CN105087627B - A kind of application of sll0659 gene in synthesis cytoalgae carotenoid - Google Patents

Abstract

The present invention relates to a kind of application of sll0659 gene in synthesis cytoalgae carotenoid.This applies related sll0659 gene, and nucleotide sequence is as shown in SEQ ID NO.1.The present invention provides a kind of applications of sll0659 gene in DNC wireless played a significant role in the synthesis of DNC wireless beta carotene, by will be after the sll0659 gene knockout in DNC wireless, the lutein content of DNC wireless increases 22.3%, luteole content increases 31.3%, and echinenone content reduces 34.3%, beta carotene percentage composition then reduces 1.3%.Meanwhile HPLC testing result shows compared with wild type DNC wireless, there is an apparent peak in sll0659 knock out mutants body in carotenoid component, through with mark product control test, determine that it is alpha-carotene.

Description

A kind of application of sll0659 gene in synthesis cytoalgae carotenoid

Technical field

The present invention relates to a kind of application of sll0659 gene in synthesis cytoalgae carotenoid, in particular to cytoalgae Sll0659 gene synthesizes the application in cytoalgae carotenoid in DNC wireless photosynthesis in PCC6803, belongs to Gene engineering technology field.

Background technique

Carotenoid (Carotenoids) is that carrotene (Carotenes) and lutein (Xanthophylls) two are big The general name of class pigment, usually hydrocarbon and its oxidized derivatives, be made of 8 isoprenoid units, be a kind of Important natural pigment, the carotenoids having now been found that are known as more than 700 kinds, common are alpha-carotene (α-Carotene), Beta carotene (β-Carotene), luteole (Zeaxanthin), lutein (Lutein) and astaxanthin (Astaxanthin) etc..

Carotenoid is widely distributed in nature, mainly micro- by plant, algae, cyanobacteria and some bacteriums, fungi etc. Biosynthesis, animal itself cannot synthesize carotenoid, can only be obtained by food chain.In photosynthetic organism, carotenoid The protective effect of photosynthesis and the participation anti-photooxidation by removing free radical is participated in as photopigment ingredient is caught, while being moved It is also played an important role in object and mankind's body.Such as carotenoid (especially beta carotene) is as vitamin in human body The important sources of A, carotenoid such as lycopene, beta carotene, astaxanthin and lutein etc. have very strong anti-oxidant work Property, the generation of free radical can be effectively inhibited, intracorporal chain type free base is blocked to react, prevents lipid peroxidation, delays to decline The formation of old and old atherosclerosis.It equally can treat disease and improves immunity of organisms.Meanwhile carotenoid Also there is biggish medical value, mankind's carotenoid of ingesting can not only prevent the generation of eye disease, cardiovascular disease, can be with Enhance the specificity and non-specific immune function of living organism, enhances tumour immunity.

Currently, at home and abroad in the market, carotenoid is mainly as medical material, functional food, dietary supplements and feeding Expect pigment, expensive, market demand is very big.The carotenoids red pigment of functional natural colorants is in medicine, food, cosmetics Etc. suffer from good application prospect, its development and application will be to relevant rows such as agricultural, food processing industry, pharmaceuticals industries Industry brings considerable economic benefit, has a vast market foreground.The production method of carotenoid includes chemical synthesis, micro- Biological fermentation process and three kinds of extracted form natural plant method.Wherein natural extraction method is just gradually becoming the main side of carotenoid production Method.It is extracted mainly from plant resources using fruits and vegetables as raw material, there are at high cost, production technology is complicated, product yield is relatively low etc. Problem, the method is not occupied an leading position at present.Compared with plant, most of algae can synthesize carotenoid, such as cyanobacteria In Arctic Ocean Synechococcus can synthesize luteole, beta carotene, kryptoxanthin etc., haematococcus (Haematococcus Pluvialis) under the conditions of nitrogen phosphate starvation, the astaxanthin accumulation in cell increases, the Du Shi algae (Dunaliella) in green alga Beta carotene can largely be accumulated.Simultaneously because microalgae does not strive ground with crops, strives water；Under the conditions ofs environment-stress such as nitrogen stress etc., Some unicellular microalgaes can largely accumulate carotenoid, much produce high added value object Quality Research using microalgae so having.And With metabolic engineering technology development and reach its maturity, by transformation, blocking and to introduce new metabolic pathway intracellular to improve The content of carotenoid can improve the yield of target product while reduce the generation of by-product.

