CN101948871A

CN101948871A - Marine microalgae chloroplast expression vector and application thereof

Info

Publication number: CN101948871A
Application number: CN 201010285197
Authority: CN
Inventors: 李宏业; 朱聪聪; 杨维东; 刘洁生
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2010-09-17
Filing date: 2010-09-17
Publication date: 2011-01-19
Anticipated expiration: 2030-09-17
Also published as: CN101948871B

Abstract

The invention discloses a marine microalgae chloroplast expression vector and application thereof. The marine microalgae chloroplast expression vector contains a homologous recombinant sequence I, a target gene expression case and a homologous recombinant sequence II, wherein the target gene expression case is positioned between the homologous recombinant sequence I and the homologous recombinant sequence II; 1st to 972nd bp of the homologous recombinant sequence I are 3' part of rns, and 973rd to 1,097th bp are 5' part of trnI; and 1st to 196th bp of the homologous recombinant sequence II are 3' part of trnA, and 197th to 1,483rd bp are 5' part of rnl. The homologous recombinant sequence ensures that the expression vector can be induced into the marine microalgae chloroplast genome by an electric shock method, which is advantageous to the way of inducing the vector into the chloroplast genome, greatly reduces conversion cost, and provides technical support for realizing that marine microalgae chloroplast is used as a bioreactor to produce high value-added products.

Description

A kind of marine microalgae chloroplast expression carrier and application thereof

Technical field

The present invention relates to little algae genetically engineered field, be specifically related to a kind of marine microalgae chloroplast expression carrier and application thereof.

Background technology

Since the first transgenic plant tobacco in 1984 was come out, gene was the main direction of studying of plant genetic engineering always to the outside the pale of civilization source of plant nucleolus transfer.But because the existence of the problems such as ecological security that foreign gene expression amount in plant nucleolus is low, unstable expression and nuclear gene are brought with the pollen diffusion easily in the offspring.Compare with traditional consideration convey gene, chloroplast(id) transforms plurality of advantages: all have a large amount of chloroplast(id) copies in each vegetable cell, make foreign gene obtain nuclear gene and transform the overexpression that is difficult to realization; Foreign gene can not produce position effect and gene silencing phenomenon by the homologous recombination site-directed integration after the chloroplast gene group; The chloroplast(id) genetic transformation has the characteristics of matrocliny, is expected to solve the biological safety problem of bringing because of the pollen diffusion in the nuclear gene conversion system; Most of chloroplast genes exist with the form of operon, can realize that a plurality of foreign genes transform simultaneously and express in the chloroplast gene group; Expressed protein can form disulfide linkage in chloroplast(id), and people's source protein also can correctly be folded after expressing.

Marine microalgae is the primary producer of Marine ecosystems, accounts for 40.86% of marine organisms species, and little algae also has the growth cycle weak point, and reproduction speed is fast, and plasticity-is strong, and nutritive factor is required simply, and chloroplast(id) accounts for characteristics such as the most of volume of microalgae cell.Diatom is to be present in unicellular plant plankton in the Marine ecosystems in a large number, thereby because its take up an area of ball primary productivity total amount 25% and and the generation of O2 is closely related is considered to specifically important effect.

The eucaryon algae that has successfully carried out at present the chloroplast(id) conversion only has Chlamydomonas reinhardtii, utilize the promotor terminator in chloroplast(id) source to regulate and control expression of exogenous gene in this transformation system, by gene gun technology carrier is imported in the chloroplast gene group, obtained successful expression, but the chloroplast(id) of marine microalgae transforms system and also is not established, and all be to finish by gene gun technology in traditional chloroplast(id) conversion carrier importing acceptor chloroplast gene group, complicated operation and cost are higher.

Summary of the invention

Primary and foremost purpose of the present invention is to overcome the deficiencies in the prior art part and defective, and a kind of marine microalgae chloroplast expression carrier is provided.

Another object of the present invention is to provide the application of described marine microalgae chloroplast expression carrier.

