CN114958899A

CN114958899A - Replicon capable of autonomously replicating in Aspergillus niger cells

Info

Publication number: CN114958899A
Application number: CN202210598320.6A
Authority: CN
Inventors: 林峻; 燕天鹤
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-08-30

Abstract

The invention discloses a replicon capable of autonomously replicating in an Aspergillus niger cell, wherein the nucleotide sequence of the replicon is shown in SEQ ID No. 2. The replicon is obtained by constructing a metagenomic library by taking 19 fungal genomes as templates and transforming aspergillus niger spores for screening, wherein the replicon is derived from a lilac paecilomyces genome. The replicon of the invention can play a replication function in an aspergillus niger expression system, can provide higher stability than an AMA1 replicon, enriches a replicon component library for replicons found in fungi other than aspergillus nidulans for the first time, and expands the selection diversity of filamentous fungi using replicons.

Description

Replicon capable of autonomously replicating in Aspergillus niger cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a replicon capable of autonomously replicating in an Aspergillus niger cell.

Background

Before fungal autonomously replicating sequences were not discovered, the major drawback of filamentous fungal transformation systems was the absence of plasmids that resemble plasmids that can autonomously replicate outside the yeast chromosome, resulting in inefficient integrative transformation. Since the discovery of ARS sequences in Saccharomyces cerevisiae by researchers, studies on replicons have been carried out in fungi, and as early as a 6.1Kb sequence AMA1 was found in Aspergillus nidulans, transformation of Aspergillus nidulans with a plasmid containing the AMA1 sequence has improved the transformation efficiency by a factor of 250 and autonomous replication in vitro, and this sequence has also been applied to other fungi, such as Penicillium chrysogenum, Aspergillus niger, Aspergillus parasiticus, etc. From the AMA1 sequence discovery in 1991 to date, the research on the autonomous replication sequence of filamentous fungi is slow, so that the screening of potential fungal replicons can provide a certain reference value for the development of synthetic biology.

Disclosure of Invention

The invention aims to provide a replicon R75flankk 3.405 capable of autonomously replicating in an Aspergillus niger cell, wherein the replicon is obtained by constructing a metagenome library by taking 19 fungal genomes as templates and screening transformed Aspergillus niger spores, and the replicon is derived from a lilac paecilomyces genome.

The nucleotide sequence of the replicon capable of autonomously replicating in the Aspergillus niger cell provided by the invention is shown in SEQ ID NO. 2.

The replicon R75flank3.405 was constructed as follows:

1) using AnEp8-AnHyg plasmid as a template, and removing an AMA1 replicon by Not I enzyme digestion to obtain an AnEp 8-AnHyg-deleetaAMA 1 vector; taking 19 fungus mixed genomes as templates, performing DNA fragmentation treatment by a Tn5 library building kit, amplifying and screening 4-5Kb target fragments by an Altama-F/R primer, and recovering and purifying amplified products; connecting an AnEp8-AnHyg-deleteAMA1 linear vector and a target fragment by using an In-fusion method, transforming the linear vector into a 10 beta super competent cell, and extracting a plasmid for later use;

2) transforming the plasmid obtained in the step 1) into aspergillus niger spores by using an HDEN technology, screening a positive transformant by using a Hyg resistance gene, taking the positive transformant spores as a template, verifying by using a Not I-F/R primer, and sequencing by using sanger to obtain a section of R75 sequence containing a part of ribosome large subunit protein L9 and a DNA replication helicase sequence;

3) using 19 fungal genomes as templates, amplifying R75-F/R primers to obtain an R75 sequence source, using an R75 sequence source as a template, amplifying R75flank-F/R to obtain an R75flank3.405 fragment, and connecting with AnEp 8-AnHyg-deletam 1 in the step 1) to obtain an AnEp8-R75flank3.405 plasmid.

The invention has the beneficial effects that: the R75flank3.405 replicon of the invention can play a replication function in an Aspergillus niger expression system, can provide higher stability than an AMA1 replicon, enriches a replicon element library for the replicon found in fungi except Aspergillus nidulans for the first time, and expands the selection diversity of filamentous fungi using the replicon.

Drawings

FIG. 1 is a graph showing the effect of different amplification cycles, Tn5 usage and treatment time on the target fragment;

m: takara 250bp DNA Ladder Marker; (A) lanes 1-5: the amplification cycle numbers were 15, 20, 25, 30, 35, respectively; (B) lanes 1-2: the dosage of Tn5 is 0.05U and 0.03U respectively; (C) 1-5 of swimming channel: the fragmentation treatment time was 10sec, 15sec, 20sec, 25sec, and 30sec, respectively.

FIG. 2 shows the Tn5 library colony PCR validation.

FIG. 3 is a Tn5 library Aspergillus niger colony PCR validation.

Fig. 4 is the R75 sequence.

FIG. 5 is an electrophoretogram showing PCR confirmation of the origin of the R75 fragment;

m: takara DL500 DNA Ladder Marker; (A) lanes 1-10 templates were: GDMCC 3.605, cic 40344, GDMCC 3.515, GDMCC 3.439, GDMCC 3.441, GDMCC 3.549, GDMCC 3.521, GDMCC 3.510, cic 40341, CBS 513.88; (B) lanes 1-9 templates were: ATCC 24919, GDMCC 3.405, CICC 40359, GDMCC 3.97, GDMCC 3.548, CICC 2397, GDMCC 2.12, GDMCC 3.413, GDMCC 3.503.

FIG. 6 shows the amplification of the R75flank3.441/3.405 fragment (A) and the validation of the AnEp8-R75flank3.441/3.405 plasmid transformant (B);

wherein, M: takara 250bp DNA Ladder Marker; (A) lane 1: R75flank 3.405; lane 2: fragment r75flank3.441 (B) lane 1-2 template: two transformants AnEp8-R75flank 3.441; lane 3 template: AnEp8-R75flank 3.405A transformant.

Fig. 7 is a sequence of r75flank 3.405.