In recent years, greater advance is achieved using the transgenic plant research of carotenogenesis related gene.Class Hu trailing plants The first step of Bu Su biosynthesis is to be catalyzed two Mang ox base Mang ox base pyrophosphoric acids (GGPP) by phytoene synthase (PSY) Molecule synthesis phytoene (phytoene).PSY, which is that one in Carotenoid biosynthetic pathway is crucial, adjusts enzyme, Its encoding gene becomes the preferred target gene in Carotenoid in Plants genetic engineering.GGPP is the reacting precursor of many approach, In tomato when overexpression PSY1 gene, GGPP largely turns to synthesis carotenoid, and the synthesis level of gibberellin declines. Carotenoid content increases 50 times in the seed of the PSY gene discovery maturation of overexpression bacterium in rape seed.In water PSY gene, LCY gene and the CRTI gene of bacterium that narcissus is co-expressed in rice endosperm, as a result in transgenic paddy rice seed endosperm A large amount of beta carotenes and its oxidation product leaf lutern, zeaxanthin etc. are had accumulated, this rice grain of rice is golden yellow, is known as " gold Color rice ".Using fruit-specific promoter in tomato excess or antisense expression Lcy-b gene, as a result, it has been found that overexpression Transgenic plant fruit in content beta-carotene increase significantly.Overexpression lycopene beta cyclase gene turns base Because of tomato, it is found that lycopene is reinforced to the conversion of beta carotene, and the content of total carotinoid also increase 50% with On.Overexpression Cartoene hydroxylase (BCH) increases the content of lutein in arabidopsis, the content of other carotenoid It is reduced there is no apparent.But, the carotenoid content and type that plant is improved by technique for gene engineering, also face Many problems, such as the balance of precursor GGPP supplied with the regulation of several metabolisms.

Cyanobacteria (cyanobacteria) is also known as cyanobacteria, is that one kind can be carried out photosynthetic prokaryotes.Cells of Blue-green Algae It can use simple inorganic matter synthesis of organic substance, expressed exogenous genes products do not form occlusion body, and most cyanobacterias And its extract is nontoxic to people and animals, is the good receptor of transgenic research.6803 (Synechocystissp.sp of cytoalgae PCC PCC6803) as a kind of unicellular blue green algae, have that the speed of growth is fast, condition of culture is simple, does not generate toxin, eucaryotic cell structure letter Single, genetic background understands, facilitates the features such as molecule manipulation, is suitable for use with wide core bioreactor large-scale production, is fine Cyanobacteria genetic engineering receptor.In view of the exogenous genes products easy purification of cyanobacteria expression, low without toxin, frustule toxigenic capacity And do not allow advantage easy to pollute etc., with the development of algal gene engineering, make using cyanobacteria as exogenous gene expression carrier with The high value added products such as production drug are possibly realized.

Four Carotenoids, i.e. beta carotene (β-carotene), luteole are significantly accumulated in DNC wireless (Zeaxanthin), echinenone (Echinenone) and myxoxanthophyll (Myxoxanthophyll).In DNC wireless Carotenoid biosynthetic pathway is shown in Fig. 1.Currently, being cloned into more than ten and carotenoid from DNC wireless Related gene is synthesized, respectively mainly there is the ipi of coding Isoprenoid isomerase, coding Mang ox base pyrophosphoric acid closes At the crtE of enzyme, the crtB of phytoene synthetase is encoded, encodes the crtP of phytoene dehydrogenase, encodes ζ-Hu The crtQ of radish element dehydrogenase encodes the crtO of beta carotene ketolase, encodes the crtR of beta carotene hydroxylase, coding is recklessly The crtH and coding for tomato red pigment cyclase/dioxygenase crtL of radish element isomerase^diox.They are taken part in respectively from isoamyl The hydroxylating of diene pyrophosphoric acid (IPP) to echinenone and luteole is reacted with ketonization.Carotenoids in DNC wireless Plain biosynthesis pathway is as shown in Figure 1.

