CN103275981A

CN103275981A - Promoter for filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, plasmid, preparation method and transformed cell

Info

Publication number: CN103275981A
Application number: CN2013102012545A
Authority: CN
Inventors: 潘力; 周斌; 王超
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2013-05-27
Filing date: 2013-05-27
Publication date: 2013-09-04
Anticipated expiration: 2033-05-27
Also published as: CN103275981B

Abstract

The invention discloses a promoter for a filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, a plasmid, a preparation method and a transformed cell. The regulation element is a base sequence of the area from (-378bp)-(-291bp) of a filamentous fungal promoter. The regulation element is related to the protein secretion pressure feedback. The promoter against feedback inhibition is a base sequence obtained by at least losing or replacing the base of the area from (-378bp)-(-291bp) on the filamentous fungal promoter, and has promoter activity. The promoter disclosed by the invention can be efficiently expressed in filamentous fungi and particularly in the protein of aspergillus oryzae.

Description

Promotor, plasmid and preparation method and the transformant of filamentous fungus protein excretion pressure feedback controlling element and anti-feedback inhibition

Technical field

The invention belongs to the genetically engineered field.Particularly, the present invention relates to filamentous fungus in the protein excretion pressure feedback regulate relevant cis element and a kind ofly remove the promotor that the albumen of secretory pressure force feedback inhibition efficiently expresses owing to lacking protein excretion pressure feedback cis-regulating element in filamentous fungus as in the aspergillus oryzae (Aspergillus oryzae).

Background technology

Filamentous fungus has very strong protein secreting ability, the processing of carrying out behind the protein translation that can be correct is modified, its glycosylation pathway differ is near Mammals, particularly its Aspergillus prolonged application is in traditional food and drink industry, it is the safety in production bacterial strain of generally acknowledging, it is desirable suitability for industrialized production recombinant protein host bacterium (Nevalainen KM etc., 2005).But compare with homologous protein output, the output that filamentous fungus produces heterologous protein is but quite low, the limitation ot it factor analysis shows, foreign gene transcribe and the mRNA level of stability is gratifying generally, bottleneck mainly was present in the stage after transcribing during it was produced, i.e. (Conesa A etc., 2001 in Secretory Pathway; Gouka RJ etc., 1997).Limiting factor analysis main in the eukaryotic protein Secretory Pathway is shown: secretory protein is topmost rate-limiting step (Raden D etc., 2005 in the folding and modification of endoplasmic reticulum; Gasser B etc., 2008).The quality control of protein folding mainly is to be participated in by two kinds of cell mechanisms in the endoplasmic reticulum: non-folded protein acknowledgement mechanism (unfolded protein response, UPR) be responsible for there is folding protein in induction and the monitoring endoplasmic reticulum and induce and activate the molecular chaperones relevant with protein folding and folding enzymes, protein modified, genetic expression that transportation is correlated with, handle the ability of non-folded protein with the reinforcement cell; Another be the relevant protein of endoplasmic reticulum fall mechanism (ER-associated protein degradation, ERAD), those do not form evil protein (Nevalainen KM etc., 2005 of correct conformation to be responsible for the cleaning degraded; Conesa A etc., 2001; Gasser B etc., 2008).To influence the correct folding of albumen in the endoplasmic reticulum at stressed conditions such as the expression of heterologous protein, culture condition change, chemicals stimulation, pathogenic infections, and cause non-folded protein accumulation in the endoplasmic reticulum.By inducing UPR mechanism and ERAD mechanism, coordinative role is kept the folding ability of mind of protein excretion approach to endoplasmic reticulum under above-mentioned pressure condition, regulates and keep the stability (Travers KJ etc., 2000) of endoplasmic reticulum autologous state under the secretion stressed condition.Mammals UPR mechanism population effect has not only increased the quantity of molecular chaperones and the protein modified enzyme relevant with protein folding, also reduce the protein content that is entered endoplasmic reticulum by rrna by the arrestin translation simultaneously, and increased not folded protein removing ability by strengthening endoplasmic reticulum proteolytic degradation (ERAD).By contrast, the UPR of yeast mechanism lacks reverse feedback adjusting effect (Patil C etc., 2001 of corresponding protein translation level; Ma Y etc., 2001).The UPR mechanism that it is worth noting filamentous fungus is similar with yeast, the same reverse feedback that lacks albumen translation skill under the endoplasmic reticulum pressure is regulated, but in filamentous fungus, but there is unique Feedback mechanism, express at transcriptional level feedback inhibition secreted protein gene at endoplasmic reticulum pressure, thereby stop or reduced negative regulation mechanism (the protein excretion pressure feedback mechanism Repression under secretion stress of the protein content that enters endoplasmic reticulum, RESS) (Al-Sheikh H etc., 2004; Pakula TM etc., 2003).It is that expression of gene all is suppressed (Guillemette T etc., 2007 that the functional gene group analysis of Aspergillus nidulans, aspergillus niger, Trichodermareesei is presented under the endoplasmic reticulum pressure main Secretases; Arvas M etc., 2006; Sims AH etc., 2005).

