CN104946688B - A kind of method for cutting off screening label and application - Google Patents

Abstract

The invention discloses a kind of method for cutting off screening label and application, belong to gene engineering technology field.The present invention be the recombinant bacterium that pyrG screening label of the both sides with loxP sequences in the same direction is incorporated using on chromosome to go out bacterium germination, bacterial strain use pyrG screening label progress genetic transformations as going out bacterium germination again using pyrG screening label of the site-specific recombination system excision between two loxP sequences.The excision of screening label can be carried out using the method for the present invention, makes screening label reusable edible, genetic modification is no longer limited by screening label deficiency, and the genetic modification for polygenes, more sites provides support.

Description

A kind of method for cutting off screening label and application

Technical field

The present invention relates to a kind of method for cutting off screening label and application, belong to gene engineering technology field.

Background technology

Aspergillus terreus is a kind of important filamentous fungi, can produce a variety of valuable compounds therefroms, including organic acid, enzyme Class, lipid and biologically active secondary metabolite.Wherein itaconic acid and Lovastatin has been carried out industrialized production, With very big market.With the continuous development of molecular biology, production bacterial strain is transformed by metabolic engineering, improves mesh The production level of product is marked, is had very important significance.

With the continuous progress of Protocols in Molecular Biology, the genetic engineering transformation of functional genomics and bacterial strain becomes to get over Come more important.In recent years, a large amount of groups learn the continuous announcement of data, and functional genomics research and genetic engineering transformation often relate to And to polygenes, more sites, but also a variety of research strategies can be used.And it is required during genetic manipulation to screen label A kind of critical elements, exogenous DNA needs to be incorporated on genome together with screening label, then again by screening the phenotype of label It is screened out.And available screening label is considerably less in Aspergillus terreus, it can not meet that carrying out heredity to polygenes, multidigit point changes The demand made.Site-specific recombination system is a kind of important molecular genetic manipulation instrument, its principle is that recombinase identifies simultaneously It is attached on the target site with special DNA sequence dna, is catalyzed recombining reaction so that the DNA fragmentation between target site is picked Remove.Based on this, the excision of screening label is being successfully applied to.Common site-specific recombination system has Cre/loxP systems System, β-rec/six systems and Flp/FRT systems, but need in traditional application process whole with shuttle plasmid form or genome Conjunction form imports recombinase Expression element into cell, gives expression to recombinase in the cell by modes such as inductions, and act on Recognition site cuts off screening label.PyrG (orotidine -5- phosphate decarboxylase genes) is that one kind is lacked with uracil nutrition The nutrition selection type label that the collocation of property bacterial strain uses is fallen into, bidirectional screening work(is had as the conversion system of screening label based on pyrG The feature of energy, screening label presence or absence can be screened by corresponding screening and culturing medium.Therefore collocation site-specific Property recombination system use, can cause screen label rejecting it is more efficient, easy.

The content of the invention

To solve during Aspergillus terreus genetic manipulation screening number of labels can be used limited, cause gene targeting native bent Using the technical problem being restricted in mould field of genetic modification, the present invention provides a kind of method for cutting off screening label, make Label reusable edible is screened, genetic modification is no longer limited by screening label deficiency, and the technical solution taken is as follows：

It is an object of the invention to provide a kind of method for cutting off screening label, this method is to be incorporated on chromosome The recombinant bacterium of pyrG screening labels is bacterium germination, enables bacterial strain again using site-specific recombination system excision pyrG screening labels It is secondary to be used as bacterium germination to use pyrG screening labels to carry out genetic transformation.

Preferably, it is described go out bacterium germination, be Aspergillus terreus (Aspergillus terreus).

It is highly preferred that it is described go out bacterium germination, to have knocked out the Aspergillus terreus of ku80 genes or lig4 genes.

Preferably, the recombinant bacterium of pyrG screening labels is incorporated on the chromosome, the both sides of pyrG screening labels carry LoxP sequences in the same direction.

Preferably, the pyrG screenings label comes from aspergillus, it is highly preferred that deriving from aspergillus niger.

Preferably, the site-specific recombination system, is Cre/loxP site-specific recombination systems；The excision PyrG screens label, is to cut off the screening label pyrG that both sides carry loxP sequences in the same direction.

Preferably, the Cre/loxP site-specific recombination systems, the dosage of Cre recombinases is 2-9U.

The step of the method, is as follows：

1) using the uracil auxotrophy Aspergillus terreus of pyrG gene delections as recipient bacterium, to come from the both ends band of aspergillus niger The pyrG gene expression elements loxP-pyrG1-loxP for having loxP sites in the same direction carries out covering experiment as screening label, obtains It is integrated with the recombinant bacterium of loxP-pyrG1-loxP screening labels；

2) preparation process 1) described in recombinant bacterium protoplast, led using DNA as assistant carrier into protoplasm somatocyte The Cre recombinases for entering 2-9U carry out screening label excision, and loxP- is screened by way of screening uracil auxotrophy The recombinant bacterium that pyrG1-loxP selection markers are removed.