Lycopene is the important branch point in carotenogenesis approach, forms one under the action of different cyclases The carrotene of series.The crtL of coding for tomato red pigment cyclase has been cloned from Synechococcus 7942.In recent years, thin in green sulphur A new class of difunctional lycopene cyclase gene C ruA is had found in bacterium, can be converted lycopene in Escherichia coli For gamma carotene.And homologous gene CruA is had also discovered in Synechococcus sp.PCC7002 (Synechococcus sp.PCC 7002) And CruP.CrtL-e is free of in DNC wireless, and there is not yet the report in relation to crtL-b.In DNC wireless Sll0659 gene (Genbank accession number WP_010873989.1) and Synechococcus CruP (Genbank accession number WP_ 012305684.1) amino acid similarity is 51.12%, and existing research shows the mutation of sll0659 to cyclization of lycopene mistake Journey influence is unobvious, but is not still improving DNC wireless alpha-carotene to sll0659 gene in DNC wireless Synthesize and promote photosynthetic aspect application report.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of sll0659 genes in synthesis cytoalgae carotenoid Application.After expression of the present invention by utilizing gene Knockout reduction sll0659 gene in DNC wireless, hair The percentage composition of myxoxanthophyll and luteole is now increased, wherein myxoxanthophyll content increases 22.3%, maize Cellulose content increases 31.3%, and echinenone content and beta carotene percentage composition then reduce 34.3% and 1.3% respectively. HPLC testing result shows compared with wild type DNC wireless, significantly increases in sll0659 knock out mutants body The content of alpha-carotene.Meanwhile normal light (40 μm of ol photons m^-2s^-1) and strong light (200 μm of ol photons m^-2s^-1) Under treatment conditions, compared with wild type DNC wireless, sll0659 gene knockout mutant strain PSI/PSII ratio obviously drops It is low.

Technical solution of the present invention is as follows:

A kind of application of sll0659 gene in synthesis cytoalgae carotenoid, the sll0659 gene nucleotide sequence Column are as shown in SEQ ID NO.1.

Above-mentioned application, steps are as follows:

(1) according to the DNC wireless sll0659 sequence amplification cytoalgae announced in GenBank database Fragment upstream is made in the upstream PCC6803 sll0659 cDNA segment, and nucleotide sequence is as shown in SEQ ID NO.1；

(2) according to the DNC wireless sll0659 sequence amplification cytoalgae announced in GenBank database Segments downstream is made in the downstream PCC6803 sll0659 cDNA segment, and nucleotide sequence is as shown in SEQ ID NO.2；

(3) fragment upstream made from step (1) is connected into carrier, with restriction enzyme BamHI and SalI double digestion, Carrier segments 1 are recycled, by restriction enzyme BamHI and the SalI double digestion of segments downstream made from step (2), recycling is made Segment 2 connects carrier segments 1 and segment 2, and sll0659BamHI carrier is made；

(4) restriction enzyme BamHI digestion pBluescript-Gm carrier is used, gentamicin resistance segment is recycled；It will After the restriction enzyme BamHI digestion of sll0659BamHI carrier made from step (3), connect with gentamicin resistance segment It connects, the carrier pKNsll0659-Gm of DNC wireless sll0659 gene knockout is made；

(5) carrier pKNsll0659-Gm made from step (4) is converted into DNC wireless, is screened, be made and turn base Because of cytoalgae, cultivated to get.