Overexpression exocrine protein or medicine irritation are as dithiothreitol (DTT) (DTT), tunicamycin, Ca ²⁺Ionophore stimulates induces generation albumen pressure, thereby produces the UPR effect.In filamentous fungus, under albumen pressure, exocrine protein mRNA transcriptional level is expressed and is suppressed, and is called protein excretion pressure feedback mechanism (RESS).At present report grinds Hashem Al-Sheikh etc., 2004 to RESS mechanism), Trichodermareesei (Tiina M.Pakula etc., 2003) and aspergillus oryzae (Bin Wang etc., 2010).Hashem Al-Sheikh etc. has studied the promotor of glucoamylase (glaA) gene and has determined at last to determine between the 1kb-2kb of promotor upstream with the machine-processed relevant promoter region of RESS in aspergillus niger.Tiina M.Pakula etc. study cellobiohydrolase 1(CBH1 in Trichodermareesei) promotor of gene determines to determine between the 161bp-2200bp of promotor upstream with the machine-processed relevant promoter region of RESS at last.Use RNA-SEQ technology such as Wang Bin are resolved the group of transcribing of aspergillus oryzae, find that a lot of exocrine protein genes are relevant with RESS mechanism, but do not have the cis transcription regulatory region of these genes is carried out deep analysis.

Aspergillus oryzae can be secreted a large amount of amylase (Taka-amylase).Diastatic expression is induced by starch, maltose, but checked by glucose (Kozo TSUCHIYA etc., 1992) simultaneously, and these effects mainly are subjected to cis-acting elements regulation and control on the promotor.To the functional analysis of amylase amyB gene (Taka-amylase B) promotor cis transcription regulatory region, mainly be the conventional func component analysis at present.For example, Kozo TSUCHIYA etc. and Yoji KANEMORI etc. determine to have region I, region II, three functional areas relevant with protein expression of region III in promotor by disappearance and convergence analysis.But up to this point, do not see report to the RESS of amyB gene promoter mechanism cis acting controlling element yet.

Summary of the invention

In view of above present situation, one of purpose of the present invention is to find out the cis transcriptional regulatory element (87bp) relevant with RESS mechanism.

Two of the object of the invention is based on the promotor that one of this mechanism construction can the secretion inhibitor pressure feedback suppresses.

Three of the object of the invention is to provide plasmid vector that is made up by above-mentioned promotor and preparation method thereof.

Four of the object of the invention is to provide a kind of transformant.

Purpose of the present invention is achieved through the following technical solutions:

Filamentous fungus protein excretion pressure feedback controlling element, this controlling element be filamentous fungus promotor-378bp～-base sequence in 291bp zone.Described base sequence is shown in SEQ ID NO.1.Controlling element has following characteristics:

(1) comprise the amyB gene promoter of SEQ ID NO.1 sequence, the transcriptional level of the external source secretory protein that this promotor connects under the protein excretion pressure condition is subjected to the feedback inhibition of protein excretion pressure;

(2) do not comprise the amyB gene promoter of SEQ ID NO.1 sequence, the transcriptional level of the external source secretory protein that this promotor connects under the protein excretion pressure condition is removed the feedback inhibition of protein excretion pressure;

(3) can hybridize under rigorous condition with the base sequence shown in the SEQ ID NO.1, and have the nucleotide sequence of protein excretion pressure feedback inhibit feature, can be that to derive from aspergillus oryzae also can be other filamentous fungus.

Above-mentioned promotor of the present invention can also can obtain by chemical synthesis process after identifying base sequence by obtaining as embodiment 1 described method.

The promotor that a kind of anti-protein excretion pressure feedback suppresses, this promotor be lack at least or replace on the filamentous fungus promotor-378bp～-base sequence of the base gained in 291bp zone and have promoter activity.Described filamentous fungus promotor is the amyB promotor.This promotor is that base sequence is shown in SEQ ID NO.2.Sequence shown in the SEQ ID NO.2, the transcriptional level of the external source secretory protein that this sequence connects under the protein excretion pressure condition is not reduced.

Promotor of the present invention not only can be the DNA shown in the SEQ ID NO.2, also can be the DNA that comprises base shown in the above-mentioned base sequence number, as long as have similar adjusting function to it.Can also be included in disappearance in the base sequence shown in the above-mentioned base sequence number, instead of or in addition to the DNA of the base sequence of a few base gained, and hybridizing under tight condition with the base sequence shown in the SEQ ID NO.2, and have similar adjusting function, from the DNA of aspergillus oryzae or

DNA of the present invention preferably comprises the base sequence that has 75% above homology with base sequence shown in the SEQ ID NO.2, and homology is preferably more than 80%, and is most preferably more than 90%, more preferably more than 95%, further preferred more than 98%.

