CN106755052B - Method for reducing aspergillus niger beta-glucosidase expression enzyme activity by light-controlled promoter - Google Patents
Method for reducing aspergillus niger beta-glucosidase expression enzyme activity by light-controlled promoter Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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Abstract
The invention discloses a method for reducing the expression enzyme activity of Aspergillus niger beta-glucosidase by using a light-controlled promoter, belonging to the field of genetic engineering. The method comprises the following steps: (1) constructing a DNA fragment consisting of a light-controlled promoter or a DNA sequence containing the light-controlled promoter, a DNA sequence expressing antisense mRNA complementary to the mRNA of the beta-glucosidase and a terminator sequence on an expression vector; (2) transforming the recombinant vector constructed in the step (1) into aspergillus niger; (3) the Aspergillus niger strains transformed with the recombinant vectors were cultured under light conditions. The promoter for photo-enhancement expression is used for regulating the complementary antisense mRNA transcription of the mRNA of the beta-glucosidase, and the translation amount and the expression activity of the beta-glucosidase are effectively reduced, so that the cellulase system combination expressed by Aspergillus niger is optimized, and the efficiency of degrading cellulose by the cellulase system is maximized.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a method for reducing the expression enzyme activity of Aspergillus niger beta-glucosidase by using a light-controlled promoter.
Background
Cellulase (beta-1, 4-glucan-4-glucan hydrolase) is a general name of a group of enzymes for degrading cellulose to generate glucose, is not a monomer enzyme, is a multi-component enzyme system with a synergistic effect, is a complex enzyme, and mainly comprises exo-beta-glucanase, endo-beta-glucanase, beta-glucosidase and the like.
When the cellulose is degraded by the cellulase, the cellulase is firstly hydrolyzed into short-chain soluble fragments by the exonuclease, the short-chain fragments are hydrolyzed into cellobiose by the endonuclease, and the cellobiose is hydrolyzed into glucose under the action of beta-glucosidase. Enzymolysis of cellulose by cellulase is a synergistic degradation process, and enzymatic hydrolysis efficiency of cellulose is reduced by too high or too low of one enzyme activity.
Aspergillus niger is a food safety bacterium, and the produced cellulase has wide application fields, such as sewage treatment, garbage utilization, food and the like. The enzymatic activity of beta-glucosidase in cellulase produced by aspergillus niger is relatively high, and the composition of three enzymes is not beneficial to the maximized catalytic efficiency of the enzymes. Reducing the activity of beta-glucosidase is an important measure for optimizing the composition of cellulase enzyme systems. The existing measures for reducing the expression efficiency of certain enzymes are mainly gene knockout, and some effects are achieved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for reducing the expression enzyme activity of Aspergillus niger beta-glucosidase by using a light-controlled promoter. The invention also aims to provide an Aspergillus niger strain with the function of photoinduced reduction of the activity of beta-glucosidase.
The purpose of the invention is realized by the following technical scheme:
a method for reducing the expression enzyme activity of Aspergillus niger beta-glucosidase by light-controlled promoter comprises the following steps:
(1) constructing a DNA fragment consisting of a light-controlled promoter or a DNA sequence containing the light-controlled promoter, a DNA sequence expressing antisense mRNA complementary to the mRNA of the beta-glucosidase and a terminator sequence on an expression vector;
(2) transforming the recombinant vector constructed in the step (1) into aspergillus niger;
(3) the Aspergillus niger strains transformed with the recombinant vectors were cultured under light conditions.
The DNA sequence containing the light-regulated promoter in the step (1) is preferably shown as SEQ ID NO. 1.
The DNA sequence of the antisense mRNA expressed complementary to the β -glucosidase mRNA described in the step (1) is preferably as shown in SEQ ID NO. 2.
The terminator sequence described in step (1) is preferably as shown in SEQ ID NO. 3.
The expression vector in the step (1) is preferably pCAMBIA1301 vector.
Step (1) is preferably: passing the DNA fragment with the sequence shown in SEQ ID NO.4 through restriction endonucleaseEcoI、KpnI and DNA ligase are constructed on a pCAMBIA1301 vector to obtain a recombinant vector.
The Aspergillus niger described in step (2) is preferably Aspergillus niger ATCC 16404.
