CN102277378B - Method for increasing total oil and fat and linoleic acid (LA) or alpha-linoleic acid (ALA) content in chlorella - Google Patents

Abstract

The invention provides the use of an AtLEC1 gene in improvement on oil and fat content in chlorella. The gene is derived from Arabidopsis thaliana which is a member of CCAAT-Box binding factor HAP3 family. The AtLEC1 gene-transformed chlorella can improve the LA, oleic acid and ALA content and can improve the total oil and fat content of the chlorella strain. The method can be used in production of unsaturated fatty acid and biodiesel by an alga expression system and by using gene engineering technology.

Description

Improve the method for the content of total grease, linolic acid, oleic acid or alpha-linolenic acid in the chlorella

Technical field

The present invention relates to the application of plant code gene in chlorella.Particularly, relate to a gene A tLEC1 with entire reading frame frame that derives from Arabidopis thaliana (Arabidopsis thaliana) is built into the chlorella expression vector, and change chlorella over to, make linolic acid (LA), oleic acid and alpha-linolenic acid (ALA) content of chlorella, and total fat content significantly improves.

Technical background

The photosynthetic efficiency height of little algae, growth cycle is short, and annual yield by area is tens times and even hundreds of times of grain, and little algae lipid content is 10%～70%, and this is that land plant is inaccessible far away.It has big, pollution-free, renewable, the easy cultivation of production capacity, contains more advantages such as lipid material.Its growth and breeding can not cause the contradiction with agricultural, the competition of animal husbandry land used in the waters.Because above many-sided advantage, little algae are considered to one of energy that has most now the exploitation future ^[1]The chlorella that the present invention uses is a kind of unicellular eucaryon algae, and reproduction speed is fast, breeds in the monogony mode, can carry out the heterotrophism of cultivation of light autotrophy and unglazed photograph and cultivate, and is produced on a large scale, and there are sophisticated cultivation, screening and transformation technology in this laboratory ^[2]

Genetic engineering technique has been widely used in crop improvement, and has obtained many significant effects, and by the research of the total fat content of genetic engineering technique raising eucaryon algae report is arranged seldom ^[3]Jarvis etc. once changed the acc1 gene of diatom C.cryptica over to diatom C.cryptica and Navicula saprophila crosses expression, on the mRNA level, all detect the overexpression of this gene, but the oil-contg of two kinds of transgenosis diatoms does not significantly increase, this is to attempt utilizing genetic engineering technique to improve the research of diatom oleaginousness first, yet does not reach desired result ^[4]Improving the most fruitful means of algae fat content at present, is to improve the oil-contg of algae by means such as regulation and control culturing process, culture condition, nutritive ingredients ^[5-6]Yet all inevitably produce a problem in these means, that is exactly the raising oleaginousness will be with the cost that is reduced to of biomass, and when biomass increased, content of oil and grease will be lower ^[7-8]And genetic engineering technique is expected to address this problem.Do not retrieve as yet at present and utilize genetic engineering means to improve algae fat content and the nondecreasing successfully report of total biomass.