Among the present invention, the promotor of " identity function " refers to that its transcriptional level by aftermentioned functional analysis test cdna is more than 50% of transcriptional level that the DNA shown in the above-mentioned base sequence number detects under the same conditions, be preferably more than 70%, most preferably more than 80%, more preferably more than 90%, further preferred more than 95%.

A kind of plasmid is connected to genetic expression unit constructed on the target nucleic acid sequence with above-mentioned promotor.

Concrete construction process is as follows: be template with SEQ ID NO.3, design two couples of primer SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, two fragments that pcr amplification is independent; Re-use primer SEQ ID NO.4 and SEQ ID NO.7, adopt the fusion PCR method two independent fragments to be fused into a gene expression frame, the big fragment phosphorylation that will merge simultaneously; PPTR I plasmid is cut and dephosphorylation with Smal I enzyme; Smal I enzyme is cut carrier and merged fragment, and connection, conversion obtain plasmid PTAF7891.The base sequence of its dna molecular is shown in SEQ ID NO.8.In addition, the used plasmid of the present invention can be PUC119, pPTR I etc.

Among the present invention, above-mentioned plasmid can be transformed into filamentous fungal host cell, make goal gene obtain expressing.Also can, whole expression unit directly is incorporated in the host chromosome with dna direct.The host here is preferably filamentous fungus, is preferably aspergillus oryzae (Aspergillus oryzae) especially.With plasmid transformed host cell of the present invention, for example use the Calcium Chloride Method transformed into escherichia coli according to the method for routine, transform aspergillus oryzae with PEG mediation protoplast transformation method.

Analyze various cracked promoter functions with resulting conversion bacterial strain, transformant can be cultivated to carry out with its utilizable carbon source and nitrogenous source.With carbon source 20 grams, nitrogenous source 3 restrains, KCl2 gram, MgSO during cultivation ₄.7H ₂The O0.5 gram, K ₂HPO ₄1 gram, FeSO ₄.7H ₂The O0.01 gram, pH5.5.The used carbon source of the present invention is preferably starch, maltose, glucose.Nitrogenous source is preferably SODIUMNITRATE, Sodium Nitrite, polyprotein peptone, peptone.Culture temperature, incubation time there are not particular restriction, as long as be fit to thalli growth, but preferably at 25-37 ℃, incubation time 12～96 hours.The thalline that cultivation is finished is analyzed the amount of reporter gene mRNA in the thalline.

The functional element analysis can be undertaken by following method, and this method can specificly detect the mRNA amount of the genetic transcription of promotor downstream connection.Analytical procedure is not had special requirement, can be that quantitative fluorescent PCR (qPCR) also can be Northern Bloting.

The screening of functional element: blocking promotor and recombinant promoter functional analysis can have the mRNA level of the reporter gene that this promotor is connected or the amount of mRNA translation product to carry out by mensuration.

It is to make this report genetic expression function that promotor of the present invention connects reporter gene in its downstream.Can be useful proteins gene of different sourcess such as Sumizyme MP, lipase, fluorescin, antibody etc. to the used reporter gene of the present invention, be preferably Palatase (rml).

Reporter gene and terminator that promotor of the present invention, downstream connect can be used as the genetic expression unit.The terminator here can use terminator commonly used, as long as can be used for expression system.As amyB, glaA, agdA etc.

Aspergillus oryzae chemical blocking agent handle as dithiothreitol (DTT) (DTT) and tunicamycin, different culture condition under can produce RESS mechanism under as endoplasmic reticulum pressure such as solid and liquid culture, carbon nitrogen source variation and heterologous protein overexpressions the protein excretion approach being influenced.So the method that the present invention adopts is under medicine irritation, examining report gene mRNA level.The promotor transformant of different lengths under the inducing of DTT, is with reference to gene with GAPDH (glyceraldehyde 3-phosphate dehydrogenase), relatively the mRNA relative expression quantity of reporter gene rml.The result is presented at and exists 87bp cis transcriptional regulatory element relevant with RESS mechanism on the promotor of amyB gene.

