CN104946647A - Chlamydomonas reinhardtii intergenic expression treating element - Google Patents
Chlamydomonas reinhardtii intergenic expression treating element Download PDFInfo
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- CN104946647A CN104946647A CN201510308371.0A CN201510308371A CN104946647A CN 104946647 A CN104946647 A CN 104946647A CN 201510308371 A CN201510308371 A CN 201510308371A CN 104946647 A CN104946647 A CN 104946647A
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- chlamydomonas reinhardtii
- gene
- treating element
- intergenic
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Abstract
The invention discloses a DNA of a chlamydomonas reinhardtii intergenic expression treating element. The sequence of the chlamydomonas reinhardtii intergenic expression treating element is shown in SEQ:1-12. The sequence can be used for chlamydomonas reinhardtii polygene operon expression, can effectively convert polycistron into monocistron through treating, and is wide in prospect.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, be specifically related to express processing element between a kind of Chlamydomonas reinhardtii gene.
Technical background
Since the first transgenic plant tobacco in 1984 is come out, become the method for a kind of routine in plant genetic engineering to plant nucleolus transfer foreign gene.But because foreign gene expression amount in plant nucleolus expression system is low, in offspring, unstable expression and nuclear gene easily spreads the problems such as the ecological security brought and are difficult in a short time overcome with pollen, and sight has been turned to again core organoid---chloroplast(id) outward by people.
1988, Boynton etc. adopted particle bombardment, are successfully imported in the chloroplast(id) of Chlamydomonas reinhardtii by foreign gene first, and obtained foreign gene stably express and hereditary transgenosis unicellular algae, thus had opened the prelude of Chloroplast Genetic Engineering.
Chlamydomonas reinhardtii is under the jurisdiction of the micro-algae of single celled eukaryotic of Chlorophyta (Chlorophyta) Chlamydomonas (Chlamydomonas), and have and only have a large-scale cup-shaped chloroplast(id), cell apical has two flagellums, and there is a photosensitive eyespot side; Can photoautotrophy, acetate heterotrophic growth can be relied on again; Fast growth, the cell quantity doubling time is 5 ~ 6 hours, can obtain a large amount of hereditary offspring at short notice.
Chlamydomonas reinhardtii chloroplast expression system is compared with nucleus expression system, the expression amount with foreign gene is high, prokaryotic organism polycistron gene can be expressed, foreign gene energy site-directed integration, biological safety is good, foreign gene can stably express and transgenic alga strain can, at short notice by large scale culturing and the advantage such as toxigenic capacity is cheap, therefore be a kind of desirable heterologous gene expression system in transfer-gen plant offspring.
The multiple gene of operon is expressed simultaneously and is considered to one of most outstanding feature of chloroplast transformation.Can successfully express in some cases, but, in most cases, unusual low of exogenous gene expression or can not express together.
Processing element is expressed between IEE (intercistronic expression element) gene, it is the sequence of a section short, usually in intergenic region, RNA stem ring secondary structure can be formed after transcribing, improve regulating mRNA stability, polycistron is processed into stable monocistron efficiently, thus gives the convertibility of downstream cistron.This IEE determined is small enough to, as a general instrument, for the operon that multiple foreign gene is stacking, express while realizing multiple protein, thus will contribute to the range of application expanding chloroplast transformation technology.
Summary of the invention
The object of this invention is to provide the DNA expressing processing element between a kind of Chlamydomonas reinhardtii gene.
The technical solution realizing the object of the invention is: express processing element between a kind of Chlamydomonas reinhardtii gene, sequence is as shown in SEQ:1-12.
Express processing element between Chlamydomonas reinhardtii gene provided by the invention and effectively polycistron can be processed into monocistron.
Embodiment
Material
Chlamydomonas reinhardtii
Major Enzymes and reagent
RTaq enzyme (Takara), restriction enzyme (Thermo Scientific), the little extraction reagent kit of plasmid (Axygen), DNA gel purification kit (Axygen), T4DNA ligase enzyme (Thermo Scientific).All the other reagent are conventional reagent.
In the present invention, utilize NCBI to retrieve Chlamydomonas reinhardtii Chloroplast gene sequence, the primer of Application Design, carries out PCR to Chlamydomonas reinhardtii and increases on a large scale, be specifically implemented as follows: get Chlamydomonas reinhardtii algae liquid, carry out genome extraction, utilize genomic dna to make pcr template.PCR program is: 95 DEG C of 3min; 95 DEG C of 1min; 49 DEG C of 50s; 72 DEG C of 30s; 72 DEG C of 5min.Amplified production identifies size in 1% sepharose leakage of electricity swimming with reference to molecule Marker.