Transcriptional control is the committed step of many biology regulation and control, and a series of Expression of Related Genes in the specific pathways metabolism that transcription factor is adjustable make this pathways metabolism change from integral level.LEC1 (Leafy Cotyledon 1) is a member of activating transcription factor HAP3 (CCAAT-Box binding factor) gene family, keep the characteristic of suspensor cell and the characteristic of cotyledon at the early stage LEC1 of fetal development, and relevant in the seed maturity processes such as acquisition of the accumulation of the later stage of fetal development LEC1 expression of gene and reserve substance, the anti-dehydration property of embryo ^[9]Found the accumulation of seed specific RNA such as oil body protein and storage protein in the seedling body of overexpression LEC1 in Arabidopis thaliana such as Loton, but it is very low to cross the rate of emergence of expressing, plant strain growth is very weak, and is final sterile or dead ^[10]Mu etc. studies show that, grow early evoking overexpression Arabidopis thaliana LEC1 gene and can influence transcription factors such as ABI3, FUS3 and WRINKLED1, improve the integral level that the lipid acid synthesis related gene is expressed, and the fat content in the blade is significantly improved ^[11]Shen etc. place corn ZwLEC1 gene under the weak promoter EPA1 of embryo-specific, obtain the transgenic corns of floorboard with high oil content, but plant leaf are reduced to about half of wild-type; Corn ZwLEC1 gene is placed under the strong promoter OLE of embryo-specific, very serious defective has taken place in seed germination and leaf growth ^[12]Tan etc. place the BnLEC1 gene in rape source under the promotor napinA of seed-specific and express in leaf mustard, and severely subnormal appears in transfer-gen plant after germination as a result, and is all dead or sterile; Therefore promotor napinA has been carried out a series of transformation, had only originally 18% the time when starting intensity, the normal and oil-contg of plant strain growth breeding is significantly improved ^[13]In higher plant, most significant events is seed development and ripe and sprouting once more, and the LEC1 gene starts the consequence of expressing under strong promoter all be lethal.Chlorella is the low eukaryote of waiting, and does not need to form seed and is vegetative propagation, does not exist the LEC1 gene to descend expression to cause lethal factor at strong promoter.Do not retrieve at present LEC1 gene transformation algae that utilizes the Arabidopis thaliana coding and the research that improves fat content as yet.

(Polyunsaturated fatty acids PUFAs) is meant and contains two or more pairs key and carbon chain length is the straight chain fatty acid of 18～22 carbon atoms polyunsaturated fatty acid, mainly comprises linolic acid (Linoleic Acid, LA, 18:2 Δ ^9,12), gamma-linolenic acid (γ-Linolenic Acid, GLA, 18:3 Δ ^6,9,12), arachidonic acid (Arachidonic Acid, AA, 20:4 Δ ^5,8,11,14), timnodonic acid (Eicosapentaenoic Acid, EPA, 20:5 Δ ^5,8,11,14,17), docosahexenoic acid (Decosahexaenoic Acid, DHA, 20:5 Δ ^{4,7,10,13,16,19}) etc.Wherein, linolic acid and linolenic acid are acknowledged as essential fatty acid (Essential fatty acids, EFA), further derivation become to have the highly unsaturated fatty acids of difference in functionality, as AA, EPA, DHA etc. ^[14]Algae contains abundant polyunsaturated fatty acid, mainly comprises linolic acid (Linoleic acid, LA, 18:2 Δ ^9,12), gamma-linolenic acid (γ-Llinolenic Acid, GLA, 18:3 Δ ^6,9,12) etc.The present invention finds that AtLEC1 makes linolic acid (LA), oleic acid and alpha-linolenic acid (ALA) content of chlorella, and total fat content significantly improves.

Summary of the invention

An object of the present invention is to provide gene A tLEC1 and be used for improving the chlorella application of the content of grease, linolic acid, oleic acid or alpha-linolenic acid always.

The detection of lipid acid mainly adopts GC-MS (gas chromatography-mass spectrography technology) to finish.Its principle is by GC (gas-chromatography) different fatty acid components to be separated, and respectively every kind of component is carried out the mass spectrometry analysis by MS (mass spectrum) detector then, thus the coupling analytical technology of the comparative maturity of the composition of definite each component and content.

Soxhlet extraction process (Soxhlet extractor method) is to generally acknowledge the classical way of measuring fat content, is generally adopted both at home and abroad at present.Utilize solvent refluxing and siphon principle, solid matter is continuously extracted by neat solvent, the material that extracts at last is enriched in the flask.

Particularly, in one aspect, the present invention relates to the application of AtLEC1 gene content of total grease, linolic acid, oleic acid or alpha-linolenic acid in improving chlorella.

In yet another aspect, the invention provides a kind of method that improves the content of total grease, linolic acid, oleic acid or alpha-linolenic acid in the chlorella, it is characterized in that the AtLEC1 gene transformation in chlorella.

In a specific embodiments of application of the present invention and method, the AtLEC1 gene is the Arabidopis thaliana source.More preferably, the sequence of AtLEC1 gene is the sequence shown in the SEQ ID NO:1, and its encoded protein sequence is the sequence shown in the SEQ ID NO:2.

