CN106632625B - Application of the protein PGR5 in regulation algae inoxidizability - Google Patents
Application of the protein PGR5 in regulation algae inoxidizability Download PDFInfo
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- CN106632625B CN106632625B CN201510727193.5A CN201510727193A CN106632625B CN 106632625 B CN106632625 B CN 106632625B CN 201510727193 A CN201510727193 A CN 201510727193A CN 106632625 B CN106632625 B CN 106632625B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
Abstract
The invention discloses application of the protein PGR5 in regulation algae inoxidizability；The protein PGR5 be a1) a2) or a3): a1) amino acid sequence is protein shown in sequence 1 in sequence table；A2) the fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；A3) the protein relevant to inoxidizability for obtaining amino acid sequence shown in sequence 1 in sequence table by the substitution and/or deletion and/or addition of one or several amino acid residues.It is demonstrated experimentally that causing to be mutated 91 couples of H of algae strain due to the missing of PGR5 gene2O2It is significantly improved with the resistance of tBOOH, the present invention to effective exploitation and utilizes algae resource, the sustainable development of algae resource is promoted to have important value to screening and obtaining with the chlamydomonas strain compared with strong anti-oxidation ability.
Description
Technical field
The present invention relates to the application of field of biotechnology more particularly to protein PGR5 in regulation algae inoxidizability.
Background technique
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) is unicellular, eucaryote, is under the jurisdiction of Chlorophyta, group
Chlamydomonas in Cutleriales, chlamydomonas section is widely used in photosynthesis and produces the important model algae of hydrogen process and mechanism study.Closely
Increasingly sharpening with the various crises such as resource, the energy and environment over year, national governments and industry all attach great importance to research and
Exploitation is not take up the microalgae living resources in arable land.It screens and obtains with the chlamydomonas strain compared with strong anti-oxidation ability to effective exploitation
With using algae resource, the sustainable development of algae resource is promoted to have important value.
Summary of the invention
The technical problem to be solved by the present invention is to how regulate and control the inoxidizability of algae.
In order to solve the above technical problems, the answering in regulation algae inoxidizability present invention firstly provides protein PGR5
With.
Protein PGR5 provided by the present invention be following a1) a2) or a3) protein:
A1) amino acid sequence is protein shown in sequence 1 in sequence table；
A2) the fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；
A3) by amino acid sequence shown in sequence 1 in sequence table by one or several amino acid residues substitution and/or
Obtained protein relevant to inoxidizability is deleted and/or added.
Wherein, sequence 1 is made of 141 amino acid residues in sequence table.
In order to make a1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 1 or
Carboxyl terminal connects upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned a3) in protein PGR5, the substitution of one or several amino acid residues and/or missing and/or add
Add as the substitution and/or deletion and/or addition no more than 10 amino acid residues.
Above-mentioned a3) in protein PGR5 can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain
It arrives.
Above-mentioned a3) in the encoding gene of protein PGR5 can be by will be lacked in DNA sequence dna shown in sequence 2
The codon of one or several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 '
The coded sequence that end and/or 3 ' ends connect label shown in table 1 obtains.
Application of the nucleic acid molecules of code for said proteins PGR5 in regulation algae inoxidizability also belongs to of the invention
Protection scope.
In above-mentioned application, the nucleic acid molecules of the code for said proteins PGR5 can be following b1) or b2) or b3) or b4)
Shown in gene:
B1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
B2) its coded sequence is DNA molecular shown in 2 nucleotide of sequence or cDNA molecule in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encoding said proteins
The cDNA molecule or genomic DNA molecule of matter PGR5；
B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and code for said proteins PGR5
CDNA molecule or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, sequence 2 is made of 426 nucleotide, the nucleotide coding sequence table sequence 1 of sequence 2
Shown in amino acid sequence.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding protein PGR5 of the invention.Those have and this hair by manually modified
The nucleotide sequence 80% of bright isolated protein PGR5 or the nucleotide of higher identity, as long as coding protein
PGR5 and related to inoxidizability is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence of the protein of the composition of amino acid sequence shown in the sequence 1 of bright polynucleotide has 75% or higher,
Or 80% or higher or 85% or higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity
It can with the naked eye or computer software is evaluated.Using computer software, identity between two or more sequences can be with
It is indicated with percentage (%), can be used to evaluate the identity between correlated series.