Preferably, the method comprises the following steps that：

1) the ku80 genes and pyrG genes of Aspergillus terreus CICC 40205 is knocked out respectively, builds uracil auxotrophy weight Group bacterium At- Δ ku80- Δs pyrG；

2) using the pyrG gene expression elements pyrG1 of aspergillus niger Co827 as screening label, by way of molecular cloning Both sides add loxP sequences in the same direction, obtain the pyrG selection markers loxP-pyrG1-loxP that both ends carry loxP sites in the same direction, Using the uracil auxotrophy recombinant bacterium At- Δs ku80- Δs pyrG constructed by step 1) as recipient bacterium, with loxP-pyrG1- LoxP carries out covering experiment for screening label, obtains the recombinant bacterium At- Δs for being integrated with loxP-pyrG1-loxP screening labels ku80-pyrG1-loxP；

3) preparation process 2) gained recombinant bacterium At- Δs ku80-pyrG1-loxP protoplast, using DNA as assistant carrier The Cre recombinases that 2-9U is imported into protoplasm somatocyte carry out screening label excision, by screening uracil auxotrophy Mode screen the recombinant bacterium that loxP-pyrG1-loxP selection markers are removed.

The either method is used to prepare the restructuring that may be repeated genetic transformation operation from screening number of labels limitation Bacterium.

The recombinant bacterium prepared using the either method is also within protection scope of the present invention.

Preferably, the method comprises the following steps that：

1) using the genome of Aspergillus terreus CICC40205 as template, with the core as shown in SEQ ID NO.12-SEQ ID NO.13 Nucleotide sequence is primer, expands the upstream sequence U-pyrG of pyrG genes；Again using the genome of Aspergillus terreus CICC40205 as mould Plate, is arranged as primer with the nucleotides sequence as shown in SEQ ID NO.14-SEQ ID NO.15, expands the downstream sequence D- of pyrG genes pyrG；Fusion DNA vaccine fusion upstream sequence U-pyrG and downstream sequence D-pyrG is recycled, using the sequence of fusion product as template, The target practice for going out pyrG genes by PCR amplification for primer is arranged with the nucleotides sequence as shown in SEQ ID NO.16-SEQ ID NO.17 Element pyrG-KO, then preparation process 1) gained recombinant bacterium At- Δs ku80 protoplast, then the target practice element by pyrG genes PyrG-KO is transformed into protoplast, and the uracil auxotrophy bacterial strain that pyrG genes are knocked is obtained after culture is screened At-Δku80-ΔpyrG；

2) the artificial synthesized DNA fragmentation for carrying two loxP sequence sites in the same direction, its nucleotide sequence such as SEQ ID are passed through Shown in NO.1, then the DNA fragmentation is cloned on carrier pUC57simple, obtains carrier PalcA-syn；With aspergillus niger Co827 Genome is template, is arranged as primer with the nucleotides sequence as shown in SEQ ID NO.18-SEQ ID NO.19, is gone out by PCR amplification The Expression element of aspergillus niger pyrG genes, carrier PalcA- is cloned into as screening label pyrG1, then by screening label pyrG1 On syn, plasmid pXH103 is obtained, obtains the pyrG1 that the both sides as shown in SEQ ID NO.26 carry loxP site sequences in the same direction Screen label loxP-pyrG-loxP；Again using pSGF957 as template, with the core as shown in SEQ ID NO.20-SEQ ID NO.21 Nucleotide sequence is primer, goes out NDA fragment gfp-TtrpC fragments by PCR amplification, then gfp-TtrpC fragments are connected to plasmid On pXH103, plasmid pXH104 is obtained；Again using Aspergillus terreus CICC40205 genomes as template, with such as SEQ ID NO.22-SEQ Nucleotides sequence shown in ID NO.23 is classified as primer, goes out ku80 downstream of gene homology arm fragments, then going out amplification by PCR amplification Ku80 downstream of gene homology arm fragments are connected on plasmid pXH104, obtain plasmid pXH105；Again with Aspergillus terreus CICC40205 bases Because group is template, is arranged as primer with the nucleotides sequence as shown in SEQ ID NO.24-SEQ ID NO.25, gone out by PCR amplification Ku80 upstream region of gene homology arm fragments, then the ku80 upstream region of gene homology arm fragments that go out of amplification are connected on plasmid pXH105, Obtain plasmid pXH106；Finally again using plasmid pXH106 as template, using sequence shown in SEQ ID NO.8 and SEQ ID NO.10 as Primer amplification goes out to practice shooting element ku80-pyrG1-gfp；Target practice element ku80-pyrG1-gfp is transformed into obtained by step 2) again In uracil auxotrophy strains A t- Δ ku80- Δs pyrG, obtained after culture is screened and carry loxP-pyrG1-loxP Recombinant bacterium At- Δs ku80-pyrG1-loxP；Uracil auxotrophy recombinant bacterium and pyrG are based on so as to prove to be successfully established Genetic transformation system of the gene as screening label.

