CN117568349A - Fungal promoter element P22 and application thereof - Google Patents
Fungal promoter element P22 and application thereof Download PDFInfo
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- CN117568349A CN117568349A CN202410051957.2A CN202410051957A CN117568349A CN 117568349 A CN117568349 A CN 117568349A CN 202410051957 A CN202410051957 A CN 202410051957A CN 117568349 A CN117568349 A CN 117568349A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The disclosure relates to the technical field of genetic engineering, in particular to a fungal-source promoter element P22 and application thereof. The method aims at obtaining a promoter element suitable for industrial expansion fermentation culture, and compared with the difference of expression of yeast transcriptome genes under different fermentation conditions, the method screens out genes with obviously increased transcription levels under the fermentation conditions easier to amplify and apply, determines the promoter element of the genes, then surveys and uses the screened promoter element to express exogenous proteins, compares the expression quantity with the existing high-yield promoter element, selects the promoter element with obviously increased exogenous protein expression, and finally proves that the promoter element obtained by the method comprises the nucleotide sequence SEQ ID No:1, and the expression quantity of the exogenous protein is higher, especially in the Kluyveromyces expression system, the expression quantity of the green fluorescent protein and beta-lactoglobulin is obviously higher than that of the inulinase promoter.
Description
Technical Field
The disclosure relates to the technical field of genetic engineering, in particular to a fungal-source promoter element P22 and application thereof.
Background
Promoters are a type of biological element that determines the transcription initiation site and controls the expression intensity of genes, and are one of the cores of gene expression systems. The control of the expression of different genes is realized by the strength of the promoter, which is a common biological regulation means. The promoter library composed of promoters with different intensities can well meet the requirements of different gene expression intensities.
Yeast (Yeast) is a good choice of protein expression host. The yeast cells have the advantages of simple fermentation, rapid growth, cheap fermentation medium, easy genetic operation, protein post-translational modification system, secretion expression of protein and simple purification process. Among them, pichia pastoris (hereinafter abbreviated PP), kluyveromyces lactis (hereinafter abbreviated KL) kluyveromyces pichia pastoris (hereinafter abbreviated KL) and kluyveromyces marxianus (hereinafter abbreviated KM) have been advantageous host cells. Wherein KM is a co-compound semi-ascomycete yeast, located in Kluyveromyces of Saccharomyces. KM is commonly found in yogurt, mare's wine, cheese, milk, and sugarcane leaves. KM is a commonly accepted food grade yeast. It has passed the GRAS authentication of FDA, the QPS authentication of European Union, and is approved by the national institutes of health as a new food raw material. Because of its high safety, KM has an unique advantage in the field of production of edible, feed and medicinal proteins. However, for the expression of foreign proteins, both PP, KL and KM currently lack efficient expression elements and supporting expression vectors, limiting the development of yeast expression systems.
Disclosure of Invention
The purpose of the present disclosure is to provide a promoter element derived from fungi and having a strong promoter activity, and to construct an exogenous protein expression cassette by using the promoter element and to introduce the exogenous protein expression cassette into a yeast expression system, so that the expression strength of exogenous genes can be improved, and the yield of exogenous proteins can be improved, thereby having industrial application potential.
In a first aspect of the present disclosure, there is provided a fungal source promoter element comprising a sequence identical to SEQ ID No:1, including but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, respectively.
In a second aspect of the present disclosure, there is provided a transcriptional expression cassette comprising a promoter element according to the first aspect of the present disclosure, and a coding gene operably linked to the promoter element.
In a third aspect of the present disclosure, there is provided a recombinant expression vector comprising a promoter element as described in the first aspect of the present disclosure, or a transcriptional expression cassette as described in the second aspect of the present disclosure.
In a fourth aspect of the present disclosure, there is provided a recombinant cell comprising a promoter element as described in the first aspect of the present disclosure, or a transcriptional expression cassette as described in the second aspect of the present disclosure, or a recombinant expression vector as described in the third aspect of the present disclosure.