Preferred according to the present invention, in the step (1), amplimer nucleotide sequence is as follows:

Sll0659-1-F:5 '-ATGAACTATGCAACTACACC-3 '；

sll0659-1-BamHI-R:5'-CGAGGATCCGGCAATGATTGGTAATT-3'；

Preferred according to the present invention, in the step (1), amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, 1 μ of forward primer sll0659-1-F L, reverse primer sll0659-1-BamHI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

Preferred according to the present invention, in the step (1), amplification system is as follows:

94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 35 recycle；72℃ 10min, 4 DEG C of preservations.

Preferred according to the present invention, in the step (2), amplimer nucleotide sequence is as follows:

sll0659-2-BamHI-F:5'-GCAGGATCCAATACTTTTGGGAAGCT-3'；

sll0659-2-SalI-R:5'-CACGTCGACTCAATGGTGGAAATCGT-3'；

Preferred according to the present invention, in the step (2), amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, forward primer sll0659-2- BamHI-F1 μ L, reverse primer sll0659-2-SalI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

Preferred according to the present invention, in the step (2), amplification system is as follows:

94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 35 recycle；72℃ 10min, 4 DEG C of preservations.

Preferred according to the present invention, in the step (3), carrier is T3 carrier.

The present invention is used to construct in the DNC wireless of the sll0659 gene knockout in DNC wireless Sll0659 gene order is as shown in SEQ ID NO:3, and amino acid sequence is as shown in SEQ ID NO:4.

Sll0659 gene order in DNC wireless is 1569 bases, encodes 323 amino acid.By It is inserted into gentamicin resistance segment in sll0659 genetic fragment, constructs sll0659 gene knockout carrier, then carry out genetic transformation DNC wireless inhibits the expression of DNC wireless endogenous gene sll0659, obtains DNC wireless The transgenosis mutation algae strain that sll0659 gene expression is suppressed.Show that sll0659 gene knockout is prominent through HPLC testing result Myxoxanthophyll content increases 22.3% in mutant, and luteole content increases 31.3%, and echinenone content reduces 34.3%, beta carotene percentage composition then reduces 1.3%.Meanwhile HPLC testing result shows and wild type cytoalgae PCC6803 is compared, and is significantly increased the content of alpha-carotene in sll0659 knock out mutants body, is reduced photosystem component PSI/PSII ratio.

Beneficial effect

1, the present invention provides a kind of collection born of the same parents played a significant role in DNC wireless β-carotenogenesis The application of sll0659 gene in algae PCC6803, by by after the sll0659 gene knockout in DNC wireless, cytoalgae The lutein content of PCC6803 increases 22.3%, and luteole content increases 31.3%, and echinenone content reduces 34.3%, beta carotene percentage composition then reduces 1.3%.Meanwhile HPLC testing result shows and wild type cytoalgae PCC6803 is compared, and has an apparent peak in sll0659 knock out mutants body in carotenoid component, through compareing with mark product Detection, determines that it is alpha-carotene.

2, present invention orientation in DNC wireless knocks out sll0659 gene, for improving in DNC wireless Myxoxanthophyll, luteole and alpha-carotene content, enhance its biomass, improve photosynthetic efficiency be of great significance, this is The research of carotenoid provides theories integration in DNC wireless.

3, the gene applied in the present invention can provide support for microalgae and Carotenoid in Plants research.

Detailed description of the invention

Fig. 1 is Carotenoid biosynthetic pathway schematic diagram in DNC wireless；

Fig. 2 is DNC wireless sll0659 gene knockout carrier structure chart；

Fig. 3 is DNC wireless sll0659 knock out mutants body PCR amplification detection figure；

Fig. 4 is DNC wireless sll0659 knock out mutants body growth curve chart；

Fig. 5 is 77k fluorescence parameter detection under the conditions of the body light processing of DNC wireless sll0659 knock out mutants Figure；

In figure: A, 40 μm of ol photons m of normal light^-2s^-177k fluorescence parameter detection figure under treatment conditions；

B, 200 μm of ol photons m of strong light^-2s^-177k fluorescence parameter detection figure under treatment conditions；

Fig. 6 is DNC wireless sll0659 knock out mutants body HPLC detection figure.

