CN101845461A - Method for improving yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes - Google Patents

Abstract

The invention belongs to a biological hydrogen production technology, particularly to a method for improving the yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes. Traditional chlamydomonas reinhardtii hydrogen production has the disadvantages that chlamydomonas reinhardtii hydrogenase is sensitive to oxygen and is easily inhabited by the oxygen to inactivate; and the hydrogen production effect of chlamydomonas is limited. The invention discloses application of soybean hemoglobin globulin subunit genes in chlamydomonas reinhardtii hydrogen production, the soybean hemoglobin globulin subunit genes lba are constructed in a chlamydomonas reinhardtii chloroplast expression vector, and the expression vector is transformed into chlamydomonas reinhardtii chloroplast, so that the lba genes are expressed in the chlamydomonas reinhardtii chloroplast. The decrease of the oxygen content in a closed culture system of the transformed chlamydomonas reinhardtii is markedly quicker than that of the chlamydomonas reinhardtii of the untransformed genes, the oxygen content can be kept at a lower level, and the hydrogen yield is markedly increased; the method is applicable for all microalgae for hydrogen production.

Description

Soybean hemoglobin genes improves the method for yield of chlamydomonas reinhardtii biological hydrogen production through

Technical field

The invention belongs to bio-hydrogen production technology, specifically soybean hemoglobin genes improves the method for yield of chlamydomonas reinhardtii biological hydrogen production through.

Background technology

Energy shortage and environmental pollution become two hang-ups that contemporary mankind's social development faces, and the development and utilization clean energy is extremely urgent.Hydrogen energy density height only produces water, does not produce the CO with Greenhouse effect after the burning ₂With other toxic gas, environmentally safe, be a kind of desirable clean energy of great exploitation potential for its.The method of prior art for preparing hydrogen is to prepare hydrogen by water electrolysis, but this method consume electric power is actually with electric energy and exchanges hydrogen as energy source for.Biological hydrogen production is one of important channel that solves hydrogen source Sustainable Production problem.Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) is a kind of unicell green alga, its hydrogen enzyme (H ₂Ase) gene can be with the H that produces in the photosynthesis under anaerobic by abduction delivering ⁺And e ^-Synthetic H ₂, be discharged into the extracellular, be the Perfected process that utilizes solar energy production hydrogen.The Chlamydomonas reinhardtii fast growth, it is low to cultivate cost, and its hydrogen enzymic activity height is little algae photosynthetic-hydrogen-production algae kind that very big potentiality are arranged.But Chlamydomonas reinhardtii hydrogen enzyme is to oxygen sensitive, is easy to be subjected to the inhibition of oxygen and loses activity; And the oxygen photosynthetic primary product that is chlamydomonas.This a pair of contradiction has limited the product hydrogen effect of chlamydomonas, has limited chlamydomonas and has produced the hydrogen The Application of Technology.Therefore, in order to improve chlamydomonas photosynthetic hydrogen production efficient, need reduce the intracellular oxygen content of chlamydomonas as much as possible.The way that reduces the intracellular oxygen content of chlamydomonas at present is by suppressing the activity of photosynthetical system II (PS II), put oxygen thereby realize suppressing photodissociation water, but this method having suppressed photosynthetic chain electron transport efficient simultaneously, has influenced the hydrogen generation efficiency of chlamydomonas; On the other hand, the growth of chlamydomonas is subjected to severe inhibition under the anoxia condition, has finally influenced hydrogen generation efficiency.

Nitrogenase in the legume nodule has very high nitrogenase activity, and contains a large amount of leghemoglobin (leghemoglobin, be called for short Lb) in the root nodule cell substantial connection is arranged.Leghemoglobin has with oxygen is reversible and combines, reduces in the cell oxygen concn and regulate respiratory characteristic, can keep oxygen content lower in the root nodule cell, can ensure the energy that nitrogen needs in oxygen that respiration needs and the fixed air again.

Leghemoglobin has two subunits to constitute, and one is the sphaeroprotein subunit, and is synthetic by leguminous plants; Another is the protoheme prothetic group, and is synthetic by symbiotic root nodule bacterium.Two subunits are combined into activated leghemoglobin.In vegetable cell, especially in chloroplast(id), protoheme prothetic group precursor can be synthetic by the chlorophyll route of synthesis.In the plant chloroplast, the former quinoline of pouncing on generates chlorophyll under the effect of magnesium chelatase; In the root nodule cell, the former quinoline synthetic protoheme prothetic group under the catalysis of root nodule bacterium synthetic ferrochelatase of pouncing on.Exist the proteolytic enzyme family that has with the pyridine nucleotide two sulfuric acid oxidation reductase enzymes (pyridine-nucleotide disulfide oxidoreductase) of ferrochelatase identity function and analog structure in the vegetable cell.

The present invention is directed to chlamydomonas and produce the key issue that hydrogen is cultivated needs oxygen free condition, be used to come from the leghemoglobin sphaeroprotein subunit gene lba of soybean (Glycine max) root nodule, solve needs to reduce the technical barrier that the interior oxygen content of cell guarantees its eubolism again as far as possible as far as possible in the chlamydomonas product hydrogen metabolic process.

Summary of the invention:

The object of the present invention is to provide a kind of leghemoglobin sphaeroprotein subunit gene lba that utilizes, solve Chlamydomonas reinhardtii and produce the interior oxygen content of reduction cell in the hydrogen, improve the hydrogenase activity, can ensure the technological method of the oxygen that respiration needs again.

The object of the present invention is achieved like this:

1, soybean hemoglobin genes improves the method for yield of chlamydomonas reinhardtii biological hydrogen production through, and step is as follows:

(1) cultivation of Chlamydomonas reinhardtii:

A, select cell walls shortcoming type Chlamydomonas reinhardtii algae kind cc849;

B, cultivation Chlamydomonas reinhardtii algae kind:

(a) liquid nutrient medium: 25 ± 1 ℃ of temperature; Fluorescent lamp intensity of illumination 100～200 micromole's photon/square metre second (μ mol photonsm ^-2s ^-1); 50～100ml Tutofusin tris-acetate-phosphoric acid salt (Tris-Acetate-Phosphate is called for short TAP) medium liquid is cultivated, initial pH7.2, horizontal shaking speed 100～130rpm, per 5～6 days 1% inoculation succeeding transfer culture;

(b) solid plate TAP substratum: agar powder 1.5%; Chlamydomonas reinhardtii mono-clonal on the picking flat board is seeded in dull and stereotyped going up by scribble method and preserves and purifying algae kind, and per 3 all subcultures once;

C, product hydrogen culture condition: in a lack of sulfur substratum, carry out, sal epsom, ferric sulfate, copper sulfate and the zinc sulfate of normal TAP substratum are changed to equimolar magnesium chloride, iron(ic) chloride, cupric chloride and zinc chloride respectively; To grow to the centrifugal 5min of Chlamydomonas reinhardtii 3000rpm room temperature in logarithmic phase later stage, collect frustule and also wash 3 times with a lack of sulfur substratum, be suspended in a lack of sulfur substratum, be contained in respectively in the culturing bottle, the space of 10ml is stayed in the culturing bottle top, and is airtight with the turned welt soft rubber ball; Dark condition was cultivated 24 hours down, was placed on then under the continuous illumination part to cultivate intensity of illumination 50～100 micromole's photon/square metre second (μ mol photons m ^-2s ^-1), 25 ± 1 ℃ of temperature; Carried out gaseous constituent and content detection in per 24 hours once;

D, measure hydrogen and oxygen content with GC: gas chromatograph is the 7890A model that U.S. Agilent Technologies company produces, molecular sieve 51/8 (OD), column length 2m, internal diameter 3mm, thermal conductivity detector TCD, with argon gas as carrier gas.Sampling volume 0.5ml, 50 ℃ of column temperatures, 200 ℃ of sample introduction temperature, 300 ℃ of thermal conductance detected temperatures;

