle-GFP expression vector.
3, the instantaneous silent carrier of Atg5 and organelle-GFP expression vector total immersion are moistened.
In some preferred embodiments, the organelle is respectively endoplasmic reticulum, mitochondria, hydrogen peroxide object and plastid.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention utilizes gene silent technology, screens two sections of specificity of Atg5 gene from Atg5 target fragment for the first time
Silencing segment SL and SS construct the instantaneous silent carrier of Atg5, the silent carrier energy effective reticence Atg5 base by RNAi technology
Because of expression, inhibit autophagy approach.
Instantaneous silent carrier constructed by the present invention can act on organelle, alleviate the albumen of different organelle positioning
Degradation, promotes the expression of different organelle foreign proteins.Present invention firstly discloses the instantaneous silent carriers of Atg5 to alleviate difference
Purposes in terms of the protein degradation of organelle positioning, the present invention are outer in influence for illustrating the autophagy approach that Atg5 gene is regulated and controled
The molecule mechanism of source protein expression aspect is of great significance, to the table for improving the foreign protein using plant as bioreactor
Up to directive significance.
Detailed description of the invention
Fig. 1 is the building of siatg5 silent carrier, wherein figure A) indicate siatg5 construction strategy, scheme B) indicate siatg5 structure
Build Vector map.
Fig. 2 is pCV1300 construction strategy.
Fig. 3 is NbAtg5 silencing specific sequence sectional analysis.
Fig. 4 is analyzed with the higher Niben101Scf02433g01001.1 conservative region of NbAtg5 homology.
Fig. 5 is the detection of siatg5 carrier silencing efficiency.
Fig. 6 is the detection of sinb1001 carrier silencing efficiency.
Fig. 7 is the detection that siatg5 carrier inhibits autophagy approach effect.
Fig. 8 is the expression for the albumen that siatg5 promotes endoplasmic reticulum positioning.
Fig. 9 is the expression for the albumen that siatg5 promotes mitochondria positioning.
Figure 10 is the expression for the albumen that siatg5 promotes peroxisome positioning.
Figure 11 is the expression for the albumen that siatg5 promotes plastid positioning.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Those skilled in the art should
Understand, under without departing from the scope of the present invention can details to technical solution of the present invention and form modify or replace
It changes, but these modifications or substitutions each fall within protection scope of the present invention.Experimental method in following embodiments, such as without special theory
It is bright, it is conventional method.Test material as used in the following examples is unless otherwise specified from conventional biochemical reagent quotient
What shop was commercially available.
The transformation of 1 pCV1300 binary expression vector of embodiment
The binary expression vector that the present invention selects pCV1300 to construct as silent carrier, structure chart are as shown in Figure 2.
Plasmid pCAMBIA1300 and PBI121 plasmid is utilized into HindIII and EcoRI double digestion, by the 35S- of pBI121
GUS-NOS section is connected into pCAMBIA1300 carrier and constitutes pCV1300 carrier.
The building of 2 siatg5 silent carrier of embodiment
1, the screening of target fragment
Screen the method with the gene silencing rnai segment of hairpin structure are as follows:
According to the Nb-Atg5 gene order (SEQ ID No.3) of NCBI (KX369397.1, overall length 1116bp), by its point
It is cut into the continuous nucleotide short-movie section that length is about 300-500bp, is analyzed by http://vigs.solgenomics.net/
Possible special silencing section is 1-300bp sequence, but sequence is analysis shows that Niben101Scf02433g01001.1 (abbreviation
Nb1001, the sequence and Atg5 homology are higher, it may have Autophagy protein Apg5 structural domain, but gene function is not
Know) gene order (SEQ ID No.4) and NbAtg5 it is higher (Fig. 3) from 450-1000 or so section conservative.And according to NCBI
Website (https: //blast.ncbi.nlm.nih.gov) analysis further confirm that NbAtg5 with
The conservative region of Niben101Scf02433g01001.1 concentrates on 450-1000bp section (Fig. 4).
The 63-552bp section of NbAtg5 is chosen respectively, the 500-1000bp section of Nb1001 is analyzed:
(1) SL segment is 63-552bp section, including 490bp base；SS segment is 63-476bp section, including 414bp
Base；
(2) SL segment is 500-1000bp section, including 501bp base；SS segment is 500-950bp section, including
451bp base；
2, the synthesis of design of primers and target fragment
According to the special silencing segment design primer of selection, base is interfered by the RNA of the special silencing Atg5 gene of PCR amplification
Because of silencing segment.And specific cleavage site is successively added respectively in the two sides of respective segments primer pair, so that acquisition will be expanded
RNA interference fragment be connected in binary expression vector specific cloning site to form hairpin structure.