In alternative embodiments, the host cell of the recombinant cell is a yeast cell selected from kluyveromyces marxianus (kluyveromyces marxianus), kluyveromyces lactis (kluyveromyces), pichia kluyveromyces (pichia luyveri) or pichia pastoris (pichia pastoris).
In a fifth aspect of the present disclosure there is provided the use of at least one of the promoter element according to the first aspect of the present disclosure, the transcription expression cassette according to the second aspect, the recombinant expression vector according to the third aspect, the recombinant cell according to the fourth aspect in (a) and/or (b):
(a) Enhancing the transcription level of the encoding gene, or preparing a reagent or a kit for enhancing the transcription level of the encoding gene;
(b) Preparing exogenous protein, or preparing reagent or kit for preparing exogenous protein.
In an alternative embodiment, the exogenous protein comprises a milk protein including at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin, or osteopontin.
In a sixth aspect of the present disclosure, there is provided a method of enhancing transcription of a gene encoding a foreign protein, the method comprising operably linking a promoter element of the first aspect of the present disclosure to a gene encoding a foreign protein and then introducing into a host cell.
In alternative embodiments, the exogenous protein comprises at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin, or osteopontin.
In alternative embodiments, the host cell is selected from kluyveromyces marxianus (kluyveromyces marxianus), kluyveromyces lactis (kluyveromyces), pichia kluyveromyces (pichia pastoris) or pichia pastoris.
In a seventh aspect of the present disclosure, there is provided a method for producing milk protein, comprising culturing the recombinant cell of the fourth aspect of the present disclosure, and isolating and collecting milk protein in the fermentation broth; wherein the recombinant cell comprises a promoter element according to the first aspect of the present disclosure operably linked to a milk protein-encoding gene.
In an alternative embodiment, the milk protein comprises at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin, or osteopontin.
The beneficial effects that this disclosure obtained are:
the method aims at obtaining a promoter element suitable for industrial expansion fermentation culture, and compared with the difference of expression of yeast transcriptome genes under different fermentation conditions, the method screens out genes with remarkably increased expression levels under the fermentation conditions easier to popularize, determines the promoter element of the genes, then inspects the promoter element screened out for exogenous protein expression, compares the expression levels with the existing high-yield promoter element, selects the promoter element with remarkably increased exogenous protein expression from the promoter element, and finally proves that the promoter element obtained by the method comprises the nucleotide sequence SEQ ID No:1, and especially in KM expression system, the expression amount of green fluorescent protein and beta-lactoglobulin is obviously higher than that of inulase promoter.
Drawings
In order to more clearly illustrate the embodiments of the present disclosure or the prior art, the drawings that are required in the detailed description or the prior art will be briefly described, it will be apparent that the drawings in the following description are some embodiments of the present disclosure, and other drawings may be obtained according to the drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 shows the results of the cell growth and sugar consumption tests of 4 fermentors in example 1;
FIG. 2 shows the results of the growth and yield measurements of 4 fermentor cells in example 1;
FIG. 3 shows the extraction results of mRNA of each sample in example 2;
FIG. 4 shows the promoters P22 and P in example 2 INU Comparison of protein amount expressing EGFP;
FIG. 5 shows the promoters P22 and P in example 2 INU Fluorescence intensity contrast expressing EGFP;
FIG. 6 shows the plasmids pHKD-P22-. Beta.LG and pHKD-P of example 3 INU Positive validation of the βlg transformed KM cells;
FIG. 7 is a diagram ofPromoters P22 and P in example 3 INU Comparison of the amount of protein expressing βlg.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present disclosure, and it is apparent that the described embodiments are some embodiments of the present disclosure, but not all embodiments.
Terms and definitions of (I)
The terms "a" and "an" when used in conjunction with the term "comprising" in the claims and/or specification, may also mean "one or more", "at least one", and "one or more". As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.