(2) clone of leghemoglobin sphaeroprotein subunit lba gene:

A, get the sophisticated root nodule liquid nitrogen grinding of soybean, extract total RNA of soybean; With DNaseI37 ℃ of water-bath 30min of DNA lytic enzyme, remove miscellaneous DNA among the RNA; Add the 25mM ethylenediamine tetraacetic acid (EDTA), 65 ℃ of water-bath 10min, the activity of termination DNA lytic enzyme; 37 ℃ of synthesizing single-stranded cDNA of water-bath 60min;

B, carry out polymerase chain reaction (PCR) clone soybean lba gene, reaction conditions: 94 ℃ of pre-sex change 5min, a circulation with archaeal dna polymerase (ExTag); 94 ℃ of sex change 1min, 62 ℃ of annealing 30s, 72 ℃ are extended 1min; Totally 30 circulations; 72 ℃ are extended 10min, and the reaction product purifying reclaims;

The Auele Specific Primer of lba gene is identified in C, order-checking:

Lba-P1:5 '-c

Aaa ttt aaa ttt atg gtt gct ttc act gag-3 ', italic is represented the restriction enzyme site sequence of restriction endonuclease sma lI, the sequence in the square frame is the ribosome bind site RBS sequence of the enhancing translation ability of adding;

Lba-P2:5 '-tcc

Ata cta att atg cct tct taa tag c-3 ', italic is represented restriction enzyme SacI restriction enzyme site sequence;

(3) structure of Chlamydomonas reinhardtii chloroplast expression carrier:

The soybean lba gene fragment and the chloroplast expression carrier cg40 that come enzyme cutting clone to obtain with restriction endonuclease sma lI and SacI; The lba gene fragment is formed the aadA-lba fusion gene afterwards with the spectinomycin resistance gene aadA that the T4DNA ligase enzyme is connected among the plasmid cg40, obtain chlamydomonas chloroplast expression carrier cg401-1-lba, transformed into escherichia coli DH5 α, extract plasmid, identify through restriction endonuclease sma lI and SacI double digestion, obtain the lba fragment of 442bp and the plasmid fragment of 5900bp;

(4) conversion of Chlamydomonas reinhardtii chloroplast(id):

A, get the Chlamydomonas reinhardtii of 100ml logarithmic phase middle and later periods, 25 ℃ of centrifugal 5min of following 3000rpm collect frustules, and it is inferior to give a baby a bath on the third day after its birth with fresh TAP, is applied on the fresh TAP solid plate; Illumination 100 micromole's photon/square metre second (μ mol photonsm ^-2S ^-1) and 25 ± 1 ℃ of conditions under cultivated 24 hours; Robbing bombardment with gene transforms; Vacuum tightness 9.48192 * 10 ⁴Pa, target distance 9cm, helium pressure 7.584236 * 10 ⁶Pa, the bronze bullet is through restriction enzyme EcoRI digested plasmid cg401-1-lba DNA parcel, 1.0 microns of bronze bullet particle diameters;

B, the chlamydomonas after transforming are under illumination and 25 ± 1 ℃ of conditions, 18h is cultivated in transition, with TAP liquid nutrient medium flushing, coat the TAP solid that contains spectinomycin 100 μ g/ml and select on the substratum, 25 ± 1 ℃ and 100 micromole's photon/square metre second (μ mol photonsm ^-2S ^-1) the continuous illumination condition under, cultivated about 7～15 days; Single algae of getting resistance falls respectively and to select on the substratum repeatedly succeeding transfer culture; Produce hydrogen respectively and cultivate, and detect hydrogen output and oxygen-consumption;

(5) integration of lba gene and expression thereof in the transgene Chlamydomonas reinhardtii:

A, lba gene integration are to the detection of Chlamydomonas reinhardtii chloroplast DNA:

Take the logarithm the growth later stage chlamydomonas nutrient solution, 4 ℃ of low-speed centrifugals are collected, the 0.1mol/L sodium-chlor that adds 350 μ lNET, the 50mmol/L ethylenediamine tetraacetic acid (EDTA), the resuspended precipitation of Tutofusin tris-hydrochloric acid of pH value 8.020mmol/L adds Proteinase K and 25 μ l, 20% sodium lauryl sulphate (SDS) of 25 μ l10mg/ml, mixes, 37 ℃ of water-baths 12 hours, cooling; Add 200 μ l 5mol/L Potassium ethanoates (KAc), leave standstill on ice, centrifugal, add isopyknic 25: 24: 1 phenol/chloroforms/primary isoamyl alcohol extracting in the supernatant liquor 2 times; Use isopyknic chloroform extracting 1 time again; Total DNA is dissolved in 30 μ l Tutofusin tris-ethylenediamine tetraacetic acid (EDTA)s (Tris-EDTA is called for short TE) damping fluid with dehydrated alcohol precipitation and 70% washing with alcohol after the drying;

The detection of lba genetic transcription in B, the transgene Chlamydomonas reinhardtii:

The Chlamydomonas reinhardtii of taking the logarithm mid-term in vegetative period, room temperature 3000rpm collected frustule in centrifugal 5 minutes; Extract the total RNA and the synthetic cDNA of Chlamydomonas reinhardtii; Utilize the Auele Specific Primer and the described PCR condition of above-mentioned clone lba gene of above-mentioned lba gene to increase, carry out the detection of lba gene transcription level, obtain the amplified band of 442bp;

The detection of Lba expression amount in C, the transgene Chlamydomonas reinhardtii:

Get and produce the Chlamydomonas reinhardtii nutrient solution that hydrogen is cultivated, the quick centrifugal 5min of room temperature 3000rpm collects frustule, suspends with 250 μ l protein extraction damping fluids, adds 2 μ l beta-mercaptoethanols, frozen-thawed three times; Under 4 ℃ of conditions, the centrifugal 20min of 14000g, the albumen supernatant liquor of getting 100 μ g adds the damping fluid (Tris-HCl) of 0.25 mole of Tutofusin tris-hydrochloric acid preparation, pH 8.0; 25% glycerine; 7.5% sodium lauryl sulphate (SDS); 0.25mg/ml bromjophenol blue (bromophenolblue); 12.5% (v/v) beta-mercaptoethanol (2-mercaptoethanol) in 10% separation gel and 5% concentrated glue carry out denaturing polyacrylamide gel electrophoresis (SDS-PAGE), change polyvinylidene difluoride (PVDF) (PVDF) film, the beans Lba of the Chinese People's Anti-Japanese Military and Political College polyclonal antibody property advanced immuning hybridization detects, antibody to horseradish peroxidase (Horseradish Peroxidase is called for short HRP) mark carries out chemiluminescence detection;

(6) analyze the influence of reorganization leghemoglobin sphaeroprotein subunit Lba to the Chlamydomonas reinhardtii growth:

A, usefulness TU-1901 twin-beam ultra-violet and visible spectrophotometer detect the absorbancy of Chlamydomonas reinhardtii at the 750nm place;

B, get the centrifugal 5min of algae liquid 3000rpm and collect frustule, add 95% ethanolic soln with volume, mix, the room temperature lucifuge is placed 20min, 4 ℃ of centrifugal 10min of following 12000rpm; Get supernatant, measure OD ₆₆₅And OD ₆₄₉Light absorption value;

C, calculating chlorophyll a, chlorophyll b and total chlorophyllous content, the mg/L of unit: chlorofucine hl (mg/l)=20.04 (OD ₆₄₉)+6.10 (OD ₆₆₅);

(7) analyze leghemoglobin sphaeroprotein subunit Lba Chlamydomonas reinhardtii produced the oxygen-consumption of hydrogen cultivation and the influence of hydrogen output:

The detection of oxygen-consumption and hydrogen output in A, a lack of sulfur substratum:

In B, the sulfuricum culture-medium to the detection of oxygen-consumption and hydrogen output.