PCR reaction system and amplification program are as follows:
Reaction system are as follows: 10 × PrimeSTAR buffer 6 μ L, dNTPs 6 μ L, each 0.5 μ L of upstream and downstream detection primer,
0.5 μ L, Prime STAR archaeal dna polymerase of template DNA 0.5 μ L, water 46.5XX μ L.Response procedures are as follows: 95 DEG C of initial denaturation 5min；
95 DEG C of denaturation 15sec, 58 DEG C of annealing 15sec, 68 DEG C of extension 15sec, after 40 recycle, 72 DEG C are continued to extend 15sec.
PCR product obtains target gene fragment through agarose gel electrophoresis, recovery purifying target fragment, and to recycling segment
It is sequenced.
3, carrier connects
It is independent with two respectively after pcr amplification product is using gel electrophoresis separation and recoveryEasy
(commercially available) connection of Vector, building obtain T-SL and T-SS；Double digestion T-SS and pCV1300, T4DNA ligase are reversed
Connection forms pCV1300-SS；Further double digestion T-SL and pCV1300-SS, T4DNAligase forward direction connect to be formed
PCV1300-SL-SS, constructed carrier are siatg5.Finally formed siatg5 carrier is the positive-sense strand of SL and bearing for SS
Adopted chain is connected, since SL segment is different from SS fragment length, SL-SS one hair clip knot of single-stranded formation after inverted repeat pairing
Structure, construction strategy such as Figure 1A) shown in.
Endonuclease reaction system are as follows: plasmid 30 μ L, corresponding 6 μ L of endonuclease digestion buffer, each 2 μ of restriction enzyme
L, 20 μ L of water.
Vector plasmid or PCR product are carried out under the optimum temperature of restriction enzyme used digestion about 12 hours.
Segment required for the product obtained after the completion of endonuclease reaction is separated and recovered from 1% (v/w) Ago-Gel is in case even
Connect use.
Connection reaction 20 μ L of total volume, target gene and carrier ratio are 15:2, T4DNA ligase 1U, 16 DEG C of connections 12~
16h。
4, Agrobacterium transient expression
The carrier built is added in Agrobacterium competent cell, the 3rd day after siatg5 agroinfiltration, screening is positive
Bacterium colony after extracting total serum IgE reverse transcription, carries out digestion verification.
Further, using UBC as internal reference, base is verified by half-quantitative detection NbAtg5 and Nb1001mRNA expression
Because of silencing efficiency.
From fig. 5, it can be seen that SL segment is 63-552bp section, it is constructed when SS segment is 63-476bp section
Siatg5 silent carrier lowers atg5mRNA obviously, does not have obvious silencing with the closer Nb1001 of its homology, illustrates this
Silent carrier can result in Atg5 gene silencing, and silencing efficiency is significant.
From fig. 6, it can be seen that SL segment is 500-1000bp section, it is constructed when SS segment is 500-950bp section
Sinb1001 silent carrier makes NbAtg5 and Nb1001mRNA have downward, illustrate the silent carrier can cause Atg5 and
Nb1001 gene silencing does not have specificity.
These results suggest that SL segment is 63-552bp section, SS segment is that 63-476bp section can be formed preferably
Hairpin structure, and generate tiny RNA and lead to Atg5 gene silencing, and show stronger specificity, the selected region of the present invention
Special silent carrier can be obtained, it can effectively special silencing Atg5 gene.
3 GFP-Atg8f of embodiment can be as the verifying of the indicator protein of autophagy approach
Atg8 is with the different homologue of quantity in different plant species, and Atg8f is monitoring autophagy way in arabidopsis, Ben Shi cigarette
Effective albumen of diameter.Since GFP is more sensitive to pH, after transient expression GFP-Atg8f, the GFP-Atg8f being modified and other
Autophagy proteins cooperate with forming autophagy vesica, and the GFP albumen then merged with Atg8f is discharged into cell by cutting under acidic environment
Matter, therefore the expressing quantity by detecting free GFP (Cleave GFP) just can detect the activation level of autophagy approach.
MV (methyl viologen) is autophagy approach inducer, and MV processing can activate autophagy approach, therefore pass through utilization
Whether MV processing is to verify GFP-Atg8f can be as the indicator protein of autophagy approach.According to XM_016638904.1 sequence, expand
Increase obtain Ben Shi cigarette Atg8f sequence (SEQ ID No.5), using pCV1300 as carrier, using nest-type PRC fusion GFP with
After Atg8f sequence, BamHI, SalI double digestion are connected into pCV1300 carrier, are named as GFP-Atg8f.