Purpose of the present invention is achieved through the following technical solutions: a kind of marine microalgae chloroplast expression carrier comprises homologous recombination sequence I, destination gene expression box and homologous recombination sequence II; The destination gene expression box is between homologous recombination sequence I and homologous recombination sequence II;

Described homologous recombination sequence I is as follows, by rns (ribosome-RNA(rRNA) small ylidene gene, coding 16s ribosome-RNA(rRNA) small subunit) 3 ' part and the 5 ' part of trnI are formed, the 1st～972bp among the homologous recombination sequence I is a 3 ' part of rns gene, and the 973rd～1097bp is the 5 ' part of trnI;

1 ACTGGGCGTA?AAGCGTCTGT?AGGTGGTCCA?ATAAGTCAAC?TGTTAAATCT?TGAGGCTCAA

61 CCTCGAAATC?GCAGTCGAAA?CTATTAGACT?AGAGTATAGT?AGGGGTAAAG?GGAATTTCCA

121 GTGGAGCGGT?GAAATGCGTA?GAGATTGGAA?AGAACACCGA?TGGCGAAGGC?ACTTTACTGG

181 GCTATTACTA?ACACTGAGAG?ACGAAAGCTA?GGGTAGCAAA?TGGGATTAGA?TACCCCAGTA

241 GTCCTAGCCG?TAAACAATGG?ATACTAGATG?TTGAACAGAT?CGACCTGTGC?AGTATCAAAG

301 CTAACGCGTT?AAGTATCCCG?CCTGGGAAGT?ATGCTCGCAA?GAGTGAAACT?CAAAGGAATT

361 GACGGGGGCC?CGCACAAGCG?GTGGAGCATG?TGGTTTAATT?CGATGCAACG?CGAAGAACCT

421 TACCAGGGTT?TGACATGATA?CGAATTTCTT?TGAAAGAAGG?AAGTGCCTTT?TGGAACGTAT

481 ACACAGGTGG?TGCATGGCTG?TCGTCAGCTC?GTGTCGTGAG?ATGTTGGGTT?AAGTCCCGCA

541 ACGAGCGCAA?CCCTCATTTT?TAGTTGCCTT?TTGGAACTCT?AAAAAGACTG?CCGGTTATAA

601 ACCGGAGGAA?GGCGGGGATG?ACGTCAAGTC?AGCATGCCCC?TTACACCCTG?GGCTACACAC

661 GTGCTACAAT?GGGCGAGACA?ATGAGACGCA?AATCTGCGAA?GACAAGCTAA?TCTATAAACT

721 CGCTCTAAGT?TCGGATTGCA?GGCTGCAACT?CGCCTGCATG?AAGTTGGAAT?CGCTAGTAAT

781 CGCTGGTCAG?CTATACAGCG?GTGAATTCGT?TCCCGGGCCT?TGTACACACC?GCCCGTCACA

841 CCATGGAAGC?TGGTTATGCC?CGAAGTCGTT?ACTCTAACCG?CTTGGAGGAG?GACGCCTAAG

901 GTAGAATTAG?TGACTAGGGT?GAAGTCGTAA?CAAGGTAACC?GTACTGGAAG?GTGCGGTTGG

961 ATCACCTCCT?TTTAAAATTA?TAAAATTTTA?AAATTAACGG?TCGATAAATA?CTAAATAGAA

1021?ACGGGCTATT?AGCTCAGTTG?GTTAGAGCGC?ACCCCTGATA?AGGGTGAGGT?CTCTGGTTCA

1081?AATCCAGAAT?GGCCCAA

Described homologous recombination sequence II is as follows, 3 ' part and rnl (the big subunit gene of ribosome-RNA(rRNA) by trnA, the big subunit of coding 23s ribosome-RNA(rRNA)) 5 ' part is formed, wherein the 1st～196bp of homologous recombination sequence II is a 3 ' part of trnA gene, and the 197th～1483bp is the 5 ' part of rnl;