Any one of in above-mentioned application, the algae is following c1)-c6): c1) Chlorophyta algae；C2) volvocales algae
Class；C3) chlamydomonas section algae；C4) Chlamydomonas algae；C5) Chlamydomonas reinhardtii；C6) Chlamydomonas reinhardtii algae (Chlamydomonas
reinhardtii)CC400(mt-)。
In above-mentioned application, the inoxidizability is anti-Exogenous Active Oxygen Species agent.
The present invention also provides a kind of methods one for cultivating recombinant algae.
A kind of method one for cultivating recombinant algae provided by the present invention, includes the following steps: to import into purpose algae
The encoding gene of the protein PGR5 obtains the recombinant algae that inoxidizability is lower than the purpose algae.
The recombinant algae can be Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) No. 91 numbers, in December, 2013
It is preserved within 31st China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: Beijing
The institute 3 of Chaoyang District North Star West Road 1), deposit number is CGMCC NO.8706.Chlamydomonas reinhardtii (the Chlamydomonas
Reinhardtii) No. 91 are the importing into Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) CC400 (mt+)
PChlamiRNA3 plasmid (purchase from Duke Univ USA's Chlamydomonas reinhardtii center (Chlamy center, http: //
Www.chlamy.org/), the recombination chlamydomonas obtained.
The present invention also provides a kind of methods two for cultivating recombinant algae.
A kind of method two for cultivating recombinant algae provided by the present invention, includes the following steps: that silencing is set out algal gene
The encoding gene of the protein PGR5 in group obtains the recombinant algae that inoxidizability is higher than the algae that sets out.
In the above method, the encoding gene of the protein PGR5 can be following d1) or d2) or d3) or d4) shown in base
Cause:
D1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
D2) its coded sequence is DNA molecular shown in 2 nucleotide of sequence or cDNA molecule in sequence table；
D3) and d1) or d2) nucleotide sequence that limits has 75% or 75% or more identity, and encoding said proteins
The cDNA molecule or genomic DNA molecule of matter PGR5；
D4) the nucleotide sequence hybridization limited under strict conditions with d1) or d2), and code for said proteins PGR5
CDNA molecule or genomic DNA molecule.
Any one of in the above method, the algae is following c1)-c6): c1) Chlorophyta algae；C2) volvocales algae
Class；C3) chlamydomonas section algae；C4) Chlamydomonas algae；C5) Chlamydomonas reinhardtii；C6) Chlamydomonas reinhardtii algae (Chlamydomonas
reinhardtii)CC400(mt-)。
In the above method, the inoxidizability is anti-Exogenous Active Oxygen Species agent.
Any of the above-described Exogenous Active Oxygen Species agent can be H2O2Or tBOOH.
It is demonstrated experimentally that PGR5 gene is transferred to mutation algae strain 91, obtain turning PGR5 gene Chlamydomonas reinhardtii.It is mutated algae strain 91
To H2O2It is higher than with the resistance of tBOOH and turns PGR5 gene Chlamydomonas reinhardtii to H2O2With the resistance of tBOOH, show due to PGR5 gene
Missing, cause be mutated algae strain 91 couples of H2O2Significantly improved with the resistance of tBOOH, this to screen and obtain have compared with strong anti-oxidation
The chlamydomonas strain of ability to effective exploitation and utilizes algae resource, the sustainable development of algae resource is promoted to have important value.
Detailed description of the invention
Fig. 1 is the protein level testing result for turning PGR5 gene Chlamydomonas reinhardtii.
Fig. 2 is H2O2Processing turns the phenotypic analysis and growth curve of PGR5 gene Chlamydomonas reinhardtii.
Fig. 3 is H2O2The plate for turning PGR5 gene Chlamydomonas reinhardtii with tBOOH processing restores experiment.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) CC400 (mt-) (referred to as Chlamydomonas reinhardtii CC400), from
Duke Univ USA's Chlamydomonas reinhardtii center (Chlamy center, http://www.chlamy.org/) purchase.
Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) 91 (for mutant strain, therefore is hereinafter referred to as mutated
Algae strain 91): on December 31st, 2013, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number are CGMCC NO.8706, classification
It is named as Chlamydomonas reinhardtii (Chlamydomonas reinhardtii).Genetic background: to Chlamydomonas reinhardtii (Chlamydomonas
Reinhardtii pChlamiRNA3 plasmid) is imported in CC400 (mt-), and (containing paromomycin resistant gene, purchase is shut out with the U.S.
Gram university's Chlamydomonas reinhardtii center (Chlamy center, http://www.chlamy.org/) obtains recombination chlamydomonas.
Plasmid pDble: being provided by University College London Saul professor purton, be recorded in " Grovenstein PB,
Wilson DA,Lennox CG et al.(2013)Identification and molecular characterization
of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll
Biosynthesis F1000Research 2013,2:138 " text, the carrier contain blasticidin resistance gene.
Ago-Gel DNA QIAquick Gel Extraction Kit is TIANGEN Biotech's product, and catalog number is
DP209-03.PEasy-blunt simple carrier, Escherichia coli (Escherichia coli) Trans-T1 competent cell
It is the product of Beijing Quanshijin Biotechnology Co., Ltd.TBOOH is Sinopharm Chemical Reagent Co., Ltd.'s product.
Culture medium and solid plate as used in the following examples:
(1) TAP fluid nutrient medium: NH4Cl 0.4g/L, MgSO4·7H2O 0.1g/L, CaC12·2H2O 0.05g/L,
K2HPO40.108g/L, KH2PO40.056g/L, Trisbase 2.423g/L, Hunter microelement (Hunter ' s trace
Elements) 1mL/L, glacial acetic acid 1ml/L；Remaining is water；
Wherein Hunter microelement (Hunter ' s trace elements) formula is as follows: H3BO411.4g/L ZnSO4·
7H2O 22.0g/L, MnCl2·4H2O 5.06g/L, CoCl2·6H2O 1.61g/L, CuSO4·5H2O 1.57g/L, (NH4)6Mo7O24·4H2O 1.10g/L, FeSO4·7H2O 4.99g/L, remaining is water.
(2) TAP solid medium: agar powder is added in TAP fluid nutrient medium and makes its concentration 1.5g/100mL.
(3) bleomycin plate: the TAP solid medium of the bleomycin containing 10 μ g/L pours into culture dish while hot,
Obtain bleomycin plate.
(4) TAP solid plate: agar powder is added in TAP fluid nutrient medium and makes its concentration 1.5g/100mL while hot
It pours into culture dish, obtains TAP solid plate.
The functional verification of embodiment 1, PGR5 albumen
One, complementing vector constructs
The construction step of complementing vector is as follows:
1, the extraction of Chlamydomonas reinhardtii CC400 total serum IgE
Using plant total RNA extraction reagent box (TIANGEN Biotech's product) and press operation instructions
Total serum IgE is extracted from Chlamydomonas reinhardtii CC400.Specific step is as follows:
(1) TAP Liquid Culture is inoculated into from picking Chlamydomonas reinhardtii CC400 monoclonal on TAP solid medium with transfer needle
In base, be placed in continuous illumination culture on the shaking table in constant temperature illumination box (25 DEG C, 60rpm, 100 μ Es-1m-2), obtain clothing
(cell concentration of Chlamydomonas reinhardtii CC400 is 4-6 × 10 to frustule culture solution 16A/mL culture solution).
(2) after 5000rpm is centrifuged 5min, 1mL lysate is added in the reinhardtii cell culture solution 1 for taking 2mL step (1) to obtain
RZ, oscillation mix, be placed at room temperature for 5min, then 4 DEG C, 12000rpm be centrifuged 5min, take supernatant, be transferred to new no RNase from
In heart pipe.
(3) add 200 μ L chloroforms into the centrifuge tube for completing step (2), cover pipe lid, acutely vibrate 15s, be placed at room temperature for
3min, then 4 DEG C, 12000rpm centrifugation 10min, is transferred to upper strata aqueous phase in the centrifuge tube of new no RNase.
(4) dehydrated alcohol of 0.5 times of volume is slowly added in the centrifuge tube equipped with upper strata aqueous phase obtained to step (3),
It mixes, obtained solution and precipitating is all transferred in adsorption column, then 4 DEG C, 12000rpm centrifugation 30s abandon waste liquid.