Step (2) preferably, the constructed recombinant vector is transformed into Aspergillus niger by Agrobacterium.
The illumination condition in step (3) is preferably blue light.
An Aspergillus niger strain with lowered photoinduced beta-glucosidase activity, which is the Aspergillus niger strain transformed with the recombinant vector and constructed in the method.
The invention has the following advantages and beneficial effects: the promoter for photo-enhancement expression is used for regulating the complementary antisense mRNA transcription of the mRNA of the beta-glucosidase, and the translation amount and the expression activity of the beta-glucosidase are effectively reduced, so that the cellulase system combination expressed by Aspergillus niger is optimized, and the efficiency of degrading cellulose by the cellulase system is maximized.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
1. Material
Aspergillus nigerAspergillus niger) ATCC16404, Escherichia coli (E.coli)Escherichia coli) DH5 alpha, Agrobacterium tumefaciens (A)Agrobacterium tumefaciens) AGL1, plasmid pCAMBIA1301 (Biovector).
An artificially synthesized DNA sequence 1 (shown in SEQ ID NO. 4) which consists of a DNA sequence containing a light-controlled promoter, a DNA sequence expressing antisense mRNA complementary with beta-glucosidase mRNA, a terminator sequence and enzyme digestion site sequences at two ends.
2. Method of producing a composite material
(1) Extraction of plasmid pCAMBIA1301
Coli DH5 a containing plasmid pCAMBIA1301 was cultured in LB medium containing 50. mu.g/mL kanamycin for 18 h. Plasmid extraction was performed according to the procedure of the one-step plasmid DNA extraction kit (product No. B518188) of Shanghai Biotech. The method comprises the following specific steps: 0.5mL of bacterial liquid is taken, centrifuged at 10000r/m for 3min to collect thalli, and the thalli are poured out or the culture medium is sucked dry. Add 700. mu.L lysine Buffer UF to the bacterial precipitation, suck or shake to completely suspend the bacterial, and leave at room temperature for 3 min. The lysate was carefully transferred to an adsorption column, left at room temperature for 1min and centrifuged at 8000rpm for 2 min. Pouring out the liquid in the collecting tube, putting the adsorption column into the same collecting tube, adding 500 μ L of Prewash Solution into the adsorption column, and centrifuging at 8000rpm for 2 min. Pouring out the liquid in the collecting tube, putting the adsorption column into the same collecting tube, adding 500 μ L Wash Solution into the adsorption column, and centrifuging at 8000rpm for 1 min. Pouring out the liquid in the collecting tube, putting the adsorption column into the same collecting tube, adding 500 μ L Wash Solution into the adsorption column again, and centrifuging at 8000rpm for 1 min. And (3) pouring out liquid in the collecting pipe, putting the adsorption column into the same collecting pipe, putting the empty adsorption column and the collecting pipe into a centrifugal machine, and centrifuging at 8000rpm for 2 min. The adsorption column was placed in a clean 1.5mL centrifuge tube, 50. mu.L of precipitation Buffer was added to the center of the adsorption membrane, left to stand at room temperature for 2min, and centrifuged at 8000r/m for 2 min. The resulting plasmid DNA solution was stored at-20 ℃ or used for subsequent experiments.
(2) Construction of recombinant plasmid
1) pCAMBIA1301 plasmid, restriction enzyme digestion and recovery of synthetic DNA sequence 1
To 20. mu.L of the extracted pCAMBIA1301 plasmid or DNA sequence 1 diluted 10-fold with double distilled water was addedEcoI andKpni (Takara) 2. mu.L of each digestion Buffer and 2.2. mu.L of each digestion Buffer were put in a water bath at 37 ℃ for 3 hours, and then 2. mu.L of Loading Buffer was added to terminate the reaction.
The enzyme-cut plasmid or DNA sequence 1 is recovered by a SanPrep nucleic acid purification kit (gel recovery) kit (B515103-0100) of Shanghai, after agarose gel electrophoresis, to obtain pCAMBIA1301 plasmid enzyme-cut recovery liquid and DNA sequence 1 enzyme-cut recovery liquid.
2) Ligation and transformation
The connecting system is as follows: pCAMBIA1301 plasmid digestion recovery liquid 17.5. mu.L, DNA sequence 1 digestion recovery liquid 2. mu.L, DNA ligase (Takara) 2.5. mu.L, ligase buffer 2.2. mu.L. Ligation was carried out at 16 ℃ for 24h and the ligation product was ready for use.