1 CGGGGGTATA?GCTCAGTTGG?TTAGAGCGCA?CCCCTGATAA?GGGTGAGGTC?TCTGGTTCAA

61 ATCCAGAATG?GCCCAACGGG?GGTATAGCTC?AGTTGGTAGA?GCACCGCCTT?TGCACGGCGG

121 TTGTCAGCGG?TTCGAATCCG?CTTACCTCCA?CATGAAAAAT?TTTAACATTT?TTTAATCATA

181 AGATTAAATA?AGTCTTAAAT?TCAAGGAATT?AAGAGTTTAT?GGGGGATACC?TTGGCATTCA

241 GAAGCGATGA?AGGACGTGGT?TACCGACGAA?ACGCTTCGGG?GAGCTGGAAA?CAAGCTATGA

301 TCCGGAGATC?TCCGAATGAG?GAAACTCTAA?TACTACTTAT?TGAATACATA?AATAAGAAAG

361 AGCGAACCTA?GGGAACTGAA?ACATCTTAGT?ACCTAGAGGA?AAAGAAAGTA?AAAACGATTC

421 CCTTAGTAGC?GGCGAGCGAA?ATGGGAACAG?CCTAAACTTA?ATTTTAAGGG?TAGCGGGACA

481 ATTATTAATG?ATTAAATGTG?ACAATTAGAC?GAACTTAGAT?GGAATGCTAA?ACCAAAGAGA

541 GTGATAGTCT?CGTAGTCGAA?AATTGAAACA?ATCAAATTGT?ATCCCAAGTA?GCATGGAGCA

601 CGTGAAATTC?CGTGTGAATC?AGCGAGGACC?ACCTCGTAAG?GCTAAATATT?CCTGAATGAC

661 CGATAGTGAA?ATAGTACCGT?GAGGGAAAGG?TGAAAAGAAC?CCCGGGAGGG?GAGTGAAAAG

721 AACATGAAAC?CATAAACTTA?CAACCAGTAG?GAGGACGACT?AAAACGTCTG?ACTGCGTGCC

781 TGTTGAAGAA?TGAGCCGGCG?ACTTATAAGT?AGTGGCAGGT?TAAGGTAGAG?AATACCGGAG

841 CCATAGTGAA?AGCGAGCCTG?AATAGGGCGT?TTGTCACTGG?TTATAGACCC?GAACCCGGAT

901 GATCTAACCA?TGGCCAGGTT?GAAGCTTAGG?TAATACTAAG?TGGAGGACCG?AACCGACTGA

961 TGTTGAAAAA?TCAGCGGATG?AGTTGTGGTT?AGGGGTGAAA?TGCCAATCGA?ATTCGGAGCT

1021?AGCTGGTTCT?CCCCGAAATG?CGTTTAGGCG?CAGCGATGAA?TGTTATAGCT?TAGGGGTAAA

1081?GCACTGTTAC?GTATGCGGGC?TGGTAATTCG?GTACCAAATC?GTTGCAAACT?AAGAATACTA

1141?AGTGTACAGT?TCATCAGTGA?GACTGTGGGG?GATAAGCTCC?ATTGTCAAGA?GGGAAACAGC

1201?CCAGAGCACC?AGTTAAGGCC?CCTAAATAAT?TGCTAAGTGA?TAAAGGAGGT?GGGAGTGCAT

1261?AGACAATCAG?GAGGTTTGCT?TAGAAGCAGC?AATCCTTTAA?AGAGTGCGTA?ATAGCTCACT

1321?GATCGAGTAA?ACCTGCGCCG?AAAATGTACG?GGACTAAGTA?ATTTGCCGAA?ACTGTGCGAT

1381?ATATAAAATA?TATCGGTAGG?GGAGCGTTCT?GTTGTAGGTT?GAAGTATTAG?CGGAAGCGGA

1441?TATGGACGAA?GCAGAAGTGA?GAATGTCGGC?TTGAGTAACG?AAA

Described marine microalgae chloroplast expression carrier also comprises resistance screening expression cassette or fluorescent protein expression box;

Described expression cassette is meant and contains the sequence that promotor, gene and terminator and gene wherein can normally transcribe and translate;

Described destination gene expression box is made up of promotor, goal gene and terminator;

The promotor of described destination gene expression box is preferably the CaMV 35S promoter;

The terminator of described destination gene expression box is preferably the NOS terminator;

Described resistance screening expression cassette is made up of promotor, resistance screening marker gene and terminator;

The resistance screening marker gene of described resistance screening expression cassette is preferably chloramphenicol resistance gene;

Described fluorescent protein expression box is made up of promotor, fluorescence protein gene and terminator;

Described fluorescence protein gene is preferably green fluorescence protein gene;

Described marine microalgae chloroplast expression carrier is applied to express foreign protein in marine microalgae;