(5) add 500 μ L protein liquid removals into the adsorption column for completing step (4), 4 DEG C, 12000rpm centrifugation 30s abandon waste liquid.
(6) add 700 μ L rinsing liquids into the adsorption column for completing step (5), be stored at room temperature 2min, 4 DEG C, 12000rpm centrifugation
30s abandons waste liquid.
(7) add 500 μ L rinsing liquids into the adsorption column of step (6), be stored at room temperature 2min, 4 DEG C, 12000rpm centrifugation 30s,
Abandon waste liquid.
(8) 4 DEG C of the adsorption column of step (7), 12000rpm are centrifuged 2min, remove residual liquid, uncaps and dry.
(9) adsorption column of step (8) is transferred in the new centrifuge tube without RNase, adds 50 μ L RNase-free
ddH2O is placed at room temperature for 2min, 4 DEG C, 12000rpm centrifugation 2min.
Liquid in the centrifuge tube of step (9) is the total serum IgE of Chlamydomonas reinhardtii CC400.
2, the first chain of cDNA synthesizes
The total serum IgE of the Chlamydomonas reinhardtii CC400 obtained using step 1 uses cDNA the first chain synthetic agent box (Beijing as template
Quan Shijin Bioisystech Co., Ltd) synthesis the first chain of cDNA.
Response procedures are as follows: 50 DEG C of warm bath 30min, then 70 DEG C of heating 15min terminate reaction.
Reaction system (20 μ L): 2 μ 10 × TSII of L mix, 1 μ l Oligo dT20 (in the reaction system final concentration of
10 μM), the total serum IgE of 1-5 μ g Chlamydomonas reinhardtii CC400 mends sterilizing distilled water to 20 μ L.
3, the acquisition of PGR5 gene
(1) primer synthesizes
Upstream primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd. and downstream primer, primer sequence are as follows:
Upstream primer: 5 '-CATATG(underscore part is the identification sequence of Nde I to CTGGCCTCCAAGCCCGTTGT-3 '
Column, 4-26 of the sequence are 1-23 of sequence 2 in sequence table)；
Downstream primer: 5 '-GAATTC(underscore part is the identification of EcoR I to TTAAGCCAGGAAGCCCAGCTTC-3 '
Sequence, the 7-28 405-426 reverse complementary sequences for sequence 2 in sequence table of the sequence).
(2) PCR amplification
The first chain of cDNA synthesized using step 2 is template, using upstream primer and downstream primer as primer, carries out PCR amplification,
Obtain the pcr amplification product of 426bp.
Response procedures: 98 DEG C of initial denaturation 2min；98 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 1min, 35 are followed
Ring；72 DEG C of extension 5min.
Reaction system (25 μ L): 2 × HS GC buffer I 12.5 μ L, dNTP mixture (each 2.5mM) 2 μ L, template
CDNA 1 μ L, PRIMERSTAR (5U/ μ L) 0.5 μ L, 1 μ L of upstream primer, 1 μ L of downstream primer mends ddH2O to 25 μ L.
(3) it is sequenced
With Ago-Gel DNA QIAquick Gel Extraction Kit recovery purifying step (2) obtain pcr amplification product, then with
The connection of pEasy-blunt simple carrier, converts Escherichia coli (Escherichia coli) Trans-T1 competent cell,
With blue hickie method screening positive clone.Picking Colony Culture carries out PCR identification with universal primer M13F and M13R, will be positive
Property clone send to Beijing three win biotechnology Co., Ltd sequencing.
Sequencing result shows containing nucleotide sequence shown in sequence 2 in ordered list in positive colony, by the positive colony
Be named as pEasy-PGR5.
It is PGR5 gene by unnamed gene shown in sequence 2 in sequence table, protein shown in sequence 1 in polynucleotide,
The albumen is named as PGR5 albumen, is made of 141 amino acid residues.
In identified the strain of mutation algae 91, PGR5 gene is silenced.
4, the pcr amplification product obtained with (2) of restriction enzyme NdeI and EcoRI double digestion step 3, uses agarose
The DNA fragmentation of gel DNA QIAquick Gel Extraction Kit recycling 426bp.
5, with restriction enzyme NdeI and EcoRI double digestion plasmid pDble, with Ago-Gel DNA QIAquick Gel Extraction Kit
Recycle the carrier framework of about 7.29kb.