It is that processing group carries out reality respectively by control group, 40 μM of MV of water after GFP-Atg8f Agrobacterium transiently transfects 2 days
Processing is tested, is changed after 12h using Western detection GFP-Atg8f expression, ImageJ is utilized to analyze Cleave GFP and GFP-
The expressing quantity of Atg8f.From figure 7 it can be seen that the ratio of MV processing group Cleave GFP/GFP-Atg8f is 0.84, control
The ratio of group Cleave GFP/GFP-Atg8f is 0.29, the results showed that the Cleave GFP that GFP-Atg8f is generated after MV processing
It is apparently higher than control group, illustrates that GFP-Atg8f can be used as the indicator protein of autophagy approach.
Detection of the 4 siatg5 silent carrier of embodiment to autophagy approach inhibiting effect
Influence for detection siatg5 silent carrier to autophagy approach is control with empty carrier gus+GFP-Atg8f,
Siatg5+GFP-Atg8f is processing group, after Agrobacterium transiently transfects 2 days, handles 12h with 40 μM of MV, is detected using Western
GFP-Atg8f expression variation, utilizes the expressing quantity of ImageJ analysis Cleave GFP and GFP-Atg8f.
From figure 7 it can be seen that the ratio of control group Cleave GFP/GFP-Atg8f is 0.69, siatg5 silent carrier
The ratio of (SL segment be 63-552bp section, SS segment is 63-476bp section) processing group Cleave GFP/GFP-Atg8f is
0.38, illustrate that control group autophagy approach after MV is handled is induced, and processing group fails effectively to induce autophagy way after MV is handled
Diameter.It can thus be appreciated that the siatg5 silent carrier can effectively inhibit autophagy approach., whereas if siatg5 is not silenced, MV can be with
Effectively induction autophagy approach.
The expression of 5 siatg5 silent carrier of embodiment promotion endoplasmic reticulum positioning protein
1, the building of endoplasmic reticulum GFP expression vector
It is had confirmed that according to what (http://nebenfuehrlab.utk.edu/markers/default.htm) was provided
Endoplasmic reticulum positioning signal peptide sequence, it is double after the C-terminal fusion ER signal peptide of GFP using the pCV1300 of this laboratory transformation as carrier
Digestion constructs ER-GFP.
According to ER signal peptide (HDEL) design primer, BamH I and SacI digestion position is introduced respectively at 5 ' ends of upper and lower primer
Point.The nucleotide sequence of the ER signal peptide is as shown in SEQ ID No.6, the alkali of the ER-GFP forward primer and reverse primer
Basic sequence is respectively as shown in SEQ ID No.7 and SEQ ID No.8.
Reaction system are as follows: 10 × PrimeSTAR buffer 6 μ L, dNTPs 6 μ L, each 0.5 μ L of upstream and downstream detection primer,
0.5 μ L, PrimeSTAR archaeal dna polymerase of template DNA 0.5 μ L, water 46.5XX μ L.Response procedures are as follows: 95 DEG C of initial denaturation 5min；
95 DEG C of denaturation 15sec, 58 DEG C of annealing 15sec, 68 DEG C of extension 15sec, after 40 recycle, 72 DEG C are continued to extend 15sec.
PCR product obtains target gene fragment through agarose gel electrophoresis, recovery purifying target fragment, and to recycling segment
It is sequenced.
Endonuclease reaction system are as follows: plasmid 30 μ L, corresponding 6 μ L of endonuclease digestion buffer, each 2 μ of restriction enzyme
L, 20 μ L of water.
Vector plasmid or PCR product are carried out under the optimum temperature of restriction enzyme used digestion about 12 hours.
Segment required for the product obtained after the completion of endonuclease reaction is separated and recovered from 1% (v/w) Ago-Gel is in case even
Connect use.
Connection reaction 20 μ L of total volume, target gene and carrier ratio are 15:2, T4DNA ligase 1U, 16 DEG C of connections 12~
16h。
2, siatg5 and ER-GFP total immersion is moistened
By the siatg5 of building and ER-GFP total immersion profit (OD600=0.500), using sigus+ER-GFP as control group, with
Actin detects the secretion table of the 2-4 days GFP using fluorescence microscope and Western blotting as internal reference albumen
Up to situation.
From figure 8, it is seen that (SL segment is 63-552bp section to siatg5 silent carrier, and SS segment is the area 63-476bp
Section) GFP expressing quantity is apparently higher than control group after processing, the 4th day processing group GFP expression quantity and the 2nd day expression quantity of control group
It is similar.