Described application comprises following steps: by gene transfer technique marine microalgae chloroplast expression carrier is changed in the marine microalgae, screening obtains the marine microalgae of destination gene expression then;

Described marine microalgae is preferably Phaeodactylum tricornutum (Phaeodactylumtricornutum);

Described gene transfer technique is preferably electric shocking method (or being called electroporation);

Described electric shocking method comprises following steps:

(1) be that f/2 substratum (no Si) is cultivated Phaeodactylum tricornutum with substratum, temperature is 21 ± 1 ℃, and intensity of illumination is 4000lx, and Light To Dark Ratio is 12h: 12h;

(2) centrifugal collection 1.0 * 10 ⁷～10 ⁸Individual Phaeodactylum tricornutum cell, the centrifugal 10min of 3000rpm;

(3) add 150 μ l 1.0mol/L NaCl suspension Phaeodactylum tricornutum cells;

(4) add 150ul 0.1mol/L N.F,USP MANNITOL, mixing is placed 30min on ice;

(5) the frustule liquid of step (4) preparation is transferred in the sharp cup of 0.4cm electricity, electricity swashs the interior algae liquid of cup is advisable to be no more than 400 μ l, adds plasmid pPtc-GFP (0.4 μ g) mixing simultaneously;

(6) electricity is swashed cup and place, transfer the electroporation apparatus (MicroPulser of U.S. BIO-RAD company ^TMElectroporation apparatus) electric capacity is to 10F, and resistance is 400, and voltage is 1.5kv, shocks by electricity;

(7) the algae liquid after will shocking by electricity is transferred in the 50ml triangular flask, adds fresh f/2 substratum 10ml, and dark place reason 2h normally cultivates 24h (12L: 12D) again;

The mode of described screening is preferably by resistance screening or by fluorescin and screens.

Principle of the present invention: the present invention adopts trnI sequence in the reverse iteron (IR) in the chloroplast gene group of marine microalgae and trnA sequence as the homologous recombination fragment, two sections sequences are positioned at the reverse iteron of chloroplast gene group, have 2 copies, can increase the integration efficiency of foreign gene, improve the expression of exogenous gene level.

The present invention has following advantage and effect with respect to prior art:

(1) homologous recombination sequence of the present invention makes and expression vector can be imported in the marine microalgae chloroplast gene group by electric shocking method, made things convenient for carrier to import the approach of chloroplast gene group and greatly reduce the conversion cost, for realizing that utilizing the marine microalgae chloroplast(id) to produce high value added product for bio-reactor provides technical support.

(2) the present invention adopts the procaryotic promotor and the terminator in non-chloroplast(id) source to regulate and control expression of exogenous gene first, makes foreign gene obtain to efficiently express in the marine microalgae chloroplast(id).

(3) the present invention can successfully obtain the little algae of chloroplast transgenic, the little algae of chloroplast transgenic can produce recombiant vaccine, Mammals antibody, the mixture that is of high nutritive value and industrial raw material such as β-Hu Luobusu, polyunsaturated fatty acid, hydrogen or biofuel etc. as bio-reactor, and this is indicating that the present invention will provide effective guarantee for producing above high value added product.

Description of drawings

Fig. 1 is the structure iron of recombinant plasmid ptrnI.

Fig. 2 is the structure iron of recombinant plasmid ptrnI-trnA.

Fig. 3 is the structure iron of recombinant plasmid ptrnI-trnA-cat.

Fig. 4 is the structure iron of recombinant plasmid pPtc-GFP.

Fig. 5 is a fluorogram of observing the Phaeodactylum tricornutum leaf that successfully transforms recombinant plasmid pPtc-GFP, wherein:

A is positive Phaeodactylum tricornutum cell observed GFP green fluorescence figure after the 488nm excitation;

B is the spontaneous red fluorescence figure of positive Phaeodactylum tricornutum cell observed chloroplast(id) after the 543nm excitation;

C is an observed positive Phaeodactylum tricornutum cytological map under the laser confocal microscope light field;

D is the Overlay figure of A, B, C, three width of cloth figure.

Fig. 6 is the average fluorescent strength analysis chart that the GFP fluorescence to Fig. 5 carries out.

Embodiment

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.