FIG. 8 is a map of the Actin and Hyg gene melting peaks.

FIG. 9 is a graph showing the relative expression amounts of Hyg genes in two samples.

FIG. 10 is a hygromycin assay after several passages of two Aspergillus niger crosses over PDA;

m: takara 250bp DNA Ladder Marker; (A) lanes 1-9: inoculating AnEp8-AnHyg Aspergillus niger for 1-9 times; lane 10: negative control; (B) lanes 1-9: inoculating AnEp8-R75flank3.405 Aspergillus niger for 1-9 times; lane 10: and (5) negative control.

FIG. 11 is a colony PCR validation of two plasmids ligated with 4Kb of exogenous fragment;

m: takara 250bp DNA Ladder Marker; (A)1-5 AnEp8-AnHyg plasmid; 6 negative control (B)1-5 AnEp8-R75flank3.405 plasmid.

FIG. 12 is a colony PCR validation of two plasmids ligated with 10Kb of exogenous fragment;

m: takara 250bp DNA Ladder Marker; (A)1-5 AnEp8-AnHyg plasmid; 6 negative control (B)1-5 AnEp8-R75flank3.405 plasmid; 6, negative control.

FIG. 13 is a colony PCR validation of two plasmid-ligated 16Kb foreign fragments;

FIG. 14 shows growth of Aspergillus niger containing exogenous fragment plasmids;

column 1: original plasmid control; column 2: connecting 4Kb exogenous fragment recombinant plasmid; column 3: connecting 10Kb exogenous fragment recombinant plasmid; column 4: connecting 16Kb exogenous fragment recombinant plasmid; line A: AnEp8-AnHyg and recombinant plasmids thereof; line B: AnEp8-R75flank3.405 and recombinant plasmid thereof.

FIG. 15 is the validation of transformants with AnEp8-AnHyg ligated to plasmids of different foreign fragments;

m is Takara 250bp DNA Ladder Marker; ABC is the result of the verification of the ligated fragments, and lanes 1 are all negative controls; DEF is the hygromycin assay, lanes 1 are all positive controls; (A) lanes 2-6, five transformants with 4Kb were ligated; (B) lanes 2-6, five transformants ligated at 10 Kb; (C) lanes 2-4 three transformants with 16Kb of ligated sequence in DEF with ABC.

FIG. 16 shows the validation of transformants with the AnEp8-R75flank3.405 plasmid ligated to different foreign fragments;

m is Takara 250bp DNA Ladder Marker; (A) lane 1 negative control; lanes 2-3 validation of two transformant fragments ligated at 4 Kb; lane 4. Single transformant fragment ligation of 16Kb verifies (B) lane 1: positive control; the remaining lane samples were sequenced as in A for hygromycin validation.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

1 fungal culture and genome extraction

1.1 fungal culture

Taking out the strain from a refrigerator at the temperature of minus 80 ℃, naturally thawing the strain on ice, dipping bacterial liquid in an inoculating loop burnt by an alcohol lamp in a super clean workbench, streaking a solid PDA culture medium by an in-line method, carrying out dark culture according to the culture conditions in the table 1 until a single bacterial colony grows out, selecting part of the bacterial body, blowing and beating the bacterial body in sterile water to be uniformly mixed, taking 200 mu L of spore suspension, carrying out dark culture in the PDA solid culture medium paved with cellophane until the culture medium is fully paved with hyphae, and storing the bacterial body in the refrigerator at the temperature of 4 ℃.

TABLE 1 fungal species and culture conditions

1.2 fungal genome extraction method

The cultured fungal hyphae are used as a template to extract a genome, and the specific steps refer to the specification of a rapid extraction kit of fungal genome DNA of biological engineering.

2 Tn5 library construction

The linear vector used in the invention is AnEp8-AnHyg-deleteAMA1, which is obtained by removing an AMA1 sequence on the basis of an AnEp8-AnHyg shuttle plasmid. The plasmid carries colibacillus screening gene ampicillin and fungus screening gene hygromycin B, so as to facilitate the screening of positive transformants.

Since the AMA1 replicon sequence is about 5Kb, the selection range of the target fragment is controlled to be 4-5 Kb. Firstly, fungus genome needs to be fragmented, and at present, the most widely used fragmenting enzyme IS Tn5, the binding site of the fragmenting enzyme IS the external ends of inverted IS50 sequences at two ends of Tn5 transposon, two transposase external ends (Tnp-OE) complexes are formed, and double-stranded DNA IS randomly inserted after the complexes are combined to fragment the genome. Primers are designed according to the homologous complementary connection principle and are connected by an Infusion method, and super competent cells are transformed to obtain a Tn5 library.

2.1 AnEp8-AnHyg plasmid extraction

Taking AnEp8-AnHyg Escherichia coli strain out of a refrigerator at-80 ℃, naturally thawing on ice, dipping a small amount of bacterial liquid by using an inoculating loop, streaking on an LB solid plate containing ampicillin resistance (working concentration is 100 mu g/mL) by a three-zone method, culturing at 37 ℃ overnight for 12-16h, picking a single colony on 5mL LB liquid culture medium containing ampicillin resistance (100 mu g/mL), placing the LB liquid culture medium in a shaking table at 37 ℃, performing shaking culture at 220rpm for 12-16h, inoculating the single colony on 10mL LB liquid culture medium containing ampicillin resistance (100 mu g/mL) according to an inoculation rate of 1 per thousand, placing the single colony on the shaking table at 37 ℃, performing shaking culture at 220rpm for 12-16h, extracting plasmids, and storing at-20 ℃. The Plasmid extraction method refers to the instruction of EndoFree Plasmid Midi Kit in the Kangji century.

2.2 AnEp 8-AnHyg-deleeAMA 1 Linear vector acquisition

The AMA1 sequence contained the same restriction enzyme site Not I at both ends, and the vector did Not contain the same restriction enzyme site at other positions, and the reaction system was as shown in Table 2, after digestion according to the NEB Not I-HF protocol.

