CN105950641A - Embedded RbcS cTP gene and expression vector and application thereof - Google Patents
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Abstract
The invention discloses an embedded RbcS cTP gene and an expression vector and application thereof. The gene is formed in the mode that the N-end of arabidopsis RbcS cTP is inserted between the N-end of corn RbcS cTP and an intermediate structure domain, and the cDNA sequence is shown as SEQ ID NO.2. The cDNA sequence of a corn RbcS cTP gene is shown as SEQ ID NO.1. The embedded RbcS cTP gene can be applied when exogenous genes are transferred into chloroplast of monocotyledon and/or dicotyledon.
Description
Technical field
The invention belongs to genetic engineering field, disclose a mosaic type RbcS cTP gene and expression vector thereof and answer
With.
Background technology
Chloroplast is that green plants is converted into the important organelle of chemical energy luminous energy, and chloroplast can only synthesize self needs
A part of protein, many chloroplast proteins are all by nuclear gene encoding, and they are translated into maturation in Cytoplasm
Functional protein or protein precursor thing, be then re-introduced in chloroplast playing respective function.Lead peptide in this process
In played particularly significant effect.It is by transit peptides (transit that most chloroplast proteins transport chloroplast from Cytoplasm
Peptide, TP) mediation realize.This transit peptides is normally at the N end being transported albumen.Tie in aminoacid sequence one-level
On structure, TP can be divided into three domains, and they are N-terminal domains, central domain and C-terminal domains respectively.Not jljl
Between the same domain planted or between the different structure territory of same species, its sequence is generally of the transmutability of height,
Lack concensus sequence or consensus motif.Each motif plays to closing in the particular procedure that chloroplast protein transports
Important effect.1,5-diphosphoribolose carboxylase/oxygenase small sub-unit (the small subunits of ribulose
1,5-bisphosphate carboxylase/oxygenase, RbcS) chloroplast transit peptides (chloroplast
Transit peptide, cTP) it is to study relatively broad plant chloroplast to lead one of peptide.
At present, the domain of the cTP of arabidopsis RbcS and the function of part motif are the clearest, but at unifacial leaf
In the cTP of plant RbcS, its domain and different motifs are how being also not very clear of function, particularly unifacial leaf and
Whether the cTP of dicotyledon has species specificity etc. and rarely has report so far.
First this research filter out Semen Maydis RbcS cTP in opening candidate's mover/enhancer checking, utilizes bioinformatics
The size of prediction transit peptides, and the cTP gene of corn clone RbcS, then use similar approach to clone in Oryza sativa L. and arabidopsis
Homologous genes, finds that the target function of the chloroplast of RbcS cTP has obvious species in unifacial leaf and dicotyledon special
The opposite sex；The mosaic type cTP that next N-end constructing Semen Maydis and arabidopsis is connected, after converting protoplast, GFP signal framing arrives
In the chloroplast of arabidopsis and Semen Maydis.
Chloroplast, as a kind of new-type bioreactor, produces the albumen that medicine has specific function with some, has height
The advantages such as efficient expression, site-directed integration, safety is good, descendant inheritting is stable, development prospect is the best.Along with plant chloroplast gene
Engineering research the most perfect, this invention favorably using chloroplast as a kind of new and effective bioreactor, will be for heredity work
Journey brings new vigor and vitality, also will bring one " Green revolution " for agricultural development.
Summary of the invention
It is an object of the present invention to provide Semen Maydis RbcS cTP gene and mosaic type RbcS cTP gene.
It is a further object to provide Semen Maydis RbcS cTP and the transient expression vector of mosaic type RbcS cTP.
A further object of the present invention is to provide Semen Maydis RbcS cTP and the transient expression vector of mosaic type RbcS cTP, wink
Time express can be used for being transferred to by exogenous gene in unifacial leaf and dicotyledon chloroplast.