Embodiment 1

(1) design primer:

The upstream and downstream primer of homologous recombination sequence I is respectively P1 and P2

P1：5’-CGAGCTCACTGGGCGTAAAGCGTCTGT-3’

SacI

P2：5’-GGGGTACCTTGGGCCATTCTGGATTTG-3’

KpnI

The upstream and downstream primer of homologous recombination sequence II is respectively P3 and P4

P3：5’

-ACGC GTCGACAACTGCAGCGGGGGTATAGCTCAGTTGG-3’

SalI

P4：5’

-AA CTGCAGACATGCATGCTTTCGTTACTCAAGCCGACATT-3’

PstI

(2) pcr amplification: with the Phaeodactylum tricornutum genomic dna is template, wherein Phaeodactylum tricornutum (Phaeodactylum tricornutum) is available from the aquatic institute in Wuhan, and the leaching process of Phaeodactylum tricornutum genomic dna is operated according to the Universal GenomicDNA Extraction Kit Ver.3.0 test kit specification sheets of the precious biotech firm in Dalian.

The reaction system of homologous recombination sequence I is as follows:

Genomic dna 2ul

dNTP?Mixture 3.2μl

10 * PCR Buffer (contains Mg ²⁺) 4 μ l

TrnI upstream primer P1 0.8 μ l

TrnI downstream primer P2 0.8 μ l

Takara?LA?Taq 0.4μl

ddH ₂O 28.8μl

Total 40μl

The reaction system of homologous recombination sequence II is as follows:

Genomic dna 2.0 μ l

dNTP?Mixture 3.2μl

10 * PCR Buffer (contains Mg ²⁺) 4.0 μ l

TrnA upstream primer P3 0.8 μ l

TrnA downstream primer P4 0.8 μ l

Takara?LA?Taq 0.4μl

ddH ₂O 28.8μl

Total 40μl

Homologous recombination sequence I and homologous recombination sequence II PCR reaction conditions are: reaction conditions is: 94 ℃ of 4min; 94 ℃ of 50s, 55 ℃ of 1min, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min.

(3) clone:

A, the homologous recombination sequence I PCR product that step (2) is obtained carry out agarose gel electrophoresis, reclaim the 1100bp fragment, carry out the TA clone, use the precious Agarose Gel DNA Purification Kit Ver.2.0 of the biotech firm test kit in Dalian to reclaim, reclaiming fragment is connected with pMD19-T, linked system is carried out according to the pMD19-T carrier working instructions of the precious biotech firm in Dalian, wherein use DH5 α competent cell (available from the biological company limited of Guangzhou ancient cooking vessel state), whole TA clone process is carried out according to DH5 α competent cell working instructions); Select positive colony, obtain the TA-trnI recombinant plasmid.Use SacI and KpnI double digestion TA-trnI recombinant plasmid and circular vectors pMD19-T (T is available from the precious biotech firm in Dalian) simultaneously again, all restriction enzymes and required enzyme are cut Buffer all available from the precious biotech firm in Dalian, TA-trnI recombinant plasmid enzyme is cut system: TA-trnI 10 μ l, SacI 7 μ l, KpnI 7 μ l, 1 * L Buffer, 2.5 μ l, ddH ₂O 1 μ l, totally 25 μ l; The enzyme of carrier pMD19-T is cut system: 49 μ l, SacI 2.5 μ l, KpnI 2.5 μ l, 1 * M Buffer, 6 μ l, totally 60 μ l; The enzyme time of cutting is 11.5h; Reclaim trnI fragment and linear pMD19-T carrier, by T4DNA ligase enzyme (the T4 dna ligase is available from the precious biotech firm in Dalian, and wherein linked system and time carry out according to specification sheets); TrnI is connected among the carrier pMD19-T, obtains containing the recombinant plasmid ptrnI (as shown in Figure 1) of trnI.