TABLE 2 AnEp8-AnHyg plasmid cleavage System

Sucking and uniformly mixing the prepared system, performing instantaneous centrifugation, placing the system in a PCR instrument for incubation for 3h at a constant temperature of 37 ℃, incubating for 20min at 65 ℃ for heat inactivation, adding 6 XLoading Buffer to the final concentration of 1X to the total enzyme digestion products, Loading the mixture into a 1% agarose Gel hole, performing 85V electrophoresis for 20min, cutting a target band by taking a lambda-Hind III digest DNA Marker as a reference, and purifying and recovering a Gel recovery product by using a GeneJET Gel Extraction and DNA clean Micro Kit to obtain a linear carrier AnEp 8-AnHyg-deleeAMA 1.

2.3 destination fragment acquisition

19 fungus mixed genomes are taken as templates, Tn5 transposase is added to break the genomes, a reaction system is shown in table 3, an Altama-F/R primer is used for amplifying genome fragments, the primer contains a sequencing joint S5 (5'-TCGTCGGCAGCGTC-3')/S7 (5'-GTCTCGTGGGCTCGG-3') sequence and 15bp sequences homologous with two ends of a linear vector Not I enzyme cutting site, and seamless connection of target fragments and the vector can be achieved. The amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and conditions for Tn5 usage, processing time, and the number of amplification cycles were investigated. All products were loaded onto agarose Gel wells and recovered by purification of Gel recovery products of 4-5Kb size using the GeneJET Gel Extraction and DNA clean Micro Kit, referenced to Takara 250bp DNA Ladder Marker.

TABLE 3 Tn5 fragmentation reaction System

Blowing the prepared system, mixing uniformly, centrifuging for a short time, placing in a PCR instrument at 55 deg.C, incubating for 10sec, 15sec, 20sec, 25sec, and 30sec, respectively, adding 15 μ L ddH into 5 μ L reaction system ₂ And O, mixing, placing in a PCR instrument, incubating for 8min at 95 ℃, and placing on ice for later use.

TABLE 4 PCR amplification System

TABLE 5 PCR amplification procedure

Sucking and uniformly mixing the prepared system, then performing instant centrifugation, placing the system in a PCR instrument for fragment amplification according to the table 5, adding 6 XLoading Buffer to the final concentration of 1X to all enzyme digestion products, Loading all the products into 1% agarose Gel holes, performing 85V electrophoresis for 20min, cutting a 4-5Kb mesh band by using a Takara 250bp DNA Ladder Marker as a reference, and purifying and recovering Gel recovery products by using a GeneJET Gel Extraction and DNA clean Micro Kit.

2.4 ligation transformation

And (3) carrying out a connection reaction on the 4-5Kb fragment recovered by cutting gel and the purified AnEp 8-AnHyg-deletamA 1 linear vector through an In-Fusion Snap Assembly, wherein the reaction system refers to the instruction of a Takara In-Fusion Snap Assembly kit, the insert fragment and the linearized vector are added into the system according to the molar ratio of 2:1, the insert fragment is calculated by 5Kb, and the system is placed In a PCR instrument for incubation at 50 ℃ for 15min after being configured as shown In Table 6.

To increase transformation efficiency, 10 β super competent cells were used for transformation. Compared with the traditional calcium chloride method for preparing competence, the conversion efficiency of the super competent cells can be 1-2 orders of magnitude higher. 10 μ L of the ligation system was transformed into 10 β super competent cells, plated with LB solid plates containing ampicillin resistance (100 μ g/mL), incubated overnight at 37 ℃ for 12-16h until single colonies grew out, single colonies were randomly picked, and ligation efficiency was verified using Not I-F/R primers, with the colony PCR system shown in Table 7 and the PCR program shown in Table 8. The 10 beta super competent cell manufacturing method refers to the technical and biological engineering super competent cell preparation reagent kit.

TABLE 6 Infusion connection System

TABLE 7 colony PCR amplification System

TABLE 8 colony PCR amplification procedure

2.5 plasmid extraction

The ligation system was expanded by the above method to transform 10. beta. super competent cells, and all single colonies were scraped off with 200mL of LB liquid medium containing ampicillin resistance (100. mu.g/mL), and cultured in a 37 ℃ constant temperature culture shaker at 220rpm for several hours to OD ₆₀₀ The plasmid was extracted in large scale at 0.2, the specific procedure was as follows.

(1) 200mL of the bacterial solution is subpackaged into 450 mL centrifuge tubes, 6000rcf is used for centrifugation for 15min, and the supernatant is discarded.

(2) 10mL of buffer P1(50mM Tris-HCl, pH 8.0; 10mM EDTA pH 8.0) that was previously incubated with ice was added to the cells, and RNAseA was added to a final concentration of 150. mu.g/mL), and the mixture was mixed by pipetting with a pipette.

(3) 10mL of BufferP2(200mM NaOH, 1% SDS) was added, gently shaken to mix thoroughly, and allowed to stand at room temperature for 5 min.

(4) 10mL of prefreezed buffer P3(3M potassium acetate, pH 5.4-5.6) was added, gently shaken until the pellet was completely white, and centrifuged at 7000rcf for 10 min.

(5) Installing a filtering device: the funnel with the sterilized paper extract was mounted on a new 50mL centrifuge tube, and the paper extract was placed layer by layer and three layers by layer.

(6) And filtering the centrifuged supernatant of the bacteria liquid through a filtering device to remove precipitates, and collecting the supernatant.

(7) Adding Buffer ER cultured in advance in the supernatant, turning upside down, mixing, and incubating on ice for 30 min.

(8) QIAGEN-tip500 was placed in a new 50mL centrifuge tube, 10mL Buffer QBT (750mM NaCl; 50mM MoPS, pH 7.0; 1% isopropanol; 0.15% Triton) was added, and the tube was emptied by gravity drip.

(9) The mixture frozen in step 7 was added to the equilibrated QIAGEN-tip500, and slowly dropped through the resin.

(10) The tip500 was washed by adding 60mL of Buffer QC (1M NaCl; 50mM MoPS, pH 7.0; 15% isopropanol).

