CN102181369A - Genetic engineering chlamydomonas having increased hydrogen producing capacity and application thereof - Google Patents

Abstract

The invention discloses a genetic engineering chlamydomonas having increased hydrogen producing capacity and application thereof. The genetic engineering chlamydomonas provided by the invention is a genetic engineering chlamydomonas which is obtained by inhibiting an expression level of ferredoxin-NADP (Nicotinamide Adenine Dinucleotide Phosphate) reductase coding genes in a starting chlamydomonas. It is determined by gas chromatography that the hydrogen producing capacity of the genetic engineering chlamydomonas is increased by 6-10 times compared with a wild strain after phosphorus deficiency treatment is carried out for 48 hours. The mutant strain is obtained to lay the basis for industrialization of a microalgal photosynthetic hydrogen production technology.

Description

Genetically engineered chlamydomonas and application thereof that hydrogen output improves

Technical field

The present invention relates to hydrogen output improves in the biological technical field genetically engineered chlamydomonas and application thereof.

Background technology

Fossil energy is non-renewable, and a large amount of use can cause environmental pollution and climate change.Therefore, press for the renewable cleaning new energy of exploitation.The Hydrogen Energy cleaning is renewable, and burning only produces water and huge energy, does not produce any pollutent, is optimal clean energy.

Little algae photosynthetic hydrogen production is the process that blue-green algae and green alga utilize decomposing water with solar energy to produce hydrogen, has obviously advantage such as renewable, hydrogen production process cleaning, is the effective way of directly utilizing sun power bio-transformation hydrogen manufacturing.Because the photodissociation of photosynthesis water is put oxygen and is produced the complex biological metabolic process that hydrogen is two not only conflicting but also close association in the microalgae cell, and people are not clear to these process mechanisms and regulative mode at present, cause genetic modification effectively not carry out, the efficient of luminous energy reforming hydrogen manufacturing can not get significantly improving, and has seriously restricted the application of little algae photosynthetic-hydrogen-production approach from the source.Therefore the structure of highly effective hydrogen yield heredity algae strain is the key that advances this approach industrial applications.

Summary of the invention

An object of the present invention is to provide a kind of genetically engineered chlamydomonas.

Genetically engineered chlamydomonas provided by the present invention is the expression level by Triphosphopyridine nucleotide photoreductase-NADP reductase enzyme encoding gene in the chlamydomonas that suppresses to set out, the genetically engineered chlamydomonas that obtains.

The expression level of the described Triphosphopyridine nucleotide photoreductase of described inhibition-NADP reductase enzyme encoding gene disturbs by RNA and realizes.

It is to import in the chlamydomonas cell by the coding DNA with double-stranded RNA to realize that described RNA disturbs; Wherein, the nucleotide sequence of a chain of described double-stranded RNA is that the T in the base sequence of sequence in the sequence table 1 is replaced with the ribonucleoside acid sequence that U obtains, another chain and its reverse complemental.

The coding DNA of described double-stranded RNA is following double chain DNA fragment: SEQ _Forward-X-SEQ _Oppositely

Wherein, SEQ _ForwardNucleotide sequence be the base sequence of sequence 1 in the sequence table;

SEQ _OppositelyWith described SEQ _ForwardReverse complemental;

X is SEQ _ForwardWith SEQ _OppositelyBetween intervening sequence, X and described SEQ _ForwardAnd SEQ _OppositelyAll not complementary.

The coding DNA of described double-stranded RNA imports the described chlamydomonas that sets out by recombinant expression vector;

The recombinant expression vector that described recombinant expression vector obtains for the multiple clone site of the coding DNA of described double-stranded RNA being inserted expression vector Maa7/XIR;

The described chlamydomonas that sets out is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) algae strain CC-400.

Another object of the present invention provides the application of described genetically engineered chlamydomonas in preparation hydrogen.

Another purpose of the present invention provides a kind of double-stranded RNA.

Double-stranded RNA provided by the present invention, for as follows:

Article one, the nucleotide sequence of chain is that the T in the base sequence of sequence in the sequence table 1 is replaced with the ribonucleoside acid sequence that U obtains, another chain and its reverse complemental.

Another purpose of the present invention provides the coding DNA of described double-stranded RNA.