3) preparation process 3) gained recombinant bacterium At- Δs ku80-pyrG1-loxP protoplast, to 100 μ prepared L concentration is 10⁸2U Cre recombinases, 1 μ g pyrG1 fragments and 10 μ L 40% are added in the protoplast solution of a/mL PEG4000, after mixing ice bath 30min, add 1ml PSTC solution after being incubated 30min at 30 DEG C, after incubation with Containing 1.2M sorbierites, it is poured into after the top agar culture medium mixing of 4g/L agaroses and 2mM uridines containing 1.2M In the CD regeneration screening and culturing medium tablets of sorbierite, 1g/L 5- fluororotic acids and 10mM uridines, at 30 DEG C, dark bar 6-10d is cultivated under part, after culture picking transformant be inoculated on CDFU tablets cultivated at 32 DEG C carry out within 5-7 days passing on it is pure Change, after passage purifies 3-5 times, obtain the uracil auxotrophy recombinant bacterium At- Δs ku80- that pyrG1 screening labels are removed ΔpyrG1-loxP；Recycled so as to fulfill screening label, break through genetic modification and limited by screening number of labels, can be with The genetic transformation based on pyrG1 screening labels is carried out again.

Expression element：The DNA fragmentation of target gene transcriptional expression, including promoter, gene can be effectively driven in the cell Code area and terminator.

Beneficial effect of the present invention：

Screening label cutting method provided by the invention, it is simple, low to genetic modification component requirements, suitable with operating process With property it is high the advantages that, including autonomous replication type filamentous fungi plasmid need not be built, it is not necessary to build the expression member of Cre recombinases Part, it is not necessary to the Expression element of Cre recombinases is directed into recipient cell, does not also have Cre recombinase Expression elements into the cell Need removal etc..Therefore, the minimizing technology for screening label is simple and practicable.

The present invention is by importing Cre recombinases into Aspergillus terreus protoplasm somatocyte, based on Cre/loxP site-specifics Property recombination system excision integrate the screening label that both sides on chromosome carry loxP sequences in the same direction, realize screening selection markers Efficient excision, so as to establish, a homologous recombination is efficient, transformation is beaten from the high efficiency gene of screening number of labels limitation Target platform.

The present invention realizes the excision of screening label in Aspergillus terreus by site-specific recombination system first, and is logical Cross and Cre recombinases are introduced directly into the excision for carrying out screening label into the cell, the screening label used can bidirectional screening PyrG genes, process are relatively easy, it is not necessary to build recombinase gene expression element, it is not necessary to Aspergillus terreus autonomously replicating plasmid and Inducible promoter, it is not necessary to undergo plasmid loss process.The present embodiments relate to the transformation system based on pyrG genes In, when uracil auxotrophy bacterial strain, obtains by way of pyrG is knocked out completely, the bacterial strain obtained relative to method of mutagenesis For genetic stability it is more preferable.Selenate is needed in the screening system of sC screening labels as screening medicine, and selenate makes There is security risk to experimenter during, be subject to control as poisonous drugs, purchase is very difficult, in filamentous fungi at present The genetic transformation system based on sC has only been successfully established in aspergillus oryzae；Nitrate reductase niaD screens the screening system of label In also to use the chlorate of high concentration, screening and culturing medium preparation process trouble, and niaD auxotrophic strains is spontaneous It is high to reply probability, false positive is high in screening process；In contrast, pyrG selection markers screening process is simple, 5 fluorine of medicine being related to The easily purchase and cheap of orotic acid, uracil or uridylic acid.But it is not all to be successfully established to be based on pyrG in all species The conversion system of label is screened, and when establishing the genetic transformation system, also difficulty differs the different strains in same species, Such as the genetic transformation system has been successfully established in several Aspergillus oryzae bacterial strains, but researcher is had found in many meter Qu The defects of pyrG gene complete deactivations can not be obtained in trichoderma strain type bacterial strain, so that the genetic transformation system can not be established.

Brief description of the drawings

Fig. 1 is the schematic diagram for knocking out the target practice element of ku80 genes and its homologous recombination knockout ku80 genes occurring；

(F1 is primer Uku80-F, and F2 is primer Cku80-F, and F3 is primer PtrA-F, and F4 is primer ptrA-F171, R1 It is primer Dku80-R, R2 is primer Cku80-R, and R3 is primer ptrA-R, and R4 is primer ptrA-R744, and F5 is primer ku80- Probe-F, R5 are primer ku80-probe-R).

Fig. 2 obtains the Genomic PCR verification result figure of transformant for knockout ku80 genes；

(figure A is primer ptrA-F/ptrA-R (F3/R3) verification result, and figure B is primer Uku80-F/ptrA-R (F1/R3) Verification result；Figure C is primer ptrA-F/Dku80-R (F3/R1) verification result, and figure D is primer ku80-probe-F/ku80- Probe-R (F5/R5) verification result；M is 1kb DNA ladder (TAKARA), and M2 is 200bp DNA ladder (TAKARA), C is that CICC40205 wild-type strains are used as control, and 1-4 is is obtained transformant).