(11) The DNA was eluted by adding 15mL of Buffer QN (1.6M NaCl; 50mM MoPS, pH 7.0; 15% isopropanol) to a new 50mL centrifuge tube.

(12) 10.5mL of isopropanol was added to the eluate to precipitate DNA, the mixture was mixed by inversion, and then centrifuged at 15000rcf at 4 ℃ for 30min, and the supernatant was discarded.

(13) 1mL of 70% ethanol was added to gently blow and resuspend the pellet, and the pellet was centrifuged at 15000rcf at 4 ℃ for 10min, and the supernatant was discarded.

(14) Standing at room temperature for 15min to air dry the residual ethanol.

(15) Add 100. mu.L of ddH ₂ The precipitate was dissolved by O and the extracted DNA was immediately subjected to the next experiment or stored at-20 ℃.

2.6 library construction results

The size of the target fragment is influenced by the Tn5 treatment time, the Tn5 dosage and the number of amplification cycles, and the larger the dosage, the longer the treatment time, the smaller the target fragment. In order to maximize the content of the target fragment, the amplification cycle number, the Tn5 dosage and the Tn5 treatment time are optimized, agarose gel electrophoresis is used for observing the amount of the 4-5Kb fragment, and the result is shown in figure 1, wherein the cycle number in the graph A is optimally 35 cycles, and the 4-5Kb fragment is the most; in the picture B, the optimal dosage of Tn5 is 0.05U; the processing time in graph C is optimally 25 sec.

The 19 fungal genomes are fragmented by using the optimal conditions, are seamlessly connected with a linearization vector and are transformed into 10 beta super competent cells, 5 single colonies are randomly picked to verify the connection efficiency by a Not I-F/R primer, the electrophoresis result is shown in figure 2, the amplification band of the Not I-F/R primer of the AnEp 8-AnHyg-deletamA 1 vector is 255bp, three bands in the 5 single colonies exceed 255bp, and the band is between 500bp and 1.5 Kb. The reaction was amplified to 200. mu.L to give about 44000 single colonies and the plasmid was amplified and stored at-20 ℃.

3 selection of transformants

The traditional exogenous nucleic acid transformation method mainly comprises a protoplast transformation method, an agrobacterium transformation method, a gene gun method and the like, but the operation is complicated, the cell survival amount is small due to great damage to the cells, and the transformation efficiency is low. In recent years, researchers have developed an HDEN technique, which is an electroporation method capable of transforming exogenous nucleic acids into mammalian cells, and which is effective in reducing the damage to cells during transformation and improving transformation efficiency, but is less studied in fungal spore transformation. Based on the precedent that HDEN technology is used for successfully transforming Aspergillus niger in the laboratory, parameters such as electric field intensity, pulse time, electric shock frequency, capacitance, buffer pH, cell concentration and exogenous nucleic acid content in the electric shock transformation process are researched, and the optimal conditions for electrically transforming Aspergillus niger spores are obtained on the basis. Specific transformation procedure the following experimental methods in the literature: huangxiaoming, study of direct transformation of fungal spores by exogenous nucleic acids [ D ]. Fuzhou: Fuzhou university, 2019.

3.1 screening method

The growth of the bacterial colony is observed after 4-5 days of electrotransformation, and under normal conditions, Aspergillus niger grows for 1-2 days to form white conidial heads, which become yellow to black thick velvet and have a yellowish brown back. If Aspergillus niger single colony grows out on the plate, selecting partial spores to be cracked in a lysate for 15min at 85 ℃ as a template, taking infusaANEp 8-F/R primer as colony PCR (polymerase chain reaction) to preliminarily verify a hygromycin fragment, and if transformants have correct bands, selecting partial thalli of the single colony to be placed in ddH ₂ And (3) blowing and beating in O to obtain spore suspension, coating 200 mu L of the suspension on a PDA solid culture medium containing hygromycin B (working concentration is 200 mu g/mL) for amplification culture, and paving cellophane on the culture medium to collect hyphae conveniently. Culturing at 28 deg.C in dark for 1-2 days until the glass paper is covered with mycelia, picking appropriate amount of mycelia with a gun head, and extracting genome with liquid nitrogen method.

The extracted genome is used as a template, Not I-F/R primers are used for amplifying a target fragment, 1% agarose gel electrophoresis is used for verification, a PCR product with a band is sent to Sanger for sequencing to obtain a new replicon sequence, meanwhile, a positive transformant is continuously transferred to a PDA plate containing hygromycin B resistance for a plurality of times to verify whether the replicon has a replication function, if the positive transformant still has a correct band after being transferred for a plurality of times, the Altama-F/R primers are used for amplifying a new replicon fragment, and an Infusion method is connected with an AnEp 8-AnHyg-deleeAMA 1 linear vector to construct a plasmid with a new replicon.

And (3) carrying out seed preservation treatment on the screened positive transformants: selecting partial thallus of positive transformant to be added into appropriate ddH ₂ Obtaining a spore suspension in ODipping the spore suspension by using an inoculating loop burned by an alcohol lamp, streaking a slant solid PDA culture medium (containing 200 mug/mL hygromycin B), culturing at 28 ℃ in the dark until black spores grow, slightly scraping the spores by 10% sterile glycerol, and subpackaging in a 1.5mL centrifuge tube for preservation at-80 ℃.

3.2 results

3.2.1 fragments of R75

A Tn5 library large-extraction plasmid constructed by a Tn5 transposase method is transformed into Aspergillus niger to obtain three transformants, a Not I F/R primer is used for amplifying a target gene, and an electrophoresis result is shown in figure 3, and only a sample No. 3 has a band of about 1000 bp.

Sanger sequencing is carried out on the sample PCR product, the sequencing result is shown in figure 4, the full length of the inserted sequence is 753bp, the sequence is found to be dissimilar to AMA1 after NCBI database Blast sequence comparison, the similarity of the sequence and the genome of Burkholderia HKI454(Burkholderia rhizoxinica HKI454) reaches 95 percent, the sequence can be aligned to 709bp, the sequence is positioned at the 1273275bp position of the genome 1272530-.