6, carrier framework is connect with the DNA fragmentation that step 4 obtains, obtains recombinant plasmid pDble-PGR5.
According to sequencing result, structure is carried out to recombinant plasmid pCT-fogac and is described as follows: the NdeI of plasmid pDble is known
It is the sequence 2 of sequence table from the end 5' the 7th that DNA small fragment between other sequence and EcoRI identification sequence, which replaces with nucleotide sequence,
Position obtains recombinant plasmid pDble-PGR5 to DNA molecular shown in the 426th.
7, with restriction enzyme Kpn I digestion recombinant plasmid pDble-PGR5, with Ago-Gel DNA QIAquick Gel Extraction Kit
Recycle the linearization plasmid pDble-PGR5 of about 7.7kb.
Two, turn the acquisition of PGR5 gene Chlamydomonas reinhardtii
In step 17 linearization plasmid pDble-PGR5 conversion is mutated algae strain 91, specific steps with glass bead method
It is as follows:
1, TAP fluid nutrient medium is inoculated into from picking mutation algae No. 91 monoclonals of strain on TAP solid medium with transfer needle
In, be placed in continuous illumination culture on the shaking table in constant temperature illumination box (25 DEG C, 60rpm, 100 μ Es-1m-2), obtain chlamydomonas
(cell concentration that mutation algae is strain 91 is 4-6 × 10 to cell culture fluid 26A/mL culture solution).
2,0.1g bead (425-600 μm of diameter, Sigma Products) is weighed, is placed in 1.5mL centrifuge tube, 121 DEG C
It sterilizes after 20min, it is stand-by to be put in room temperature.
3, the μ l step 1 of reinhardtii cell culture solution 2 and 10 that 100 μ l steps 1 obtain is added to the centrifuge tube for completing step 2
In 7 linearization plasmid pDble-PGR5 (5 μ g/ μ l), then vortex viberating 15s, 25 DEG C of placement 4min of room temperature.
4, after completing step 3, the algae solution in centrifuge tube is transferred in fresh TAP fluid nutrient medium, is placed in constant temperature light
According to behind on the shaking table in incubator continuous illumination culture 24 hours (25 DEG C, 60rpm, 110 μ Es-1m-2), 2500rpm centrifugation is received
Collection precipitating is resuspended with TAP fluid nutrient medium, is coated on bleomycin plate, and after two weeks, acquisition is quasi- to turn PGR5 gene Rhein clothing
Algae.
Linearization plasmid pDble-PGR5, other same step 1-4 are replaced to obtain turning empty carrier Rhein clothing with plasmid pDble
Algae, as control.
Three, turn the molecular level identification of PGR5 gene Chlamydomonas reinhardtii
(Comp-18 is respectively designated as with quasi- two plants for turning to randomly select in PGR5 gene Chlamydomonas reinhardtii that step 2 obtains
And Comp-50), turn empty carrier Chlamydomonas reinhardtii, mutation algae strain No. 91 and Chlamydomonas reinhardtii CC400 be experimental material.In transcriptional level
Detect the expression quantity of PGR5 gene in each algae strain.According in step 11 and 2 method, the total serum IgE of each experimental material and anti-is extracted
Transcription obtains cDNA.
Using cDNA as template, it is opposite in each experimental material that PGR5 gene is analyzed by real time fluorescence quantifying PCR method
Expression quantity (using CBLP gene as reference gene).Simultaneously with isometric ddH2O is template, and other steps are all the same, and setting is negative
Control.Expand the primer sequence of PGR5 gene are as follows: primer 1:5 '-CCTCTGGTCATCGTTGTTCGC-3 '；Primer 2: 5 '-
TTAAGCCAGGAAGCCCAGC-3'.Expand the primer sequence of internal reference CBLP gene are as follows: primer 3:5 '-
ATGACCACCAACCCCATCATC-3 ', primer 4:5 '-GGTCCCACAGCATGGCAATG-3 '.