In another specific embodiment, described AtLEC1 gene is included in the plant expression vector.Described expression vector can be any expression vector that the guiding foreign gene is expressed in plant.Preferably, described expression vector can be plant expression vector pBIN19, pBI121, pBI221, and pCambia 1300, pGreen.Be preferably pGreen0029.

Carrier of the present invention also can contain suitable promotor.Can use any strong promoter in the present invention.These promotors include but not limited to cauliflower mosaic virus (CaMV 35S), Ubiqutin, Actin promotor.It can use separately or be used in combination with other plant promoter.

Expression vector of the present invention can be by the use Ti-plasmids, the Ri plasmid, and plant viral vector, directly DNA transforms, microinjection, modes such as electroporation import vegetable cell.

In another specific embodiment, described chlorella is selected from chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella vulgaris (Chlorella vulgaris) and Chlorella pyrenoidesa (Chlorella pyrenoidosa).

The annex explanation

Fig. 1 contains the carrier pEB-AtLEC1 of AtLEC1 gene.

Fig. 2 contains the carrier figure building process of no terminator.

Fig. 3 zero load (UN_CK) plant expression vector figure building process.

The plant expression vector figure building process of Fig. 4 gene A tLEC1.

The PCR of Fig. 5 transgenic alga strain detects.M:Marker, 1-3: positive algae strain, CK: negative control.

The RT-PCR of Fig. 6 transgenic alga strain detects.M:Marker, 1-4: positive algae strain, CK: negative control.

The Southern of Fig. 7 transgenic alga strain detects.The 4th swimming lane is contrast, other all positive algae strain.1：LEC1-1(EcoR I)，2：LEC1-1(Xba I)，3：LEC1-3(Xho I)，4：UN_CK(Xho I)，5：LEC1-3(EcoR I)，6：LEC1-8(Xba I)，7：LEC1-8(Xho I)，8：LEC1-9(EcoR I)，9：LEC1-9(Xba I)。

Strain of Fig. 8 transgenic alga and contrast growth curve.

Fig. 9 purifying polybasic transgenic alga strain lipid acid GC-MS measurement result relatively.

SEQ ID NO:1.AtLEC1 nucleotide sequence.

SEQ ID NO:2.AtLEC1 aminoacid sequence.

The used forward primer of SEQ ID NO:3.nos terminator fragment cloning.

The used reverse primer of SEQ ID NO:4.nos terminator fragment cloning.

The used forward primer of clone of SEQ ID NO:5.ubi promoter fragment.

The used reverse primer of clone of SEQ ID NO:6.ubi promoter fragment.

The forward primer of SEQ ID NO:7. amplification gene AtLEC1 from the pEB-AtLEC1 carrier.

The reverse primer of SEQ ID NO:8. amplification gene AtLEC1 from the pEB-AtLEC1 carrier.

SEQ ID NO:9. is from the forward primer of the inner amplification of gene A tLEC1.

SEQ ID NO:10. is from the reverse primer of the inner amplification of gene A tLEC1.

Embodiment

Describe the present invention in detail below with reference to embodiment and accompanying drawing.What those having ordinary skill in the art will appreciate that is, following embodiment is illustrational purpose, and it should not be interpreted as limitation of the present invention by any way.Protection scope of the present invention is limited by accompanying Claim.

The structure of embodiment 1.AtLEC1 gene chlorella carrier

1. according to disclosed sequence (NCBI Reference Sequence:NM_102046.4) synthetic gene AtLEC1, and be connected into T carrier (available from the Beijing Quanshijin Biotechnology Co., Ltd), constitute the carrier pEB-AtLEC1 that contains gene A tLEC1, sequence verification gene A tLEC1 sequence is SEQ IDNO:1, expressed protein sequence is SEQ ID NO:2, and this plasmid vector figure sees Fig. 1.

2. be carrier is carrier with pGreen0029 (available from the John Innes Centre), make up the control vector pG29-UN-CK that contains Ubiquitin promotor and no terminator.