And transforming the ligation product into escherichia coli DH5 alpha competent cells, selecting a transformant for culturing, carrying out enzyme digestion and sequencing identification on the quality-improved plasmid, and identifying the correct recombinant plasmid to be named as pCAMBIA 01.
(3) Preparation of aspergillus niger spore protoplast and transformation of recombinant plasmid
1) Bevel activation
In a sterile operating platform, the preserved Aspergillus niger ATCC16404 strain is inoculated into a sterilized slant culture medium, and is placed in a constant temperature incubator at 30 ℃ for 3 days, and spores grow on the slant.
The slant culture medium is PDA culture medium, and its preparation method comprises: 60g of fresh potatoes are taken, peeled and cut into filaments, a proper amount of distilled water is added, the fresh potatoes are placed in a cooking pot to be boiled and are continuously stirred by a glass rod to be pasty, eight layers of gauze are used for filtering to obtain filtrate, 2g of glucose and 2g of agar which are weighed by balance are sequentially added, the mixture is stirred uniformly, finally, the volume is fixed to 100mL by distilled water, and the mixture is heated and dissolved. Subpackaging in test tubes to about one third of the test tubes, then filling in a plug, wrapping, and sterilizing at 121 deg.C under 0.1Mpa for 20 min. And after the sterilization is finished, placing an inclined plane for cooling for standby.
2) Preparation of spore suspension
The activated Aspergillus niger spores are scraped from the test tube into a triangular flask filled with normal saline, and the flask is placed on a shaking table at 30 ℃ and shaken at 150r/m for 10 min. Taking out, filtering with 4 layers of mirror paper, centrifuging the filtrate at 3000r/m, discarding supernatant, and washing with normal saline for 1-2 times. The suspension was then microscopically examined and counted to adjust the spore concentration to approximately 5X 108Culturing at 50 deg.C for 1min and 30 deg.C for 8 hr to obtain spore germination suspension.
3) Preparation of protoplast suspension
Adopting hypertonic solution, namely 0.8mol/L NaCl solution to prepare 10mL of mixed enzyme solution of 0.5% helicase and 1% cellulase, adding 10mL of spore germination suspension, carrying out water bath at 30 ℃ for 4h, filtering by using a microporous filter membrane, repeatedly washing the spores on the filter membrane by using 0.8mol/L NaCl solution, and finally dissolving the spores on the filter membrane into 10mL of 0.8mol/L NaCl solution to obtain the protoplast suspension.
4) Preparation of agrobacterium tumefaciens competence
Agrobacterium tumefaciens AGL1 was picked and inoculated into 3mL of LB liquid medium and cultured overnight at 28 ℃ at 180 r/min. Inoculating 2mL of the culture medium into 100mL of LB liquid medium, and continuing the culture until OD is reached600About 0.5. Placing the culture solution in ice bath for 40min, centrifuging at 4 deg.C and 5000r/min for 10min, and discarding supernatant. The supernatant was discarded after 1 centrifugation and washing with 10mL of 4 ℃ pre-cooled sterile water. Then 10mL of 10% glycerol precooled at 4 ℃ is used for suspending the thalli, centrifugation is carried out for 5min at 4 ℃ and 5000r/min, and supernatant is discarded. Finally using 1mL of 4 DEG CPre-cooled 10% glycerol was suspended and split into 70. mu.L portions per tube and stored at-70 ℃.
5) Transformation and culture of Agrobacterium tumefaciens
Adding 1 μ L of recombinant plasmid pCAMBIA01 into 70 μ L of Agrobacterium tumefaciens AGL1 competent cells, mixing, sucking, adding into an electric rotating cup with a distance of 0.2cm, wiping, adjusting the electric shock voltage to 2.5kV, and electric shock for 5 ms. After the electric transformation, Agrobacterium tumefaciens was plated on LB plate (containing 50. mu.g/mL kanamycin) to perform screening. Positive clones obtained from the selection were streaked on LB plates (containing 50. mu.g/mL kanamycin) and cultured at 28 ℃ for 2 days. A single colony is picked and inoculated in 5mL LB liquid medium (containing 50 ug/mL kanamycin), and cultured for 16-20 h at 28 ℃ and 180 r/min. Inoculating 1mL of Agrobacterium tumefaciens bacterial liquid into 100mL of LB liquid culture medium containing corresponding resistance, and culturing at 28 ℃ and 180r/min to OD600About 0.8 for standby.