The result shows that the protein degradation of endoplasmic reticulum positioning can be effectively relieved in the silent carrier, promote the outer of endoplasmic reticulum positioning
The expression of source protein.It is on the contrary, it may be determined that Atg5 may betide endoplasmic reticulum for adjusting the starting of autophagy approach, at least demonstrate,prove
Endoplasmic reticulum is an important place of autophagy approach in clear-cells.Although certain functional gene silencings can be made by carrier,
Be that silent carrier of the invention leads to atg5 silencing, autophagy approach caused to be suppressed, thus alleviate be positioned at it is a certain specific thin
Protein degradation on born of the same parents' device delays.It is generally acknowledged that micro- autophagy is to wrap up intracellular organic matter by vacuole membrane invagination to degrade in plant
Effect, huge autophagy is then the autophagy vesicle to form duplicature, is combined by carrying out receptor on cell membrane so as to cause signal
Conduction.But present invention discloses the inhibition autophagocytosis of siatg5 can occur in endoplasmic reticulum, autophagy caused by possible Atg5
It is at least related with endoplasmic reticulum.Since endoplasmic reticulum is the main place of albumen synthesis, it is also current that foreign protein, which is positioned at endoplasmic reticulum,
Increase the Critical policies of exogenous protein expression.Since foreign protein can cause to coerce in endoplasmic reticulum overexpression, activation autophagy way
Diameter and foreign protein of degrading.Therefore the degradation for the foreign protein for inhibiting endoplasmic reticulum to position by siatg5, is conducive to external source egg
White further accumulation.
The expression of 6 siatg5 silent carrier of embodiment promotion mitochondria positioning albumen
1, the building of mitochondria GFP expression vector
It is had confirmed that according to what (http://nebenfuehrlab.utk.edu/markers/default.htm) was provided
Mitochondria positioning signal peptide sequence, it is double after the N-terminal fusion Mt signal peptide of GFP using the pCV1300 of this laboratory transformation as carrier
Digestion constructs Mt-GFP.
According to Mt signal peptide (LSLRQSIRFFKPATRTLCSSRYLLQQKP) design primer, at 5 ' ends point of upper and lower primer
It Yin Ru not BamH I and SacI restriction enzyme site.The nucleotide sequence of the Mt signal peptide is as shown in SEQ ID No.9, the Mt-
The base sequence of GFP forward primer and reverse primer is respectively as shown in SEQ ID No.10 and SEQ ID No.11.
Pcr amplification reaction and connection response procedures are the same as embodiment 5.
2, siatg5 and Mt-GFP total immersion is moistened
By the siatg5 of building and Mt-GFP total immersion profit (OD600=0.500), using sigus+Mt-GFP as control group, with
Actin detects the secretion table of the 2-4 days GFP using fluorescence microscope and Western blotting as internal reference albumen
Up to situation.
From fig. 9, it can be seen that (SL segment is 63-552bp section to siatg5 silent carrier, and SS segment is the area 63-476bp
Section) siatg5+Mt-GFP total immersion moistens the 3rd day expressing quantity and is higher than control sample after processing.3 days albumen of control sample position is
It degrades, and siatg5 processing group continues to expression albumen.Illustrate that mitochondria positioning can be effectively relieved in the silent carrier
Protein degradation, promote the expression of the foreign protein of mitochondria positioning.
Current study show that mitochondria is also the important place that autophagy vesica is formed and acted on, mitochondria positioning albumen is a large amount of
Expression, cell be to keep the balance of interior environment that can activate autophagy approach, and foreign protein of degrading, to limit mitochondria positioning
Foreign protein accumulation.The degradation process of mitochondria foreign protein can be alleviated to a certain degree by siatg5 inhibition autophagy approach,
Increase the yield of albumen to greatest extent.
The expression of 7 siatg5 silent carrier of embodiment promotion peroxisome positioning protein
1, the building of peroxisome GFP expression vector
It is had confirmed that according to what (http://nebenfuehrlab.utk.edu/markers/default.htm) was provided
Peroxisome positioning signal peptide sequence, using the pCV1300 of this laboratory transformation as carrier, the C-terminal of GFP merges Px signal
Double digestion constructs Px-GFP after peptide.
According to Px signal peptide (RSKL) design primer, BamH I and SacI digestion position is introduced respectively at 5 ' ends of upper and lower primer
Point.The nucleotide sequence of the Px signal peptide as shown in SEQ ID No.12, the Px-GFP forward primer and reverse primer
Base sequence is respectively as shown in SEQ ID No.13 and SEQ ID No.14.
Pcr amplification reaction and connection response procedures are the same as embodiment 5.
2, siatg5 and Px-GFP total immersion is moistened
By the siatg5 of building and Px-GFP total immersion profit (OD600=0.500), using sigus+Px-GFP as control group, with
Actin detects the secretion table of the 2-4 days GFP using fluorescence microscope and Western blotting as internal reference albumen
Up to situation.