The purpose of the present invention can be achieved through the following technical solutions:
One mosaic type RbcS cTP gene, is inserted into the N-end of Semen Maydis RbcS cTP by the N-end of arabidopsis RbcS cTP
And formed between central domain, its cDNA sequence is as shown in SEQ ID NO.3.
The transit peptides of described mosaic type RbcS cTP gene code, its aminoacid sequence is as shown in SEQ ID NO.4.
Transient expression vector containing described mosaic type RbcS cTP gene.
Described transient expression vector is preferably with pJIT163-hGFP as initial carrier, by described mosaic type RbcS cTP
Insert gained between HindIII and the BamHI restriction enzyme site of pJIT163-hGFP.
Exogenous gene is being transferred to unifacial leaf and/or dicotyledon chloroplast by described mosaic type RbcS cTP gene
In application.
Transient expression vector containing described mosaic type RbcS cTP gene exogenous gene is transferred to unifacial leaf and/
Or the application in dicotyledon chloroplast.
Semen Maydis RbcS cTP gene, its cDNA sequence is as shown in SEQ ID NO.1.
Transient expression vector containing the Semen Maydis RbcS cTP gene shown in SEQ ID NO.1.
Described transient expression vector is preferably with pJIT163-hGFP as initial carrier, by the jade shown in SEQ ID NO.1
Gained between HindIII and the BamHI restriction enzyme site of rice RbcS cTP gene insertion pJIT163-hGFP.
Exogenous gene is being transferred to unifacial leaf and/or dicotyledonous by the Semen Maydis RbcS cTP gene shown in SEQ ID NO.1
Application in plant chloroplast.
Exogenous gene is being transferred to by the transient expression vector containing the Semen Maydis RbcS cTP gene shown in SEQ ID NO.1
Application in monocotyledon chloroplast.
Beneficial effect:
The present invention first from Semen Maydis clone obtain RbcS cTP gene and coded protein thereof, in Semen Maydis first
Report.The RbcS cTP of Semen Maydis and arabidopsis is inserted transient expression vector pJIT163-hGFP, is transformed into Semen Maydis, Semen Tritici aestivi, water
In rice and protoplasts of Arabidopsis thaliana broken by ultrasonic, the GFP signal of the RbcS cTP instantaneous conversion carrier of Semen Maydis can navigate to Semen Maydis, Semen Tritici aestivi or
In the chloroplast of Oryza sativa L., it is not possible to navigate in the chloroplast of arabidopsis；And the RbcS cTP instantaneous conversion carrier of arabidopsis
GFP signal may only navigate in arabidopsis chloroplast, it is not possible to navigates in the chloroplast of Semen Maydis or Oryza sativa L..Arabidopsis
The N-terminal domains of RbcS cTP is inserted into the N-end of Semen Maydis RbcS cTP and the mosaic type cTP that central domain is middle, converts
During after protoplast, GFP signal can navigate to the chloroplast of arabidopsis and Semen Maydis simultaneously.
Accompanying drawing explanation
Fig. 1 Semen Maydis RbcS cTP transient expression vector builds schematic diagram.
Three domains of Fig. 2 cTP sequence divide.
Fig. 3 TP-DNTP structural representation.
Fig. 4 Semen Maydis RbcS cTP subcellular localization signal in Oryza sativa L., Semen Tritici aestivi, arabidopsis chloroplast.
Fig. 5 arabidopsis RbcScTP subcellular localization signal in arabidopsis and Semen Maydis.
The N-end series hybrid cTP of Fig. 6 RbcScTP subcellular localization signal in Semen Maydis and arabidopsis.
Detailed description of the invention
Embodiment 1 clones RbcScTP gene and vector construction
Semen Maydis is monocotyledon, and genome has completed order-checking.Semen Maydis RbcScTP gene is by the first applicant (invention
People) Zhang Wenli identifies DHSs (DNase I hypersensitive according to mDNase-seq (modified DNase-seq)
Sites), the analysis in combination with RNA-seq obtains alternate promoters/enhancer, then with GFP as reporter gene, utilizes
Protoplast transformation screens from these alternate promoters/enhancers and obtains.NCBI finds the same of arabidopsis with blast
Source gene.