Specific embodiment

With reference to the accompanying drawings of the specification and embodiment the present invention will be further described, but institute's protection scope of the present invention is unlimited In this.Following examples set forth the present invention in building DNC wireless sll0659 gene knockout and Overexpression vector And the method for conversion DNC wireless.

It is as follows to apply experimental material source used in example:

Coli strain (Escherichia coli) DH5 α and carrier T3 is limited purchased from the full formula gold biotechnology in Beijing Company,

Wild type DNC wireless is purchased from Chinese Academy of Sciences's American Type Culture Collection committee fresh water algae library；

Plasmid pBluescript-Gm is purchased from Chinese plasmid vector strain cell pnca gene collection；

Enzyme used in other, reagent and kit etc. are commercial product.

Embodiment 1

Separation clone is for constructing the sll0659 gene cDNA fragment for turning DNC wireless sll0659 gene knockout:

Log in: http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn& PAGE_TYPE=Blast

Search&LINK_LOC=blasthome, (Genbank is logged in input Synechococcus sp.PCC7002 CruP nucleotide sequence Number: EF529627.1), obtain the sll0659 gene order in the DNC wireless of Serial No. CP003265.1.

For constructing the sll0659 gene upstream and downstream fragment amplification of DNC wireless sll0659 gene knockout carrier:

According to announced in GenBank database DNC wireless sll0659 sequence (accession number are as follows: CP003265.1) the design amplification upstream DNC wireless sll0659 cDNA fragment primer:

Sll0659-1-F:5 '-ATGAACTATGCAACTACACC-3 '；

sll0659-1-BamHI-R:5'-CGAGGATCCGGCAATGATTGGTAATT-3'；

Amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, 1 μ of forward primer sll0659-1-F L, reverse primer sll0659-1-BamHI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

Amplification program is as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, and 35 After circulation, 72 DEG C of 10min, 4 DEG C of preservations.

PCR carries out electrophoresis gel extraction after reaction, converts bacillus coli DH 5 alpha after being connected to T3 carrier cloning carrier, Screening positive clone is simultaneously sequenced, and fragment upstream is made, nucleotide sequence is as shown in SEQ ID NO.1；

Expand the downstream DNC wireless sll0659 cDNA fragment primer:

sll0659-2-BamHI-F:5'-GCAGGATCCAATACTTTTGGGAAGCT-3'；

sll0659-2-SalI-R:5'-CACGTCGACTCAATGGTGGAAATCGT-3'；

Amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, forward primer sll0659-2- BamHI-F1 μ L, reverse primer sll0659-2-SalI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

Amplification program is as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, and 35 After circulation, 72 DEG C of 10min, 4 DEG C of preservations.

PCR carries out electrophoresis gel extraction after reaction, converts bacillus coli DH 5 alpha after being connected to T3 carrier cloning carrier, Screening positive clone is simultaneously sequenced.Segments downstream is made, nucleotide sequence is as shown in SEQ ID NO.2.

Embodiment 2

The building and conversion that DNC wireless sll0659 gene knockout carries

By being inserted into inactivation technique, it is mutated sll0659 genetic fragment in DNC wireless, is lacked according to sll0659 gene Lose mutation algae strain phenotype and carotenoid composition research DNC wireless sll0659 gene in carotenogenesis and Application in photosynthesis.