From fig. 10 it can be seen that siatg5 carrier (SL segment is 63-552bp section, and SS segment is 63-476bp section)
The 2-4 days expressing quantities of siatg5+Px-GFP total immersion profit are above control sample after processing, and expressing quantity is higher than within the 3rd day
Nearly 3 times of control sample.The result shows that the silent carrier can effectively facilitate the expression of the albumen of peroxisome positioning.
The effect of peroxisome mainly has in plant: photorespiration is 1. participated in, by photosynthetic by-product
Glycolic is oxidized to glyoxalic acid and hydrogen peroxide, 2. in the seed of sprouting, carries out the beta oxidation of fat, generates acetyl coenzyme A,
It is recycled through glyoxalic acid, glyoxalic acid and succinic acid is cracked by isocitric acid, the latter leaves peroxisome and is further transformed into
Glucose, this process is known as glyoxalic acid circulation, therefore the peroxisome of plant cell is also known as glyoxysome
(glyoxysome).Peroxisome is the important organelle for participating in plant cell physiological reaction, and during autophagy approach
Peroxisome is main endocytosis object in the autophagy vesica of formation, is sent out by the research of siatg5 and peroxisome
Existing, siatg5 can make the foreign protein for being positioned at peroxisome alleviation slow down.This side light siatg5 silent carrier energy
Leading to the autophagy approach degradation rate of peroxisome reduces, to preferably guarantee that it executes biological function.
The expression of 8 siatg5 silent carrier of embodiment promotion plastid positioning protein
1, the building of plastid GFP expression vector
It is had confirmed that according to what (http://nebenfuehrlab.utk.edu/markers/default.htm) was provided
Plastid positioning signal peptide sequence, using the pCV1300 of this laboratory transformation as carrier, double enzymes after the N-terminal fusion Pt signal peptide of GFP
Cut building Pt-GFP.
According to Pt signal peptide (ASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC
MQVWPPINKKKYETLSYLPDLS) design primer introduces BamH I and SacI restriction enzyme site at 5 ' ends of upper and lower primer respectively.
The nucleotide sequence of the Pt signal peptide is as shown in SEQ ID No.15, the base of the Pt-GFP forward primer and reverse primer
Sequence is respectively as shown in SEQ ID No.16 and SEQ ID No.17.
Pcr amplification reaction and connection response procedures are the same as embodiment 5.
2, siatg5 and Pt-GFP total immersion is moistened
By the siatg5 of building and Pt-GFP total immersion profit (OD600=0.500), using sigus+Pt-GFP as control group, with
Actin detects the secretion table of the 2-4 days GFP using fluorescence microscope and Western blotting as internal reference albumen
Up to situation.
It can be seen from figure 11 that siatg5 carrier (SL segment is 63-552bp section, and SS segment is 63-476bp section)
The 2-4 days expressing quantities of siatg5+Pt-GFP total immersion profit are above control sample after processing, and 3 days albumen of control sample position is
Through degrading, and siatg5 processing group continues to expression albumen.The result shows that plastid can be effectively relieved in the silent carrier
The protein degradation of positioning promotes the expression of the foreign protein of plastid positioning.
Plastid is a kind of organelle closely related with storage with the synthesis of carbohydrate, it is that plant cell is distinctive
Structure.According to the difference of pigment, plastid can be divided into three types: chloroplaset (chloroplast), chromoplast (or chromoplast)
(chromoplast) and leucoplast (leucoplast).Plastid is bilateral membrane structure, and duplicature is the precursor that autophagy vesica is formed
Substance, therefore plastid is also the potential place of autophagy approach, current study show that chloroplaset also has autophagy process, research is found
The degradation of ageing leaves Chloroplast may be related with autophagy approach, and chloroplaset may have two ways by the degradation of autophagy approach
Diameter, respectively RCB (Rubisco-containing bodies) approach and chlorophagy approach.This research and utilization siatg5
Silent carrier inhibit autophagy approach so as to cause plastid positioning protein degradation slows down, it was demonstrated that siatg5 silent carrier to chloroplaset from
The approach of biting may there is also inhibiting effect.
Based on the above embodiments, the SL segment for preferably constructing the instantaneous silent carrier of Atg5 is the 63- of Atg5 gene order
552bp section, including 490bp base, restriction enzyme site are followed successively by BamHI, KpnI；SS segment is the 63- of Atg5 gene order
476bp section, including 414bp base, restriction enzyme site are followed successively by SacI, KpnI.Siatg5 silent carrier can delay to a certain degree
The Heterologous protein degradation that endoplasmic reticulum, plastid, mitochondria, peroxisome position is solved, this also illustrates the autophagy of siatg5 silencing
Approach is related to most cells such as endoplasmic reticulum, plastid, mitochondria, peroxisome place.Therefore siatg5 may also be right
The autophagy approach in a variety of places, organelle in plant cell has adjustment effect.Current study show that plant expression system is still
So there is a problem of that target protein expression amount is lower, research find protein degradation process be influence exogenous protein expression it is important because
One of element.About more than 2000 direct or indirect enzyme for participating in protein degradation in plant, at present just for a kind of or a kind of
The inhibitor or silencing strategies of enzyme can not have the effect of the inhibition Heterologous protein degradation of wide spectrum.By the study show that
The autophagy approach that siatg5 positions various kinds of cell device is inhibited, causes autophagy approach to press down by siatg5 silencing Atg5
System, it may be possible to which a kind of potential wide spectrum inhibits the strategy of Heterologous protein degradation.