The transformant that makes up by aforesaid method relatively is under different culture condition: foreign protein pressure condition (DTT induces) and normal culture condition (not adding DTT induces), the transcriptional level of the mRNA of reporter gene rml changes, and comes the transcriptional activity of more above-mentioned promotor.The result shows, by promotor P _{△ F378-291}The expression unit constructed with rml, under the protein excretion pressure condition, the mRNA transcriptional level of reporter gene rml is not to be subjected to suppress, and is that anti-protein excretion pressure feedback regulation and control suppress so show this promotor.This promotor along with the increase of induction time, has raising in various degree under the normal culture condition of the relative expression quantity of the mRNA of reporter gene rml under the albumen pressure condition simultaneously.Its concrete numerical value is embodied in, and the relative expression quantity of reporter gene rml improved 1.45 times at 1 hour, improves 2.93 times at 2 hours, improves 8.97 times at 4 hours, improves 11.95 times at 6 hours.Simultaneously to P _{△ F378-291}Promotor and P _AmyB1110The aspergillus oryzae transformant AO3821 of the expression unit that promotor and rml are constructed and AO1110 carry out shake flask fermentation (fermentation condition is not optimized).Fermented 72 hours, measure the lipase enzyme and live: the work of AO3821 enzyme is 1.8513U/mL; The work of AO1110 enzyme is 1.6884U/mL.The transformant lipase activity of the promotor that the transformant of this anti-feedback inhibition promotor is not more transformed has improved about 10%, illustrates that this promotor is that anti-protein excretion pressure feedback regulation and control suppress.The enzyme work of this two strains bacterium simultaneously will be higher than the expression of lipase in yeast of this type.Research such as ZhangW-G etc. (2008) and Jiang Fengchun (2010) lipase is when expressing in yeast, and the enzyme work that obtains is respectively 0.3U/mL and 1.4U/mL.As seen, promotor of the present invention can be filamentous fungus, and particularly albumen can efficiently express in aspergillus oryzae.

Description of drawings

The plasmid PTA1110 that sets out that Fig. 1 uses for the present invention.

Fig. 2 is the transparent circle of primary dcreening operation transformant in the step 7 of the present invention.

Fig. 3 is the vector construction process of different promoters length in the step 3 of the present invention, 4.

Fig. 4 identifies tactful synoptic diagram for blocking promotor transformant PCR in the step 7 of the present invention.

Fig. 5 identifies tactful synoptic diagram for merging disappearance promotor transformant PCR in the step 7 of the present invention.

Fig. 6 is mRNA agarose electrophoresis figure in the step 8 of the present invention.

Fig. 7 is haciA gene (a) and the transcribe situation of amyB gene (b) under albumen pressure in the step 10 of the present invention.

Fig. 8 blocks the reporter gene rml that promotor connects, the transcriptional level of mRNA under albumen pressure for amyB gene in the step 10 of the present invention.

Fig. 9 merges the reporter gene rml that the disappearance promotor connects, the transcriptional level of mRNA under albumen pressure for amyB full length gene in the step 10 of the present invention.

Embodiment

Embodiment 1

1, the extraction of PTA1110 and pPTR I plasmid DNA

Containing the plasmid PTA1110 of total length amyB gene promoter and reporter gene rml and agdA gene terminator and have a PT(pyrithiamine) r plasmid pPTR I (available from Takara company) all can adopt commercially available plasmid extraction kit to extract.

2, the structure of cracked promotor and fusion disappearance promotor

2.1 the structure of cracked promotor

According to the aspergillus oryzae amyB gene promoter sequence of having reported, the design primer is transformed the amyB gene promoter.Design cracked promoter primer the amyB gene promoter is blocked into the genetic expression unit of 14 different fragments: P successively from 5 ＇ end _AmyB941(promoter region-941bp～-1bp, SEQ ID NO.9 and SEQ ID NO.7); P _AmyB871(promoter region-871bp～-1bp, SEQ ID NO.10 and SEQ ID NO.7); P _AmyB766(promoter region-766bp～-1bp, SEQ and SEQ ID NO.7); P _AmyB674(promoter region-674bp～-1bp, SEQ ID NO.12 and SEQ ID NO.7); P _AmyB582(promoter region-582bp～-1bp, SEQ ID NO.13 and SEQ ID NO.7); P _AmyB499(promoter region-499bp～-1bp, SEQ ID NO.14 and SEQ ID NO.7); P _AmyB419(promoter region-419bp～-1bp, SEQ ID NO.15 and SEQ ID NO.7); P _AmyB378(promoter region-378bp～-1bp, SEQ ID NO.16 and SEQ ID NO.7); P _AmyB358(promoter region-358bp～-1bp, SEQ ID NO.17 and SEQ ID NO.7); P _AmyB329(promoter region-329bp～-1bp, SEQ ID NO.18 and SEQ ID NO.7); P _AmyB291(promoter region-291bp～-1bp, SEQ ID NO.19 and SEQ ID NO.7); P _AmyB234(promoter region-234bp～-1bp, SEQ ID NO.20 and SEQ ID NO.7); P _AmyB182(promoter region-182bp～-1bp, SEQ ID NO.21 and SEQ ID NO.7); P _AmyB73(promoter region-73bp～-1bp, SEQ ID NO.22 and SEQ ID NO.7).