Burkholderia HKI454 is an intracellular symbiont of the plant pathogenic fungus Rhizopus microsporus (Rhizopus microsporus), in which symbiotic life forms the host fungus produces sporangia and spores depending on the presence of burkholderia, which in turn produces endophytes that produce endophytes that nourish the decaying plant. Ribosomal Protein (RP) is an important component constituting the ribosome, and is attached to the ribosomal RNA backbone to form an intact ribosome, which is involved in protein biosynthesis. The RPL9 protein is a component of ribosome large subunit, has a slender shape, is connected with an N-terminal domain and a C-terminal domain by an alpha-helix, the two domains are alpha-beta globulin, wherein the N-terminal domain contains 56 residues, the C-terminal domain contains 92 residues, the alpha-helix length of the RPL9 protein in different species is different, but the distance between the two 23S rRNA domains can be stabilized, and the RPL9 protein plays an important structural function in ribosome. Meanwhile, the RPL9 protein is one of the most basic proteins combined with RNA, plays an important role in the rapid movement of ribosome in the translation process, and ensures the accuracy of translation. The DNA replication helicase plays an important role in the prophase of replication, the core part is an MCM complex consisting of six subunits (Mcm 2-7), the prophase of replication is loaded on a replication origin, dsDNA (Double-Stardand DNA) is encircled under the participation of a loading factor to form an MCM Double hexamer complex in an inactivated state, the S phase is activated into two CMG (Cdc 45-MCM-GINS) complexes under the participation of a protein factor and is encircled on ssDNA (Single-Stardand DNA), the two CMG Double hexamer complexes move from the 3 'direction to the 5' direction of a guide chain and assemble a replicator, and the two CMG Double hexamer complexes are depolymerized and end of replication in the replication termination stage.

The 753bp sequence was named R75 and cloned into AnEp 8-AnHyg-deleeAMA 1 vector to obtain a novel plasmid AnEp 8-R75. In order to compare the strong and weak transformation efficiency of two replicons to Aspergillus niger, the two plasmids AnEp8-AnHyg and AnEp8-R75 were electroporated at 6. mu.g, 12. mu.g and 24. mu.g, respectively, and the transformation results showed that AnEp8-AnHyg had the best electroporation effect at 12. mu.g of plasmid, the highest transcriptions could grow 98 transformants, while AnEp8-R75 had the best transformation effect at 6. mu.g of plasmid, and the highest records could grow 128 transformants. To confirm the stability of the R75 replicon again, the AnEp8-R75 plasmid was electroporated several times, but the results of the repeated experiments were unstable. The sequence analysis shows that the AT content of the R75 sequence 249-259bp is up to 91% (5'-TTTTTTTTGTT-3'), the difference between the AT content and the 11bp conserved sequence [5 '- (A/T) TTTA (T/C) (A/G) TTT (A/T) -3' ] in ARS is only 3 bases, which is probably an active region with the replication function, and compared with the special structure of AMA1, the R75 sequence does not have an inverted repeat structure and other special structures, and the sequence is shorter and is not enough to maintain the stable replication function in the Aspergillus niger.

3.2.2 improvement of fragment R75

After alignment, the R75 sequence was found to be derived from the HKI454 of Burkholderia, and therefore attempts were made to isolate this fungal endophyte from fungi. Firstly, because the R75 sequence is obtained by screening 19 fungal genomes, the R75 sequence is used as a template to design a primer R75-F/R, the primer can amplify a 373bp fragment, the 19 fungal genomes are used as templates to respectively amplify target fragments, the electrophoresis result is shown in FIG. 5, the brightest band is GDMCC 3.441 (FIG. 5A lane 5), the next band is GDMCC 3.405 (FIG. 5B lane 2), and the rest bands are weaker and are not considered.

Marking out a PDA flat plate without antibiotics on the screened GDMCC 3.405, respectively picking partial hyphae after the hyphae grow out, carrying out shake flask culture in a liquid LB culture medium at 37 ℃ for 1-2 days, then taking partial bacterial liquid, centrifuging and collecting thalli, and verifying 16S rRNA, wherein the result shows that the endophyte is not successfully separated.

The transformation effect of the AnEp8-R75 plasmid is unstable, so that a new sequence R75flank is obtained by adding a partial RPL9 sequence and a DNA replication helicase sequence at both ends of the R75 sequence, and the new sequence comprises the complete RPL9 and DNA replication helicase sequences which are positioned at the 1274378bp positions of 1272234 and 1274 of the genome of HKI454 of Burkholderia. Designing a connecting primer R75flank-F/R according to R75flank and a linear vector, amplifying an R75flank fragment by taking GDMCC 3.405 as a template, obtaining a 1.5Kb fragment as shown in a figure 6A lane 1, cloning the fragment to an AnEp 8-AnHyg-deleeAMA 1 linear vector to obtain a new plasmid AnEp8-R75flank3.405 (derived from the GDMCC 3.405), electrically transforming Aspergillus niger, growing 1 transformant after transformation, verifying the transformant by a Not I-F/R primer, and carrying out Sanger sequencing on a PCR product, wherein the electrophoresis result is shown in a figure 6B lane 3 and a band of about 1.8Kb is formed.

The sequencing result is shown in FIG. 7, the full length of R75flank3.405 is 1543bp, after NCBI alignment, the similarity between 520bp base and GDMCC 3.405 pentapeptide repeat protein (PPR) is found to be 98%, the comparison between the other sequences has no result, at 855-865bp (5'-ATTTTAATCTT-3'), 1340-1350bp (5'-TCGTAAATTGA-3'), the difference between the other sequences and ARS conserved sequence is 3 and 4 bases, and no special structure exists. The PPR protein consists of 2-30 continuous motifs, each motif comprises 35 residues, and specific residues can be combined with nucleotides, are in hairpin structures, have RNA binding capacity, can maintain RNA stability or promote the translation of the RNA, and influence the metabolism in organelles from multiple aspects such as editing, shearing and the like.