Fig. 3 is knockout Aspergillus terreus pyrG gene principle schematics；

(wherein, pdc is gene to be knocked out, and Up is upstream homology arm U-pyrG, and Down is downstream homology arm D-pyrG, arrow Head is design of primers site).

Fig. 4 is the Genomic PCR verification result for knocking out transformant obtained by Aspergillus terreus pyrG genes；

(it is primer hph-F/DpyrG-R verification results wherein, to scheme A, and figure B is primer C-F4/C-R verification results, and figure C is Primer S-pyrG-F/S-pyrG-R verification results；M is 200bp DNA ladder (TAKARA), and Δ ku80 is engineered strain At- Δ ku80 obtains transformant as control, 1#, 2# for knockout pyrG genes).

Fig. 5 is the structure diagram of plasmid PalcA-syn.

Fig. 6 for covering element ku80-pyrG1-gfp and its with ku80 sites site-directed integration principle schematic；

(wherein, ku80-UP, ku80-Dw are respectively the upstream and downstream homology arm of ku80 genes, and pyrG1 is as selection markers Come from aspergillus niger pyrG gene expression elements (including promoter, pyrG genes and terminator), arrow is design of primers position Point).

Fig. 7 is obtained by ku80-pyrG1-gfp elements covering uracil auxotrophy strains A t- Δ ku80- Δs pyrG The Genomic PCR verification result of transformant；

(it is primer Uku80-F/pyrG1-R verification results wherein, to scheme A, and figure B is primer pyrG1-F/pyrG1-R verification knots Fruit, figure C are primer pyrG1-F/TtrpC-R verification results, and figure D is primer pyrG1-F/Dku80-R verification results；M is 1kb DNA ladder (TAKARA), M2 are 200bp DNA ladder (TAKARA), and C is that engineered strain At- Δ ku80- Δs pyrG makees For control, 1-6 is tested by covering and is obtained transformant).

Fig. 8 is the Genomic PCR proof diagram for being introduced directly into transformant obtained by Cre recombinases excision pyrG1 selection markers；

(M is 1kb DNA ladder (TAKARA), and C is engineered strain At- Δs ku80-pyrG1-loxP as control, 1- 8 are obtained transformant by covering experiment).

Fig. 9 is the DNA sequencing result for having cut off pyrG1 screening label engineered strain At- Δ ku80- Δs pyrG1-loxP；

(wherein, ku80-UP is ku80 upstream region of gene homology arms, and loxP is the loxP sequences of Cre recombinase specific recognitions Row, PalcA is PalcA promoter sequences, and pyrG1 sheets are located at ku80-UP in engineered strain At- Δs ku80-pyrG1-loxP Between PalcA sequences).

Embodiment

With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.

Material therefor, reagent, instrument and method in following embodiments, are the routine in this area without specified otherwise Material, reagent, instrument and method, can be obtained by commercial channel.

Plasmid extraction uses OMEGA companies Plasmid Mini Kit I kits (D6942-01), DNA pieces in the present invention Duan Huishou is to use OMEGA companies Cycle-Pure Kit kits (D6492-01), and gel recycling is to use OMEGA companies Gel Extraction Kit kits (D2500-01).

The composition of CD tablets is：3g/L NaNO₃, 2g/L KCl, 1g/L KH₂PO₄, 0.5g/L MgSO₄·7H₂O, 0.02g/L FeSO₄·7H₂O, 10g/L glucose.

IPM fluid nutrient mediums：60g L^-1Glucose, 2g L^-1NH₄NO₃, 20mg L^-1NH₄HPO₄, 20mg L^-1FeSO₄, 0.4g L^-1MgSO₄, 4.4mg L^-1ZnSO₄, 0.5g/L corn pulps, pH 3.5.

The structure of embodiment 1 knocks out the NHEJ approach missing gene engineered strain At- Δs ku80 of ku80 genes