2.2 merge the structure of disappearance promotor

Make up the reorganization amyB gene promoter of disappearance part fragment: P by the method design primer of disappearance and fusion PCR _{△ F378-291}(merge the new promotor that disappearance promoter region-378～-291 backs make up, SEQ ID NO.4 and SEQ ID NO.5; SEQ ID NO.6 and SEQ ID NO.7); P _{△ F378-358}(merge the new promotor that disappearance promoter region-378～-358 backs make up, SEQ ID NO.4 and SEQ ID NO.23; SEQ ID NO.24 and SEQ ID NO.7); P _{△ F358-291}(merge the new promotor that disappearance promoter region-358～-291 backs make up, SEQ ID NO.4 and SEQ ID NO.25; SEQ ID NO.26 and SEQ ID NO.7); P _{△ F378-329}(merge the new promotor that disappearance promoter region-378～-329 backs make up, SEQ ID NO.4 and SEQ ID NO.27; SEQ ID NO.28 and SEQ ID NO.7); P _{△ F329-291}(merge the new promotor that disappearance promoter region-329～-291 backs make up, SEQ ID NO.4 and SEQ ID NO.29; SEQ ID NO.30 and SEQ ID NO.7); P _{△ F342-318}(merge the new promotor that disappearance promoter region-342～-318 backs make up, SEQ ID NO.4 and SEQ ID NO.31; SEQ ID NO.32 and SEQ ID NO.7); P _{△ F419-358}(merge the new promotor that disappearance promoter region-419～-358 backs make up, SEQ ID NO.4 and SEQ ID NO.33; SEQ ID NO.34 and SEQ ID NO.7).

3, the structure of amyB gene cracked promoter expression unit and carrier

Being primer with 14 groups of DNA in the above-mentioned steps 2.1, is that template is carried out pcr amplification and obtained 14 fragments with the prepared PTA1110 of step 1.That amplification is used is the PrimeSTAR of Takara company ^{R zero}HS DNA Polymerase PCR archaeal dna polymerase.Amplification condition, 95 ℃ 5 minutes, 98 ℃ 15 seconds, annealing temperature is set 15 seconds according to different primer, 72 ℃ according to different expanding fragment length setting-up times, 30 circulations, 72 ℃ 10 minutes, 16 ℃ of insulations.Obtain the promoter expression unit of different lengths, simultaneously amplification PCR products is carried out phosphorylation, the phosphorylation process is with reference to Takara T4PNK specification sheets.Use Smal I enzyme to cut and dephosphorylation to the pPTR I plasmid in the step 1 simultaneously, what this process was used is Thermo Scientific FastDigest Smal I restriction enzyme and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase dephosphorylation enzyme (available from Thermo Scientific company), and concrete use is with reference to the working instructions of this product.14 PCR products of above-mentioned processing are cut carrier pPTR I with enzyme respectively, use the connection test kit DNA Ligation Kit Ver.2.0 of Takara company, 16 ℃, the connection of spending the night.Afterwards, (Escherichia coli E.coli), extracts plasmid and checks order the ligation liquid transformed into escherichia coli that will spend the night.Sequencing result shows, the expression unit sequence of a series of cracked amyB gene promoter that the present invention makes up is consistent with the base sequence of amyB gene promoter in the corresponding position, and above-mentioned promotor is blocked the corresponding plasmid name of fragment and is called PTA941, PTA871, PTA766, PTA674, PTA582, PTA499, PTA419, PTA378, PTA358, PTA329, PTA291, PTA234, PTA182, PTA73.The concrete process that makes up as shown in Figure 3.

4, the structure of amyB gene fusion disappearance promoter expression unit and carrier

Being primer with 7 groups of DNA in the above-mentioned steps 2.2 respectively, is that template is carried out pcr amplification and obtained 7 groups of DNA with the prepared PTA1110 of step 1, and every group of DNA has two fragments.That particularly, amplification is used is the PrimeSTAR of Takara company ^{R zero}RHS DNA Polymerase PCR archaeal dna polymerase.Every group of primer, amplification condition, 95 ℃ 5 minutes, 98 ℃ 15 seconds, annealing temperature is set 15 seconds according to different primer, 72 ℃ according to different expanding fragment length setting-up times, 30 circulations, 72 ℃ 10 minutes, 16 ℃ of insulations.Amplification obtains two fragments respectively.Being primer with the DNA shown in SEQ ID NO.4 and the SEQ ID NO.7 again, is that template is carried out recombinant PCR with two above-mentioned fragments, specifically be 95 ℃ 5 minutes, 98 ℃ 15 seconds, 68 ℃ 4 minutes, 30 circulations, 72 ℃ 10 minutes, 16 ℃ of insulations.Obtain 7 different disappearance promoter expression unit that merge.Simultaneously amplification PCR products is carried out phosphorylation, the phosphorylation process is with reference to Takara T4PNK specification sheets.Use Smal I enzyme to cut and dephosphorylation to the pPTR I plasmid in the step 1 simultaneously, it is Thermo Scientific FastDigest Smal I restriction enzyme and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase dephosphorylation enzyme that this process is used, and concrete use is with reference to the working instructions of this product.PCR product and the enzyme of above-mentioned processing are cut carrier pPTR I, use the connection test kit DNA Ligation Kit Ver.2.0 of Takara company, 16 ℃, the connection of spending the night.Afterwards, the ligation liquid transformed into escherichia coli that will spend the night extracts plasmid and checks order.