From carrier pGreen0029 amplification no terminator, use primer SEQ ID NO:3 (forward primer) and SEQ ID NO:4 (reverse primer) with high-fidelity enzyme Pfu (available from the Beijing Quanshijin Biotechnology Co., Ltd).

The PCR reaction system is as follows:

The PCR reaction conditions is as follows:

1.95℃ 5min

2.94℃ 30s

3.55℃ 40s

4.72 a ℃ 1min returns back to 2,32cycles

5.72℃ 10min

6.4℃ pause

Amplified production directly uses restriction enzyme Not I and Sac I to carry out double digestion, is connected with the pGreen0029 of same double digestion, makes up intermediate carrier pGreen0029-nos (Fig. 2), sequence verification.

Go up amplification Ubi promotor, the primer SEQ ID NO:5 (forward primer) and SEQ ID NO:6 (reverse primer) with high-fidelity enzyme Pfu (it is the same to originate) from the carrier (pC1303 is available from Cambia company) that contains the Ubi promotor.

The PCR reaction system is as follows:

The PCR reaction conditions is as follows:

1.95℃ 5m

2.94℃ 30s

3.55℃ 40s

4.72 a ℃ 4min returns back to 2,32cycles

5.72℃ 10min

6.4℃ pause

Amplified production directly uses restriction enzyme Hind III and BamH I to carry out double digestion, be connected with the pGreen0029-nos of same double digestion, structure contains the control vector pGreen0029-Ubi-Nos (writing a Chinese character in simplified form UN-CK) of Ubiquitin promotor and no terminator, sequence verification, its carrier figure sees Fig. 3.

3.AtLEC1 the structure of gene plant expression vector

With high-fidelity enzyme Pfu (it the is the same to originate) AtLEC1 that from the carrier pEB-AtLEC1 that contains gene A tLEC1, increases, use primer to be SEQ ID NO:7 (forward primer) and SEQ ID NO:8 (reverse primer), reaction system and reaction conditions are with the clone of Ubi promotor, utilize Spe I, the NotI restriction enzyme site, two backs of cutting obtain the AtLEC1 gene, directed cloning is in the chlorella medial expression vector UN-CK that cuts through same enzyme, obtain chlorella expression plasmid p29-UN-LEC1, transformed into escherichia coli DH-5 α (available from the Beijing Quanshijin Biotechnology Co., Ltd), picking mono-clonal at random, use primer to carry out the PCR checking as SEQ ID NO:9 and SEQ ID NO:10, reaction system and reaction conditions be with the clone of Ubi promotor, proves the plant expression vector of the AtLEC1 gene that has obtained to contain SEQ ID NO:1.The vector construction program is seen Fig. 4.

Embodiment 2. changes acquisition and the detection of the chlorella of AtLEC1 gene

1. the conversion of chlorella

Used chlorella is chlorella ellipsoidea (Chlorella ellipsoidea is from Inst. of Hydrobiology, Chinese Academy of Sciences), reference literature ^[2]Cultivate chlorella to suitable growth conditions, carry out height behind the centrifugal collection frustule and ooze processing, adopt above-mentioned document ^[2]The sharp conversion condition of identical electricity carries out electricity to chlorella ellipsoidea and swashs, and chlorella cells was applied to and contains 30mg/L G418 solid medium (reference literature after electricity was swashed ^[2]) the middle cultivation, the algae that grows on about 4～6 weeks back detection flat boards falls.

2. PCR being carried out in the transgenic alga strain detects

Single algae on the picking flat board falls cell inoculation in 15mg/L G418 liquid nutrient medium (reference literature ^[2]) (concentration reached and was about 1 * 10 middle 2 weeks of cultivation ⁸Individual/mL), the centrifugal 10min of 5000r/min collects frustule, cell is ground reference literature in liquid nitrogen ^[2]In method extract the total DNA of chlorella, the DNA that obtains is dissolved in the distilled water of sterilization and places-80 ℃ of preservations.