1.1 structure ku80 target practice elements

According to Aspergillus terreus NIH2624 genome databases announce information design primer Uku80-F (5 '- gtcgtagctcttcttgccatc-3’)、Uku80-R(5’- aatgggatcccgtaatcaattgccctcaatcaccatctcccttatc-3’)、Dku80-F(5’- caagagcggctcatcgtcaccccattccggcctcgatgtggatgc-3’)、Dku80-R(5’- Tccacgcggccatcaccgagc-3 '), using the genome of Aspergillus terreus bacterial strain CICC40205 as template, it is polymerize using pfu DNA Enzyme (Fermentas, Catalog No.:EP0501 PCR amplification) is carried out, acquisition can be expanded with primer Uku80-F/Uku80-R The upstream sequence U-ku80 for the ku80 that size is about 1.5kb, can expand acquisition size with primer Dku80-F/Dku80-R is The downstream sequence D-ku80 of the ku80 of 1.5kb.With plasmid pPTR II (TAKARA, Catalog No.:3621) it is template, with PtrA-F (5 '-gggcaattgattacgggatc-3 ') and ptrA-R (5 '-atggggtgacgatgagccgc-3 '), which is used as, to be drawn Thing amplification obtains the ptrA fragments that size is about 2.0kb, is detected through 1.0% agarose gel electrophoresis and be tapped and recovered pure Change.U-ku80 fragments, D-ku80 are merged with ptrA fragments with the method for fusion DNA vaccine, and made with the product of the fusion DNA vaccine For template, with Cku80-F (5 '-gggtttctagaagtcacatc-3 ') and ptrA-R744 (5 '- Atggcccatcgtgaccagtggtac-3 ') obtain the ku80 that size is about 3.0kb as primer amplification and knock out element ku80- A, with Cku80-R (5 '-atcaccgaccctacgctgtg-3 ') and ptrA-F171 (5 '- Ggtctctcgtgccatgaccagacg-3 ') obtain the ku80 that size is about 2.6kb as primer amplification and knock out element ku80- B, such as Fig. 1.

It is prepared by 1.2 protoplasts

By the spore inoculating of Aspergillus terreus CICC40205 into 50mL liquid IPM culture mediums, it is about 10 to make spore concentration⁷A/ ML, in 200rmp, 32 DEG C of culture 12-18h.It is collected by filtration the mycelia grown with sterile 500 mesh nylon cloth of individual layer, and with sterilizing 0.6M MgSO₄Solution flushes three times, and press dry and is placed in sterile 50ml triangular flasks；By 1g mycelia is weighed, 10ml enzymes are added Liquid is solved, in 30 DEG C, 60rpm processing 1-3h.Enzymolysis liquid component is：0.8% cellulase (Sigma, Catalog No.： C1184), 0.8% lyases (Sigma, Catalog NO.：L1412), 0.4% glusulase (gives birth to work, Catalog No. in Shanghai： SB0870)、0.6M MgSO₄, via 0.22 μm of sterilizing filter filtration sterilization.By the mixed liquor after above-mentioned enzymolysis first with 8 layers Lens wiping paper filters, and collects filtrate.4 DEG C are collected by centrifugation protoplast, washed once with precooling 1.0M sorbitol solutions, then use precooling STC (STC is 1.0M sorbierites, 50mM TrisHCl-pH 8.0,50mM CaCl₂) washed once, finally plasm Weight is suspended from the STC of precooling, and protoplast concentration is adjusted to 5 × 10 with STC⁷A/mL, obtains protoplast suspension.

1.3 protoplast transformation

Add the ku80-A fragments (about 2 μ g) of 5 μ l and the ku80-B of 5 μ l at the same time into the 150 above-mentioned protoplast suspensions of μ l Fragment (about 1.5 μ g), adds 50 μ l PSTC, gently mixes, ice bath 30min.1mL PSTC are added, room temperature is placed after mixing 20min；Then with being poured into after 15mL top-layer agars (CD+1.2M sorbierite+4g/L agaroses, 48 DEG C of insulations after sterilizing) mixing On 5 pieces of regeneration screening and culturing medium tablet CDSP (CD tablet+1.2M sorbierite+0.1mg/L pyrithiamines), in 30 DEG C, dark bar Cultivated 5 days under part.

The screening and verification of 1.4 transformants

There to be pyrithiamine resistant transformants to be forwarded on screening flat board CDP (CD tablet+0.1mg/L pyrroles from tablet Pyridine thiamines), carry out passage purifying within 5 days in 32 DEG C of cultures, this passage purification experiment is repeated twice.Random 4 plants of transformants of picking in Cultivated in IPM fluid nutrient mediums, collect mycelia extraction genome, primer sites have carried out Genomic PCR with reference to shown in Fig. 1 Verification.As shown in Figure 2 A, PCR detections are carried out by primer pair of F3/R3, transformant has all amplified the ptrA mesh of size about 2kb Band, and starting strain Aspergillus terreus CICC40205 does not have the band, this illustrates to incorporate in this 4 transformants complete PtrA selection markers.As shown in Fig. 2 B, 2C, PCR detections are carried out respectively with primer pair F1/R3 and primer pair F3/R1, as a result 2#, 3# transformants have all amplified the purpose band that size is about 3.5kb respectively, other transformants are then without band, this explanation 2#, 3# Transformant is that there occurs homologous recombination on ku80 sites for target practice element.As shown in Figure 2 D, with design in ku80 gene internals Primer pair ku80-probe-F (5 '-gcactctcgaacaggcagtgtc-3 ') and ku80-probe-R (5 '- Atcagtcgtgtcgatgtgaactgc-3 ') PCR verifications are carried out, 2#, 3# transformant fail to expand as wild-type strain WT Go out the band of 600bp sizes, which is defined as by successful knockout by the ku80 genes of this explanation 2#, 3# transformant The Aspergillus terreus engineering strain At- Δs ku80 of NHEJ approach missing.