Sequencing result shows, the genetic expression unit of the amyB gene promoter of a series of fusion disappearances that the present invention makes up is consistent with the base sequence of amyB gene promoter in the corresponding position, corresponding plasmid name was called PTAF7891, PTAF7858, PTAF2991, PTAF4218, PTAF1958, PTAF5891, PTAF7829 after above-mentioned promotor merged deletion fragment, and the concrete process that makes up as shown in Figure 3.

5, the importing of aspergillus oryzae foreign gene

Use PEG-CaCl ₂Method mediated transformation aspergillus oryzae (Aspergillus oryzae niaD300 is available from Japanese NRIB institute)

Get the fresh spore inoculating of an amount of aspergillus oryzae to DPY substratum (2% glucose, 1% peptone, 0.5% yeast extract, the 0.5%K of 50ml ₂HPO ₄, 0.05%MgSO ₄) in, 30 ℃, cultivated 20-24 hour.Filter, and with behind the 0.8M NaCl flushing thalline, after adding in the enzyme liquid of preparing with 0.8M NaCl and 10mM phosphate buffered saline buffer (pH6.0) (1% Mierocrystalline cellulose+1% helicase+0.5% N,O-Diacetylmuramidase), 30 ℃ of temperature are bathed and were collected protoplastis in 3 hours, the plasmid that

step

2,3 is obtained is respectively with protoplastis in proportion behind the mixing, add the PEG mediated transformation, the detailed process of conversion is with reference to the pPTRI DNA(Code No.3621 of Takara company), working instructions.Cultivated 5-10 days for 30 ℃, select the transformant of PT resistance.Obtain the transformant that comprises different promoters length of structure in the

step

2,3 by above-mentioned conversion.

6, the extraction of aspergillus oryzae genomic dna

Get and shake bottle liquid culture aspergillus oryzae mycelia in right amount and filter and drain, the mycelia that obtains arrives finely powdered with liquid nitrogen grinding, simultaneously ground mycelia is transferred to lysis buffer (the 50mM Tris-HCl pH8.0 that 700ul is housed, 50mM EDTA, 3%SDS, 1% mercaptoethanol) in the 2ml centrifuge tube, 60～65 ℃, water-bath 1h.Then, add isopyknic Tris-phenol: chloroform: behind the primary isoamyl alcohol (25: 24: 1), put upside down mixing up and down gently.The centrifugal 15min of 12000g/min.Get supernatant in the another one centrifuge tube, use equal-volume Tris-phenol again: chloroform: after primary isoamyl alcohol (25: 24: the 1) extracting, get in supernatant to the new centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, put upside down mixing after, place 30min for-20 ℃.12000g/min，4℃，15min。Abandon supernatant, after twice of 75% washing with alcohol precipitation, drying at room temperature.Aqueous solution dissolution precipitation with an amount of RNase that contains 50ug/ml.Be kept at behind the resolution of precipitate-20 ℃ standby.

7, the evaluation of aspergillus oryzae transformant

(1) primary dcreening operation

In the genetic expression unit that different lengths promotor among the present invention makes up, used reporter gene is the lipase gene of Rhizomucor miehei (rml).Transformant is chosen the CD(0.2%PVA-124 that contains tributyrin with toothpick, 0.5% 3 0.3%NaNO ₂, 0.2%KCl, MgSO ₄.7H ₂O0.05%, 0.001%FeSO ₄.7H ₂O, 0.1%K ₂PHO ₄, 2% glucose, 0.25% Triton-100, pH5.5,2% agar powder) and on the flat board, 30 ℃ of overnight incubation see whether can produce transparent circle, as shown in Figure 2, have transparent circle explanation reporter gene rml to express in the host.

Go out the transformant of different promoters by the aforesaid method preliminary screening.