4 transformant validation

4.1 plasmid copy number validation

Real-time fluorescent quantitative PCR (qpcr) the fluorescent signal during PCR is monitored using intercalating dyes such as SYBR Green I, and the initial DNA levels of different samples are quantified by Ct values (initial thresholds), the higher the Ct value, the less the initial amount of template. The fluorescent dye added into the PCR system can only be combined with double-stranded DNA to excite green fluorescence, and the fluorescent signal is gradually enhanced along with the increase of target fragments, but the fluorescent dye can be combined with any double-stranded DNA, so that primer dimer or non-specific amplification products cannot be distinguished, and the primer specificity needs to be detected by utilizing a melting curve. The melting curve is a relation graph of fluorescence signal change and temperature, in the PCR process, when the melting temperature (Tm) of a double-stranded product is reached, the fluorescence signal can be rapidly reduced along with the unwinding of the double strands, the fluorescence signal is gradually reduced to the lowest along with the continuous increase of the temperature, when the primer is subjected to specific amplification, only a single melting peak graph can be generated, otherwise, the primer is subjected to non-specific amplification. In order to analyze the relative expression difference of hygromycin genes after the four plasmids are transformed into the Aspergillus niger, an internal reference gene needs to be selected for standardization. For some common sample types such as peripheral blood, lung cancer tissue, liver cancer tissue and the like, a great deal of research has been carried out on selection of reference genes, the common reference genes comprise Actin, GAPDH, 18S rRNA and the like, while the research on the reference genes in the aspergillus niger genome is less, K.Bolhe and the like compare 10 candidate genes in the aspergillus niger genome, wherein three genes of the Actin, sarA and cox5 are stably expressed, and the Actin is highly conserved in eukaryotic cells and is also a main component of a cytoskeleton.

Actin is used as an Aspergillus niger internal reference gene, qPCR primers are designed on NCBI aiming at the gene and the Hyg gene, a positive Aspergillus niger genome obtained by carrying out electrotransformation on AMA1 and R75flank3.405 replicon plasmids is used as a template, and Takara TB is referred to

Premix DimerEraser ^TM (Perfect Real Time) kit instruction, normalization treatment is carried out by taking Actin as an internal reference gene, Hyg gene expression in two samples is relatively quantified, and each sample isMaking three multiple wells with ddH ₂ And taking O as a template as a negative control, carrying out data analysis by LightCyler 96 software, and calculating the relative expression difference by a delta CT method. The calculation formula is shown in formula 1.

Relative expression amount of 2 ^-ΔΔCT Equation 1

Wherein Δ Δ CT ═ CT Hyg _{(unknown sample)} -Ct Actin _{(unknown sample)} ]-[Ct Hyg _{(control sample)} -Ct Actin _{(control sample)} ]。

The qPCR system is shown in table 9 and the qPCR program is shown in table 10.

TABLE 9 qPCR System

TABLE 10 qPCR amplification procedure

4.2 plasmid stability verification

Taking out two kinds of Aspergillus niger strains after preservation at-80 deg.C, thawing naturally on ice, streaking PDA plate without hygromycin resistance, culturing at 28 deg.C in dark until large single strain grows out, picking part of strain to 200 μ L sterile ddH in ultraclean bench ₂ And (4) blowing and uniformly mixing in O, sucking 200 mu L of spore suspension, coating on a PDA nonreactive solid culture medium paved with glass paper, performing dark culture at 28 ℃ for 1-2d until white hyphae grow on the glass paper, and extracting a genome by a liquid nitrogen grinding method.

Taking the extracted fungal genome as a template, amplifying an infusaneap 8-F/R primer, verifying hygromycin fragments by 1% agarose gel electrophoresis, if bands are correct, picking part of hyphae growing after the first transformation of PDA to perform second transfer to a PDA solid culture medium without hygromycin, picking hyphae to extract the genome to verify the hygromycin fragments, repeating the steps until no hygromycin fragments exist, and verifying the drug resistance of the two plasmids to hygromycin.

4.3 plasmid load verification

Compared with the Bacterial Artificial Chromosome (BAC) which can bear 300Kb of exogenous fragment, the current Fungal Artificial Chromosome (FAC) can be connected with exogenous fragment with the maximum of 150Kb, and the larger the exogenous fragment which can be connected with replicon, the larger the variety and the potential of the expressed exogenous protein.

4.3.1 plasmid construction

Selecting a bacillus subtilis genome as a template to design an Infusion primer to amplify 4Kb, 10Kb and 16Kb fragments, and carrying out electrophoresis verification on 1% agarose gel to recover target fragments. M4i-F/R primer was used for 4Kb of target fragments, M10i-F/R primer was used for 10Kb of target fragments, M16i-F/R primer was used for 16Kb of target fragments, and the primer sequences are shown in Table 13. The STE method is used for extracting the bacillus subtilis genome, and the specific steps are as follows.

(1) Taking a proper amount of bacillus subtilis liquid cultured overnight into a 1.5mL centrifuge tube, centrifuging for 1min at 12000rcf, discarding the supernatant, and controlling the wet weight of the bacteria to be about 30 mg.

(2) Add 600. mu.L of 1 XSTE buffer and 60. mu.L of lysozyme (200mg/mL) to the cells, blow-suck them well, add grinding tube and grind for 20s (10s for 1 time of ice incubation), centrifuge briefly, and take the supernatant to a new centrifuge tube.

(3) 1% SDS, 1.2. mu.L RNAseA (100mg/mL), 9.6. mu.L proteinase K (25 mg/mL) were added to the supernatant and mixed by pipetting.

(4) Placing the centrifuge tube in 65 deg.C water bath for 30min, and mixing every 5min or more.

(5) Adding equivalent phenol into the centrifuge tube, centrifuging at 20000rpm at 4 ℃ for 10min, and taking the supernatant to a new centrifuge tube.