6) Induction culture and transformation
And (3) induction culture of agrobacterium tumefaciens: take 4mL (OD)6000.8) was centrifuged at 5000r/min for 5min to collect the cells, and the cells were suspended in 6mL of an IM medium containing 400. mu. mol/L acetosyringone and cultured at 28 ℃ for 5 hours. IM medium: KH (Perkin Elmer)2PO4 1.45 g，K2HPO4 2.05g，NH4NO3 0.5g，CaCl2 0.01g，MgSO4·7H20.6g of O, 0.3g of NaCl, 2g of glucose, 5g of glycerol, 1000mL of water and hydrochloric acid to adjust the pH value to 5.4.
And (3) transformation: 250 mu L of aspergillus niger protoplasm suspension, adjusting the concentration of the induced agrobacterium tumefaciens liquid, uniformly mixing the aspergillus niger spores and the agrobacterium tumefaciens in equal volume according to the quantitative ratio of 1:100, and coating the mixture on an IM solid culture medium of a transformation medium acetic acid and nitric acid mixed membrane.
7) Screening for transformants
And (3) carrying out light-shielding culture for 48h at 24 ℃ on Aspergillus niger coated on an IM solid culture medium of a transformation medium acetic acid and nitric acid mixed membrane, diluting spores by using sterile water, coating the diluted spores on a plate containing 150 mu g/mL hygromycin B for primary screening, culturing for 4d, transferring grown hypha to an IM culture medium containing 200 mu g/mL hygromycin B for secondary screening, and obtaining a transformant which is successfully transformed.
(4) Photoinduced reduction of beta-glucosidase expression activity
1) Enzyme producing culture
Preparing an enzyme production culture medium: 10g of bran, 1g of peptone, 1g of beef extract and 1L of tap water, and sterilizing at 115 ℃ for 30 min. A250 mL Erlenmeyer flask was filled with 150mL of liquid enzyme-producing medium.
Inoculating Aspergillus niger or selected transformant into sterilized slant PDA culture medium in aseptic operation table, and culturing in 30 deg.C incubator for 3d to obtain slant spore. Inoculating the slant spore into enzyme-producing culture medium with an inoculum size of 1 × 105And (3) coating the whole triangular flask with kraft paper, culturing at 30 ℃ for 36h at 150r/m, filtering the culture solution with filter paper, and measuring the enzyme activity of the filtrate beta-glucosidase. The enzyme activity of the original strain is 22U/mL, and the enzyme activity of the selected transformant is 21.0U/mL.
The enzyme activity of the beta-glucosidase is determined according to the method in the literature (Zhang Li, Jing, Yang Ting. producing beta-glucosidase saccharomyces cerevisiae strain ultraviolet mutagenesis breeding and enzymological property analysis, 2015 in food industry science and technology).
2) Light-regulated enzyme production culture
A40W blue fluorescent lamp was used as a blue light source, and the triangular flask was irradiated with blue light through a blue filter (No. 73, cut-off filter manufactured by Japan K.K.) so that the strongest wavelength of blue light was 450nm and the full width at half maximum was 50 nm.
According to the method, after the slant spores are inoculated to the enzyme production culture medium, a triangular flask filled with the enzyme production culture medium is wrapped without kraft paper, the triangular flask is arranged right below a blue fluorescent lamp and is vertically spaced at 40cm, cultured at 150r/m and 30 ℃ for 36h, a culture solution is filtered by filter paper, and the enzyme activity of the filtrate beta-glucosidase is measured. The enzyme activity of the original strain is 23.1U/mL, and the enzyme activity of the selected transformant is 8.1U/mL.