The term "promoter" as used in this disclosure refers to a nucleic acid molecule, typically located upstream of the coding sequence of the gene of interest, which provides a recognition site for RNA polymerase and is located 5' upstream of the transcription initiation site of mRNA. It is a nucleic acid sequence that is not translated, and RNA polymerase, when bound to this nucleic acid sequence, initiates transcription of the gene of interest. In ribonucleic acid (RNA) synthesis, a promoter can interact with a transcription factor that regulates gene transcription, controlling the start time and extent of gene expression (transcription), including core promoter and regulatory regions, like "on-off", to determine the activity of the gene and thus which protein the cell begins to produce. The polynucleotide sequence of the promoter of the present disclosure contains SEQ ID No:1, or a nucleotide sequence having an identity of 80% or more, preferably at least 81% or more, preferably at least 82% or more, preferably at least 83% or more, preferably at least 84% or more, preferably at least 85% or more, preferably at least 86% or more, preferably at least 87% or more, preferably at least 88% or more, preferably at least 89% or more, preferably at least 90% or more, preferably at least 91% or more, preferably at least 92% or more, preferably at least 93% or more, preferably at least 94% or more, preferably at least 95% or more, preferably at least 96% or more, preferably at least 97% or more, preferably at least 98% or preferably at least 99% or more.
The SEQ ID No:1 is as follows: GGACAACGATACTGCCTTGCTGGGGGCCTTGGCCTTGGCCCCTGAATCATTGCCCTCTGCCTTGCCGCCTGGCAAGGCAGAGGGAGAAACCAGTACCACCTCAAACGGACACTGCTGGTCAAACGTTTCCAGCTCATCGCTGGACACATCCTGGAACGTGTCTCCCGTGAACACCACCACCCGCACGCGCATCTTGCGCTTACCGACAAACTCCTTGAGCTGGAGTACCAACACTAGCAGTGCATTAAACAGCGATGAGGAGGCGTCCGTCTCCTCATCTATCTCCTCACCTTCCTCATCCTTGGGGTTTCTAAAGTCCCCAGATCTCTCCAAATGTCTAGAAACCCTCCCCACCCACTCTCCAACATCCTTGACGCTTATCAATGGCCTTGGTTGCGGGCCCGCGAGCATCGCCATGTTCACGGGCAGCGCGGGGTCGCGGTCTGGGTCGTTTGAGCTGAGGTTTGCGAGCGCGACCTGGACGTAGTCTGTCTTTCTTTGGGCATGGGCCTTGTTGAGAAGGGTGTACTCGAGGTAGGAGAGGCATTGGCTGTTGCCTGCTGCCACGTCGATTAGAAACGCTGTGAGCTGGGACATGGCTTTGTTCTGGGGGTCTTTTCACTGTCTCCTGTCTCCTTGTTCTGGGGTCTCTCTTTTCTTGGAGAAGCTGATGCTGTAATCAGTAGGCCAGGCCTGGCCTGGCCTTAATTAAGGTGGTTATCTAGTGGTATTAATAAGGAAAGGCCAGGGGTATCCCTTGGGCCATCCCAGGGGTATTGTTGCCCCTTGTCAATGACTAAGACAAGGGGTGCTCCAGGCATCCCTTGTTTATCCCCTGCCTTTCCCCTGGGTATTCCTGGCAAGGCCGATGTATTATCTAGAACCTTATATAAAGACAAAGCTGAGGCCTTGACCTCAGCTTGGGATGGTTAGACCTTCCCTCTCCTATTGGCAGTCGCATGCAGCAAGCTGCACACGACTGCACACGACTACTAATACA.