(6) Adding equal amount of chloroform, centrifuging at 20000rpm at 4 deg.C for 10min, and collecting supernatant to new centrifuge tube.

(7) Adding two times volume of cold ethanol and 4 μ L nucleic acid precipitation promoter, centrifuging at 20000rpm at 4 deg.C for 10min, and discarding the supernatant.

(8) The precipitate was added with 1mL of 70% ethanol and gently blown, centrifuged at 20000rpm at 4 ℃ for 1min, and the supernatant was discarded.

(9) Drying and precipitating to be transparent at room temperature after uncovering, and 100 mu L of sterile ddH ₂ O dissolution, concentration by NanoDrop, -20 ℃ storage.

The two plasmids were digested with Takara Mlu I endonuclease under the conditions described In the Takara Specification, and Takara In-Fusion Snap Assembly cloning kits were used to ligate the desired fragment to the linear vector at a molar ratio of 2: 1. the ligation system was transformed into stbl3 chemocompetent cells, the ligated fragments were verified by colony PCR, the Not I-F/R primer (4503bp) was used for 4Kb fragment verification, the 10K-F/R primer (1102 bp) was used for 10Kb fragment verification, the 16Kb fragment was verified by 16K-F/R primer (1894bp), and the primer sequences are shown in Table 13.

4.3.2 transformation of Aspergillus niger and validation of transformants

After all plasmids are successfully constructed, electric shock is transferred to aspergillus niger spores, each treatment is repeated for 3 times, the growth condition and the number of transformants are observed after culturing for 5-6 days, and hygromycin and connecting fragment verification is carried out on the transformants, wherein the hygromycin verification uses an infusanneP 8-F/R primer (1366bp), the 4Kb fragment verification uses a 4K-F/R primer (1261 bp), and the 10Kb and 16Kb fragment verification primers are the same as 4.3.1.

4.4 plasmid copy number analysis

Two Aspergillus niger genomes are used as templates, target fragments are respectively amplified by qPCR Act-F/R and qPCR Hyg-F/R primers to verify the specificity of the primers, the sequences of the primers are shown in a table 13, the amplification products are respectively 105bp and 150bp, a melting peak diagram is shown in a figure 8, a single peak appears at 83 ℃ and 88 ℃ respectively, and the primers are proved to be specifically amplified.

The results of statistical analysis of Ct values of the reference gene and the Hyg gene of the two samples are shown in Table 11, the positive control sample AnEp8-AnHyg Aspergillus niger has the highest Hyg gene expression abundance, the average Ct value of the three repetitions is 13.45 + -0.12, the average Ct value of the three repetitions of the Hyg gene in AnEp8-R75flank3.405 Aspergillus niger is 15.68 + -0.22, and 2 is calculated according to the formula 1 ^{-ΔΔCt(3.405/AnHyg)} 0.075. As shown in fig. 9, the expression amount ratio of Hyg genes in two aspergillus niger was 1: 0.075, results show that the R75flank3.405 replicon can also play a replication function in A.niger.

TABLE 11 analysis of Actin and Hyg Gene Ct values for two samples

4.5 plasmid stability analysis

The result of the transfer is shown in FIG. 10, the resistance gene is lost after 9 times of transferring the AnEp8-AnHyg plasmid on the PDA plate without hygromycin B screening resistance, while the resistance gene still exists after nine times of transferring the AnEp 8-R75flnk3.405 plasmid, which shows that the R75flnk3.405 replicon has stronger stability compared with the AMA1 replicon.

4.6 plasmid load Capacity analysis

4.6.1 plasmid construction results

The Plasmid construction results are shown in FIGS. 11, 12 and 13, the two plasmids are successfully connected with exogenous fragments of 4Kb, 10Kb and 16Kb, the bands of target fragments are the same as the theoretical size, the sequencing result shows that no base mutation exists, and the Plasmid is extracted by using the EndoFree Plasmid Midi Kit for later use.

4.6.2 growth of transformants

All plasmids were successfully transformed into A.niger and the results after five days of growth are shown in FIG. 14 and the statistical table in Table 12. From the growth situation, 72, 13 and 3 transformants were respectively grown after the AnEp8-AnHyg plasmid carrying exogenous fragments of 4Kb, 10Kb and 16Kb was transformed into Aspergillus niger, and the number of transformants was gradually reduced compared with 112 transformants grown after the AnEp8-AnHyg original plasmid was transformed; after the AnEp8-R75flank3.405 plasmid carrying 4Kb and 16Kb exogenous fragments is transformed, 2 transformants and 1 transformant are respectively grown, after the Aspergillus niger is transformed by carrying 10Kb plasmid, no transformant is grown, and after the original plasmid is transformed, 1 transformant is grown.

TABLE 12 number of Aspergillus niger transformants containing exogenous fragment plasmids

The data are shown as mean values + -standard deviation, which indicates statistical significance, P is less than 0.05 (ANG), < 0.01 (ANG), < 0.001 (ANG), < 0.0001 (ANG), and ns indicates no significant difference.

4.6.3 transformant verification

The verification results are shown in FIGS. 15 and 16, four selected single colonies of five AnEp8-AnHyg-4Kb and five AnEp8-AnHyg-10Kb Aspergillus niger are positive transformants, the hygromycin verification and the connecting fragment verification are consistent with the theoretical size of the target fragment, the hygromycin fragment in the selected single colonies of three AnEp8-AnHyg-16Kb Aspergillus niger is correct, only 1 in the fragment verification is a positive transformant, and the conjecture that the 16Kb fragment is too large is lost in the process of converting Aspergillus niger; the AnEp8-R75flank3.405-4Kb/16Kb picked all appeared to have the hygromycin fragment correct but the ligation fragment was lost, but overall the replicon could carry a 4Kb foreign fragment.