Chlamydomonas reinhardtii mono-clonal inoculation method is the scribble method inoculation among step of the present invention (1) B (b).

The bronze bullet wraps up through restriction enzyme EcoRI digested plasmid cg401-1-lba DNA among step (4) A.

The frozen-thawed number of times is three times among step (5) C.

The spectrophotometric that uses among step (6) A is counted TU-1901 twin-beam ultraviolet-visible photometer.

The application method of leghemoglobin gene of the present invention in green alga or blue-green algae product hydrogen, make to reduce the interior oxygen content of microalgae cell and improve hydrogen output and become possibility, overcome a great technical barrier in the biological hydrogen production, opening up new forms of energy for the mankind has great promotion and pushing effect.

By discovering, after soybean hemoglobin sphaeroprotein subunit gene lba expressed in the chloroplast(id) of Chlamydomonas reinhardtii, the render transgenic chlamydomonas produced the oxygen content fast speed reduction in the hydrogen culture system and maintains lower oxygen content level, and hydrogen output increases.And this result is unexpected before being, its purposes also is that people seek in the biological hydrogen production field and unexpected always.

A leghemoglobin sphaeroprotein of the present invention subunit gene lba derives from soybean, the nucleotide sequence of this lba gene and aminoacid sequence are known, its gene pool (NCBI GenBank) sequence number is V00453, and those skilled in the art can retrieve the information that obtains this lba gene by the data message in the gene order.The present invention preferably has the nucleotide sequence shown in this sequence number information, and more preferably coding has the gene of the aminoacid sequence shown in this sequence number information.In the example of the present invention, a leghemoglobin sphaeroprotein subunit gene lba who mentions especially clones the cDNA in soybean nodulation, utilization well known to a person skilled in the art method, can directly from the soybean nodulation cell, extract mRNA, reverse transcription becomes cDNA again, is used for leghemoglobin sphaeroprotein subunit gene lba of the present invention with the PCR method clone then.Selectively according to known nucleotide sequence and synthetic this gene of aminoacid sequence.Selectively utilize in the existing plasmid that contains this gene and extract this goal gene.

Expression vector of the present invention is except the top leghemoglobin sphaeroprotein subunit gene lba gene of mentioning especially, also comprise promotor, terminator, the selected marker, increase the regulator of transcript and expression stability and can make goal gene be incorporated on the host cell DNA and the gene fragment of stably express etc., these all are gene expression elements commonly used in plant and the work of algae transgenosis.In another specific examples of the present invention, the expression vector that is used for leghemoglobin sphaeroprotein subunit gene lba comprises that psbB promotor and terminator, 3 '-rbcL transcribe stability and strengthen element, aadA screening property marker gene and help this expression vector to be incorporated into cpDNA-1 sequence area that contains the atpB gene order and cpDNA-2 sequence area on the Chlamydomonas reinhardtii chloroplast DNA.

Promotor provided by the invention, terminator, increase are transcribed the regulator of stability and help goal gene to be incorporated into the kind of the gene fragment on the chloroplast DNA unrestricted, as long as can import and be incorporated on the Chlamydomonas reinhardtii chloroplast DNA, can express this stable gene just passable at leghemoglobin sphaeroprotein subunit gene lba gene.Preferably psbB promotor and terminator, 3 '-rbcL gene order and the Chlamydomonas reinhardtii chloroplast(id) native gene expression regulation elements such as gene order fragment that contain the atpB gene order.

The conversion of indication of the present invention comprises particle gun conversion method and electric shock conversion method, more preferably particle gun conversion method.Concrete being beneficial under the condition that expression vector imports,, will be contained the expression vector that comprises leghemoglobin sphaeroprotein subunit gene lba and import in the chloroplast(id) of Chlamydomonas reinhardtii cell by the bronze of expression vector dna bombardment chlamydomonas cell with bag.

The present invention goes out to express the transgene Chlamydomonas reinhardtii of leghemoglobin sphaeroprotein subunit Lba by antibiotic selective screening, and further succeeding transfer culture repeatedly on the microbiotic flat board, increase the integration rate of goal gene in the transgene Chlamydomonas reinhardtii chloroplast DNA, and the transgene Chlamydomonas reinhardtii mono-clonal that oxygen content is lower in the hydrogen culture system, hydrogen output is higher is produced in screening.

The present invention expresses and is interpreted as that the genetic information with initiate dna or RNA is transferred to gene product polypeptide or protein, i.e. leghemoglobin sphaeroprotein subunit.

Transgenosis of the present invention or reorganization be interpreted as genetic information be transferred to organism, particularly Chlamydomonas reinhardtii.This means to comprise all methods of introducing the absorption of for example particle bombardment, electroporation, chemistry mediation or the mediation of purulence bacillus, microinjection etc. to the known information of skilled work personnel.Genetic information can be introduced cell, for example with DNA, RNA, plasmid or with any alternate manner, and mixes the mode of host genome by reorganization.Transgene Chlamydomonas reinhardtii of the present invention is the Chlamydomonas reinhardtii through genetic modification.

The present invention provides new capability and the new purposes that the leghemoglobin gene is undiscovered and put down in writing in the world first; Provide a kind of first and reduced oxygen content and the novel method that improves hydrogen output in the green alga product hydrogen culture system, so the present invention has novelty and creativeness.The present invention for the mankind provide cleaning the desirable energy, is to solve the human energy scarcity that faces, the invention of the great difficult problem of environmental pollution that great practical value is arranged by biological hydrogen production.

Advantage of the present invention is as follows:

1, provides a transgene Chlamydomonas reinhardtii algae kind that increases with hydrogen output that in chloroplast(id), express leghemoglobin sphaeroprotein subunit gene lba first.

2, the Chlamydomonas reinhardtii hydrogen output obviously increases.

3, the inventive method is applicable to little algae of all product hydrogen.

For the organism of the object of the invention be have the little algaes of product hydrogen of photosynthetic activity and need to breathe provide energy to produce metabolic little algae of hydrogen or microorganism, as miniature green alga, blue-green algae, photosynthetic bacterium.Preferred algae is miniature green alga such as Chlamydomonas reinhardtii and blue-green algae such as synechocystis (Synechocystis).

Description of drawings

Fig. 1 transforms carrier cg401-1-lba oligogene component structure figure for the Chlamydomonas reinhardtii chloroplast(id).

Fig. 2 cuts the evaluation electrophorogram for the enzyme of Chlamydomonas reinhardtii chloroplast expression carrier cg40-1-1-lba.

Fig. 3 is containing 100mg.L for the Chlamydomonas reinhardtii that transforms the lba gene in the chloroplast(id) ^-1(figure below) screening in (last figure) and the TAP liquid nutrient medium on the TAP flat board of spectinomycin.A: the Chlamydomonas reinhardtii that transforms the lba gene; B: the Chlamydomonas reinhardtii (positive controls) that transforms the cg40 carrier; C: the Chlamydomonas reinhardtii of conversion carrier cg401-1 (positive controls); D: not genetically modified Chlamydomonas reinhardtii 849 (negative control group).

Fig. 4 is that the PCR that transforms the lba gene in the Chlamydomonas reinhardtii chloroplast(id) identifies figure.A figure: with total DNA is the PCR electrophorogram of masterplate; 1 transgene Chlamydomonas reinhardtii; 2 transgene Chlamydomonas reinhardtiis not; 3 molecule marker DL2000; B figure: with cDNA is the PCR electrophorogram of masterplate; The 1-3 transgene Chlamydomonas reinhardtii; 4 transgene Chlamydomonas reinhardtiis not; 5 molecule marker DL2000.