The term "polynucleotide" of the present disclosure refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments or may be an integral part of a larger nucleotide sequence structure, derived from nucleotide sequences that are separated at least once in number or concentration, and capable of identifying, manipulating and recovering sequences and their constituent nucleotide sequences by standard molecular biological methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
The term "expression" of the present disclosure includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "transcriptional expression cassette" of the present disclosure refers to a class of expression elements comprising a transcriptional regulatory element and a target gene, with which expression of the target gene is regulated. In the present disclosure, the transcription regulatory element includes a promoter, and may further include an enhancer, a silencer, an insulator, and the like. In the present disclosure, the target gene is specifically a protein-encoding gene. By "operably linked" a target gene to a polynucleotide is meant that the polynucleotide having promoter activity is functionally linked to the target gene to initiate and mediate transcription of the target gene in any manner described by one of skill in the art.
The term "gene of interest" of the present disclosure relates to a gene of any one of which is linked to a polynucleotide having a promoter activity in the present disclosure to regulate its transcription level. In some embodiments, the target gene refers to a gene encoding a protein of interest in a microorganism. Illustratively, the target gene is a gene encoding an enzyme associated with biosynthesis of the target compound, a gene encoding an enzyme associated with reducing power, a gene encoding an enzyme associated with glycolysis or TCA cycle, or a gene encoding an enzyme associated with release of the target compound, or the like.
The term "expression vector" of the present disclosure refers to bacterial plasmids, phages, yeast plasmids, viruses or other vectors well known in the art. The plasmid may be a linear or closed circular plasmid. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. Expression vectors generally include (from 5 'to 3') orientation: promoters directing transcription of genes of interest and genes of interest. The recombinant vector may further comprise, if necessary: downstream of the promoter a multiple cloning site or at least one cleavage site, a ribosome binding site for translation initiation, a3 '-transcription terminator, a 3' -polynucleotide signal, other untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, a resistance selection marker, an enhancer or an operator. The gene of interest is ligated into the appropriate multiple cloning site or cleavage site, thereby operably linking the gene of interest to a promoter. Ribosome binding sites, 3' -polynucleotide acidification signals, other untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers, enhancers or operators, which are commonly used in the art for various translation initiation, can be used in the present disclosure.
The vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may comprise any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids, or transposons, may be used which together comprise the total DNA to be introduced into the host cell genome. The vectors of the present disclosure preferably comprise one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. Selectable markers are genes, the products of which provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophs, and the like. The vectors of the present disclosure preferably comprise elements that allow the integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome. The expression vector may contain more than one copy of the gene of interest to increase the yield of the gene product. The increase in copy number of the gene of interest can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene and the gene of interest, wherein cells comprising amplified copies of the selectable marker gene and thereby comprising additional copies of the gene of interest can be screened by culturing the cells in the presence of an appropriate selection agent. Methods for preparing recombinant expression vectors are well known to those skilled in the art and include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
The term "identity" of the present disclosure refers to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or by deleting nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or by deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
The term "host cell" as used in the present disclosure has the meaning commonly understood by one of ordinary skill in the art, i.e., a cell into which the present disclosure promoter-mediated expression cassette can be introduced, which is referred to as a recombinant host cell after introduction. In other words, the present disclosure may utilize any host cell as long as the cell contains the promoter sequence of the present disclosure therein. The host cell of the present disclosure may be a yeast cell, including but not limited to any of the strains kluyveromyces marxianus (kluyveromyces marxianus), kluyveromyces lactis (kluyveromyces), pichia kluyveromyces (pichia pastoris) or pichia pastoris, and mutants or strains of the enzyme preparations and organic acids produced from the above yeasts.
The "recombinant host cell" described in the present disclosure is in particular achieved, for example, by transformation. "transformation" here has the meaning generally understood by those skilled in the art, i.e.the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl) 2 ) Precipitation, microinjection, polyethylene glycol (PEG), DEAE dextran, cationic liposome, and lithium acetate DMSO.
In the present disclosure, the cultivation of the host cells may be performed according to conventional methods in the art, including, but not limited to, well plate cultivation, shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, and the like, and various cultivation conditions such as temperature, time, and pH value of the medium, and the like, may be appropriately adjusted according to actual situations.