The coding DNA of described double-stranded RNA is following double chain DNA fragment:

SEQ _Forward-X-SEQ _Oppositely

Wherein, SEQ _ForwardNucleotide sequence be the base sequence of sequence 1 in the sequence table;

SEQ _OppositelyWith described SEQ _ForwardReverse complemental;

X is SEQ _ForwardWith SEQ _OppositelyBetween intervening sequence, X and described SEQ _ForwardAnd SEQ _OppositelyAll not complementary.

The recombinant expression vector that contains described coding DNA also belongs to protection scope of the present invention.

The recombinant expression vector that described recombinant expression vector obtains for the multiple clone site of described coding DNA being inserted expression vector Maa7/XIR.

The present invention is a material with photosynthetic hydrogen production pattern green alga Chlamydomonas reinhardtii, adopt RNA perturbation technique (RNAi, RNAinterference) expression of the photosynthetic key gene FNR1 of downward modulation, obtain mutant strain system by the resistance label filtration, and with the FNR1 gene downward modulation in the strain of transcriptional level checking RNAi algae of RT-PCR method, the hydrogen output of RNAi algae strain improves 6-10 doubly than the strain of wild-type algae when the gas Chromatographic Determination a lack of sulfur is handled 48 hours.The acquisition of this sudden change algae strain is that the realization of little algae photosynthetic-hydrogen-production industrialization is laid a good foundation.

Description of drawings

Fig. 1 is a FNR1 RNAi vector construction synoptic diagram.Wherein, Pro: promotor; Sp: transcribed spacer; Ter: terminator; AphVIII: paromycin resistance sequence label; S, Xh, E, Xb, the different restriction enzyme digestion sites of B representative are respectively: SacI, XhoI, EcoRI, XbaI, BamHI; Maa7 3 ' UTR:5-FI resistance sequence label.

Fig. 2 is that wild-type chlamydomonas (CC-400) and RNAi clone (F1 and F3) at the growing state that contains on 5-FI (5mM) solid medium.

Fig. 3 is that wild-type chlamydomonas (CC-400) and RNAi clone's (F1 and F3) FNR1 gene expression dose changes.

Fig. 4 is the measurement result of wild-type chlamydomonas (CC-400) and RNAi clone (F1 and F3) hydrogen desorption capacity.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1, reduce the FNR1 gene expression dose, obtain the sudden change chlamydomonas that hydrogen output improves by the RNA perturbation technique

CC-400 is available from algae kind storehouse, Duke University (Chlamycenter) in Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) algae strain;

The Maa7/XIR carrier is available from algae kind storehouse, Duke University (Chlamy center);

E.coli DH5 α bacterial strain is available from precious biotechnology (Dalian) company limited, and catalog number is D9057.

One, method

1, material is cultivated

The preparation substratum:

TAP liquid nutrient medium: NH ₄Cl 0.4g/L; MgSO ₄7H ₂O 0.1g/L; CaCl ₂2H ₂O 0.05g/L; K ₂HPO ₄0.108g/L; KH ₂PO ₄0.056g/L; Trisbase 2.423g/L; The Hunter trace element (Hunter ' s trace elements) 1ml/L, glacial acetic acid 1ml/L, all the other are water;

Hunter trace element: EDTA 50g/L; BO ₃H ₃11.4g/L; ZnSO ₄7H ₂O 22.0g/L; MnCl ₂4H ₂O 5.06g/L; FeSO ₄7H ₂O 4.99g/L; CoCl ₂6H ₂O 1.61g/L; CuSO ₄5H ₂O 1.57g/L; Mo ₇O ₂₄(NH ₄) ₆4H ₂O 1.1g/L adjusts the pH value to 6.5-6.8 with 20%KOH.

A lack of sulfur TAP liquid nutrient medium: the vitriol in the above-mentioned TAP liquid nutrient medium is replaced to identical cationic chlorate in molar ratio, and all the other compositions are all identical with above-mentioned TAP liquid nutrient medium with content.

TAP solid medium: in above-mentioned TAP liquid nutrient medium, add 1.5% agar powder.

To buy Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) algae strain CC-400 and in Bechtop, rule to the TAP solid medium, place 25 ℃, 200 μ Em with inoculating needle ^-2S ^-1Cultivated about 7 days under the condition.With inoculating needle from the TAP solid medium the picking mono-clonal to the TAP liquid nutrient medium, at 25 ℃, 200 μ Em ^-2S ^-1The condition low suspension cultivated frustule 5-7 days, to the OD value be OD ₇₅₀During ≈ 1.5 left and right sides, obtain being in logarithmic phase CC400 cell.