Response procedures: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 20s, 35 recycle；
72 DEG C of extension 5min.
Reaction system: 2 × GC I buffer I, 10 μ L, dNTP mixture (each 2.5mM) 2 μ L, template cDNA (1 μ g/
μ L) 1 μ L, LA-Taq (5U/ μ L) 0.5 μ L, 1 μ L of upstream primer (10 μM), 1 μ L of downstream primer (10 μM), mend ddH2O to 20 μ L.
The result shows that being mutated algae strain 91 and turning not expand PGR5 gene in empty carrier Chlamydomonas reinhardtii；And Comp-18 and
The expression quantity of PGR5 gene and the expression quantity of PGR5 gene in Chlamydomonas reinhardtii CC400 are almost the same in Comp-50, and no statistics is poor
It is different.
Four, turn the protein level identification of PGR5 gene Chlamydomonas reinhardtii
With Comp-18, Comp-50, turn empty carrier Chlamydomonas reinhardtii, mutation algae strain No. 91 and Chlamydomonas reinhardtii CC400 as experiment
Material.The expression quantity of PGR5 albumen in each algae strain is detected in protein level.
With Peterson GL (1977) Simplification of protein assay method of Lowry et
The method that al-which is more generally Applicable.Anal Biochem 83:346-356 is recorded is extracted each
The total protein of experimental material carries out SDS-PAGE separation, then by the coomassie brilliant blue staining concentration of total protein (instruction) and
Western blot experiment (detects PGR5 albumen using PGR5 antibody as primary antibody), and experimental result is shown in Fig. 1, (A is in Fig. 1
Western blot experimental result, B is coomassie brilliant blue staining as a result, wherein CC400 is Chlamydomonas reinhardtii CC400 in Fig. 1,
Hpm91 is mutation algae strain 91, and Comp-18 and Comp-50 are mutation algae strain 91 two complementary algaes for being transferred to PGR5 gene
Strain).
PGR5 antibody is to be prepared by Beijing Bo Ermai Bioisystech Co., Ltd, and preparation method is specially with 2mgPGR5 weight
Rabbit is immunized in histone, collects serum and obtains PGR5 antibody.PGR5 recombinant protein the preparation method is as follows:
(1) the pEasy-PGR5 monoclonal in above-mentioned steps one is inoculated into the LB liquid of 900mL kanamycins containing 50mg/L
Body culture medium, 37 DEG C of cultures, as the OD of the culture bacterium solution of Escherichia coli600Reach 0.6~0.8, it is thio that isopropyl ss-D-1- is added
Galactoside (IPTG) makes IPTG concentration 0.5mM in system, 28 DEG C of induction 6h, the culture bacterium solution after being induced.
(2) by culture bacterium solution 4000rpm, the centrifugation 20min after step (1) induction, thallus is collected, high pressure fine is then used
Born of the same parents are crushed instrument cracking, obtain full cell pyrolysis liquid, and 30000g is centrifuged 50min, through detecting, inclusion body of the recombinant protein in precipitating
In, with washing buffer (20mM Tris-HCl pH 8.0,500mM NaCl, 0.2mM DTT, 2%Triton, 2M Urea)
Wash inclusion body, with dissolution buffer (20mM Tris-HCl pH 8.0,500mM NaCl, 0.2mM DTT, 2%Triton,
8M Urea) dissolution inclusion body, centrifuging and taking supernatant loading to nickel ion metal affinity chromatography column (nickel-ammonia ethanedioic acid (Nickel-
Nitri lotriacetic acid, Ni-NTA) agarose medium filling chromatographic column, chromatograph column type number E0001V, knob Great Britain
Biotech company's product), with 5mL rinsing liquid (20mM Tris-HCl pH 8.0,500mM NaCl, 10mM imidazole)
Rinsing removal foreign protein, then uses 5mL elution buffer (20mM Tris-HCl pH 8.0,500mM NaCl, 300mM
Imidazole it) elutes and collects recombinant protein.With dialyzate (50mM Tris-HAc pH 8.0,1mM
Dithiothreitol) to the recombinant protein of elution dialysed overnight on ice.The PGR5 weight that 12%SDS-PAGE detection purifying obtains
Histone.
The result shows that being mutated algae strain 91 and turning in empty carrier Chlamydomonas reinhardtii without PGR5 protein expression；And Comp-18 and
The expression quantity of PGR5 albumen is almost the same with the expression quantity of PGR5 albumen in Chlamydomonas reinhardtii CC400 in Comp-50, no statistics
Difference.As it can be seen that Comp-18 and Comp-50 are the complementary algae strain for being mutated algae strain 91.