TABLE 13 primer sequences

Sequence listing

<110> Fuzhou university

<120> a replicon autonomously replicable in A.niger cells

<130> 2022

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 753

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tctcgtgggc tcggtcacga atcaggacat cgccgtcgcg ctcaagcaac aaggcttcga 60

cgtcgaaaag gcacaggtgc gcctgtcgca aggtccgctg aagatcgtgg gtgatcatcc 120

ggtgcaagtg gcgctgcaca ccgacgtggt gacggacatt acggtgtcgg tgctcggcga 180

gcacgcgtaa gatcgctgcg caccttcggg tgtcgcatgc aagcaggatc cgggtaaccg 240

gttcctgctt ttttttgttt catgccattg cgtgaatggc tgaatgaccg ataatcggtg 300

cccatgaacg cttcaaatca cgatccgcaa cttgagtcac tgaaggtccc gccgcattcg 360

atcgaggccg agcagtcggt gcttggcggt ttactgctcg acaacggcgc gtgggaccgg 420

attgcggact tcctgggtca ccaggatttc tatcgctttg atcaccggct gatctacgag 480

cacatcggca agctgattgc cacgtcgcgg ccggccgatg tgatcacggt gttcgagtca 540

ttgtccaccg cgggcaaggc ggacgatgtc ggcggccttg cctatctgaa tgcgctcgcg 600

cagaacacgc cgagcgcggc gaacatccgc cggtatgccg agatcgtgcg cgaccgtgca 660

gtgttgcgca agctggtgac cgttgccgac gagattgccg gcgacgcgtt caatccgcag 720

ggcaaggagg tccggacgct gccgacgagg ccg 753

<210> 2

<211> 1543

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggcgtcgatc aagtcgagtc cgtcgtccgc aaggtccttg aggatcgtga acgcagtctc 60

cttgtgggcc atgtcggttg ccagcttgcg agcgaagtga atcaaagtgt tggtgtgcat 120

cctgcgccct gccctcttca cgatgctgta gagttcctcc acctggtcgg cgggcagctt 180

ctgggcaaga agtccaaagc tcctctggct gaacggaaca tgtcccgctg gcgtctcctc 240

gatgaccttg ctcaccagat gcagcacgtc ctctgcctcg attgtccgtt cccttgtgga 300

tttgaagcct tccaggtcca tccgccgcgt gacgaagagc aagacgtcgt gtatcgcata 360

gcctgggggt aaagagtcca aggtagcttc aagcactagc ctggcctttt ctggacagcg 420

gtgcatggtt gaaagcatca ccgacggcca ttttcgctgc cgctcttcca cgctgagtga 480

ctcccaagcc ttgcgcatgg acgccacgga ttcaagctca aacagcatag ctccctcctc 540

acgccacggc caggaagaag ggttgatcgg ctccaagacc ttctgcaact tgaccttcca 600

ggacctaaag gccctcctgg cctgcaccac ctcggatttc ggatactggt gacgaacgta 660

tccccattgc tggtatatga gtcgctgttc cttgagtcgg ctccaagctt ctgccgtcaa 720

gcgaccttgg ccagacaaat cgagctttgc gaaatccttt gccgcaacag gtactcgacg 780

accactgact ctggcctgcc gcggtgctgg ccgtgcccgg cggctgggag actgactccg 840

cccttgccct tcagatttta atcttctgtg accagtgcgg ccatcggcgg ctgcgctcgt 900

cgagtcggta gtgggcgcga tcttgctgcc tcgggatagg tcgggggaat catgttcgcc 960

ataggcaagt cggttcgacg aagttgaagg cgagcgctct ggcgtgcact ccggcgcatt 1020

gcgattgctt gaactttctc gatcctcaag gctcggctct cgcgccgggt gaggcgcgag 1080

gcaatgcctc catcctgtaa agggtcgcaa ctgttgtgct ggagatgccg gtagcttcgg 1140

gacagccaac tgcaaccgca aaaagcccag gcgtgagaaa tgagccgtta tcatctcggg 1200

ggctccactt ccagacagtc ggaggctgcc ttcatcgtgc gaggtcgcgc gggtgctcgg 1260

agagacttgg ggtgtggccg cgaacgttgg ctgtttgaag atcgcaaagg tgagtcggga 1320

cgaaattctg cgccatccat cgtaaattga catcccgcgt tacaggggcc gtcggcgccg 1380

ctctagcccg tacgttaacc gcatccgcgc gccaatctca gcgtccctcc agccactcca 1440

cccatccact gacggcgctg gggagtcgag cctggcagct cgagttttgc cccaccaacg 1500

cgcaaacctc caacctagac cttgatcgac gcccgcggcc gcg 1543

Claims

1. A replicon capable of autonomously replicating in cells of Aspergillus niger, wherein the nucleotide sequence of said replicon is as shown in SEQ ID No. 2.

2. The method of claim 1, comprising the steps of:

1) using AnEp8-AnHyg plasmid as template throughNotI, carrying out enzyme digestion to remove an AMA1 replicon to obtain an AnEp8-AnHyg-deleteAMA1 vector; taking 19 fungus mixed genomes as templates, carrying out DNA fragmentation treatment by a Tn5 library construction kit, carrying out amplification screening on 4-5Kb target fragments by an Altama-F/R primer, and recovering and purifying amplification products; connecting an AnEp 8-AnHyg-deleeAMA 1 linear vector and a target fragment by using an In-fusion method, transforming the linear vector into 10 beta super competent cells, and extracting plasmids for later use;

2) transforming the plasmid obtained in the step 1) into aspergillus niger spores by using an HDEN technology,Hygscreening a positive transformant by the resistance gene, taking the spore of the positive transformant as a template,NotI-F/R primer verification and sanger sequencing to obtain an R75 sequence comprising a partial ribosome large subunit protein L9 and a DNA replication helicase sequence;

3) taking 19 fungal genomes as templates, amplifying R75-F/R primers to obtain a source of an R75 sequence, taking a source of an R75 sequence as a template, amplifying R75flank-F/R to obtain a fragment of R75flank3.405, and connecting the fragment with AnEp 8-AnHyg-deleeAMA 1 in the step 1) to obtain an AnEp8-R75flank3.405 plasmid.