With high-fidelity enzyme Pfu (it is the same to originate) amplification gene AtLEC1, the primer SEQ ID NO:7 (forward primer) and SEQ ID NO:8 (reverse primer), reaction system and reaction conditions are referring to the clone of the Ubi promotor of embodiment one, Fig. 5 is a part transgenic alga strain PCR detected result, 52 transgenic positive algae strain systems have been obtained altogether, be numbered LEC1-1 ,-2 ,-3 ... up to LEC1-52.

3. RT-PCR being carried out in the transgenic alga strain detects

Picking PCR detects the single algae of the male algae strain cell that falls, and it is the same to cultivate collection method, and getting about 100mg, to place liquid nitrogen to be ground to material broken fully, adds TRIzol (available from Invitrogen company) room temperature placement 5 minutes.Add chloroform extracting twice, get supernatant,, after the dry air, be dissolved in DEPC H with the total RNA of isopropanol precipitating ₂O.With test kit (FSK-100, TOYOBO Co., the Random primer that LTD.) carries carry out reverse transcription and obtain cDNA, and carry out RT-PCR (concrete operations are referring to FSK-100, TOYOBO Co., LTD.).

The RT-PCR reaction system is as follows

Carry out following operation

1.30℃ 10min

2.42℃ 30min

3.90℃ 5min

4.5℃ 5min

Get an amount of above-mentioned reverse transcription product and carry out pcr amplification with primer SEQ ID NO:9 and SEQ ID NO:10, reaction system and reaction conditions are referring to the clone of the Ubi promotor of embodiment 1, and Fig. 6 is a part transgenic alga strain RT-PCR detected result.

4. Southern being carried out in the transgenic alga strain detects

Southern hybridization is molecular biological classical experimental technique, its ultimate principle is that DNA sample to be detected is fixed on the solid phase carrier, hybridize with the nucleic acid probe of mark, demonstrate hybridization signal having on the position of solid phase DNA of homologous sequence with probe.Can judge whether have in the detected DNA sample and probe homologous fragment by Southern hybridization.This experimental implementation and agents useful for same main reference document ^[15]

4.1 probe mark

Use primer SEQ ID NO:9 and SEQ ID NO:10 from carrier pEB-AtLEC1 (Fig. 1) pcr amplification gene A tLEC1, reaction system and reaction conditions are referring to embodiment one, probe mark uses Random Primer DNA Labeling Kit ver.2.0 (TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.Code D6045).Get 10ng-1 μ g after the PCR product purification reclaims and be put in the 0.2mL centrifuge tube, add Random Primer 2 μ L, ddH as template ₂O polishing 16 μ L, 95 ℃ of sex change 5min put ice bath 5min immediately.

In another 1.5mL centrifuge tube, add other component that test kit DNALabeling Kit ver.2.0 carries:

The sex change template DNA is joined in the pipe room temperature or 37 ℃ of 1hr.

4.2 chlorella total DNA extraction and enzyme are cut

Picking changes fall cell and the contrast (algae that changes UN-CK fall cell) of the single algae of AtLEC1 and is inoculated in 15mg/L G418 liquid nutrient medium (reference literature respectively ^[2]) the middle cultivation for 2 weeks, the centrifugal 10min of 5000r/min collects frustule, cell is ground reference literature in liquid nitrogen ^[2]In method extract the total DNA of chlorella, the DNA of acquisition carries out enzyme with the restriction enzyme enzyme and cuts.LEC1-1-2 uses EcoR I and Xba I single endonuclease digestion respectively; LEC1-3-3 uses EcoR I and Xho I single endonuclease digestion respectively; UN_CK Xho I single endonuclease digestion; LEC1-8-4 uses Xba I and Xho I single endonuclease digestion respectively; LEC1-9-5 uses EcoR I and Xba I single endonuclease digestion respectively.Restriction enzyme is all available from Takara company.

Reaction system: comprise DNA 10～20 μ g, buffer 10 μ L, restriction enzyme 4 μ L in the 100 μ L reaction systems, all the other use ddH ₂O mends flat.