(2) PCR of transformant identifies

Extract the genome of generation transparent circle transformant and as template, identify transformant with the method in the step 5.The methods and strategies of identifying is shown in Fig. 4,5.Fig. 4 when identifying that the amyB gene blocks the transformant of promotor, uses primer 1, primer 2, primer 3,4,4 pairs of primers of primer.Primer 1 is included in and blocks in the promoter region so one amplify surely and transform the fragment band that is more or less the same; Primer 2 is blocking Auele Specific Primer and can just amplifying transformant and change fragment over to of this promotor; Primer 3 and primer 4 are not included in the fragment promoter region (dotting among the figure) so can not get amplified production, except the non-specific amplification.Fig. 5 when identifying the transformant of amyB gene fusion disappearance promotor, uses primer 1, primer 2, primer 3, primer 4,5,5 pairs of primers of primer.Primer 1 is included in and merges in the disappearance promoter region so one amplify surely and transform the fragment band that is more or less the same; Primer 2 is that this promotor merges disappearance Auele Specific Primer and can just amplify transformant and change fragment over to; Primer 3 and primer 4 are not included in and merge in the disappearance promoter region (dotting among the figure) so can not get amplified production, except the non-specific amplification, primer 5 outside corresponding circle of sensation so can amplify the purpose band.

Simultaneously with this amplification PCR products order-checking.Sequencing result shows that the base fragment sequence of order-checking is consistent with self-designed sequence.

8, the extraction of aspergillus oryzae RNA

With the spore inoculating of transformant to CD-P liquid nutrient medium (0.3% peptone, 0.2%KCl, 0.05%MgSO ₄.7H ₂O, 0.001%FeSO ₄.7H ₂O, 0.1%K ₂PHO ₄, 2% glucose, pH5.5) in (200ml/1000ml), make the spore final concentration reach 10 ⁶Individual/ml, cultivate after 24 hours, under aseptic condition, filter and collect thalline.Thalline is all assigned to 10 bottles of fresh CD-P substratum (50ml/250ml), it is divided into 5 groups simultaneously.Cultivate after 6 hours, 1 bottle adds freshly prepared DTT and makes final concentration reach 20mM in every group, and contrast adds isopyknic water.Induce liquid nitrogen grinding behind 30min, 60min, 120min, 240min, the 360min, add TRIZO dissolving thalline (100mg thalline/1ml TRIZO).The extraction step of RNA is with reference to the product RNAiso Plus(D9108B of Takara company) specification sheets.The RNA that extracts is dissolved with no RNA enzyme ddH2O and be diluted to below the 1ug/ul-80 ℃ of preservations.Carry out common agarose electrophoresis simultaneously, determine integrity and the pollution condition of RNA, as shown in Figure 6.

9, the RT-PCR of aspergillus oryzae RNA

Be template with the RNA in the step 7, use the Takara primeScript of company ^{Zero R}RT reagent Kit With gDNA Eraser (Perfect Real Time) reverse transcription test kit synthesizes cDNA, and reverse transcription reaction is finished in two steps, and the first step reaction is to remove the genome that pollutes among the RNA, 42 ℃ of reaction times, 2 minutes; The reaction of second step is to carry out reverse transcription on first one basis, response procedures: 37 ℃, and 15 minutes; 85 ℃, 5 seconds.Specific operation process is with reference to this test kit specification sheets.

10, the functional analysis of promotor

With reference to (YE, L etc., 2008) such as Wang Bin etc. (Bin Wang etc., 2010) and Ye Lei with the GAPDH(glyceraldehyde 3-phosphate dehydrogenase) be with reference to gene, detecting primer is SEQ ID NO.35 and SEQ ID NO.36.Reporter gene rml gene test primer SEQ ID NO.37 and SEQ ID NO.38.The haciA gene is the directly related crucial transcription factor of UPR mechanism, and under the albumen pressure condition, haciA gene mRNA level can raise, as shown in Figure 7.HaciA gene test primer SEQ ID NO.39 and SEQ ID NO.40.The amyB gene is the effector relevant with RESS mechanism, and the mRNA of amyB gene can reduce under the albumen pressure condition, as shown in Figure 7, and amyB gene test primer SEQ ID and SEQ ID NO.42.The mRNA level of the said gene of different transformants is carried out qPCR to be analyzed.8 combs that reaction is used are available from AXYGENE, and fluorescent quantitation reagent is SYBR ^{R zero}Premix Ex Tap ^TMII (Tli RNaseH Plus); Reaction system is: each composition proportion of 10ul(is with reference to specification sheets); Program: 95 ℃ 30 seconds, 95 ℃ 5 seconds, 60 ℃ 34 seconds, 40 circulations are carried out the solubility curve analysis to amplified production after circulation is finished.The instrument that uses: ABI7500 quantitative real time PCR Instrument.Show that at Fig. 8 and 9 the 87bp cis-acting elements relevant with RESS mechanism passes through to merge resulting promotor P in the concrete zone on the amyB gene promoter with after removing this element _{△ F378-291}At the following mRNA expression amount that connects reporter gene rml of albumen pressure.As Fig. 8 by block the length of analyzing promotor as can be seen-378bp～-during 1bp, under albumen pressure, the mRNA horizontal down-regulation of rml; But at-291～-1 o'clock, under albumen pressure, the mRNA of rml did not reduce in the length of promotor.Thereby illustrate that 87bp cis acting control region is relevant with RESS mechanism.Find by merging deletion analysis that as Fig. 9 amyB full length gene promotor has only the new promotor P that has made up when having lacked-378～-291 these zones _{△ F378-291}, under albumen pressure, the mRNA of rml is uncontrolled.