Sequence table
<110>Zhejiang Academy of Agricultural Science
<120>the instantaneous silent carrier of a kind of Atg5
<130> 18-100070-00005069
<141> 2018-05-14
<160> 21
<170> SIPOSequenceListing 1.0
<210> 2
<211> 490
<212> DNA
<213>Ben Shi cigarette (Nicotiana benthamiana)
<400> 2
actacagatt caccttcata aatccgaagt cactactctc cctcctcctc cccctgctct 60
gattttagct cctcgaatag gttacctgcc tcttttagga acaaaaataa aacctttctt 120
cagtaattca cttcctcctg gtgtagatac cgtatggttt gagtacaaag gcttccctct 180
caaatggtat ataccaactg gggtactctt tgatcttctt tgtgcagaac ctgaacgtcc 240
ttggaatctg acggtacatt ttagaggata tcctggaaat attttaacac cttgtgaagg 300
agaagatagt gcaaagtgga gttttatcaa ttcactgaaa gaggcggcat acataatcaa 360
tgggaactgc aagaatgtaa tgaatatgtc ccaacctgac caattagaac tctggcgctc 420
cattatggat ggtaatttag aagcttatct ccgaatctcg tctaagctta aacttggcct 480
attagtggat 490
<210> 2
<211> 414
<212> DNA
<213>Ben Shi cigarette (Nicotiana benthamiana)
<400> 2
actacagatt caccttcata aatccgaagt cactactctc cctcctcctc cccctgctct 60
gattttagct cctcgaatag gttacctgcc tcttttagga acaaaaataa aacctttctt 120
cagtaattca cttcctcctg gtgtagatac cgtatggttt gagtacaaag gcttccctct 180
caaatggtat ataccaactg gggtactctt tgatcttctt tgtgcagaac ctgaacgtcc 240
ttggaatctg acggtacatt ttagaggata tcctggaaat attttaacac cttgtgaagg 300
agaagatagt gcaaagtgga gttttatcaa ttcactgaaa gaggcggcat acataatcaa 360
tgggaactgc aagaatgtaa tgaatatgtc ccaacctgac caattagaac tctg 414
<210> 3
<211> 1116
<212> DNA
<213>Ben Shi cigarette (Nicotiana benthamiana)
<400> 3
atgggaagta aaggggcagg agaaagtgaa gcacagaaat acatatggga aggagcaatt 60
ccactacaga ttcaccttca taaatccgaa gtcactactc tccctcctcc tccccctgct 120
ctgattttag ctcctcgaat aggttacctg cctcttttag gaacaaaaat aaaacctttc 180
ttcagtaatt cacttcctcc tggtgtagat accgtatggt ttgagtacaa aggcttccct 240
ctcaaatggt atataccaac tggggtactc tttgatcttc tttgtgcaga acctgaacgt 300
ccttggaatc tgacggtaca ttttagagga tatcctggaa atattttaac accttgtgaa 360
ggagaagata gtgcaaagtg gagttttatc aattcactga aagaggcggc atacataatc 420
aatgggaact gcaagaatgt aatgaatatg tcccaacctg accaattaga actctggcgc 480
tccattatgg atggtaattt agaagcttat ctccgaatct cgtctaagct taaacttggc 540
ctattagtgg atgatttttc aatacaactt agctcctcct cttctaaatc accggaaagc 600
actcaaaatg cagatggcac aggaccagct aaaacaggta gaataccggt tcgactgtat 660
gttaggacta tcaatgagga ttttgatgac ttgcaagatg cacctgcagt tgaaagttgg 720
gacaaaatct cttacataaa cagacctgtt gagattcatg gagatggagg caaatgcttc 780
accttgtgtg acgcaataac aaagcttctg ccggagctgt ttggggaaaa actgatgcca 840
atggatgacg tttctggaga ggaagttgag gttggacaga gattatctct tgaagaaaca 900
agcaagagca gcacgggaca aagtggagag atgttgaatg agcgcattgt ctcctgctcc 960
gtgtcagatg gtgctgagat taagctcata cgcattcagg gaattgaacc aaagatggag 1020
attccttttg catgggtggt aaacaatttg atgaaccctg aatactttct tcatatctgt 1080
gtatatgtca aaattcaaga acccatcacc atatag 1116
<210> 4
<211> 834
<212> DNA
<213>Ben Shi cigarette (Nicotiana benthamiana)
<400> 4
atgatgtctc gccatattgg acttgttgtt tactttctga tatttctact ggatgatctg 60
caggcagcat acataatcaa tgggaactgc aagaatgtaa tgaacatgtc ccaacctgac 120
caattagaac tctggcgctc cattatggat ggtaatttag aagcttatct ccgaacctcg 180
tctaagctta aacttggcct agtggtggat gatttttcaa cacaacttag ctcttcctct 240
cctaaatcac cggaaagcac tcaaaatgca gatggcacag gaccagctaa aacaggtgtc 300
acatgggtgt atggtactcc gtatggattg ttcatacacc ctagtccatt tgacactggt 360
agaaaaccgg ttcgactgta tgttaggact ataaatgagg attttgataa cttgcaagat 420
gcacctgcag atgaaagttg ggacaaaatc tcttacataa acagacctgt tgagattcat 480