Reaction conditions: 37 ℃ are spent the night, and add 1 μ L enzyme continuation enzyme and cut 3-5 hour, and 1% agarose electrophoresis detects enzyme and cuts effect then, and DNA need be cut into a band of disperse, does not have master tape.The enzyme system of cutting adds ddH ₂O is to volume 200 μ L, adds ethanol alcohol precipitation after the extracting of equal-volume chloroform, and 70% ethanol is washed, and dries Hou Jiashui and is no more than 50 μ L, and 65 ℃ of hot 10min are standby.

4.3 the preparation of glue and commentaries on classics film

The sepharose of preparation 0.8% adds enzyme and cuts back to close product 50 μ L, and in 1 * TAE electrophoretic buffer, electrophoresis to tetrabromophenol sulfonphthalein arrives the glue lower end under the 30V voltage, uses ddH ₂O washes twice with glue, is dipped in jog 15min in the ealkaline buffer of several times volume, changes the primary alkali damping fluid and soaks and sway 20min gently.Cut a nylon membrane than the big 1mm of gel (CAT.No:RPN303B, Amersham Biosciences UK Limited) simultaneously, soak in distilled water in advance and make it drenched, the nylon membrane after the infiltration immerses in the alkaline transfering buffering liquid 15min at least.Under the NaOH and 1MNaCl alkalescence transfering buffering liquid condition of 0.4M, change film instrument (U.S. Bole Bio-rad 785 type vacuum transfer printing instrument) with vacuum the DNA on the sepharose is forwarded on the positively charged nylon membrane, changeing the film condition is that vacuum tightness 50m bar changes 1h-2h.Change and after film is finished film is soaked in room temperature 15min among the neutralization buffer II.Place on the filter paper and dry, UV-crosslinked (1.245J/cm) 8min, 80 ℃ of baking 1h are used for nylon membrane immediately hybridization at last or wrap with preservative film that to be placed on-20 ℃ of refrigerators stand-by.

4.4 prehybridization and hybridization

In the hybridization bottle, add hybridization solution 10-20mL (0.5M Na ₃PO ₄(pH7.2), 1mM EDTA (pH8.0) 7%SDS), is adjacent to a hybridization bottle wall with the back side of film, and is positive towards hybridization solution, notes not producing bubble, puts into hybrid heater, makes the hybridization system be raised to 65 ℃.Behind the prehybridization 2-3h, pour out the hybridization solution of prehybridization, change to the hybridization solution about 5mL.The NaOH that adds 0.4N in the probe of above-mentioned making makes its sex change, is added to rapidly in the hybridization bottle, and 65 ℃ of hybridization are spent the night.

4.5 wash film and compressing tablet

Taking out nylon membrane uses following condition to wash film at 65 ℃ successively:

2×SSC，0.5％SDS，15min

1×SSC，0.5％SDS，15min

0.5×SSC，0.1％SDS，15min

In washing the process of film, constantly jolting, and to keep film be moistening state always, constantly uses the intensity of radioactivity on the radiation survey meter detection membrane.Wash the power that detects hybridization signal behind the film 2 times with monitor during this time, get final product when being reduced to the 20 μ Gy/h left and right sides.The film that washes blots the moisture on film surface with filter paper, and wraps up with preservative film.

Film is faced up, put into magazine (adding the bilateral intensifying screen), under the ruddiness in darkroom, paste an X-ray sheet facing up of film, the magazine that closes places-80 ℃ of cryogenic refrigerators to expose.According to the signal strong and weak decision time shutter, the general time is about 2 days.

4.6 developing and fixing

The magazine that takes out from-70 ℃ takes out the X-ray sheet after developing room is opened, soak 1min in the developing solution, during constantly rock mating plate, take out drop and fall some moisture, be dipped into 1min in the stop bath, ddH ₂Twice of O flushing also taken a picture, and sees accompanying drawing 7.The point sample order is as follows: 1:LEC1-1 (EcoR I), 2:LEC1-1 (Xba I), 3:LEC1-3 (Xho I), 4:UN_CK (Xho I), 5:LEC1-3 (EcoR I), 6:LEC1-8 (Xba I), 7:LEC1-8 (Xho I), 8:LEC1-9 (EcoRI), 9:LEC1-9 (Xba I).