Strains for use in the present disclosure include: kluyveromyces marxianus CJ3113, deposited with China center for type culture Collection with a deposit number of CCTCC No: m20211265, the preservation date is 2021, 10 and 13, the preservation address is university of Wuhan in Wuhan, china, and the classification name is Kluyveromyces marxianus CJ3113.
Some embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
The experimental techniques and methods used in the examples of the present disclosure are conventional techniques unless otherwise specified, such as those not noted in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.
The composition of the medium used was as follows:
LB: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, and solid culture medium with 2% agar powder.
YPD: yeast extract 10g/L, tryptone 20g/L, D-glucose 20g/L, and 2% agar powder added into the solid culture medium.
YD: yeast extract 20g/L, D-glucose 40g/L, pH 5.6.
Example 1
In this example, K.marxianius CJ3113 strain was cultured in a fermenter under different conditions, and then the transcript level of the genome under different conditions was examined by transcriptome analysis.
1.1 The experimental group settings are shown in the following table, with a fermenter volume of 5L, wherein the conventional process is: referring to the fermentation process in example 1 of the patent (application number: CN202111359573. X), the results of cell growth and sugar consumption detection are shown in FIG. 1, and the results of cell growth and yield detection are shown in FIG. 2.
Table 1: design of experiment
1.2 According to the fermentation test results of FIG. 1, samples after 12 hours in each tank were selected for transcriptome analysis, the sample numbers are shown in the following table, wherein A1, A2, A3 and A4 represent 1 to 4# tanks, respectively, and 12, 20 and 26 represent the time points (h) of sampling:
1.3 Transcriptome analysis
(1) RNA preparation
The RNA extraction was performed on each of the above groups of samples according to the instructions using a yeast RNA extraction kit (alkaline method) (Shanghai organism, NE 0251), and the extraction results are shown in Table 2 and FIG. 3. It can be seen that the prepared RNA has better quality and meets the preparation requirement of eukaryotic non-strand specific transcriptome library.
Table 2: preparation of eukaryotic non-strand specific transcriptome library to prepare a summary of RNA detection results
RIN value description: RIN values are quantitative values that reflect RNA integrity, automatically generated by 2100 BioAnalyzer, with greater values providing better integrity. However, some samples cannot generate RIN values due to species or purity, and are not qualified, and such samples can be judged to be qualified or not according to the experience accumulation of a company.
(2) Analysis of expression level
The Read Count value was aligned to each gene using HTSeq statistics as the original expression level of the gene. Reads counts correlated positively with the true expression level of the gene, as well as the length of the gene and sequencing depth. In order to make the gene expression levels of different genes and different samples comparable, FPKM is used for normalizing (normalization) the expression level, FPKM (Fragments Per Kilo bases per Million Fragments) is the number of Fragments from a certain gene per kilobase length in each million Fragments, two Reads are generated for each fragment for Pair-End sequencing, and the FPKM only calculates the number of Fragments of which two Reads can be aligned to the same transcript. Among the transcriptomes of the present invention, the gene FPKM > 1 is considered to be expressed. This threshold is recommended by the mainstream journal and also can well reflect the expression level of the gene. The formula of the FPKM is as follows:
inulase promoter (P) INU ) Has stronger expression activity and is often used for constructing the expression of Kluyveromyces marxianus protein. Inulase promoter (P) INU ) The nucleotide sequence of (2) is shown as SEQ ID No:2 is shown as follows:
CAAACACAAACACAAACACAAAAACGCTAAATTATGCACACAAGGGCCGGCGGGGCTGCCGGAAAAAAAAAGGGAAAAATACACAGACGAGCGCGCACAGATGGGGTTACCACTGCAAGTTACAAGTTGCAAGTTGCACGCTGGAATCAGAATTGGAATCAGAATTGGAATTGGAATTAGAATTAGAATTAAACTTGGGGTAGCCACGGGAACGGGATAACTCAGGAATCGCTCGCAGGCGTCTCCGTCTAGGCAATCCCAAGGTAAGCCTAGGCACTCCCACAGGGGAAAGAACGGTTGAAGGCAAAGTAGTGCTAACAATTGGTAACGAATGGTAACAAGTGTGTCCGTCTCCACCTGACATTTGCTAGAGCTGGGGATTCCACATTCTTGTGCTCTGAATTCTCAAACCGAAATGGGGCGTTGTTACCCCAGGTATCCGGTTGTAGTTGGCACTGGGGATGGAAAAAAATGATGTTGATGTTGAGTTAGTTGGGTTGAGTCAATTAGTGCGTGAAAGTATCACCACTTTTGTCATCCGGCGTTTCTGTGCGAATCACACACACACACACAGTTTATTGGAGCACTTGTTTCTGGCGTATTCGTAATTGTTCTGCGGTGCGGTTCTGTGTGCATTTTTCCTGGGGTGTCTGCCGCACCTACTCATCACCCACGCCGTGGGTTTGAGCCATGGCGGAGGTACGACTGACTGGCTGCCTGCCTGCCTGACTGACTGCCTGACTGCAGGAAAAGAGGGTTTCGAAGGAAAAACTTTTCCTGTGTAAATCCGGCCGTGCGCCGCTGCTCCAAAATCCACCTTCATGAGAAGGAGTTTGAAAAAACAAAAAAATTCACATATAAAAAGCGTATCTCGAGATCTCAAAGTCTCCCTTGAATCGTGTTTGCCAGTTGTAACTCATCCTTTATTCTTCTATTCTATCTCTCTCTTTCCTTCCCCTAATCAGCAATTAAATCCGGGGTAAGGAAGAATTACTACTGTGTGTAACGGTTATATTTCGGTTAGAT。
with P INU The transcriptional activity FPKM was used as a reference, and the analysis of the transcriptional data table 3 revealed that the gene encoding the stationary phase Spg4 was found at 20 hours and 26 hours, compared to P INU The transcription activities of the fermentation tubes are obviously improved by 2 times, 25.6 times, 0.6 times and 5.4 times respectively in 20 hours compared with the fermentation tubes; the increase was 88.7 times, 19.4 times, 27.5 times and 24.4 times, respectively, at 26 hours. The promoter of Spg4 in Kluyveromyces marxianus was defined as P22.
Table 3: differential analysis of Gene expression
EXAMPLE 2 intensity analysis of EGFP expressed by P22 promoter
2.1 Preparation of recombinant plasmid vector pHKD-noP-EGFP
The recombinant plasmid vector pHKD-noP-EGFP is obtained by replacing a URA3 gene on a pUKD plasmid with a hygromycin resistance gene (Hyg), and the specific preparation process is as follows: using a synthetic hygromycin resistance Gene (Hyg, gene ID:9697146 in NCBI database) as a template, hygromycin resistance Gene fragments were amplified using primer pairs Hyg-F and Hyg-R. pUKD plasmid ((SEQ ID No: 20) XX) is used as a template, pUKD-F and pUKD-R are used for amplification to obtain a pUKD plasmid skeleton, GJ-F and GJ-R are used for amplification to obtain a fragment A, and EGFP-F and EGFP-R are used for amplification to obtain a fluorescent protein gene fragment. And then connecting the obtained pUKD plasmid skeleton, fragment A and fluorescent protein gene fragment to obtain a recombinant vector plasmid pUKD-noP-EGFP. And then, carrying out enzyme digestion on the recombinant vector plasmid, and connecting the recombinant vector plasmid with the hygromycin resistance gene fragment to obtain the recombinant vector plasmid pHKD-noP-EGFP, wherein the specific primer sequences are shown in the following table.