2, FNR1 IR RNAi vector construction

According to the gene order (GeneBank of this gene number is 5722854) (FNR1) of known Triphosphopyridine nucleotide photoreductase-NADP reductase enzyme (Ferredoxin-NADP reductase) among the Gene Bank, design three primers, respectively called after:

FNR1-UTR-F: GaattcGCAGCGAGGAGCTTCTGCTCT, the line part is the EcoRI restriction enzyme site,

FNR1-UTR-R1: AgatctAGTCCTCCAGCCCTTCAAGCCA, the line part is the BglII restriction enzyme site,

FNR1-UTR-R2: AgatctCACAAACGACTCCATCCACTCC, the line part is the BglII restriction enzyme site.

CDNA with Chlamydomonas reinhardtii algae strain CC-400 is a template, and FNR1-UTR-F and FNR1-UTR-R1 are that primer amplification goes out corresponding to FNR1 3 ' UTR zone 404bp short-movie section; Be that primer amplification is corresponding to FNR1 3 ' UTR zone 610bp long segment with FNR-UTR-F and FNR1-UTR-R2 simultaneously.The fragment that amplifies is linked on the cloning vector pEASY-Blunt Simple Cloning Vector (available from the Beijing Quanshijin Biotechnology Co., Ltd, catalog number is CB111) sequence verification respectively.Sequencing result shows that amplification obtains corresponding to FNR1 3 ' UTR zone 404bp short-movie section (FNR1-S), and its nucleotide sequence is shown in sequence in the sequence table 1; Amplification obtains corresponding to FNR1 3 ' UTR zone 610bp long segment (FNR1-L), and its nucleotide sequence is shown in sequence in the sequence table 2.Obtain the purpose band of 404bp and 610bp by EcoRI and BglII double digestion from the correct carrier that checks order respectively and reclaim; Simultaneously by utilizing the EcoRI enzyme to cut the Maa7/XIR carrier and reclaiming carrier segments.Utilize the T4 ligase enzyme that the fragment of long and short two clauses and subclauses is connected (Fig. 1) with the Maa7/XIR carrier segments, obtain the purpose recombinant vectors.The recombinant vectors that obtains is transformed DH5 α bacterial strain, screen by penbritin, obtain resistance clone, extract the resistance clone plasmid and utilize EcoRI and BglII double digestion, carrying out 1.0% agarose gel electrophoresis detects, obtain the big or small purpose fragment that is respectively 404bp and 610bp, then be defined as positive colony; Extract the positive colony plasmid and carry out sequence verification, sequencing result shows: the DNA that inserts the EcoRI restriction enzyme site in this recombinant vectors is SEQ _Forward-X-SEQ _OppositelyWherein, SEQ _ForwardNucleotide sequence be the base sequence of sequence 1 in the sequence table; SEQ _OppositelyWith described SEQ _ForwardReverse complemental; X is SEQ _ForwardWith SEQ _OppositelyBetween intervening sequence, X and described SEQ _ForwardAnd SEQ _OppositelyAll not complementary.The nucleotides sequence of X is classified in the sequence table the 405th of sequence 2 as to shown in 610.This dna encoding shRNA, shRNA are cut into double-chain small disturbance RNA by the intravital enzyme of chlamydomonas again, disturb to realize RNA.This sequencing result explanation construction of recombinant vector is correct, with this recombinant vectors called after FNR1 IR RNAi carrier.