4.7 interpretation of result

Can analyze from the restriction enzyme site of plasmid vector Fig. 4,, use these three kinds of enzymes to carry out single endonuclease digestion respectively inserting the restriction enzyme site that does not have EcoR I, Xba I and Xho I on the genomic fragment of chlorella.Swimming lane 4 among Fig. 7 is the strains of contrast algae, does not have the band explanation not hybridize to endogenous gene, and other swimming lane all respectively has a band, and the AtLEC1 gene that has only inserted a copy in the genome the inside of each transgenic alga strain is described.

The growth curve of embodiment 3. chlorellas

The offspring LEC1-1 and the LEC1-3 of the positive algae strain of Molecular Detection among the embodiment 2, and control vector transforms algae strain CK, reference literature ^[2]Enlarged culturing in 15mg/L G418 liquid nutrient medium, wild algae (WT) enlarged culturing in not containing antibiotic liquid nutrient medium simultaneously.Use the 50mL triangular flask to cultivate after 4-6 days, by initial concentration enlarged culturing in the new substratum of 150mL of the about 0.2g/L of dry weight, every day, same time sampling was measured biomass, continuous 8 days records, and measurement result sees Table 1.

According to measurement result, make the growth curve of transgenic alga and wild algae, see Fig. 8.

Can find that in Fig. 8 chlorella entered plateau (increment reaches running balance) since the 6th day, begin to descend to the biomass of the 8th day algae.Use SPSS software (IBM Corporation) that the algae biomass of measuring is carried out the Student-Newman-Keuls check respectively.Assay shows that the biomass of transgenic alga and contrast (wild algae and control vector transform algae strain CK) does not have significant difference (P＞0.05).

The GC-MS that embodiment 4.AtLEC1 gene improves the different fatty acid content of chlorella analyzes

Reference literature is pressed in the positive algae strain of Molecular Detection among the embodiment 2 ^[2]Stroke plate, some plate carry out purifying, enlarged culturing in 15mg/L G418 liquid nutrient medium continuously on 30mg/L G418 solid medium.Use the 50mL triangular flask to cultivate after 4-6 days, the initial concentration of pressing dry weight 0.1g/L is in the new substratum enlarged culturing of 150mL, and centrifugal collection frustule uses GC-MS to measure each fatty acid component content after 7 days.

1. the extraction of lipid acid

Centrifugal collection growth reaches the chlorella (OD540=16) of plateau, uses deionized water wash, and-80 ℃ of lyophilizes in the oven dry of spending the night of 85 ℃ of baking ovens, grind to form uniform fine powder after the freeze-drying in mortar.Take by weighing the 50mg chlorella powder, add the confidential reference items of the margaric acid (Heptadecanoic Acid, d17:0 available from Sigma company, are dissolved in methanol concentration 100 μ M/mL) of 80 μ L as fatty acid content mensuration.5mL 5%KOH-CH ₃OH adds HCl and is acidified to its pH value and reaches 2.0 behind 70 ℃ of water-bath 5h.Add 4mL 14%BF again ₃-CH ₃OH (available from Aldtich company) solution, 70 ℃ of water-bath 1.5h.Add 2mL 0.9%NaCl solution, static a moment behind the mixing.Add the 2mL chloroform: normal hexane (V/V 1: 4) extracting, draw extract, N ₂Dry up.Be dissolved in 150 μ L ethyl acetate at last.

2. end product GC-MS check and analysis experiment

Used GC-/MS instrument is TurboMass (a PerkinElmer company), pillar: BPX-70,30m * 0.25mm * 0.25vm, 120 ℃ of column temperatures, 230 ℃ of vaporizer temperature.Get sample on the 1 μ L end product, splitting ratio 10: 1.

3.GC-MS interpretation of result

Analytical results shows, in obtaining that purifying is many and falling for algae, the C16:0 of these two algae strains (palmitinic acid) all compare according to improving, and wherein LEC1-1 improves 64%, LEC1-3 raising 27%; C18:1 (oleic acid) all compares according to improving, and the LEC1-1 comparison is according to improving 94%, and LEC1-3 improves 75%; The C18:2 of LEC1-3 (linolic acid) comparison is according to improving 21%; The C18:3 of LEC1-3 (linolenic acid) comparison is according to improving 36% (Fig. 9), more than these differences all significantly (P＜0.01).