The results show that under the induction of blue light, the light-controlled promoter starts the transcription of beta-glucosidase antisense mRNA of a transformant, the combination of the antisense mRNA and the beta-glucosidase mRNA inhibits the translation of the beta-glucosidase mRNA, and the translation efficiency of the beta-glucosidase of the transformant is reduced, so that the expression level of the beta-glucosidase is reduced.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Hubei university of industry
<120> method for reducing Aspergillus niger beta-glucosidase expression enzyme activity by light-controlled promoter
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1430
<212> DNA
<213> Artificial
<220>
<223> DNA sequence containing light-regulated promoter
<400> 1
attggaatgc tacagacgct atatgtaccc cttctgttat gtacataaag taacatttca 60
catctggctc tcttatgcag cataaaaaaa gattgttctg cgctccaatt cggtccatgc 120
tgacatgagc gggaggtgcg cgtgagggtt gacaaaagtc gagaggtcta gctttcagct 180
ctcagatatt agattcttgg cgactgcgtc ctgctcccta ttaaatgctc accccttgat 240
cggaaaatca aacctgcctg cgcaattacc taactatcag ttttaaaggc actgtattct 300
attccaacca ggtatccacg tagctcccac cgggcctccc atcgctctcc gttcgctccg 360
cccactaagg agctaccagc atcccactag tactccattt ttgtgaatta acaatacaat 420
actaaactgg cgtcgtggga ctagagtaaa atagatatgg tgataacatt gtctagataa 480
ggataaaaaa aataatatat aaaaatatat aatatgatag tagaagtact tggaggctct 540
tgtctcttac tactgggcag gagagactta tatttggcag tagatcccgc tgcgattggt 600
tgttctacct ggttattctc ttgatttgat ggagcggaaa ggttgtttgg tcccagtact 660
tacgacatac catgtaataa taaatattat tggagaaaag ggtgggaggg gacaaaaggg 720
agttctttca tgcgccaggc agagcgaaag atggaggaac agaagacatt tgggggggct 780
ggtggtggga agaatcagca gacaccgtcc taatggctac ctcactggtc catggccgag 840
cgagagaccc tgaccaactt atgcgcgttt ggcttttccc gctcggaact acgtaaaaca 900
gggcaagaaa gtccgagcag cctgcagctg cttgcgcggt ttcagtattt gcagggataa 960
ctatcggctt atcttaacgt tgctcatgct atttattggt gggtgatgtc gagcaaccta 1020
agagctagat ggatcggacc tttttgcaac aggtatgaga tacatataag tcgactacac 1080
tacgctcgag cagactgcac tctcatttca ctagcaatgg ttgtccttat ctcggctggt 1140
ggtatcggct atttgcgcca tcgggagtat tcatttagct agtagtaaac cgcagtggct 1200
atagactata catcttctac cacgaatttc atgaaattac gatgcgctag tcaggaaggt 1260
ccatgacagt tccattgctg gatctctttc actattaaag ttattacacg gcgcgcctct 1320
ttccactact tttatctttg cctttatttc ttctctgaaa cgctttagta tgtcatccca 1380
tcaaaccagc cccagaggaa tgtcgtgtct tgcattgtgc tcatccgaca 1430
<210> 2
<211> 110
<212> DNA
<213> Artificial
<220>
<223> DNA sequence expressing antisense mRNA complementary to beta-glucosidase mRNA
<400> 2
acagacacaa agaccggtaa agttgaggcg cgggatctcg gcaatgaagc cggagcagcc 60
agtcttcgag ctcactccag ccgtgagatt gaccttttcg tcgtccgtca 110
<210> 3
<211> 214
<212> DNA
<213> Artificial
<220>
<223> terminator sequence
<400> 3
cgacaagctc gacctcgagt ttctccataa taatgtgtga gtagttccca gataagggaa 60
ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat 120
ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagta 180
ctaaaatcca gatcccccga attaattcgg cgtt 214
<210> 4
<211> 1770
<212> DNA
<213> Artificial
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<223> DNA sequence 1
<400> 4
ggggtaccat tggaatgcta cagacgctat atgtacccct tctgttatgt acataaagta 60
acatttcaca tctggctctc ttatgcagca taaaaaaaga ttgttctgcg ctccaattcg 120
gtccatgctg acatgagcgg gaggtgcgcg tgagggttga caaaagtcga gaggtctagc 180
tttcagctct cagatattag attcttggcg actgcgtcct gctccctatt aaatgctcac 240
cccttgatcg gaaaatcaaa cctgcctgcg caattaccta actatcagtt ttaaaggcac 300