First with primers F 1CGATAAGGCGACAAGTGGTG (SEQ ID NO.5)/R1:CTGCATGCACCGGATCCTT
(SEQ ID NO.6) and F2:AGTAGTAATGGCTTCCTCTATGC (SEQ ID NO.7)/R2:
RbcScTP gene is divided from Semen Maydis and arabidopsis gene group by ACCTTCATGCAGCTAACTCTTC (SEQ ID NO.8) respectively
It is not cloned on pMD19-T carrier, after order-checking, uses NCBI aligned sequences.Then with Semen Maydis RbcScTP gene primer F3:
GCCCTTGCTCACCATGGATCCATGGCGCCCACCGTGATGAT (SEQ ID NO.9)/R3:
GCCCTTGCTCACCATGGATCC GGACCGGATCCTTCCGCCGT (SEQ ID NO.10) and arabidopsis RbcS cTP gene
Primers F 4:TGGAGAGGACAGCCCAAGCTTATGGCTTCCTCTATGCTCTC (SEQ ID NO.11)/R4:
GCCCTTGCTCACCATGGATCCGCAGCTAACTCTTCCCCCGT (SEQ ID NO.12) carries out PCR amplification, and uses fast Suke
Grand test kit ClonExpressTMII (Nanjing Nuo Weizan company) links transient expression vector pJIT163-hGFP's PCR primer
Between Hind III and BamH I (as shown in Figure 1), use NCBI aligned sequences after order-checking, and be respectively designated as M-TP (maize
And A-TP (ArabidopsisRbcS-TP) RbcS-TP).Simultaneously by identical method, first the middle junction of Semen Maydis RbcS cTP
Structure territory and C-terminal domains (as shown in Figure 2) are linked between the Hind III and BamH I of transient expression vector pJIT163-hGFP:
(F5:TGGAGAGGACAGCCCAAGCTTGTCGCCCCGTTCCAGGGTCT(SEQ ID NO.13)/R5:
GCCCTTGCTCACCATGGATCCGCACCGGATCCTTCCGCCGT (SEQ ID NO.14), template M1:
GTCGCCCCGTTCCAGGGTCTCAAGTCCGCCGCCAGCCTCCCCGTCGCCCGCCGCAGCACCAGGAGCCTCGGCAACGT
CAGCAACGGCGGAAGGATCCGGTGC (SEQ ID NO.15)), then it is inserted into the N-terminal domains of arabidopsis RbcScTP
(F6:TGGAGAGGACAGCCCAAGCTTATGGCTTCCTCTATGCTCTC(SEQ ID NO.16)/R6:
GAGACCCTGGAACGGGGCGACCATGGTGGCCTGAGCCGGGG (SEQ ID NO.17), template M2:
ATGGCTTCCTCTATGCTCTCCTCCGCCGCTGTGGTTACATCCCCGGCTCAGGCCAC CATG (SE Q ID NO.18)),
Finally it is inserted into the N-terminal domains (F7:TGGAGAGGACAGCCCAAGCTTATGGCGCCCACCGTGATG of Semen Maydis RbcS cTP
(SEQ ID NO.19)/R7:GGAGAGCATAGAGGAAGCCATGGCGGTTGCCGACGAGGC (SEQ ID NO.20), template
M3:ATGGCGCCCACCGTGATGATGGCCTCGTCGGCAACCGCC (SEQ ID NO.21)), it is built into arabidopsis RbcS
The N-terminal domains of cTP is inserted into the N-end of Semen Maydis RbcS cTP and the instantaneous conversion of the mosaic type cTP of central domain centre
Expression vector (as shown in Figure 3), and named TP-DNTP.