Five, inoxidizability compares
Reinhardtii cell, detection reinhardtii cell (Comp-18, Comp-50, mutation algae strain are handled by additional active oxygen reagent
No. 91 or Chlamydomonas reinhardtii CC400) inoxidizability.Experiment in triplicate, is averaged.
1, it is inoculated into TAP fluid nutrient medium with transfer needle from picking reinhardtii cell monoclonal on TAP solid medium,
Be placed in continuous illumination culture on the shaking table in constant temperature illumination box (25 DEG C, 60rpm, 100 μ Es-1m-2), obtain reinhardtii cell
(cell concentration of reinhardtii cell is 5 × 10 to culture solution6A/mL culture solution).
Reinhardtii cell culture solution is taken, adjusting reinhardtii cell absorbance value at 750nm is 0.05, and H is then added2O2, make
H2O2Final concentration 1mM in system, is placed on the shaking table in constant temperature illumination box, 25 DEG C, 60rpm, 60 μ Es-1m-2Continuously
Illumination cultivation.From addition H2O2Start timing, sample within every 12 hours during the cultivation process, measures reinhardtii cell light at 750nm and inhale
Receipts value.Using incubation time as abscissa, reinhardtii cell absorbance value at 750nm is ordinate, draws the growth of reinhardtii cell
Curve.
Experimental result is shown in Fig. 2, (A is reinhardtii cell 1mM H in Fig. 22O2Processing is for 24 hours with 48h growth conditions, and B is in Fig. 2
Reinhardtii cell 1mM H2O2Handle the growth curve of 0h-48h；Wherein CC400 is Chlamydomonas reinhardtii CC400, and hpm91 is mutation algae
Strain 91, Comp-18 and Comp-50 are two strains for turning PGR5 gene Chlamydomonas reinhardtii).The result shows that with 1mM H2O2Processing
Reinhardtii cell, mutation algae strain 91 cells of the cell growth rate greater than Chlamydomonas reinhardtii CC400, Comp-18 and Comp-50 are raw
Long rate.
2, it is inoculated into TAP fluid nutrient medium with transfer needle from picking reinhardtii cell monoclonal on TAP solid medium,
Be placed in continuous illumination culture on the shaking table in constant temperature illumination box (25 DEG C, 60rpm, 100 μ Es-1m-2), suspend culture chlamydomonas
Cell, obtaining reinhardtii cell culture solution, (cell concentration of reinhardtii cell is 2 × 106A/mL culture solution)
24 orifice plates are taken, every hole is added 1mL reinhardtii cell culture solution, H is then added2O2, make H2O2Concentration in system is
0mM, 1mM, 3mM or 5mM are placed on the shaking table in constant temperature illumination box, 25 DEG C, 80rpm, 50 μ Es-1m-2Continuous illumination training
It supports, after culture for 24 hours, every hole takes 5 μ L to be uniformly coated on TAP solid plate, is placed in constant temperature illumination box continuous illumination culture
(25 DEG C, 60 μ Es-1m-2), since the timing being coated on TAP solid plate observed recovery situation after a week and photographed to record,
Obtain various concentration H2O2Treated, and plate restores experimental result.Each H2O25 multiple holes are arranged in concentration.
According to the method described above, by H2O2Replace with tBOOH, H2O2Concentration in system is that 0mM, 1mM, 3mM or 5mM are replaced
Being changed to concentration of the tBOOH in system is 0 μM, 60 μM, 80 μM or 100 μM, and other steps are constant, obtain various concentration
TBOOH treated plate restores experimental result.
Experimental result is shown in Fig. 3, (left figure is various concentration H in Fig. 32O2Treated, and plate restores experimental result, the right side in Fig. 3
Figure is that various concentration tBOOH treated plate restores experimental result；Wherein CC400 is Chlamydomonas reinhardtii CC400, and hpm91 is prominent
Become algae strain 91, Comp-18 and Comp-50 are mutation algae strain 91 two complementary algae strains for being transferred to PGR5 gene).As a result table
It is bright, 91 couples of H of mutation algae strain2O2All it is higher than Chlamydomonas reinhardtii CC400, Comp-18 and Comp-50 to H with the resistance of tBOOH2O2With
The resistance of tBOOH, it is seen then that due to the missing of PGR5 gene, cause to be mutated 91 couples of H of algae strain2O2It is significantly improved with the resistance of tBOOH.