B, the homologous recombination sequence II PCR product that step (2) is obtained carry out agarose gel electrophoresis, reclaim the fragment of 1500bp, carry out the TA clone, use the precious Agarose Gel DNA Purification Kit Ver.2.0 of the biotech firm test kit in Dalian to reclaim, the system that the recovery fragment is connected with pMD19-T is carried out according to the pMD19-T carrier working instructions of the precious biotech firm in Dalian, wherein use DH5 α competent cell (available from the biological company limited of Guangzhou ancient cooking vessel state), whole TA clone process is carried out according to DH5 α competent cell working instructions); Select positive colony, obtain the TA-trnA recombinant plasmid.Carry out double digestion with SalI and PstI again, all restriction enzymes and required enzyme are cut Buffer all available from the precious biotech firm in Dalian; TA-trnA recombinant plasmid enzyme is cut system: TA-trnA 19 μ l, SalI 4.0 μ l, PstI 4.0 μ l, 1 * H Buffer, 3.0 μ l, ddH ₂O 4.0 μ l, totally 30 μ l; The enzyme of recombinant plasmid ptrnI is cut system: 22 μ l, SalI 7.0 μ l, PstI 7.0 μ l, 1 * H Buffer, 4.0 μ l, totally 40 μ l; The enzyme time of cutting is 11.5h; The recombinant plasmid ptrnI of double digestion TA-trnA recombinant plasmid and steps A preparation simultaneously, reclaim trnA fragment and linear ptrnI carrier, by T4DNA ligase enzyme (available from the precious biotech firm in Dalian), wherein linked system and time carry out according to specification sheets) trnA is connected among the carrier ptrnI, obtain recombinant plasmid ptrnI-trnA (as shown in Figure 2).

C, (BL21 (DE3) bacterial strain that contains plasmid pLysS is available from the modern bio tech ltd that steps in Shanghai with plasmid pLysS, the DsbioDW01-1 plasmid a small amount of extraction reagent kit that uses new east station of Guangzhou to contain company extracts plasmid pLysS, leaching process carries out according to explanation) be the terminator of the about 150bp length of promotor and back of template pcr amplification E.C. 2.3.1.28 (CAT) gene and this about 260bp length in gene front, the upstream and downstream primer of E.C. 2.3.1.28 (CAT) expression casette is respectively P5 and P6:

P5：5’-CG GGATCCCGGGATCCAGCATCACCCGACGCACT-3’

BamHI

P6：5’-GC TCTAGAGCTCTAGATAACGACCCTGCCCTGAAC-3’

XbaI

The PCR reaction system is as follows:

Plasmid pLysS 2 μ l

dNTP?Mixture 3.2μl

10 * PCR Buffer (contains Mg ²⁺) 4 μ l

CAT expression cassette upstream primer P5 0.8 μ l

CAT expression cassette downstream primer P6 0.8 μ l

Takara?LA?Taq 0.4μl

ddH ₂O 28.8μl

Total 40μl

Reaction conditions is: 94 ℃ of 4min; 94 ℃ of 50s, 58 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.

The PCR product carries out the TA clone after electrophoresis, recovery, use the precious Agarose Gel DNA Purification Kit Ver.2.0 of the biotech firm test kit in Dalian to reclaim, the system that the recovery fragment is connected with pMD19-T is carried out according to the pMD19-T carrier working instructions of the precious biotech firm in Dalian, wherein use DH5 α competent cell (available from the biological company limited of Guangzhou ancient cooking vessel state), whole TA clone process is carried out according to DH5 α competent cell working instructions, select positive colony, obtain the TA-CAT recombinant plasmid; With BamHI and XbaI while double digestion TA-CAT recombinant plasmid and carrier ptrnI-trnA, reclaim CAT fragment and linear ptrnI-trnA carrier, by the T4DNA ligase enzyme trnA is connected among the carrier ptrnI-trnA, obtains recombinant plasmid ptrnI-trnA-CAT (as shown in Figure 3).

D, with plasmid pBI121-GFP, plasmid pBI121 is the CaMV 35S promoter of template pcr amplification GFP gene and this gene front 830bp length and the NOS terminator of the 250bp length that this gene has later, wherein plasmid pBI121-GFP obtains as follows: the plasmid pGEM (available from Guangzhou Rui Zhen Bioisystech Co., Ltd) that contains the GFP gene, with plasmid pGEM is template, has designed PCR primer upstream and downstream primer and has been respectively P7 and P8:

P7：5’-GCTCTAGAGGATCCCTCGAGATGGTGAGCAAGGGCGAGGAGCTGTT-3’

P8：5’-AGTGCCGTACCTGCTCGACATGTTCATTGAGCTCC-3’

The reaction system of PCR is as follows:

Plasmid pGEM 2 μ l

dNTP?Mixture 3.2μl

10 * PCR Buffer (contains Mg ²⁺) 4 μ l

GFP upstream primer P9 0.8 μ l

GFP downstream primer P10 0.8 μ l

Takara?LA?Taq 0.4μl

ddH ₂O 28.8μl

Total 40μl

The PCR GFP that comes out with XbaI and SacI double digestion PCR product and pBI121 plasmid (available from Jin Weike (China) biotechnology center), is connected the GFP fragment again with the pBI121 carrier, so just GUS has been replaced to GFP, has obtained plasmid pBI121-GFP.