Embodiment 2 protoplast extracts and converts
The extraction of protoplast and method for transformation referring especially to delivered method (list of references: Yang Zhang,
Jianbin Su, etc., A highly efficient rice green tissue protoplastsystem for
Transient gene expression andstudying light/chloroplast-related processes,
Plant Methods, 2011), and do suitably modified, it is extracted the primary of Semen Maydis, Oryza sativa L., Semen Tritici aestivi and arabidopsis by the method
Plastid.
Step:
1) cultivate the greening seedling of normal growth, choose Semen Maydis, Oryza sativa L. and the wheat growth seedling of about ten days, intend Nan Sanzhou
The seedling of left and right, two kinds of transient expression plasmids 20ul (1ug/ul) that the embodiment 1 that Simultaneous purification converts builds.
2) preparation enzyme liquid 10ml, 55 DEG C of heating 10min.
3) with blade, blade (Semen Maydis, Semen Tritici aestivi and arabidopsis) or stem (Oryza sativa L.) are switched to about 0.5-1mm, immerse enzyme immediately
In liquid.
4) evacuation 30min, at 28 DEG C in dark about 30-50rpm shaken cultivation 4-6h.
5) add equal-volume W5 and shake up, then filtering enzyme liquid by 35 μm (200 mesh) nylon membrane,.
6) 100g room temperature is centrifuged 2min, abandons supernatant, slowly adds 4ml W5 solution along wall, suspends gently, enter
Row 100g x 2min room temperature is centrifuged.
7) supernatant discarded, adds the centrifugal rear supernatant discarded of MMG solution 4ml, 100g x 2min room temperature of pre-cooling along tube wall,
Rejoin 4ml MMG solution, ice bath 30min after fully suspending.
8) 100g room temperature is centrifuged 2min to precipitate protoplast, supernatant discarded, and add MMG adjusting protoplast concentration is 1-2
×105/ml。
9) take 2ml tube, add 10 μ l plasmids by 100 μ l protoplasts, after flicking mixing, add 110 μ l40%PEG,
Flick mixing.Room temperature stands 20min.
10) adding 1ml W5 solution, mixing is cleaned, and 100g x 2min room temperature is centrifuged.
11) take supernatant, add 500 μ l W5, flick mixing, 28 DEG C of dark incubated overnight.
12) second day, 100g, 3min room temperature was centrifuged, and removes supernatant, leaves and takes 20-40 μ l, and after flicking mixing, film-making is observed.
Example 3RbcScTP gene mapping
Protoplast laser confocal fluorescence microscope after cultivation is observed.Set different excitation wavelengths, utilize GFP
Fluorescin sends green fluorescence and chloroplast spontaneous red light district sub-signal, and carries out signal observation.Result shows, Semen Maydis and water
The GFP signal of the RbcS cTP instantaneous conversion carrier of rice can navigate in the chloroplast of Semen Maydis, Semen Tritici aestivi or Oryza sativa L., it is not possible to fixed
Position is in the chloroplast of arabidopsis；And the GFP signal of the RbcS cTP instantaneous conversion carrier of arabidopsis may only navigate to intend south
In mustard chloroplast, it is not possible to navigate in the chloroplast of Semen Maydis or Oryza sativa L..The N-terminal domains of arabidopsis RbcS cTP is inserted
Mosaic type RbcS cTP in the middle of the N-end and central domain of Semen Maydis RbcS cTP, after converting protoplast, GFP signal is fixed
Position is in the chloroplast of arabidopsis and Semen Maydis.Exogenous gene can be navigated to grass family by above-mentioned description of test Semen Maydis RbcS cTP
In monocotyledonous chloroplast, and exogenous gene can be navigated to dicotyledonous and monocotyledonous by mosaic type RbcS cTP
In chloroplast.
Claims (10)
1. a mosaic type RbcS cTP gene, it is characterised in that be inserted into Semen Maydis RbcS by the N-end of arabidopsis RbcS cTP
Being formed between N-end and the central domain of cTP, its cDNA sequence is as shown in SEQ ID NO.3.