The building of DNC wireless sll0659 gene knockout carrier:

The fragment upstream positive colony plasmid of DNC wireless sll0659 gene made from embodiment 1 is limited Property restriction endonuclease BamHI and SalI double digestion, recycle carrier segments 1；With same method double digestion DNC wireless The segments downstream positive colony plasmid of sll0659 gene recycles segment 2.It is anti-that connection is done with the segment 2 and carrier segments 1 of recycling It answers, converts bacillus coli DH 5 alpha.By screening positive clone, sll0659BamHI carrier is obtained, restriction enzyme is then used BamHI single endonuclease digestion sll0659BamHI carrier recycles carrier segments sll0659BamHI；With the mono- enzyme of restriction enzyme BamHI PBluescript-Gm carrier is cut, gentamicin resistance segment is recycled.With the gentamicin resistance segment and carrier segments of recycling Sll0659BamHI does connection reaction, conversion conversion bacillus coli DH 5 alpha.By screening positive clone, cytoalgae is obtained PCC6803 sll0659 gene knockout carrier is named as pKNsll0659-Gm (as shown in Figure 1).

Pass through Natural Transformation method (Williams, J.G.K, 1988.Construction of specific mutationsin photosystem II photosynthetic reaction center by genetic Engineering methods inSynechocystis6803.Methods Enzymol.167:766-778.) it will build Vector introduction DNC wireless in, through antibiotic-screening, identification obtains transgenic positive cytoalgae, and steps are as follows:

Take logarithm culture period (OD₇₃₀=0.6) for DNC wireless 30ml in room temperature, 4500g is centrifuged 8min, in abandoning Clearly；After adding fresh BG-11 fluid nutrient medium washed once, add fresh BG-11 fluid nutrient medium to final concentration OD730=4.8, and It is used to convert at once；The algae solution of collection is dispensed into 1.5ml EP pipe (every 400 μ l of pipe), every pipe adds 5-10 μ g plasmid, weak Illumination incubates 6 hours under the conditions of light, during which rocks primary.Mixture is coated onto containing gentamicin antibiotic (10 μ g/ml) On BG-11 plating medium.About 10 days visible conversion.The obtained pKNsll0659-Gm of gene knockout carrier containing sll0659 is made Transgenic Synechocystis PCC6803.

Embodiment 3

The algae strain PCR detection of sll0659 gene knockout

Using the transgenic Synechocystis PCC6803 and wild type DNC wireless of the pKNsll0659 containing knockout carrier as material Material extracts total DNA and carries out PCR detection and analysis.The specific method is as follows:

Using neutral phenol reagent (being purchased from Invitrogen company) and method is shaken from sll0659 gene knockout using bead DNA is extracted in the cytoalgae and wild type cytoalgae of overexpression.Specific steps are as follows:

Take 50ml OD₇₃₀=1.8 cyanobacteria, 4 DEG C, 5000rpm is centrifuged 10min and collects frustule, and 0.4mL neutral phenol is added Reagent (being purchased from Invitrogen company) connects 0.4mL BG-11 fluid nutrient medium, and it is 0.17mm left that suitable diameter, which is then added, There is the suspension of 0.5mL above right bead (being purchased from sigma company) to bead interface.By turbula shaker with maximum Rate shakes 1min, and 4 DEG C, 11900rpm is centrifuged 10min, takes supernatant into new 1.5ml centrifuge tube, and addition 0.5mL phenol/ Chloroform/isoamyl alcohol (volume ratio 25:24:1), is mixed by inversion 15s, stands 3~5min, and 4 DEG C, 11900rpm centrifugation 10~ 15min.It takes supernatant into new 1.5ml centrifuge tube, 0.5mL isopropanol is added, is stored at room temperature 10min after being mixed by inversion.4 DEG C, 11900rpm is centrifuged 10min.Supernatant is removed, is added 75% ethyl alcohol of 1ml (v/v), overturns oscillation several times, 4 DEG C, 7500rpm centrifugation 10min.Abandon supernatant, room temperature lower open mouth dry to precipitate it is transparent, appropriate ddH is added₂O dissolution precipitating, is made sll0659 base respectively Because of the cytoalgae and wild type cytoalgae total DNA for knocking out and being overexpressed.