The upstream and downstream primer of GFP expression cassette is respectively P9 and P10:

P9：5，

-GG ACTAGT

CTCGAGCAGGTCCCCAGATTAGCC-3’

SpeI NotI XhoI

P10：5’-ACGC GTCGACCAGGGTTTTCCCAGTCAC-3’

SalI

The reaction system of PCR is as follows:

Plasmid pBI121-GFP 2 μ l

dNTP?Mixture 3.2μl

10 * PCR Buffer (contains Mg ²⁺) 4 μ l

GFP expression cassette upstream primer P9 0.8 μ l

GFP expression cassette downstream primer P10 0.8 μ l

Takara?LA?Taq 0.4μl

ddH ₂O 28.8μl

Total 40μl

Reaction conditions is: 94 ℃ of 4min; 94 ℃ of 50s, 60 ℃ of 1min, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min.

The PCR product carries out the TA clone after electrophoresis, recovery, the same, selects positive colony, obtains containing the TA-GFP recombinant plasmid of GFP expression cassette.With SalI and SpeI double digestion TA-GFP recombinant plasmid, reclaim GFP expression cassette fragment, with SalI and XbaI double digestion carrier ptrnI-trnA-cat, by the T4DNA ligase enzyme GFP expression cassette is connected among the carrier ptrnI-trnA-cat, obtain recombinant plasmid pPtc-GFP.

(4) transform: utilize electric shocking method that conversion carrier pPtc-GFP is imported in the Phaeodactylum tricornutum chloroplast gene group, embodiment is as follows: with substratum is that f/2 substratum (no Si) is cultivated Phaeodactylum tricornutum, temperature is 21 ± 1 ℃, intensity of illumination is 40001x, and Light To Dark Ratio is in the artificial intelligence weather incubator of 12h: 12h;

1. centrifugal collection 1.0 * 10 ⁷～10 ⁸Individual Phaeodactylum tricornutum cell, the centrifugal 10min of 3000rpm.

2. add 150 μ l 1.0mol/LNaCl suspension Phaeodactylum tricornutum cells.

3. add 150 μ l 0.1mol/L N.F,USP MANNITOL, mixing is placed 30min on ice.

4. the frustule liquid that 3. step is prepared is transferred to the 0.4cm electricity and is swashed in the cup, and algae liquid is advisable to be no more than 400 μ l in the sharp cup of electricity, adds plasmid pPtc-GFP (0.4 μ g) mixing simultaneously.

5. electricity is swashed cup and place, transfer the electroporation apparatus (MicroPulser of U.S. BIO-RAD company ^TMElectroporation apparatus) electric capacity is to 10F, and resistance is 400, and voltage is 1.5kv, shocks by electricity.

6. the algae liquid after will shocking by electricity is transferred in the 50ml triangular flask, adds fresh f/2 substratum 10ml, and dark place reason 2h normally cultivates 24h (12L: 12D) again.

(5) screening: after electric shocking method imports Phaeodactylum tricornutum chloroplast gene group with carrier pPtc-GFP, on flat board, carry out the chlorampenicol resistant screening, embodiment: it is 400 μ g/ml that paraxin screening concentration is set, in the solid medium except that paraxin, ammonia benzyl and Ka Na microbiotic have also been added with the strain of purifying algae, the antibiotic ultimate density of ammonia benzyl and Ka Na is 100 μ g/ml, the electric shock back is cultivated the centrifugal 5min of whole algae liquid 1000rpm of 24h, with f/2 substratum suspension frustule, coated plate is cultivated, and establishes a contrast and two experimental group.