2. the transit peptides of the mosaic type RbcS cTP gene code described in claim 1, it is characterised in that its aminoacid sequence is such as
Shown in SEQ ID NO.4.
3. contain the transient expression vector of mosaic type RbcS cTP gene described in claim 1.
Transient expression vector the most according to claim 3, it is characterised in that with pJIT163-hGFP as initial carrier, will power
Profit requires that the mosaic type RbcS cTP described in 1 inserts gained between HindIII and the BamHI restriction enzyme site of pJIT163-hGFP.
5. contain the transient expression vector of the Semen Maydis RbcS cTP gene shown in SEQ ID NO.1.
Transient expression vector the most according to claim 5, it is characterised in that with pJIT163-hGFP as initial carrier, will
Institute between HindIII and the BamHI restriction enzyme site of the Semen Maydis RbcS cTP gene insertion pJIT163-hGFP shown in SEQ ID NO.1
?.
7. exogenous gene is being transferred to unifacial leaf and/or dicotyledonous is planting by the mosaic type RbcS cTP gene described in claim 1
Application in thing chloroplast.
8. exogenous gene is being transferred to unifacial leaf and/or dicotyledon leaf by the transient expression vector described in claim 3 or 4
Application in green body.
Exogenous gene is being transferred to unifacial leaf and/or dicotyledonous is planting by the Semen Maydis RbcS cTP gene shown in 9.SEQ ID NO.1
Application in thing chloroplast.
10. exogenous gene is being transferred to by the transient expression vector containing the Semen Maydis RbcS cTP gene shown in SEQ ID NO.1
Application in unifacial leaf and/or dicotyledon chloroplast.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987591A (en) * | 2017-03-31 | 2017-07-28 | 江苏省农业科学院 | A kind of light-inducible promoter gene and its application |
| CN108977414A (en) * | 2018-08-16 | 2018-12-11 | 中国科学院昆明植物研究所 | The artificial synthesized mutant and its coded sequence of a kind of beta carotene assimilation enzyme and application |
| CN110349624A (en) * | 2019-05-30 | 2019-10-18 | 山东省农业科学院玉米研究所 | The method of sam file f lag tag location T-DNA insertion point |
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| CN102858982A (en) * | 2010-02-04 | 2013-01-02 | 拜尔农科股份公司 | A method for increasing photosynthetic carbon fixation using glycolate dehydrogenase multi-subunit fusion protein |
| CN102372767A (en) * | 2010-08-19 | 2012-03-14 | 北京大学 | Plant trait related protein, coding gene and application thereof |
| CN102533844A (en) * | 2011-12-21 | 2012-07-04 | 中国农业科学院生物技术研究所 | Corn chloroplast transgene expression vector containing herbicide resistant gene and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106987591A (en) * | 2017-03-31 | 2017-07-28 | 江苏省农业科学院 | A kind of light-inducible promoter gene and its application |
| CN106987591B (en) * | 2017-03-31 | 2020-01-24 | 江苏省农业科学院 | Photoinduction type promoter gene and application thereof |
| CN108977414A (en) * | 2018-08-16 | 2018-12-11 | 中国科学院昆明植物研究所 | The artificial synthesized mutant and its coded sequence of a kind of beta carotene assimilation enzyme and application |
| CN108977414B (en) * | 2018-08-16 | 2020-09-04 | 中国科学院昆明植物研究所 | Artificially synthesized mutant of beta-carotene ketolase and coding sequence and application thereof |
| CN110349624A (en) * | 2019-05-30 | 2019-10-18 | 山东省农业科学院玉米研究所 | The method of sam file f lag tag location T-DNA insertion point |
| CN110349624B (en) * | 2019-05-30 | 2021-09-21 | 山东省农业科学院玉米研究所 | Method for positioning T-DNA insertion site by sam file flag tag |
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