Embodiment 2 builds uracil auxotrophy Aspergillus terreus engineering strain

2.1 structure pyrG target practice elements

According to Aspergillus terreus NIH2624 genome databases announce information design primer UpyrG-F (5 '- tccatccgatggccattcgtggc-3’)、UpyrG-R(5’- ctttacgcttgcgatcccgaaaaggtcaattgagacttggacgac-3’)、DpyrG-F1(5’- tttcgggatcgcaagcgtaaagatgatgagcatgaagaattatgc-3’)、DpyrG-R(5’- Cgaggtcctaccgatgatgttgc-3 '), using the genome of Aspergillus terreus bacterial strain CICC40205 as template, gathered using pfu DNA Synthase (Fermentas, Catalog No.:EP0501 PCR amplification) is carried out, can be expanded and obtained with primer UpyrG-F/UpyrG-R The upstream sequence U-pyrG for the pyrG that size is about 2.5kb is obtained, acquisition size can be expanded with primer DpyrG-F1/DpyrG-R For the pyrG downstream sequence D-pyrG of 2.5kb, detected through 1.0% agarose gel electrophoresis and carry out being tapped and recovered purifying.With melting The method for closing PCR is merged U-pyrG fragments with D-pyrG fragments, and using the product of the fusion DNA vaccine as template, with CpyrG-F (5 '-gtcgtaaatcgttctttgtactg-3 ') and CpyrG-R (5 '-ggaaccccagataatgatacatgc- 3 ') obtain the pyrG that size is about 3kb as primer amplification and knock out element pyrG-KO fragments (Fig. 3).

2.2 protoplast transformations and screening verification

The spore of inoculation said gene engineered strain At- Δs ku80 carries out mycelia training in the IPM fluid nutrient mediums of 50mL Support, protoplast is prepared with reference to 1.2 described method of above-described embodiment, add 10 μ l at the same time into above-mentioned protoplast suspension Element pyrG-KO fragments (about 3 μ g) are knocked out, 50 μ l PSTC is added, gently mixes, ice bath 30min.1mL PSTC are added, are mixed Room temperature places 20min after even, and then (CD+1.2M sorbierite+4g/L agarose+2mM Uridine, go out with 15mL top-layer agars 48 DEG C of insulations after bacterium) (CD tablet+1.2M sorbierites+1g/L on 5 pieces of regeneration screening and culturing medium tablet CDSFU is poured into after mixing 5 ' fluororotic acid+10mM Uridine), cultivated 7 days under 30 DEG C, dark condition.

There to be 5 ' fluororotic acid resistant transformants to be forwarded on screening flat board CDFU (CD tablets+1g/L 5 ' from tablet Fluororotic acid+10mM Uridine), carry out passage purifying within 7 days in 32 DEG C of cultures, this passage purification experiment is repeated twice.Picking The transformant grown on CDFU tablets is inoculated on CD tablets, and 32 DEG C are cultivated 7 days.2 plants of transformants are randomly selected to put down in CDFU Normal growth on plate and be unable on CD tablets normal growth transformant be inoculated in IPMU fluid nutrient mediums (IPM nutrient solutions+ 10mM Uridine) in cultivated, collect mycelia extraction genome, with reference to Fig. 3 shown in primer sites progress Genomic PCR test Card.Carried out with distinctive primer hph-F on primer target practice element and the primer DpyrG-R collocation on the exosome of homology arm PCR is detected, and as shown in Figure 4 A, two transformants all expand to have obtained the band that size is about 2.5kb, and starting strain At- Δs Ku80 is then without this band, and there occurs homologous recombination on pyrG sites for this explanation target practice element.As shown in Figure 4 B, with C-F4 (5 '- Cggatacgtctactccggtaaggc-3 ')/C-R (5 '-agaaactagcgtggagactttgcc-3 ') be primer pair carry out PCR is detected, and transformant has all amplified the band of size about 1.2kb, and the band that starting strain At- Δs ku80 is expanded is big Small is 2.1kb, and the pyrG bands that this explanation size is about 0.9kb are knocked.As shown in Figure 4 C, with positioned at pyrG gene internals Primer pair S-pyrG-F (5 '-actcgcgcccgcacgcacccgaac-3 ')/S-pyrG-R (5 '- Tccttctggtaccgctggacagc-3 ') PCR detections are carried out, as a result starting strain At- Δs ku80 amplifications have arrived size and have been about The pyrG bands of 0.8kb, and two transformants all fail to amplify the band, this explanation pyrG gene in the two transformants Knocked out completely.The uracil auxotrophy Aspergillus terreus engineering strain is denoted as At- Δ ku80- Δs pyrG.