Fig. 5 is for carrying out WesternBlot detected result figure to Lba expression amount in the Chlamydomonas reinhardtii that transforms the lba gene in the chloroplast(id).C: the protein sample (positive controls) of expressing the e. coli bl21 of cg401-1-lba.Preceding set of number 0,3,5 and 7 refers to that respectively transgene Chlamydomonas reinhardtii begins to produce the fate that hydrogen is cultivated; Back set of number 3 and 5 refers to that respectively not genetically modified Chlamydomonas reinhardtii begins to produce the fate (negative control group) that hydrogen is cultivated.

Fig. 6 transforms the Chlamydomonas reinhardtii of lba and the comparison diagram of the growing state of transgene Chlamydomonas reinhardtii 849 not in the chloroplast(id).

Fig. 7 is the comparison diagram of oxygen-consumption and hydrogen output under the product hydrogen culture condition that transforms the Chlamydomonas reinhardtii of lba and transgene Chlamydomonas reinhardtii 849 not in the chloroplast(id) contain 0,12.5,50,100 μ M vitriol respectively in substratum.

Embodiment

Embodiment 1:

Chlamydomonas reinhardtii and cultivation thereof:

Because Chlamydomonas reinhardtii fast growth, low, the active height of hydrogenase of cultivation cost are so be the representative species of little algae photosynthetic-hydrogen-production.For render transgenic is operated easily, the Chlamydomonas reinhardtii algae kind cc849 of preferred cell wall shortcoming type of the present invention.

1. normal culture condition: according to Harris chief editor's " The Chlamydomonas Sourcebook:a comprehensive guide to biology and laboratory use.New York:AcademicPress.1989 ", preferably at 25 ± 1 ℃, fluorescent lamp intensity of illumination (100～200mol photonsm ^-2s ^-1), liquid culture is at 50～100ml Tris-Acetate-Phosphate (being called for short TAP) substratum, initial pH7.2, horizontal shaking speed 100～130rpm, per 5～6 days 1% inoculation succeeding transfer culture; Solid TAP (flat board) substratum contains 1.5% agar powder, and the preservation of algae kind and purifying are that the Chlamydomonas reinhardtii mono-clonal on the picking flat board is seeded on the flat board by scribble method, and per 3 all subcultures once.

2. produce the hydrogen culture condition: (it is to carry out in a lack of sulfur substratum (being called for short TAP-S) that the product hydrogen of Chlamydomonas reinhardtii is cultivated for Plant Physiology.2000, method 122:127-136) according to Melis etc.A lack of sulfur substratum (TAP-S) is that sal epsom, ferric sulfate, copper sulfate and the zinc sulfate of normal TAP substratum are changed to equimolar magnesium chloride, iron(ic) chloride, cupric chloride and zinc chloride respectively.The present invention can use in suitability for industrialized production for this technology in the future, the autotelic operation steps of having simplified its product hydrogen cultivation: the centrifugal 5min of Chlamydomonas reinhardtii 3000rpm room temperature that is about to grow to the logarithmic phase later stage, collecting frustule also washes 3 times with the TAP-S substratum, be suspended in TAP-S then or contain in the TAP-S substratum of sulfate with different, be contained in respectively in the culturing bottle of 50ml, the space of 10ml is stayed in the culturing bottle top, use the airtight cultivation of turned welt soft rubber ball jam-pack culturing bottle at once, be placed on earlier under the dark condition and cultivated 24 hours, be placed on then under the continuous illumination part and cultivate intensity of illumination 50～100 μ mol photons m ^-2s ^-1, 25 ± 1 ℃ of temperature.Carried out gaseous constituent and content detection with gas in the airtight sampler extraction of the trace bottle in per 24 hours.

3. according to Ran Chunqiu etc. (SCI, 2006, method 27:62-66.) is measured hydrogen and oxygen content with GC.Gas chromatograph is the 7890A that U.S. Agilent Technologies company produces, molecular sieve 5 * 1/8 (OD), column length 2m, internal diameter 3mm, thermal conductivity detector TCD, with argon gas as carrier gas.Sampling volume 0.5ml, 50 ℃ of column temperatures, 200 ℃ of sample introduction temperature, 300 ℃ of thermal conductance detected temperatures.Calculate hydrogen and oxygen volume with the known external standard method of those skilled in the art.

Embodiment 2:

The clone of leghemoglobin sphaeroprotein subunit lba gene:

Product description (QIAGEN by known standard method of those skilled in the art (Sambrook etc., MolecularCloning.New York:Cold Spring Harbor Laboratory Press.1998) and agents useful for same manufacturers ^TM, Promega ^TM) extract total RNA of soybean and synthetic cDNA and RNA is carried out concentration and purity detecting.Specific implementation method is: get the sophisticated root nodule 0.1g of soybean, liquid nitrogen grinding is extracted total RNA of soybean according to QIAGEN RNeasy Plant Mini Kit reagent manufacturer specification, and its concentration and purity detect with ultraviolet spectrophotometry.And use DNase I

37 ℃ of water-bath 30min remove miscellaneous DNA among the RNA; Add 25mM EDTA then, 65 ℃ of water-bath 10min, the activity of termination DNase I.The synthetic of strand cDNA is according to Promega ^TMAMVRT protocol manufacturer specification carries out: contain total RNA and the 1 μ gRadom Primer of 2 μ g in 25 μ l reaction systems, 37 ℃ of water-bath 60min.

The present invention designs Auele Specific Primer according to the nucleotide sequence of soybean lba gene (sequence number V00453) among the NCBI GenBank, and adds specific enzymes respectively and cut site sequence before primer, carries out PCR reaction clone soybean lba gene with ExTag.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min, a circulation; 94 ℃ of sex change 1min, 62 ℃ of annealing 30s, 72 ℃ are extended 1min; Totally 30 circulations; 72 ℃ are extended 10min, and the PCR product reclaims with the test kit purifying that QIAGEN company produces, and exactness is identified in order-checking.The Auele Specific Primer of lba gene is as follows:

Lba-P1:5 '-c

Aaa ttt aaa ttt atg gtt gct ttc actgag-3 ', italic is represented restriction endonuclease sma l I restriction enzyme site sequence, the sequence in the square frame is the ribosome bind site RBS sequence of the enhancing translation ability of adding.

Lba-P2:5 '-tcc

Ata cta att atg cct tct taa tag c-3 ', italic is represented restriction enzyme SacI restriction enzyme site sequence.

Embodiment 3:

The structure of Chlamydomonas reinhardtii chloroplast expression carrier:

By the known standard method (Sambrook etc. of those skilled in the art, MolecularCloning.New York:Cold Spring Harbor Laboratory Press.1998) and method (The Plant Journal that Chlamydomonas reinhardtii chloroplast expression carrier cg40 is described such as Vaistij, 2000,21 (5): 469-482) carry out vector construction.Specific implementation method is: come enzyme to cut soybean lba gene fragment and carrier cg40 that above-mentioned clone obtains with restriction endonuclease sma lI and SacI respectively, the lba gene fragment of purifying is formed the aadA-lba fusion gene by above-mentioned two restriction enzyme sites afterwards with the aadA gene that the T4DNA ligase enzyme is connected among the plasmid cg40, obtain chlamydomonas chloroplast expression carrier cg401-1-lba (Fig. 1), transformed into escherichia coli DH5 α, extract plasmid, identify through Restriction enzyme Sma I and SacI double digestion, obtain the lba purpose fragment of the correct 442bp of size and the plasmid fragment (Fig. 2) of 5900bp.