3, cell transformation and FNR1 RNAi colony screening checking

The FNR1 IR RNAi carrier that step 2 is obtained with the SacI enzyme cut make it linearizing after, this linearized vector (10 μ g) and step 1 are cultivated the logarithmic phase CC-400 cell (1*10 that obtains ⁸Cells) and diameter be that the granulated glass sphere (0.1g) of 425-600 μ m mixes, placed on the vortex vibrator (7 grades) vibration 15 seconds, remove granulated glass sphere, obtain transformant.With the cell after transforming be coated on contain paromycin (5 μ g/mL) and 5-fluoro indole (5-FI, on TAP solid medium 5mM), 25 ℃, less than 50 μ Em ^-2S ^-1The low light level is cultivated down, grows mono-clonal after about three weeks.With toothpick wild-type (CC400) and RNAi mono-clonal algae strain (F1 and F3) are chosen in the 50ml triangular flask of about 10ml TAP liquid nutrient medium and are cultured to logarithmic phase, with cell dilution to different concns (1*10 ⁶Cellsml ^-1And 1*10 ⁵Cells ml ^-1) drop on the TAP solid medium that contains 5-FI (5mM) screening verification once more, serve as the strain of contrast algae with wild-type CC400, can contain the positive algae strain of algae strain of growing on the solid TAP substratum of 5-FI (5mM).Observe after 7 days, the result as shown in Figure 2, RNAi clone F1 and F3 all can grow containing on 5-FI (5mM) solid medium, wild-type CC400 then can not be survived, provable thus two RNAi clones (F1 and F3) are positive colonies.

4, sxemiquantitative RT-PCR checking FNR1 expression of gene

(1) extracts the total RNA of sample

It is centrifugal that (centrifugal rotation speed is 2,500g, temperature is 4 ℃, time is 2min) collect F1 positive colony and F3 positive colony and wild-type CC400 chlamydomonas cell that step 3 obtains, use TIANGEN Biotech's plant total RNA extraction reagent box to extract total RNA of sample, with total RNA of RNase-free DNase I (available from Takara company) digestion preparation, to remove the wherein residual DNA of possibility.

(2) sxemiquantitative RT-PCR

Be template with the F1 positive colony of said extracted and F3 positive colony and wild-type CC400 chlamydomonas cell total rna respectively, carry out reverse transcription with Oligo (dT) reverse transcription primer and Quant ThermoScript II (available from sky root biochemical technology company limited), obtain cDNA respectively.Design special primer according to the 3 ' terminal fragment that will detect gene:

Upstream primer: GCCTGGACTACGCCCTGTC,

Downstream primer: GCTGCTTTCGCCGCTGTTA is template with cDNA respectively, carries out pcr amplification gene FNR1, compares with 18s rRNA, and amplified production detects sxemiquantitative RT-PCR triplicate with 1.5% agarose gel electrophoresis.

The result as shown in Figure 3.The FNR1 gene expression dose of RNAi clone F1 and F3 is obvious than wild-type CC-400 downward modulation, and this result confirms that these two RNAi clones' (F1 and F3) FNR1 expression of gene is suppressed really.

5, measure different algae strain hydrogen desorption capacities

(1) a lack of sulfur is handled

F1 positive colony that step 3 is obtained and F3 positive colony and wild-type CC400 chlamydomonas are inoculated into respectively in the TAP liquid nutrient medium of 1L, are that 25 ℃ and light intensity are 200 μ Em in temperature ^-2S ^-1The continuous illumination condition under stir culture 3-5 days, to OD ₇₅₀During ≈ 1.5, obtain being in the cell of logarithmic phase.2, centrifugal 2 minutes of 500g, (cell density is 2 * 10 to the cell of collection logarithmic phase ⁶-5 * 10 ⁶Individual ml ^-1), with the washing of TAP-S substratum once, will precipitate with a small amount of TAP-S substratum suspension, get 20ul suspension algae liquid, add 980ul a lack of sulfur substratum.Mix the back and add 4ml acetone, centrifugal 5 minutes of 5000rpm, the light absorption value at mensuration supernatant liquor 663nm and 645nm place, (Chl (a+b)=8.02*OD663+20.21*OD645) calculates total chlorophyll content according to formula.

With suspension transfer to 100mL put the hydrogen bottle (Schott type, Germany) in, to 100mL, to adjust the initial chlorophyll concentration of culture system be 30 μ gml by calculating resulting total chlorophyll content and volume of culture with TAP-S culture medium supplemented volume ^-1Seal a bottle with plug, seal bottleneck, leak outside to prevent gas with paraffin.To place temperature be 25 ℃ and be 200 μ Em in intensity of illumination with culturing bottle ^-2S ^-1Cultivate in the incubator, get different time point determining hydrogen desorption capacities respectively.

Adopt SHIMADZU GC-2014 gas Chromatographic Determination hydrogen content, carrier gas is a nitrogen.Put hydrogen bottle gas part 500 μ L with the absorption of 1mL syringe during mensuration, inject sample holes, calculate gas volume with the methane external standard method.