Embodiment 3 establishes the genetic transformation system based on uracil auxotrophy

Target practice element of 3.1 structures using pyrG1 as selection markers

SEQ ID NO.1 are synthesized by gene chemical synthesis mode, which opens including two loxP sites in the same direction, PalcA Mover and restriction endonuclease sites, the sequence of the synthesis are finally cloned on pUC57simple carriers, obtain carrier PalcA-syn (Fig. 5).With pyrG1-F (5 '-ctaat gctagc cagcagggaatacgagctcca-3 ')/pyrG-R (5 '- Ctaat gctagc gcatcaaatcgtcgtaccgc-3 ') primer is used as, carried out by template of aspergillus niger Co827 genomes PCR amplification, obtains the Expression element that size is about the pyrG genes of 1.5kb aspergillus nigers, as selection markers pyrG1, passes through Nhe PyrG1 fragments are cloned into PalcA-syn carriers by I restriction enzymes, obtain plasmid pXH103.With primer Sgfp-F1 (5’-gat ccatggtgagcaagggcgaggagc-3’)/TtrpC-R1(5’-gag ctcgag Ttactattgtatacccatcttag-3 '), with pSGF957 (referring to Jeong-Gu Kim, Yang Do Choi, Yung-Jin Chang, Soo-Un Kim, Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003,25:1509-1514 structure) it is that template carries out PCR amplification gfp-TtrpC fragments, The band that size is about 1.3kb is cloned into plasmid pXH103 by Nco I and Xho I restriction enzymes, obtains plasmid pXH104.With Dw2-ku80-F (5 '-cat ctcgag tccggcctcgatgtggatgc-3 ')/Dw2-ku80-R (5 '-cta Gaattc tccacgcggccatcaccgagc-3 ') primer is used as, carry out PCR by template of Aspergillus terreus CICC40205 genomes Amplification, passes through restriction enzyme Xho I and Eco RI gram by the ku80 downstreams homology arm fragment that obtained size is about 1.5kb In the grand pXH104 to plasmid, plasmid pXH105 is obtained.With Up2-ku80-F (5 '-tca gcggccgc gtcgtagctcttcttgccatc-3’)/Up2-ku80-R(5’-ctaat gctagc tcaatcaccatctcccttatc- 3 ') primer is used as, PCR amplification is carried out by template of Aspergillus terreus CICC40205 genomes, is about 1.5kb's by obtained size Ku80 upstreams homology arm fragment, after being carried out digestion with restriction enzyme Not I and Nhe I and recycled, be cloned into through Not I and In pXH105 carriers after Spe I digestions, plasmid pXH106 is obtained.Cku80-F/Cku80-R is primer, and pXH106 is as mould Plate, amplification obtain DNA fragmentation that size is about 6.3kb as target practice element ku80-pyrG1-gfp, such as Fig. 6.

3.2 uracil auxotrophy At- Δs ku80- Δs pyrG are tested as the covering of host strain

The spore of said gene engineered strain At- Δ ku80- Δs pyrG is inoculated with the IPMU fluid nutrient mediums (IPM of 50mL 20mM Uridine are supplemented in culture medium) in, 30 DEG C, 200rpm, cultural hypha is carried out, is described with reference to above-described embodiment 1.2 Method prepare protoplast, into above-mentioned protoplast suspension at the same time add 10 μ l knock out element ku80-pyrG1-gfp fragments (about 3 μ g), adds 50 μ l PSTC, gently mixes, ice bath 30min.1mL PSTC are added, room temperature places 20min after mixing, Then with being poured into regeneration screening training after top-layer agar (CD+1.2M sorbierite+4g/L agaroses, 48 DEG C of insulations after sterilizing) mixing Support on base tablet CDS (CD tablet+1.2M sorbierites), cultivated 7 days under 30 DEG C, dark condition.

Obtained transformant is subjected to passage purifying in 5-7 days in 32 DEG C of cultures, this passage purification experiment repeats five times, these The transformant that growth can be stablized on CD tablets is all to have regained uracil synthesis capability.The primer sites with reference to shown in Fig. 6 Genomic PCR verification is carried out, as shown in the electrophoresis detection result in Fig. 7,6 is obtained and incorporates loxP- in the genome The positive transformant of pyrG1-loxP selection markers, and these transformants be all in ku80 sites there occurs homologous recombination, this Illustrate that pyrG1 selection markers are arranged in pairs or groups with uracil auxotrophy Aspergillus terreus bacterial strain At- Δ ku80- Δs pyrG, one can be established The genetic transformation system of a high homology recombination efficiency.Wherein 1# transformants are denoted as At- Δs ku80-pyrG1-loxP.

4 practical site specific recombination systems of embodiment carry out the excision of selection markers

It is prepared by the protoplast of 4.1 engineering bacteria At- Δs ku80-pyrG1-loxP

For the spore inoculating of picking engineering bacteria At- Δs ku80-pyrG1-loxP in IPM culture mediums, 32 DEG C of culture 14-20 are small When, the method with reference to described by embodiment 1.2 carries out protoplast preparation after collecting mycelia, and final protoplast is resuspended in STC, And protoplast concentration is adjusted as 10⁸A/ml.