Claims (13)
1. application of the protein PGR5 in regulation algae inoxidizability；The protein PGR5 is that amino acid sequence is sequence table
Protein shown in middle sequence 1；The algae is Chlamydomonas reinhardtii.
2. encoding application of the nucleic acid molecules of protein PGR5 described in claim 1 in regulation algae inoxidizability；The algae
Class is Chlamydomonas reinhardtii.
3. application as claimed in claim 2, it is characterised in that: the nucleic acid of protein PGR5 described in the coding claim 1
Molecule be following b1) or b2) shown in gene:
B1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
B2) its coded sequence is DNA molecular shown in 2 nucleotide of sequence or cDNA molecule in sequence table.
4. application a method according to any one of claims 1-3, it is characterised in that: the algae is that the Chlamydomonas reinhardtii is following c1)
Or c2):
C1) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) CC400(mt-)；
C2) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) mutant 91, deposit number is CGMCC NO.
8706。
5. application a method according to any one of claims 1-3, it is characterised in that: the inoxidizability is anti-Exogenous Active Oxygen Species agent.
6. a kind of method for cultivating recombinant algae includes the following steps: to import protein described in claim 1 into purpose algae
The encoding gene of PGR5 obtains the recombinant algae that inoxidizability is lower than the purpose algae；The algae is Chlamydomonas reinhardtii.
7. method as claimed in claim 6, it is characterised in that: the nucleic acid of protein PGR5 described in the coding claim 1
Molecule be following b1) or b2) shown in gene:
B1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
B2) its coded sequence is DNA molecular shown in 2 nucleotide of sequence or cDNA molecule in sequence table.
8. method according to claim 6 or 7, it is characterised in that: the Chlamydomonas reinhardtii is following c1) or c2):
C1) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) CC400(mt-)；
C2) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) mutant 91, deposit number is CGMCC NO.
8706。
9. method according to claim 6 or 7, it is characterised in that: the inoxidizability is anti-Exogenous Active Oxygen Species agent.
10. a kind of method for cultivating recombinant algae includes the following steps: claim 1 institute that silencing is set out in algal gene group
The encoding gene for stating protein PGR5 obtains the recombinant algae that inoxidizability is higher than the algae that sets out；The algae is Rhein
Chlamydomonas.
11. method as claimed in claim 10, it is characterised in that: the core of protein PGR5 described in the coding claim 1
Acid molecule be following b1) or b2) shown in gene:
B1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
B2) its coded sequence is DNA molecular shown in 2 nucleotide of sequence or cDNA molecule in sequence table.
12. method as described in claim 10 or 11, it is characterised in that: the Chlamydomonas reinhardtii is following c1) or c2):
C1) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) CC400(mt-)；
C2) Chlamydomonas reinhardtii algae (Chlamydomonas reinhardtii) mutant 91, deposit number is CGMCC NO.
8706。
13. method as described in claim 10 or 11, it is characterised in that: the inoxidizability is anti-Exogenous Active Oxygen Species agent.
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| CN103833835A (en) * | 2012-11-22 | 2014-06-04 | 中国科学院植物研究所 | Antioxidation and nutrient stress resistance associated protein and coding gene and application thereof |
| CN103880934A (en) * | 2014-03-21 | 2014-06-25 | 中国科学院植物研究所 | Chlamydomonas reinhardtii protein capable of highly yielding hydrogen as well as encoding gene and application thereof |
| WO2013056212A3 (en) * | 2011-10-14 | 2015-06-18 | Phycal Llc | Chlorophyte genes for the optimization of productivity |
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| CN103833835A (en) * | 2012-11-22 | 2014-06-04 | 中国科学院植物研究所 | Antioxidation and nutrient stress resistance associated protein and coding gene and application thereof |
| CN103880934A (en) * | 2014-03-21 | 2014-06-25 | 中国科学院植物研究所 | Chlamydomonas reinhardtii protein capable of highly yielding hydrogen as well as encoding gene and application thereof |
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