tgtattctat tccaaccagg tatccacgta gctcccaccg ggcctcccat cgctctccgt 360
tcgctccgcc cactaaggag ctaccagcat cccactagta ctccattttt gtgaattaac 420
aatacaatac taaactggcg tcgtgggact agagtaaaat agatatggtg ataacattgt 480
ctagataagg ataaaaaaaa taatatataa aaatatataa tatgatagta gaagtacttg 540
gaggctcttg tctcttacta ctgggcagga gagacttata tttggcagta gatcccgctg 600
cgattggttg ttctacctgg ttattctctt gatttgatgg agcggaaagg ttgtttggtc 660
ccagtactta cgacatacca tgtaataata aatattattg gagaaaaggg tgggagggga 720
caaaagggag ttctttcatg cgccaggcag agcgaaagat ggaggaacag aagacatttg 780
ggggggctgg tggtgggaag aatcagcaga caccgtccta atggctacct cactggtcca 840
tggccgagcg agagaccctg accaacttat gcgcgtttgg cttttcccgc tcggaactac 900
gtaaaacagg gcaagaaagt ccgagcagcc tgcagctgct tgcgcggttt cagtatttgc 960
agggataact atcggcttat cttaacgttg ctcatgctat ttattggtgg gtgatgtcga 1020
gcaacctaag agctagatgg atcggacctt tttgcaacag gtatgagata catataagtc 1080
gactacacta cgctcgagca gactgcactc tcatttcact agcaatggtt gtccttatct 1140
cggctggtgg tatcggctat ttgcgccatc gggagtattc atttagctag tagtaaaccg 1200
cagtggctat agactataca tcttctacca cgaatttcat gaaattacga tgcgctagtc 1260
aggaaggtcc atgacagttc cattgctgga tctctttcac tattaaagtt attacacggc 1320
gcgcctcttt ccactacttt tatctttgcc tttatttctt ctctgaaacg ctttagtatg 1380
tcatcccatc aaaccagccc cagaggaatg tcgtgtcttg cattgtgctc atccgacaac 1440
agacacaaag accggtaaag ttgaggcgcg ggatctcggc aatgaagccg gagcagccag 1500
tcttcgagct cactccagcc gtgagattga ccttttcgtc gtccgtcacg acaagctcga 1560
cctcgagttt ctccataata atgtgtgagt agttcccaga taagggaatt agggttccta 1620
tagggtttcg ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa 1680
aatacttcta tcaataaaat ttctaattcc taaaaccaaa atccagtact aaaatccaga 1740
tcccccgaat taattcggcg ttgaattcgg 1770
Claims (6)
1. A method for reducing the expression enzyme activity of Aspergillus niger beta-glucosidase by light-controlled promoter is characterized in that: the method comprises the following steps:
(1) constructing a DNA fragment consisting of a DNA sequence containing a light-controlled promoter, a DNA sequence expressing antisense mRNA complementary to the mRNA of the beta-glucosidase and a terminator sequence on an expression vector; the DNA sequence containing the light-controlled promoter is shown as SEQ ID NO. 1; the DNA sequence of the antisense mRNA which is complementary to the mRNA of the beta-glucosidase in expression is shown as SEQ ID NO. 2; the terminator sequence is shown as SEQ ID NO. 3;
(2) transforming the recombinant vector constructed in the step (1) into aspergillus niger;
(3) the aspergillus niger strain transformed with the recombinant vector is cultured under the illumination condition, wherein the illumination condition is blue light.
2. The method of claim 1, wherein: the expression vector in the step (1) is a pCAMBIA1301 vector.
3. The method of claim 1, wherein: the step (1) is as follows: passing the DNA fragment with the sequence shown in SEQ ID NO.4 through restriction endonucleaseEcoI、KpnI and DNA ligase are constructed on a pCAMBIA1301 vector to obtain a recombinant vector.
4. The method of claim 1, wherein: the Aspergillus niger in step (2) is Aspergillus niger ATCC 16404.
5. The method of claim 1, wherein: and (2) transforming the constructed recombinant vector into Aspergillus niger by agrobacterium.
6. An Aspergillus niger strain characterized by: an A.niger strain transformed with a recombinant vector constructed according to any one of claims 1-5.
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