4.2 are introduced directly into the excision that Cre recombinases carry out selection markers

100ul protoplast solutions are taken, add 2U Cre recombinases (NEB, Catalog No.:M0298L)、1μg The PEG4000 of pyrG1 fragments and 10 μ l 40%, ice bath 30min after mixing.Add 1ml PSTC solution, 30 DEG C of incubation 30min. Then with being poured into after top-layer agar (CD+1.2M sorbierite+4g/L agarose+2mM Uridine, 48 DEG C of insulations after sterilizing) mixing In regeneration screening and culturing medium tablet CDSFU on (5 ' fluororotic acid+10mM Urdine of CD tablet+1.2M sorbierites+1g/L), 30 DEG C, cultivate 6-10 days under dark condition.The transformant switching that picking 27 grows out cultivates 5-7 on CDFU at 32 DEG C It carries out passage purifying, this passage purification experiment repeats 3-5 times.The transformant that picking is grown on CDFU tablets is inoculated in CD and puts down On plate, 32 DEG C are cultivated 5-7 days.Randomly select 8 plants on CDFU tablets normal growth and be unable to normal growth on CD tablets Transformant is inoculated in IPMU fluid nutrient mediums (IPM nutrient solution+10mM Uridine) and is cultivated, and collects mycelia extraction gene Group, primer U-ku80-F and TtrpC-R carry out Genomic PCR verification with reference to shown in Fig. 8.Can if pyrG1 labels are removed The fragment that size is 3.3kb is amplified, if pyrG1 fails to cut off, the fragment that size is 4.8kb can be amplified.Such as Fig. 8 Shown, the pyrG1 labels of transformant 1#, 3#, 4#, 6#, 7#, 8# are all cut off completely, by the verification PCR product of 1# transformants Gene sequencing is sent after middle 3.3kb bands recovery purifying, sequencing primer is S-ku80-F (5 '-ctcctaaacagatataccactc- 3 ') a loxP site (Fig. 9), is left behind between the results show ku80-UP and PalcA, pyrG1 labels are removed really, The engineered strain is designated as At- Δ ku80- Δs pyrG1-loxP.Found in implementation process, using 10U and 2U Cre recombinases all Efficiently cutting off and obtain enough positive transformants for selection markers can be realized, in view of cost consideration prioritizing selection uses on a small quantity Cre recombinases carry out selection markers excision.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this The people of technology, is not departing from spirit and scope of the invention, can do various change and modification, therefore, guarantor of the invention Shield scope should be subject to what claims were defined.

Claims (2)

1. A kind of 1. method for cutting off screening label, it is characterised in that be to be incorporated on chromosomepyrGScreen the restructuring of label Bacterium is bacterium germination, is cut off using site-specific recombination systempyrGScreening label enables bacterial strain to use again as going out bacterium germinationpyrGScreen label and carry out genetic transformation；It is described go out bacterium germination, be Aspergillus terreus（Aspergillus terreus）；The chromosome On incorporatepyrGThe recombinant bacterium of label is screened,pyrGThe both sides of screening label also incorporate in the same directionloxPSequence；It is describedpyrG Screening label comes from aspergillus, is carried in the same direction in both sidesloxPSequence；The site-specific recombination system, is Cre/loxPPosition Point specific recombination systems；The excisionpyrGLabel is screened, is that excision both sides carry in the same directionloxPThe screening label of sequencepyrG；The Cre/loxPSite-specific recombination system, the dosage of Cre recombinases is 2-9U；The method, step are as follows：

   Above-mentioned steps are specific as follows：

   1) knock out Aspergillus terreus CICC40205's respectivelyku80Gene andpyrGGene, builds uracil auxotrophy recombinant bacterium At-∆ku80-∆pyrG；

   2) with aspergillus niger Co827'spyrGGene expression elementpyrG1To screen label, and by the method for molecular cloning two Side adds loxP sequences in the same direction, obtains both ends with the same directionloxPSitepyrG1Selection markers loxP-pyrG1-loxP, With step 1）Constructed uracil auxotrophy recombinant bacterium At- ku80- pyrG are recipient bacterium, with loxP-pyrG1- LoxP carries out covering experiment for screening label, obtains the recombinant bacterium At- for being integrated with loxP-pyrG1-loxP screening labels ku80-pyrG1-loxP；

   3) preparation process 2）The protoplast of gained recombinant bacterium At- ku80-pyrG1-loxP, is assistant carrier to original using DNA The Cre recombinases that 2-9U is imported in raw plastid cell carry out screening label excision, by the side for screening uracil auxotrophy Formula is screenedpyrG1The recombinant bacterium that selection markers are removed.
2. 2. claim 1 the method, it is characterised in that be used to prepare and may be repeated screening from screening number of labels limitation Recombinant bacterium.