Using sll0659 knockout transgenic cytoalgae genomic DNA and wild type cytoalgae DNA as template, with Sll0659-1-F and sll0659-2-SalI-R is primer, carries out PCR amplification, and specific amplification program is as follows:

94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 35 recycle, 72 DEG C 10min, 4 DEG C of preservations.

Reaction system:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, 1 μ of forward primer sll0659-1-F L, reverse primer sll0659-2-SalI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

Electrophoresis detection PCR product result is as shown in Figure 3.

Embodiment 4

The algae strain growth curve measurement of DNC wireless sll0659 knock out mutants

Wild type DNC wireless and the algae strain of sll0659 gene knockout are inoculated in 50ml BG-11 fluid nutrient medium In, adjust OD₇₃₀=0.1, in 30 DEG C, 40 μ E.m^-2.s^-1Shaken cultivation (180rpm) under the conditions of continuous light, respectively at 1 day, 2 It, 3 days, 4 days, 5 days, 6 days, samples, by measuring OD for 7 days₇₃₀Monitor its growing state, as a result as shown in Figure 4.

As seen from Figure 4, initial inoculation concentration OD₇₃₀=0.1, after inoculation wild type DNC wireless and The sll0659 gene knockout mutant strain speed of growth gradually increases.But the sll0659 knock out mutants body speed of growth is obvious low In wild type DNC wireless, reaching maximum in both the 5th day concentration difference, concentration difference gradually becomes smaller both after the 6th day, and the 7th It when, mutant strain has been approached wild type concentration.Illustrate that sll0659 gene knockout affects the life of mutant strain to a certain extent It is long, but after cultivating a period of time, the two gap is gradually decreased.

Embodiment 5

The algae strain 77k fluorescence emission spectrum detection of DNC wireless sll0659 knock out mutants

Wild type DNC wireless and the algae strain of sll0659 knock out mutants are inoculated in the training of 50ml BG-11 liquid It supports in base, adjusts OD₇₃₀=0.1, in 30 DEG C, 40 μ E.m^-2.s^-1Shaken cultivation (180rpm) under the conditions of continuous light, in the 7th day 1ml liquid medium is taken, supernatant is abandoned in 13,000rpm centrifugations 5 minutes, and adding 1ml DMF, (n,N-Dimethylformamide is purchased from traditional Chinese medicines Chemical reagent Co., Ltd of group), it mixes, 13,000rpm centrifugations 3 minutes detect OD using spectrophotometer₆₂₅And OD₆₆₄Value. Chlorophyll content (Chlorophyll a, μ g/ml) is calculated according to chlorophyll calculation formula:

[Chlorophyll a]=12.1OD₆₆₄-0.17OD₆₂₅

Frond is collected by centrifugation, adjustment chlorophyll content is 15mg, adds 1ml BG-11 fluid nutrient medium, and mixing, Yu Ye are beaten in suction Green element fluorescence analyser detects 77k value, as a result as shown in Figure 5.

As seen from Figure 5, under normal light treatment conditions, compared with wild type DNC wireless, sll0659 gene Knockout mutant strain PSI/PSII ratio obviously increases；And under the conditions of strong light processing, compared with wild type, sll0659 gene knockout Mutant strain PSI/PSII ratio is substantially reduced.

Embodiment 6

The algae strain class HPLC detection of DNC wireless sll0659 knock out mutants

Wild type DNC wireless and sll0659 gene are crossed into knockout mutations algae strain and are inoculated in 50ml BG-11 liquid In culture medium, frustule is collected by centrifugation in logarithmic growth phase, is freeze-dried in liquid nitrogen.It is dry from freezing with methanol (HPLC rank) Pigment is extracted in dry wild type DNC wireless and sll0659 knock out mutants algae strain cell.Pass through high-efficient liquid phase color Spectrometry (HPLC) separates carotenoid, and column model used is Spherisorb ODS24.0mm × 250mm C18 chromatography Column, ethyl acetate gradient is 0-100% in 15 minutes, and second cyanogen-water-triethylamine (volume ratio 9:1:0.01) flow velocity is 1ml/ min.The absorption spectrum at individual peaks is obtained using photodiode array detector.β-carotene standard items are purchased from the U.S. Sigma company, as a result as shown in Figure 6.