The gas-chromatography parameter is set to when measuring hydrogen desorption capacity:

Detector: TCD detector; Nitrogen flow rate: 60mlmin ^-1Detector temperature: 100 ℃; Column temperature: 60 ℃; Injector temperature: 100 ℃.

The result:

The photosynthetic hydrogen production situation of utilizing the wild-type algae strain (CC400) of gas Chromatographic Determination and RNAi clone (F1, F3) as shown in Figure 4.As seen from the figure, two RNAi clones' hydrogen desorption capacity is apparently higher than the strain of wild-type algae.Use H ₂And CH ₄Peak area ratio, calculate H ₂Volume, as shown in table 1.A lack of sulfur is in the time of 24 hours as can be seen, and the strain of wild-type algae has just begun to produce hydrogen, and hydrogen output only is 31.8 μ l/100mL substratum, and this moment, the hydrogen output of F1 and F3 then all was higher than 130 μ l/100mL substratum, was more than 4 times of wild-type algae strain.When a lack of sulfur was handled 48 hours, the hydrogen output of F1 and F3 reached 299.5 μ l/100mL substratum and 500.2 μ l/100mL substratum respectively, was 6-10 times of wild-type.

Table 1 wild-type algae strain (CC400) and RNAi clone's (F1, F3) photosynthetic hydrogen production amount

Claims (10)

1. genetically engineered chlamydomonas is the expression level by Triphosphopyridine nucleotide photoreductase-NADP reductase enzyme encoding gene in the chlamydomonas that suppresses to set out, the genetically engineered chlamydomonas that obtains.

2. genetically engineered chlamydomonas according to claim 1 is characterized in that: the expression level of the described Triphosphopyridine nucleotide photoreductase of described inhibition-NADP reductase enzyme encoding gene disturbs by RNA and realizes.

3. genetically engineered chlamydomonas according to claim 1 and 2 is characterized in that:

It is to import in the described chlamydomonas that sets out by the coding DNA with double-stranded RNA to realize that described RNA disturbs; Wherein, the nucleotide sequence of a chain of described double-stranded RNA is that the T in the base sequence of sequence in the sequence table 1 is replaced with the ribonucleoside acid sequence that U obtains, another chain and its reverse complemental.

4. according to arbitrary described genetically engineered chlamydomonas among the claim 1-3, it is characterized in that: the coding DNA of described double-stranded RNA is following double chain DNA fragment: SEQ _Forward-X-SEQ _Oppositely

Wherein, SEQ _ForwardNucleotide sequence be the base sequence of sequence 1 in the sequence table;

SEQ _OppositelyWith described SEQ _ForwardReverse complemental;

X is SEQ _ForwardWith SEQ _OppositelyBetween intervening sequence, X and described SEQ _ForwardAnd SEQ _OppositelyAll not complementary.

5. according to arbitrary described genetically engineered chlamydomonas among the claim 1-4, it is characterized in that:

The coding DNA of described double-stranded RNA imports the described chlamydomonas that sets out by recombinant expression vector;

The recombinant expression vector that described recombinant expression vector obtains for the multiple clone site of the coding DNA of described double-stranded RNA being inserted expression vector Maa7/XIR;

The described chlamydomonas that sets out is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) algae strain CC-400.

6. the application of arbitrary described genetically engineered chlamydomonas in preparation hydrogen among the claim 1-5.

7. double-stranded RNA, for as follows:

Article one, the nucleotide sequence of chain is that the T in the base sequence of sequence in the sequence table 1 is replaced with the ribonucleoside acid sequence that U obtains, another chain and its reverse complemental.

8. the coding DNA of the described double-stranded RNA of claim 7 is following double chain DNA fragment:

SEQ _Forward-X-SEQ _Oppositely

Wherein, SEQ _ForwardNucleotide sequence be the base sequence of sequence 1 in the sequence table;

SEQ _OppositelyWith described SEQ _ForwardReverse complemental;

X is SEQ _ForwardWith SEQ _OppositelyBetween intervening sequence, X and described SEQ _ForwardAnd SEQ _OppositelyAll not complementary.

9. the recombinant expression vector that contains the described coding DNA of claim 8.

10. recombinant expression vector according to claim 9 is characterized in that: the recombinant expression vector that described recombinant expression vector obtains for the multiple clone site of described coding DNA being inserted expression vector Maa7/XIR.

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