Embodiment 5.AtLEC1 gene improves the analysis of the total fat content of chlorella

Algae strain and the same embodiment 4 of cultural method are used in this experiment, and total fat content analytical procedure is that the Soxhlet extraction process is referring to document ^[16]Apparatus,Soxhlet's is made up of extraction flask, extraction tube, condenser three parts, and the extraction tube both sides have siphon pipe and pipe connecting respectively.During extraction, testing sample is wrapped in the fat-free filter paper bag, put into extraction tube.Add the low boiling point organic solvent that is fit to this testing sample in the extraction flask, the heating extraction flask, organic solvent gasifies, and being risen by pipe connecting enters condenser, and the liquid that congeals into splashes in the extraction tube, the lipid material in the lixiviate sample.The organic solvent liquid level reach a certain height in the pipe to be extracted, and the organic solvent that is dissolved with crude fat flows into extraction flask through siphon pipe.The organic solvent that flows in the extraction flask continues to be heated gasification, rising, condensation, splashes in the extraction tube, so moves in circles, and makes solid matter constantly get, the material that extracts is enriched in the flask for pure solvent institute apple.

At first that the algae of collecting is centrifugal, place 90 ℃ of-110 ℃ of oven dry of spending the night of glass dish, add liquid nitrogen grinding and become uniform powder.Use the low boiling point organic solvent chloroform: methyl alcohol (V/V=3: 1) leach, dry the calculating fat content of weighing with the infiltration of algae powder and with grease.Centrifugal collection growth reaches the chlorella (OD540=16) of plateau, uses deionized water wash, and-80 ℃ of lyophilizes in the oven dry of spending the night of 85 ℃ of baking ovens, grind to form uniform fine powder after the freeze-drying in mortar.Take by weighing the 0.5g chlorella powder, use apparatus,Soxhlet's (article No.: ST-80 is available from skill company limited of Beijing Sai Fulai Boke) to detect algae strain grease total content.The strain of subordinate list statistics demonstration transgenic alga is all compared according to improving a lot.Change AtLEC1 algae strain LEC1-1 comparison according to improving 31.36%, the total fat content comparison of algae strain LEC1-3 is according to improving 32.87% (table 3), significant difference (P＜0.01).

Table 1 transgenic alga and control group increment are measured

Annotate: unit is g/L

Table 2 changes the flow measurement of LEC1 algae strain total oil content

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Claims (8)

1. the application of the content of total grease, linolic acid, oleic acid or alpha-linolenic acid in improving chlorella of the protein sequence shown in nucleotide sequence shown in SEQ ID NO:1 or the SEQ ID NO:2.

2. improve the method for the content of total grease, linolic acid, oleic acid or alpha-linolenic acid in the chlorella, it is characterized in that the nucleotide sequence shown in SEQ ID NO:1 is transformed in the chlorella.

3. the method for the application of claim 1 or claim 2, the nucleotide sequence shown in the wherein said SEQ ID NO:1 is from Arabidopis thaliana (Arabidopsis thaliana).

4. the method for the application of claim 1 or claim 2, the nucleotide sequence shown in the wherein said SEQ ID NO:1 is included in the plant expression vector.

5. the application of claim 4 or method, wherein said expression vector is selected from plant expression vector pBIN19, pBI121, pBI221, pCambia1300, pGreen.

6. the application of claim 4 or method, wherein said expression vector is pGreen0029.

7. claim 5 or 6 application or method, wherein said carrier comprises promotor, and described promotor is selected from cauliflower mosaic virus promoter CaMV 35S, Ubiqutin promotor or Actin promotor.

8. the method for the application of claim 1 or claim 2, wherein said chlorella is selected from chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella vulgaris (Chlorella vulgaris) or Chlorella pyrenoidesa (Chlorella pyrenoidosa).

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