ggagatggag gcaaatgctt caccttgtgt gacgcaataa cgaagcttct gccggagctg 540
tttggggaaa aactgctgcc gaaagatatt tctggagaag aagttgaggt tggacagaga 600
ttacctcttg aagaaacaaa caagagcagc acgggacaaa gtggagagat gttgaatgag 660
cgtgttgtct cctgctctgt gtcagatggt gctgagatta agctaatacg cattcagggc 720
attgaaccaa agatggagat tccttttgca tgggtggtaa acaatttgat gaaccctgaa 780
tactttcttc atatctgtgt atatgtcaaa attgaagaac ccatcaccat atag 834
<210> 5
<211> 369
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atggttaaga gctcattcaa gcaggagcat gatcttgaga agaggcgcgc cgaggctgct 60
gggattaggg aaaaatactc agataggatt ccggtgatag ttgaaaaggc tgaaaaaagt 120
gatattccca acatcgacaa gaaaaagtat ctcgtgccag ctgacttgac agttgggcaa 180
tttgtctatg tcattcgcaa gagaatcaaa ttgagtgcag aaaaggcaat attcatattt 240
attgacaatg tcctaccgcc aacaggggca atcatgtctg caatctatga cgagaagaag 300
gatgaagatg gtttccttta tgttacttac agtggagaaa acacattcgg ggacctgaac 360
gagctgtag 369
<210> 6
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
catgatgagc tt 12
<210> 7
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gggatccatg aagactaatc tttttctctt t 31
<210> 8
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ggagctctta gagttcgtca tgtttgtata g 31
<210> 9
<211> 84
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ctttcactac gtcaatctat aagatttttc aagccagcca caagaacttt gtgtagctct 60
agatatctgc ttcagcaaaa accc 84
<210> 10
<211> 118
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gggatccatg ctttcactac gtcaatctat aagatttttc aagccagcca caagaacttt 60
gtgtagctct agatatctgc ttcagcaaaa acccatgaag actaatcttt ttctcttt 118
<210> 11
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ggagctctta tttgtatagt tcatccatgc ca 32
<210> 12
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
agatcgaagc tg 12
<210> 13
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gggatccatg aagactaatc tttttctctt t 31
<210> 14
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ggagctctta cagcttcgat ctgagttcgt catgtttgta tag 43
<210> 15
<211> 261
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
atggcttcct cagttctttc ctctgcagca gttgccaccc gcagcaatgt tgctcaagct 60
aacatggttg cacctttcac tggccttaag tcagctgcct cattccctgt ttcaaggaag 120
caaaaccttg acatcacttc cattgccagc aacggcggaa gagtgcaatg catgcaggtg 180
tggccaccaa ttaacaagaa gaagtacgag actctctcat accttcctga tttgagcatg 240
aagactaatc tttttctctt t 261
<210> 16
<211> 292
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gggatccatg gcttcctcag ttctttcctc tgcagcagtt gccacccgca gcaatgttgc 60
tcaagctaac atggttgcac ctttcactgg ccttaagtca gctgcctcat tccctgtttc 120
aaggaagcaa aaccttgaca tcacttccat tgccagcaac ggcggaagag tgcaatgcat 180
gcaggtgtgg ccaccaatta acaagaagaa gtacgagact ctctcatacc ttcctgattt 240
gagcatgaag actaatcttt ttctctttat gaagactaat ctttttctct tt 292
<210> 17
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ggagctctta tttgtatagt tcatccatgc ca 32
<210> 18
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gggatccact acagattcac cttcataaa 29
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gggtaccatc cactaatagg ccaagtttaa 30
<210> 20
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gggtacccag agttctaatt ggtcaggtt 29
<210> 21
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ggagctcact acagattcac cttcataaa 29
Claims (7)
1. a kind of silent carrier for silencing Atg5 gene, which is characterized in that the silent carrier include first dual-expression vector with
And the SL segment and SS segment of the insertion binary expression vector.