Embodiment 2

1, aspergillus oryzae transformant fermentation

With P _{△ F378-291}Promotor and P _AmyB1110The flask with indentation that the aspergillus oryzae transformant AO3821 of the expression unit that promotor and rml are constructed and AO1110 are inoculated into 50mL(500mL) fermention medium (0.3% peptone, 0.2%KCl, 0.05%MgSO ₄.7H ₂O, 0.001%FeSO ₄.7H ₂O, 0.1%K ₂PHO ₄, 2% maltose, pH5.5) in, the spore final concentration reaches 10 ⁶Individual/ml.At 30 ℃, 200r/min condition bottom fermentation.

2, enzyme measuring method alive

PNPC enzyme activity determination method is as follows:

(1) add the Tris-HCl damping fluid (50mM pH8.0) of 480 μ L and the PNPC(4-nitrophenyl phenol ester of 500 μ L in the centrifuge tube of 1.5mL) solution, behind the mixing 40 ℃ of water-baths 5 minutes;

(2) add the fermented liquid of 20 μ L in (1), behind the mixing 40 ℃ of water-baths 10 minutes;

(3) add the TCA(trichoroacetic acid(TCA) of 20 μ L5% in (2)), behind the mixing 40 ℃ of water-baths 2 minutes;

(4) get reaction solution 200 μ L in (3) to enzyme plate, measure A with microplate reader ₄₀₅Light absorption value.

3, enzyme activity unit calculation formula:

U = \frac{A_{405} \times 0.12 \times 1000 uL}{V \times T}

Enzyme unit alive (U) is defined as: the per minute hydrolysis substrate generates 1 μ mol and has the required enzyme amount of color p-NP.

V: the volume (L) that adds fermented liquid in the reaction system.

T: add the fermented liquid afterreaction time

4, enzyme activity determination

Cultivate and stopped fermentation in 72 hours.Get fermented liquid supernatant and measure enzyme work according to the method described above.The result shows: the work of AO3821 enzyme is 1.8513U/mL; The work of AO1110 enzyme is 1.6884U/mL.

Claims

1. filamentous fungus protein excretion pressure feedback controlling element is characterized in that, this controlling element be filamentous fungus promotor-378bp～-base sequence in 291bp zone.

2. according to the described filamentous fungus protein excretion of claim 1 pressure feedback controlling element, it is characterized in that described base sequence is shown in SEQ ID NO.1.

3. the promotor of anti-feedback inhibition is characterized in that, this promotor be lack at least or replace on the filamentous fungus promotor-378bp～-base sequence of the base gained in 291bp zone and have promoter activity.

4. promotor according to claim 3 is characterized in that, described filamentous fungus promotor is the amyB promotor.

5. according to claim 3 or 4 described promotors, it is characterized in that this promotor is the base sequence shown in SEQ ID NO.2.

6. a plasmid is characterized in that, claim 3 or 4 or 5 described promotors are connected to genetic expression unit constructed on the target nucleic acid sequence.

7. plasmid according to claim 6 is characterized in that, its DNA base sequence is shown in SEQ ID NO.8.

8. the construction process of the described plasmid of claim 7 is characterized in that, comprises the steps:

Be template with SEQ ID NO.3, design two couples of primer SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, two fragments that pcr amplification is independent; Re-use primer SEQ ID NO.4 and SEQ ID NO.7, adopt the fusion PCR method two independent fragments to be fused into a gene expression frame, the big fragment phosphorylation that will merge simultaneously; PPTR I plasmid is cut and dephosphorylation with Smal I enzyme; Smal I enzyme is cut carrier and merged fragment, and connection, conversion obtain plasmid.

9. a transformant is characterized in that, obtains by filamentous fungal host cell importing claim 6 or 7 described plasmids.

10. the described transformant of claim 9, it is characterized in that, described host cell is aspergillus oryzae (Aspergillus oryzae), aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori) or Aspergillus nidulans (Aspergillus nidulans).