Embodiment 4:

The conversion of Chlamydomonas reinhardtii chloroplast(id):

(Science, 1988,240 (4858): method 1534-1538) transforms the Chlamydomonas reinhardtii chloroplast(id), gets 100ml and grows to the logarithmic phase middle and later periods (about 4～5 * 10 by Boynton etc. ⁶) Chlamydomonas reinhardtii, the centrifugal 5min of 25 ℃ of following 3000rpm collect frustules, it is inferior to give a baby a bath on the third day after its birth with fresh TAP, is applied on the fresh TAP solid plate, at illumination 100 μ molm ^-2S ^-1With cultivated under 25 ± 1 ℃ of conditions 1 day, with gene rob (

Model:PDS1000/He Biolistic particle delivery system)) bombard conversion.Parameter is: vacuum tightness 9.48192 * 10 ⁴Pa, target distance 9cm, helium pressure 7.584236 * 10 ⁶Pa,, bronze particle diameter 1.0 μ m.The bronze bullet wraps up with linearizing plasmid cg401-1-lba DNA (cutting through restriction enzyme EcoRI enzyme).

Chlamydomonas after the conversion is washed with the TAP liquid nutrient medium after cultivating through the 18h transition under 25 ℃ and illumination, coats on the TAP solid selection substratum that contains spectinomycin 100 μ g/ml, in 25 ℃ and 100 μ molm ^-2S ^-1The continuous illumination condition under; cultivated about 7～15 days; picking has single algae of resistance to fall respectively to select on the substratum repeatedly succeeding transfer culture (Fig. 3); produce hydrogen then respectively and cultivate and detect hydrogen output and oxygen-consumption, use the composition and the content of hydrogen, oxygen and nitrogen standard substance (Shanghai Jiliang Standard Gas Co., Ltd.) the definition gas of surveying.

Embodiment 5:

Analyze the integration and the expression thereof of lba gene in the transgene Chlamydomonas reinhardtii:

1. the lba gene integration is to the detection of Chlamydomonas reinhardtii chloroplast DNA:

Mensuration lba gene integration is the PCR method of implementing hereinafter described to the appropriate method of Chlamydomonas reinhardtii chloroplast DNA, if the lba gene is incorporated on the Chlamydomonas reinhardtii chloroplast DNA by the mode of homologous recombination exchange, be that the PCR product of template should be 5892bp then with the total DNA of transgenosis chlamydomonas, and the PCR product of not integrating should be 5400bp (Fig. 4, A).Be cpDNA-P1:5 '-aga cag cca aca ttt tgt ta-3 ' wherein with Chlamydomonas reinhardtii chloroplast DNA bonded primer; CpDNA-P2:5 '-gct tca aaa aca aaatca aa-3 '.

Method (Trends in Biochemistry Science, 1988,13:56-59) the total DNA of extraction chlamydomonas by Rochaix and Erickson.Particularly, get the chlamydomonas nutrient solution that 10ml is in the logarithmic growth later stage, 4 ℃ of low-speed centrifugals are collected, add 350 μ l NET (0.1mol/L NaCl, 50mmol/L EDTA, 20mmol/L Tris-HCl, pH 8.0) resuspended precipitation, the Proteinase K (Proteinase K) and the 25 μ l 20%SDS that add 25 μ l 10mg/ml, mixing also spends the night in 37 ℃ of water-baths, and cooled on ice adds 200 μ l 5mol/L KAc, it is centrifugal to leave standstill the back on ice, add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting 2 times in the supernatant, use isopyknic chloroform extracting 1 time again, total DNA precipitates and 70% washing with alcohol with dehydrated alcohol, be dissolved in 30 μ l TE damping fluids after the drying, be used for above-mentioned PCR and detect.

2. the detection of lba genetic transcription in the transgene Chlamydomonas reinhardtii:

Detect appropriate method that whether the lba gene transcribe be in transgene Chlamydomonas reinhardtii according to the known reverse transcription PCR of those skilled in the art standard method (Sambrook etc., Molecular Cloning.New York:Cold Spring Harbor Laboratory Press.1998) or with reference to the enforcement part of above-mentioned soybean lba gene clone method.The sampling of Chlamydomonas reinhardtii is about the Chlamydomonas reinhardtii 1ml that takes the logarithm mid-term in vegetative period, and room temperature 3000rpm collected frustule in centrifugal 5 minutes.Product description (QIAGEN according to test kit manufacturers ^TM, Promega ^TM) extract total RNA of Chlamydomonas reinhardtii and according to Promega ^TMAMV RTProtocol specification sheets synthesizes cDNA, utilize the Auele Specific Primer and the described PCR condition of above-mentioned clone lba gene of above-mentioned lba gene to increase, carry out the detection of lba gene transcription level, the result obtains amplified band (Fig. 4 of 442bp, B), order-checking back proof lba gene is correctly transcribed in transgene Chlamydomonas reinhardtii.

3. the detection of Lba expression amount in the transgene Chlamydomonas reinhardtii:

With reference to Hemschemeier etc. (Planta, 2008, method 227:397-407) is extracted the Chlamydomonas reinhardtii total protein and is carried out Western blot and detect.Particularly, get 20-30ml and produce the Chlamydomonas reinhardtii nutrient solution that hydrogen is cultivated, the centrifugal 5min of room temperature 3000rpm collects frustule fast, suspends with 250 μ l protein extraction damping fluids (50mM Tris/HCl pH 8.3,1%Triton-X 100), add 2 μ l beta-mercaptoethanols, behind the frozen-thawed three times, 4 ℃, the centrifugal 20min of 14000g, the albumen supernatant liquor of getting 100 μ g add the sample damping fluid (0.25M Tris-HCl, pH 8.0; 25%glycerol; 7.5%SDS; 0.25mg ml-1bromophenolblue; 12.5% (v/v) 2-mercaptoethanol) after separation gel in 10% and 5% concentrated glue carry out the SDS-PAGE gel electrophoresis, change pvdf membrane (Millipore, USA), one is anti-with the beans Lba of Chinese People's Anti-Japanese Military and Political College polyclonal antibody (go up marine section protein antibodies and the prepare company) property advanced immuning hybridization detection, according to ECL Plus (Amersham company) specification sheets the antibody of horseradish peroxidase HRP mark is carried out chemiluminescence detection, the result shows in the beginning in the 3rd day of producing the hydrogen cultivation can detect Lba, the 5th day expression amount maximum, almost detected less than (Fig. 5) again in the 7th day.For proteinic quantitatively and commentaries on classics film, sealing in testing of SDS-PAGE gel electrophoresis and western blot, to hybridize, wash operative techniquies such as film, development be the known standard method of those skilled in the art (Sambrook etc., Molecular Cloning.New York:Cold Spring HarborLaboratory Press.1998) or according to test kit manufacturer specification (Bio-Rad ^TMBradfordkit and GE Healthcare).