Behind 7～10 days resistance screening, on solid medium, grow macroscopic algae tongue, picking algae tongue places the f/2 substratum enlarged culturing of liquid, the paraxin, ammonia benzyl and the Ka Na that also add same concentrations in the liquid nutrient medium, after 10～15 days enlarged culturing, whether get 20 μ l left and right sides algae liquid and make slide, observing by laser confocal microscope has fluorescence to send.Through growing the algae tongue after the chlorampenicol resistant screening, Fig. 5 transforms the confocal figure (focused view altogether) of GFP reporter gene for chloroplast gene, the expression level of reporter gene very high (shown in Fig. 5 a), and be positioned to express in the chloroplast(id), locate (shown in Fig. 5 d) fully altogether with the red autofluorescence (shown in Fig. 5 b) of chloroplast(id), Fig. 5 c is an observed positive Phaeodactylum tricornutum cell under the laser confocal microscope light field, Fig. 5 illustrates the reporter gene successful expression, utilizes the Phaeodactylum tricornutum chloroplast(id) successfully to be set up as the method for bio-reactor.

Fig. 6 analyzes the average fluorescent strength (Meanfluorescence intensity) that the GFP fluorescence on Fig. 5 carries out, utilize AxioVision Rel.4.7 (Zeiss, Germany) software carries out, the Meanfluorescence intensity of frustule that has as can be seen from the figure successfully transformed GFP is very high, the value of average each transgenic alga cell is 100.41, the value of not changeing the Phaeodactylum tricornutum of GFP only is 20.37, the Mean fluorescence intensity of frustule that has as can be seen from the figure successfully transformed GFP is very high, illustrate that foreign gene obtains to efficiently express (gray scale when Grey is meant that fluorescence changes into black and white, just fluorescence intensity) in the marine microalgae chloroplast(id).

The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims

1. a marine microalgae chloroplast expression carrier is characterized in that this marine microalgae chloroplast expression carrier comprises homologous recombination sequence I, destination gene expression box and homologous recombination sequence II; The destination gene expression box is between homologous recombination sequence I and homologous recombination sequence II;

Described homologous recombination sequence I is shown in SEQ ID No.1, and wherein the 1st～972bp is a 3 ' part of coding 16s ribosome-RNA(rRNA) small ylidene gene, and the 973rd～1097bp is the 5 ' part of trnI;

Described homologous recombination sequence II is shown in SEQ ID No.2, and wherein the 1st～196bp is a 3 ' part of trnA gene, and the 197th～1483bp is a 5 ' part of the big subunit gene of coding 23s ribosome-RNA(rRNA).

2. according to the described marine microalgae chloroplast expression of claim 1 carrier, it is characterized in that: this marine microalgae chloroplast expression carrier also comprises resistance screening expression cassette or fluorescent protein expression box.

3. according to claim 1 or 2 described marine microalgae chloroplast expression carriers, it is characterized in that: described destination gene expression box is made up of promotor, goal gene and terminator.

4. according to the described marine microalgae chloroplast expression of claim 3 carrier, it is characterized in that: the promotor of described destination gene expression box is the CaMV 35S promoter; The terminator of described destination gene expression box is the NOS terminator.

5. according to the described marine microalgae chloroplast expression of claim 2 carrier, it is characterized in that: described resistance screening expression cassette is made up of promotor, resistance screening marker gene and terminator; Described fluorescent protein expression box is made up of promotor, fluorescence protein gene and terminator.

6. according to the described marine microalgae chloroplast expression of claim 5 carrier, it is characterized in that: the resistance screening marker gene of described resistance screening expression cassette is a chloramphenicol resistance gene; Described fluorescence protein gene is a green fluorescence protein gene.

7. the application of claim 1 or 2 described marine microalgae chloroplast expression carriers is characterized in that: described marine microalgae chloroplast expression carrier is applied to express foreign protein in marine microalgae.

8. application according to claim 7 is characterized in that comprising following steps: by gene transfer technique marine microalgae chloroplast expression carrier is changed in the marine microalgae, screening obtains the marine microalgae of destination gene expression then.

9. application according to claim 8 is characterized in that: described gene transfer technique is an electric shocking method;

The mode of described screening is for screening by resistance screening or by fluorescin.

10. application according to claim 9 is characterized in that: described marine microalgae is Phaeodactylum tricornutum (Phaeodactylum tricornutum).