2. silent carrier according to claim 1, which is characterized in that its construction method includes the following steps: (1) basis
Nb-Atg5 gene order designs SL segment and SS fragment amplification primer；(2) pcr amplification product constructs T-SL, T-SS load respectively
Body；(3) double digestion T-SS and binary expression vector, connection obtain recombinant plasmid；(4) the recombination matter that double digestion step (3) obtains
Grain and T-SL, are attached；(5) connection product that step (4) obtains is converted to Agrobacterium competent cell, screening obtains sun
Property clone, extract plasmid, obtain silent carrier.
3. silent carrier according to claim 1, which is characterized in that the nucleotide sequence of the SL segment such as SEQ ID
Shown in No.1, the nucleotide sequence of the SS segment is as shown in SEQ ID No.2.
4. silent carrier according to claim 1, which is characterized in that the SL segment by Atg5 gene order 63-
552 nucleotide compositions, including 490bp base；The SS segment is made of 63-476 nucleotide of Atg5 gene order,
Including 414bp base.
5. silent carrier according to claim 1, which is characterized in that the connection of the SS segment and binary expression vector is
Opposite direction connection；The connection of the SL segment and binary expression vector is positive connection.
6. silent carrier construction method according to claim 2, which is characterized in that the base sequence of the SL fragment primer
It is as follows:
Forward primer sequence is 5 '-GGGATCCACTACAGATTCACCTTCATAAA-3 '；
Reverse primer sequences are 5 '-GGGTACCATCCACTAATAGGCCAAGTTTAA-3 '；
The base sequence of the SS fragment primer is as follows:
Forward primer sequence is 5 '-GGGTACCCAGAGTTCTAATTGGTCAGGTT-3 '；
Reverse primer sequences are 5 '-GGAGCTCACTACAGATTCACCTTCATAAA-3 '.
7. a kind of purposes of instantaneous silent carrier of Atg5 in terms of the protein degradation for alleviating organelle positioning, which is characterized in that should
Silent carrier can act on organelle region, promote the exogenous protein expression of organelle positioning.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852866A (en) * | 2021-03-09 | 2021-05-28 | 中国科学院西北高原生物研究所 | Method for cultivating plant male sterile line by utilizing mitochondrial gene editing system |
| WO2024075729A1 (en) * | 2022-10-06 | 2024-04-11 | 国立大学法人北海道大学 | Protein manufacturing method, plant, and plant viral vector production method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012571A (en) * | 2012-12-05 | 2013-04-03 | 北京师范大学 | Gene capable of reducing nicotine content of tobacco and application thereof |
| CN103484480A (en) * | 2013-09-25 | 2014-01-01 | 中国热带农业科学院热带生物技术研究所 | Papaya ringspot virus genes and method for constructing RNA interference (RNAi) expression vectors of papaya ringspot virus genes |
-
2018
- 2018-05-14 CN CN201810458343.0A patent/CN110484557A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012571A (en) * | 2012-12-05 | 2013-04-03 | 北京师范大学 | Gene capable of reducing nicotine content of tobacco and application thereof |
| CN103484480A (en) * | 2013-09-25 | 2014-01-01 | 中国热带农业科学院热带生物技术研究所 | Papaya ringspot virus genes and method for constructing RNA interference (RNAi) expression vectors of papaya ringspot virus genes |
Non-Patent Citations (4)
| Title |
|---|
| CHIHIRO TAKATSUKA,ET AL: "3-Methyladenine Inhibits Autophagy in Tobacco Culture Cells under Sucrose Starvation Conditions", 《PLANT CELL PHYSIOL》 * |
| PING WANG ET AL: "New advances in autophagy in plants: Regulation, selectivity and function", 《SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY》 * |
| YAKUPJAN HAXIM ET AL: "Autophagy functions as an antiviral mechanism against geminiviruses in plants", 《PLANT BIOLOGY》 * |
| 陈思涵 等: "农杆菌介导的外源基因在本氏烟中瞬时表达体系优化研究", 《西南林业大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852866A (en) * | 2021-03-09 | 2021-05-28 | 中国科学院西北高原生物研究所 | Method for cultivating plant male sterile line by utilizing mitochondrial gene editing system |
| WO2024075729A1 (en) * | 2022-10-06 | 2024-04-11 | 国立大学法人北海道大学 | Protein manufacturing method, plant, and plant viral vector production method |
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