As seen from Figure 6, by by after the sll0659 gene knockout in DNC wireless, DNC wireless Lutein content increase 22.3%, luteole content increases 31.3%, and echinenone content reduces 34.3%, β- Carrotene percentage composition then reduces 1.3%.Meanwhile HPLC testing result shows compared with wild type DNC wireless, Have an apparent peak in sll0659 knock out mutants body in carotenoid component, through with mark product control test, determine it For alpha-carotene.

Claims (8)

1. a kind ofsll0659Gene is in improving cytoalgae carotenoid Lutein, luteole and alpha-carotene content Application, it is describedsll0659Gene nucleotide series are as shown in SEQ ID NO.1, and steps are as follows:

(1) according to the DNC wireless announced in GenBank database sll0659Sequence amplification DNC wirelesssll0659Fragment upstream is made in upstream cDNA segment, and nucleotide sequence is as shown in SEQ ID NO.1；

(2) according to the DNC wireless announced in GenBank database sll0659Sequence amplification DNC wirelesssll0659Segments downstream is made in downstream cDNA segment, and nucleotide sequence is as shown in SEQ ID NO.2；

(3) fragment upstream made from step (1) is connected into carrier, with restriction enzyme BamHI and SalI double digestion, is returned Carrier segments 1 are received, by restriction enzyme BamHI and the SalI double digestion of segments downstream made from step (2), recycling is made Segment 2 connects carrier segments 1 and segment 2, is madesll0659BamHI carrier；

(4) restriction enzyme BamHI digestion pBluescript-Gm carrier is used, gentamicin resistance segment is recycled；By step (3) obtainedsll0659It after the restriction enzyme BamHI digestion of BamHI carrier, connect, makes with gentamicin resistance segment Obtain DNC wireless sll0659The carrier pKNsll0 of gene knockout659-Gm；

(5) by carrier pKNsll0 made from step (4)659- Gm converts DNC wireless, is screened, and transgenosis collection is made Born of the same parents algae, cultivated to get.

2. application as described in claim 1, which is characterized in that in the step (1), amplimer nucleotide sequence is as follows:

Sll0659-1-F:5 '-ATGAACTATGCAACTACACC-3 '；

sll0659-1-BamHI-R: 5’-CGAGGATCCGGCAATGATTGGTAATT-3’。

3. application as described in claim 1, which is characterized in that in the step (1), amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L of 1 μ L, forward primer sll0659-1-F, instead To primer sll0659-1-BamHI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

4. application as described in claim 1, which is characterized in that in the step (1), amplification reaction condition is as follows:

94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 35 recycle；72 DEG C of 10min, 4 DEG C of preservations.

5. application as described in claim 1, which is characterized in that in the step (2), amplimer nucleotide sequence is as follows:

sll0659-2-BamHI-F: 5'-GCAGGATCCAATACTTTTGGGAAGCT-3'；

sll0659-2-SalI-R: 5’-CACGTCGACTCAATGGTGGAAATCGT -3’。

6. application as described in claim 1, which is characterized in that in the step (2), amplification system is as follows:

2 × Hifi PCR Mix, 10 μ L, genome of E.coli DNA profiling 1 μ L, forward primer sll0659-2-BamHI-F 1 μ L, reverse primer sll0659-2-SalI-R 1 μ L, ddH₂7 μ L of O, overall reaction system are 20 μ L.

7. application as described in claim 1, which is characterized in that in the step (2), amplification reaction condition is as follows:

94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 35 recycle；72 DEG C of 10min, 4 DEG C of preservations.

8. application as described in claim 1, which is characterized in that in the step (3), carrier is T3 carrier.