Embodiment 6:

Analyze the influence of reorganization leghemoglobin sphaeroprotein subunit Lba to the Chlamydomonas reinhardtii growth:

The present invention comes comparison to express the influence of leghemoglobin sphaeroprotein subunit Lba to the Chlamydomonas reinhardtii biomass in chloroplast(id) by the cell count of measuring transgenic alga front and back Chlamydomonas reinhardtii and the variation of chlorophyll content.The mensuration of frustule number and chlorophyll content is with reference to Harris chief editor " The ChlamydomonasSourcebook:a comprehensive guide to biology and laboratory use.NewYork:Academic Press.1989 " method, because Chlamydomonas reinhardtii is at the absorbancy (OD at 750nm place ₇₅₀) with the cell count positive correlation of Chlamydomonas reinhardtii, so the Chlamydomonas reinhardtii that directly detects cultivation with spectrophotometer (TU-1901 twin-beam ultraviolet-visible photometer) is in the absorbancy at the 750nm place growth indexes as the Chlamydomonas reinhardtii cell count.The mensuration of chlorophyll content is to get 1ml algae liquid, and the centrifugal 5min of 3000rpm collects frustule, adds 95% ethanolic soln with volume, mixing, and after the room temperature lucifuge was placed 20min, 4 ℃ of centrifugal 10min of following 12000rpm got supernatant liquor, measured OD ₆₆₅And OD ₆₄₉Light absorption value, calculate chlorophyll a according to following formula, chlorophyll b and total chlorophyllous content, unit is mg/L.Chlorofucine hl (mg/l)=20.04 * (OD ₆₄₉)+6.10 * (OD ₆₆₅).

The result shows that the growth of expressing leghemoglobin sphaeroprotein subunit Lba render transgenic Chlamydomonas reinhardtii in chloroplast(id) is subjected to slight inhibition (Fig. 6).

Embodiment 7:

Analyze leghemoglobin sphaeroprotein subunit Lba Chlamydomonas reinhardtii produced the oxygen-consumption of hydrogen cultivation and the influence of hydrogen output:

1. in a lack of sulfur substratum to the detection of oxygen-consumption and hydrogen output:

Because in a lack of sulfur substratum, the activity of Chlamydomonas reinhardtii PSII is suppressed, but respiration is unaffected, oxygen content under air-proof condition in the Chlamydomonas reinhardtii liquid nutrient medium descends fast because of respiration consumption, cause and occur anoxic condition in the liquid nutrient medium, induce Chlamydomonas reinhardtii hydrogen expression of enzymes and begin to produce hydrogen, by detecting oxygen consumption and the product hydrogen situation that the airborne oxygen sealed up for safekeeping sealing bottle top and hydrogen content changing conditions just can reflect Chlamydomonas reinhardtii.The result shows (Fig. 7, A, B), oxygen content in the Chlamydomonas reinhardtii culture system of commentaries on classics lba gene just descends soon from producing hydrogen cultivation beginning, in the 3rd day, reach minimum, be 3.3-4.8%, and the oxygen content in the not genetically modified Chlamydomonas reinhardtii culture system reaches minimum the 5th talent, is 6.3-7.2%, than the high 50-90% that changes lba gene Chlamydomonas reinhardtii.The hydrogen output of Chlamydomonas reinhardtii culture system that changes the lba gene is similar with the hydrogen output of genetically modified Chlamydomonas reinhardtii not at preceding 4 days that produce that hydrogen cultivates, but rose rapidly at the 5th day, reached maximum at the 6th day, be 178 μ l, than transgene Chlamydomonas reinhardtii is not high by 48% at the maximum 120 μ l of accumulation in the 8th day.Accordingly, the product hydrogen maximum rate that changes lba gene Chlamydomonas reinhardtii appears at the 5th day, is 11.25 μ lmg ^-1Chlh ^-1, than transgene Chlamydomonas reinhardtii not at 3 days maximum hydrogen-producing speed, 3.47 μ lmg ^-1Chlh ^-1High 2.2 times.

Western blot detect to find (Fig. 5), and the expression amount that changes reorganization Lba in the lba gene Chlamydomonas reinhardtii can be detected producing the beginning in the 3rd day that hydrogen cultivates, and reached maximum at the 5th day, began again to quickly fall in the 7th day almost detect less than.This result shows that the fast expression amount with reorganization Lba of oxygen content decline that changes lba gene Chlamydomonas reinhardtii is proportionate; And the maximum hydrogen-producing speed that changes lba gene Chlamydomonas reinhardtii appears at that to produce the 5th day this time that hydrogen cultivates consistent with the time that the maximum expression amount of reorganization Lba occurs, and oxygen consumption and the product hydrogen generation certain influence of reorganization Lba to transgene Chlamydomonas reinhardtii be described.

2. in sulfuricum culture-medium to the influence of oxygen-consumption and hydrogen output:

Though in a lack of sulfur substratum, can improve the hydrogen generation efficiency of Chlamydomonas reinhardtii, also suppressed photosynthetic electronic efficiency when lacking element sulphur, it is low finally to cause producing the hydrogen rate.And in substratum, recover a certain amount of element sulphur, can partly recover the activity of PSII, if the concentration of control element sulphur makes the respiration oxygen consumption rate greater than photosynthetic oxygen evolution speed, still can make culture system be in anoxic condition, but photosynthetic electronic efficiency can increase, and can increase hydrogen output in theory.

Result of the present invention shows (Fig. 7, C, D), when in substratum, containing 12.5 μ M vitriol, the oxygen content lowering speed of changeing lba gene Chlamydomonas reinhardtii culture system is still very fast, when cultivating the 4th day, product hydrogen drops to lower-most point, be 3.3%, than the lower-most point 5.9% of transgene Chlamydomonas reinhardtii not low 78%, and the hypoxia that maintains 3.3-4.3% just made oxygen content go back up to 21% at the 8th day gradually because the element sulphur in the substratum has recovered part PSII activity for two days afterwards; And the oxygen content of transgene Chlamydomonas reinhardtii after lower-most point second day does not just go back up to 21% rapidly, afterwards several days even reach 24-26%.The hydrogen output that changes lba gene Chlamydomonas reinhardtii culture system reached maximum at the 7th day, be 137 μ l, and the maximum hydrogen output of transgene Chlamydomonas reinhardtii culture system only is not 98 μ l.The maximum hydrogen-producing speed that changes lba gene Chlamydomonas reinhardtii also appears at the 5th day, is 7.13 μ lmg ^-1Chlh ^-1, be about 2 times of transgene Chlamydomonas reinhardtii not.

(Fig. 7 when in substratum, containing 50-100 μ M vitriol, E, F, G, H), the oxygen content lowering speed of changeing lba gene Chlamydomonas reinhardtii culture system is still very fast, all can drop to Schwellenwert 3.7% or 3.4% at the 3rd day or the 4th day that produces the hydrogen cultivation, and all can maintain such hypoxemia level for two days, oxygen content just was elevated to 21-23% gradually at the 7th day or the 6th day that cultivates then.And the oxygen content of transgene Chlamydomonas reinhardtii culture system did not all only drop to 9.9%-16% at the 3rd day or the 2nd day, just was elevated to 24-26% then rapidly.The maximum hydrogen output that changes lba gene Chlamydomonas reinhardtii culture system maintains 138-145 μ l, and the maximum hydrogen output of transgene Chlamydomonas reinhardtii culture system does not drop to 66-51 μ l gradually.The maximum hydrogen-producing speed that changes lba gene Chlamydomonas reinhardtii maintains 7.98-7.07 μ lmg ^-1Chlh ^-1, and the maximum hydrogen-producing speed of transgene Chlamydomonas reinhardtii does not drop to 3.47-2.80 μ lmg ^-1Chlh ^-1

Result of the present invention shows, the hydrogen output that changes lba gene Chlamydomonas reinhardtii recovers can be stabilized under the condition of Determination of Trace Sulfur element the level of 137 μ l-145 μ l in substratum, and the hydrogen output of transgene Chlamydomonas reinhardtii does not drop to 51 μ l under the 100 μ M vitriol conditions gradually from 120 μ l under a lack of sulfur condition.