Design of primers is as follows:
IEE1
IEE1-F:5’-CGTCCAGGCGCGCCGCACCACCAACTCTGTAG-3’
IEE1-R:5’-CATGTCGACCGAGTATAAAACCACTCTGC-3’
IEE2
IEE2-F:5’-CGTCCAGGCGCGCCTATTTAATTTTTTGTAGGGCTG-3’
IEE2-R:5’-CATGTCGACATTTATTTATCCGTTAATTTTCAA-3’
IEE3
IEE3-F:5’-CGTCCAGGCGCGCCGAGTATTAACATAGGCAGTGG-3’
IEE3-R:5’-CATGTCGACGCTCCGCAGTATTAACATC-3’
IEE4
IEE4-F:5’-CGTCCAGGCGCGCCACTGCCTCCTTCGGAGT-3’
IEE4-R:5’-CATGTCGACTTTATTTATCCGTTAATTTTCAATAAAACT-3’
IEE5
IEE5-F:5’-CGTCCAGGCGCGCCTATTTAATTTTTTGTAGGGCTGC-3’
IEE5-R:5’-CATGTCGACACTTTAGTTGCCCAATATTTATATT-3’
IEE6
IEE6-F:5’-CGTCCAGGCGCGCCAGGAACTCGGTATATGCT-3’
IEE6-R:5’-CATGTCGACCTTTGTACTGTATAGTTAATATAATTT-3’
IEE7
IEE7-F:5’-CGTCCAGGCGCGCCTTTATAGTATTTGGAAAAGCAAAA-3’
IEE7-R:5’-CATGTCGACATAACGATGAACGTTTTTCTG-3’
IEE8
IEE8-F:5’-CGTCCAGGCGCGCCAGCATATACGTGCGACC-3’
IEE8-R:5’-CATGTCGACTTTTTATTTTTCATGATGTTTATGTG-3’
IEE9
IEE9-F:5’-CGTCCAGGCGCGCCGTCCCCTTACGGGAATATAAAT-3’
IEE9-R:5’-CATGTCGACCAAGTTCCATAGCCTAACAGA-3’
IEE10
IEE10-F:5’-CGTCCAGGCGCGCCTTTACGAGTATTAGGAACTCAT-3’
IEE10-R:5’-CATGTCGACGTGGCGGTACCACTG-3’
IEE11
IEE11-F:5’-CGTCCAGGCGCGCCATATATAAAATAAAAAAAACGTTAGTA-3’
IEE11-R:5’-CATGTCGACATTTTAAAAGATTGCCATACTTA-3’
IEE12
IEE12-F:5’-CGTCCAGGCGCGCCAAAATATTTAAGAAAATTAAGAGCATAAG-3’
IEE12-R:5’-CATGTCGACTCAACACTATGCTTTTCCCTA-3’
5 ' end of IEE-F primer adds Asc I restriction enzyme site, and 5 ' end of IEE-R primer adds Sal I restriction enzyme site.
PCR reaction system and reaction conditions as shown in table 1:
Table 1
Claims (2)
1. express processing element between Chlamydomonas reinhardtii gene, it is characterized in that, express the sequence of processing element as shown in SEQ:1-12.
2. as claimed in claim 1 express processing element in Chlamydomonas reinhardtii polycistronic expression on application.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510308371.0A CN104946647A (en) | 2015-06-08 | 2015-06-08 | Chlamydomonas reinhardtii intergenic expression treating element |
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| CN201510308371.0A CN104946647A (en) | 2015-06-08 | 2015-06-08 | Chlamydomonas reinhardtii intergenic expression treating element |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001098335A2 (en) * | 2000-06-20 | 2001-12-27 | Phycotransgenics, Llc | Transgenic algae for delivering antigens to an animal |
| CN101023174A (en) * | 2004-05-06 | 2007-08-22 | 株式会社大熊 | Preparation method for the production of active and soluble proteins in prokaryotes and polycistronic vectors therefor |
| CN103627725A (en) * | 2013-11-25 | 2014-03-12 | 舒海燕 | Plant chloroplast poly-gene transformation vector, and construction method and application thereof |
-
2015
- 2015-06-08 CN CN201510308371.0A patent/CN104946647A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001098335A2 (en) * | 2000-06-20 | 2001-12-27 | Phycotransgenics, Llc | Transgenic algae for delivering antigens to an animal |
| CN101023174A (en) * | 2004-05-06 | 2007-08-22 | 株式会社大熊 | Preparation method for the production of active and soluble proteins in prokaryotes and polycistronic vectors therefor |
| CN103627725A (en) * | 2013-11-25 | 2014-03-12 | 舒海燕 | Plant chloroplast poly-gene transformation vector, and construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘国宪等: "衣藻叶绿体表达系统的研究", 《生物技术通报》 * |
| 登录号: "X64066.1", 《NCBI GENBANK》 * |
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Application publication date: 20150930 |
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