To sum up the result shows, expressing the lba gene in the Chlamydomonas reinhardtii chloroplast(id) can accelerate the oxygen depletion speed of Chlamydomonas reinhardtii product hydrogen culture system significantly and keep lower oxygen content in culture system, can obviously improve the hydrogen output that changes lba gene Chlamydomonas reinhardtii, the influence that not increased by sulphate content even in the substratum that recovers certain sulphate content, still can make hydrogen output maintain higher level.And the reorganization maximum expression amount time of occurrence of Lba in Chlamydomonas reinhardtii is consistent with the time that its maximum hydrogen-producing speed occurs, and the increase that the reduction of the increase of changeing keto consumpting speed in the lba gene Chlamydomonas reinhardtii product hydrogen culture system, oxygen content and hydrogen output be describeds is that the expression with the Lba that recombinates links together.If can improve the expression amount of Lba in this transgene Chlamydomonas reinhardtii, further increase the oxygen-consuming capacity that produces in the hydrogen culture system, this method can be used for further improving the hydrogen production potential of miniature green alga and blue-green algae.Below annex be the nucleotides sequence tabulation of the soybean 1ba gene (gene pool sequence number V00453) that the present invention relates to.

Annex:

1, the nucleotide sequence of the soybean 1ba gene that the present invention relates to (gene pool sequence number V00453) is:

1 aagctttggt?tttctcactc?tccaagccct?ctatataaac?aaatattgga?gtgaagttgt

61 tgcataactt?gcatcgaaca?attaatagaa?ataacagaaa?attaaaaaag?aaatatggtt

121 gctttcactg?agaagcaaga?tgctttggtg?agtagctcat?tcgaagcatt?caaggcaaac

181 attcctcaat?acagcgttgt?gttctacact?tcgtaagttt?tctctctaag?catgtgtctt

241 ccattctatg?tttttctttt?ggaaatttgt?tgtgtttgaa?aaaagatata?ttgttaatgt

301 gagtggtttt?ggtttgatta?aaaatgaata?ggatactgga?gaaagcacct?gcagcaaagg

361 acttgttctc?atttctagca?aatggagtag?accccactaa?tcctaagctc?acgggccatg

421 ctgaaaagct?ttttgcattg?gtaagtatca?gccaactaaa?attataacta?ttttatgtga

481 ttaattttaa?gattaagcat?catgtatttt?aacactctta?aaacatcaat?gaacattaat

541 tgtttgaatt?gtattttata?tttttgccat?atcttgaact?aggaatagta?tataaatttc

601 tattagtatt?tgttgataat?tatttttctt?tcataactat?cttgtcacat?attatatatt

661 ttttgaattg?taggtgcgtg?actcagctgg?tcaacttaaa?gcaagtggaa?cagtggtggc

721 tgatgccgca?cttggttctg?ttcatgccca?aaaagcagtc?actgatcctc?agttcgtggt

781 atgataaata?atgaaatgtt?ataataaatt?atgcatactt?caatttttca?tggagcagta

841 taatgatcaa?cacacacttc?ttttgtttca?tgcatttgat?aactacaatc?ttaaaatgtt

901 gcaatcttaa?aaatagtatt?aaaaatataa?catttaatta?gctcatcaat?atttttctgt

961 tgcaattttt?tatgaaaaaa?ttataattat?gaattctttg?agcaatgttt?aattaaaaaa

1021 ttgatttaat?aatgaaataa?ctaagctacc?tctgtctcgt?ttttcattta?aactatgaca

1081 taaacaatga?ataaagtaaa?ctaaaccatg?acatgtttat?ttttgaatga?ggttattaat

1141 aatttttttt?cactatctat?tgcaatgttc?attgattatc?aattatcttg?gttgcattga

1201 ttctctcgat?ttttttcttg?aggttaagct?tcagttcaat?atatattcat?tttttgataa

1261 aaaaaaatag?tacaatatat?tttcatttag?ctgatcatat?ttatttaagt?tcaacttaaa

1321 attttataga?tgttaattga?tataatttgt?tgagatgatg?agaagaccaa?taccattacg

1381 tactcttttg?aaagtgttat?atggatttta?attataagga?aaaatgtaag?agctaaacca

1441 ttgctgatga?ttttgaaggt?ggttaaagaa?gcactgctga?aaacaataaa?ggcagcagtt

1501 ggggacaaat?ggagtgacga?gttgagccgt?gcttgggaag?tagcctacga?tgaattggca

1561 gcagctatta?agaaggcata?attagtatct?attgcagtaa?agtgtaataa?ataaatcttg

1621 tttcactata?aaacttgtta?ctattagaca?agggcctgat?acaaaatgtt?ggttaaaata

1681 atggaattat?atagtattgg?ataaaaatct?taaggttaat?attctatatt?tgcgtaggtt

1741 tatgcttgtg?aatcattatc?ggtatttttt?ttcctttctg?ataattaatc?ggtaaattat

1801 acaaataagt?tcaaaatgat?ttatatgttt?caaaattatt?ttaacagcag?gtaaaatgtt

1861 atttggtacg?aaagctaatt?cgtcga

Wherein: the exon of expression (exon) is nucleotide sequence 115bp-212bp, 332bp-440bp, 674bp-778bp and 1459bp-1588bp.

2, the aminoacid sequence of the soybean 1ba gene that the present invention relates to (gene pool sequence number V00453) is:

MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAEKL

FALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEV

AYDELAAAIKKA

Claims (1)

1. soybean hemoglobin genes improves the method for yield of chlamydomonas reinhardtii biological hydrogen production through, and step is as follows:

(1) cultivation of Chlamydomonas reinhardtii:

A, select cell walls shortcoming type Chlamydomonas reinhardtii algae kind cc849;

B, cultivation Chlamydomonas reinhardtii algae kind:

(a) liquid nutrient medium: 25 ± 1 ℃ of temperature; Fluorescent lamp intensity of illumination 100～200 micromole's photon/square metre. second (μ mol photons m ^-2s ^-1); 50～100ml Tutofusin tris-acetate-phosphoric acid salt (Tris-Acetate-Phosphate is called for short TAP) medium liquid is cultivated, initial pH7.2, horizontal shaking speed 100～130rpm, per 5～6 days 1% inoculation succeeding transfer culture;

(b) solid plate TAP substratum: agar powder 1.5%; Chlamydomonas reinhardtii mono-clonal on the picking flat board is seeded in dull and stereotyped going up by scribble method and preserves and purifying algae kind, and per 3 all subcultures once;

C, product hydrogen culture condition: in a lack of sulfur substratum, carry out, sal epsom, ferric sulfate, copper sulfate and the zinc sulfate of normal TAP substratum are changed to equimolar magnesium chloride, iron(ic) chloride, cupric chloride and zinc chloride respectively; To grow to the centrifugal 5min of Chlamydomonas reinhardtii 3000rpm room temperature in logarithmic phase later stage, collect frustule and also wash 3 times with a lack of sulfur substratum, be suspended in a lack of sulfur substratum, be contained in respectively in the culturing bottle, the space of 10ml is stayed in the culturing bottle top, and is airtight with the turned welt soft rubber ball; Dark condition was cultivated 24 hours down, was placed on then under the continuous illumination part to cultivate intensity of illumination 50～100 micromole's photon/square metre second (μ mol photons m ^-2s ^-1), 25 ± 1 ℃ of temperature; Carried out gaseous constituent and content detection in per 24 hours once;

D, measure hydrogen and oxygen content with GC: gas chromatograph is the 7890A model that U.S. Agilent Technologies company produces, molecular sieve 51/8 (OD), column length 2m, internal diameter 3mm, thermal conductivity detector TCD, with argon gas as carrier gas.Sampling volume 0.5ml, 50 ℃ of column temperatures, 200 ℃ of sample introduction temperature, 300 ℃ of thermal conductance detected temperatures;

(2) clone of leghemoglobin sphaeroprotein subunit 1ba gene:

A, get the sophisticated root nodule liquid nitrogen grinding of soybean, extract total RNA of soybean; With I37 ℃ of water-bath 30min of DNA lytic enzyme DNase, remove miscellaneous DNA among the RNA; Add the 25mM ethylenediamine tetraacetic acid (EDTA), 65 ℃ of water-bath 10min, the activity of termination DNA lytic enzyme; 37 ℃ of synthesizing single-stranded cDNA of water-bath 60min;

B, carry out polymerase chain reaction (PCR) clone soybean 1ba gene, reaction conditions: 94 ℃ of pre-sex change 5min, a circulation with archaeal dna polymerase (ExTag); 94 ℃ of sex change 1min, 62 ℃ of annealing 30s, 72 ℃ are extended 1min; Totally 30 circulations; 72 ℃ are extended 10min, and the reaction product purifying reclaims;

The Auele Specific Primer of 1ba gene is identified in C, order-checking:

Lba-P1:5 '-c gag ctc Aaa ttt aaa ttt atg gtt gct ttc act gag-3 ', italic is represented the restriction enzyme site sequence of restriction endonuclease sma lI, the sequence in the square frame is the ribosome bind site RBS sequence of the enhancing translation ability of adding;

Lba-P2:5 '-tcc ccc ggg ata cta att atg cct tct taa tag c-3 ', italic is represented restriction enzyme SacI restriction enzyme site sequence;

(3) structure of Chlamydomonas reinhardtii chloroplast expression carrier:

The soybean 1ba gene fragment and the chloroplast expression carrier cg40 that come enzyme cutting clone to obtain with restriction endonuclease sma lI and SacI; The 1ba gene fragment is formed the aadA-1ba fusion gene afterwards with the spectinomycin resistance gene aadA that the T4 dna ligase is connected among the plasmid cg40, obtain chlamydomonas chloroplast expression carrier cg401-1-1ba, transformed into escherichia coli DH5 α, extract plasmid, identify through restriction endonuclease sma lI and SacI double digestion, obtain the 1ba fragment of 442bp and the plasmid fragment of 5900bp;

(4) conversion of Chlamydomonas reinhardtii chloroplast(id):

A, get the Chlamydomonas reinhardtii of 100ml logarithmic phase middle and later periods, 25 ℃ of centrifugal 5min of following 3000rpm collect frustules, and it is inferior to give a baby a bath on the third day after its birth with fresh TAP, is applied on the fresh TAP solid plate; Illumination 100 micromole's photon/square metre second (μ mol photonsm ^-2S ^-1) and 25 ± 1 ℃ of conditions under cultivated 24 hours; Robbing bombardment with gene transforms; Vacuum tightness 9.48192 * 10 ⁴Pa, target distance 9cm, helium pressure 7.584236 * 10 ⁶Pa, the bronze bullet is through restriction enzyme EcoRI digested plasmid cg401-1-1ba DNA parcel, 1.0 microns of bronze bullet particle diameters;

B, the chlamydomonas after transforming are under illumination and 25 ± 1 ℃ of conditions, 18h is cultivated in transition, with TAP liquid nutrient medium flushing, coat the TAP solid that contains spectinomycin 100 μ g/ml and select on the substratum, 25 ± 1 ℃ and 100 micromole's photon/square metre second (μ mol photonsm ^-2S ^-1) the continuous illumination condition under, cultivated about 7～15 days; Single algae of getting resistance falls respectively and to select on the substratum repeatedly succeeding transfer culture; Produce hydrogen respectively and cultivate, and detect hydrogen output and oxygen-consumption;

(5) integration of 1ba gene and expression thereof in the transgene Chlamydomonas reinhardtii:

A, 1ba gene integration are to the detection of Chlamydomonas reinhardtii chloroplast DNA:

Take the logarithm the growth later stage chlamydomonas nutrient solution, 4 ℃ of low-speed centrifugals are collected, the 0.1mol/L sodium-chlor that adds 350 μ lNET, the 50mmol/L ethylenediamine tetraacetic acid (EDTA), the resuspended precipitation of Tutofusin tris-hydrochloric acid of pH value 8.020mmol/L adds Proteinase K and 25 μ l, 20% sodium lauryl sulphate (SDS) of 25 μ l10mg/ml, mixes, 37 ℃ of water-baths 12 hours, cooling; Add 200 μ l 5mol/L Potassium ethanoates (KAc), leave standstill on ice, centrifugal, add isopyknic 25: 24: 1 phenol/chloroforms/primary isoamyl alcohol extracting in the supernatant liquor 2 times; Use isopyknic chloroform extracting 1 time again; Total DNA is dissolved in 30 μ l Tutofusin tris-ethylenediamine tetraacetic acid (EDTA)s (Tris-EDTA is called for short TE) damping fluid with dehydrated alcohol precipitation and 70% washing with alcohol after the drying;

The detection of 1ba genetic transcription in B, the transgene Chlamydomonas reinhardtii:

The Chlamydomonas reinhardtii of taking the logarithm mid-term in vegetative period, room temperature 3000rpm collected frustule in centrifugal 5 minutes; Extract the total RNA and the synthetic cDNA of Chlamydomonas reinhardtii; Utilize the Auele Specific Primer and the described PCR condition of above-mentioned clone 1ba gene of above-mentioned 1ba gene to increase, carry out the detection of 1ba gene transcription level, obtain the amplified band of 442bp;

The detection of Lba expression amount in C, the transgene Chlamydomonas reinhardtii:

Get and produce the Chlamydomonas reinhardtii nutrient solution that hydrogen is cultivated, the quick centrifugal 5min of room temperature 3000rpm collects frustule, suspends with 250 μ l protein extraction damping fluids, adds 2 μ l beta-mercaptoethanols, frozen-thawed three times; Under 4 ℃ of conditions, the centrifugal 20min of 14000g, the albumen supernatant liquor of getting 100 μ g adds the damping fluid (Tris-HCl) of 0.25 mole of Tutofusin tris-hydrochloric acid preparation, pH 8.0; 25% glycerine; 7.5% sodium lauryl sulphate (SDS); 0.25mg/ml bromjophenol blue (bromophenolblue); 12.5% (v/v) beta-mercaptoethanol (2-mercaptoethanol) in 10% separation gel and 5% concentrated glue carry out denaturing polyacrylamide gel electrophoresis (SDS-PAGE), change polyvinylidene difluoride (PVDF) (PVDF) film, the beans Lba of the Chinese People's Anti-Japanese Military and Political College polyclonal antibody property advanced immuning hybridization detects, antibody to horseradish peroxidase (Horseradish Peroxidase is called for short HRP) mark carries out chemiluminescence detection;

(6) analyze the influence of reorganization leghemoglobin sphaeroprotein subunit Lba to the Chlamydomonas reinhardtii growth:

A, usefulness TU-1901 twin-beam ultra-violet and visible spectrophotometer detect the absorbancy of Chlamydomonas reinhardtii at the 750nm place;

B, get the centrifugal 5min of algae liquid 3000rpm and collect frustule, add 95% ethanolic soln with volume, mix, the room temperature lucifuge is placed 20min, 4 ℃ of centrifugal 10min of following 12000rpm; Get supernatant, measure the light absorption value of OD665 and D649;

C, calculating chlorophyll a, chlorophyll b and total chlorophyllous content, the mg/L of unit: chlorofucine hl (mg/l)=20.04 (OD ₆₄₉)+6.10 (0D ₆₆₅);

(7) analyze leghemoglobin sphaeroprotein subunit Lba Chlamydomonas reinhardtii produced the oxygen-consumption of hydrogen cultivation and the influence of hydrogen output:

The detection of oxygen-consumption and hydrogen output in A, a lack of sulfur substratum:

In B, the sulfuricum culture-medium to the detection of oxygen-consumption and hydrogen output.

Images