Table 4: primers for P22 promoter strength analysis
SEQ ID No:20 is as follows:
GGGGATGATCCACTAGTCAAACACAAACACAAACACAAAAACGCTAAATTATGCACACAAGGGCCGGCGGGGCTGCCGGAAAAAAAAAGGGAAAAATACACAGACGAGCGCGCACAGATGGGGTTACCACTGCAAGTTACAAGTTGCAAGTTGCACGCTGGAATCAGAATTGGAATCAGAATTGGAATTGGAATTAGAATTAGAATTAAACTTGGGGTAGCCACGGGAACGGGATAACTCAGGAATCGCTCGCAGGCGTCTCCGTCTAGGCAATCCCAAGGTAAGCCTAGGCACTCCCACAGGGGAAAGAACGGTTGAAGGCAAAGTAGTGCTAACAATTGGTAACGAATGGTAACAAGTGTGTCCGTCTCCACCTGACATTTGCTAGAGCTGGGGATTCCACATTCTTGTGCTCTGAATTCTCAAACCGAAATGGGGCGTTGTTACCCCAGGTATCCGGTTGTAGTTGGCACTGGGGATGGAAAAAAATGATGTTGATGTTGAGTTAGTTGGGTTGAGTCAATTAGTGCGTGAAAGTATCACCACTTTTGTCATCCGGCGTTTCTGTGCGAATCACACACACACACACAGTTTATTGGAGCACTTGTTTCTGGCGTATTCGTAATTGTTCTGCGGTGCGGTTCTGTGTGCATTTTTCCTGGGGTGTCTGCCGCACCTACTCATCACCCACGCCGTGGGTTTGAGCCATGGCGGAGGTACGACTGACTGGCTGCCTGCCTGCCTGACTGACTGCCTGACTGCAGGAAAAGAGGGTTTCGAAGGAAAAACTTTTCCTGTGTAAATCCGGCCGTGCGCCGCTGCTCCAAAATCCACCTTCATGAGAAGGAGTTTGAAAAAACAAAAAAATTCACATATAAAAAGCGTATCTCGAGATCTCAAAGTCTCCCTTGAATCGTGTTTGCCAGTTGTAACTCATCCTTTATTCTTCTATTCTATCTCTCTCTTTCCTTCCCCTAATCAGCAATTAAATCCGGGGTAAGGAAGAATTACTACTGTGTGTAACGGTTATATTTCGGTTAGATATGAAGTTAGCATACTCCCTCTTGCTTCTATTGGCAGGAGTCAGTGCTTCAGTTATCAATTACGAGAAGAGAGAGGCTGAAGCTTTGATCGTTACCCAAACTATGAAGGGTTTGGACATTCAAAAGGTTGCTGGTACTTGGTACTCTTTGGCTATGGCTGCTTCTGACATCTCCTTGTTGGATGCTCAATCCGCTCCATTGAGAGTTTACGTTGAAGAATTGAAGCCAACCCCAGAGGGTGACTTGGAAATTTTGTTGCAAAAGTGGGAAAACGGTGAATGTGCTCAAAAGAAGATCATTGCTGAAAAGACCAAGATCCCAGCTGTTTTCAAGATTGATGCTTTGAACGAAAACAAGGTTTTGGTTTTGGACACTGATTACAAGAAGTACTTGTTGTTCTGTATGGAAAACTCTGCTGAACCAGAACAATCCTTGGCTTGTCAATGTTTGGTTAGAACCCCAGAAGTTGACGATGAAGCTTTGGAAAAGTTCGATAAGGCTTTGAAGGCTTTGCCAATGCACATCAGATTGTCTTTCAACCCAACTCAATTGGAAGAACAATGTCACATTTGAGTTTAAACCCGCTGATCCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGCCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCCCTTCCCTTTGCAAATAGTCCTCTTCCAACAATAATAATGTCAGATCCTGTAGAGACCACATCATCCACGGTTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAAACCCACACCGGGTGTCATAATCAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCCCACCACACCGTGTGCATTCGTAATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGAGTACTGCAATTTGACTGTATTACCAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAAATTGTACTTGGCGGATAATGCCTTTAGCGGCTTAACTGTGCCCTCCATGGAAAAATCAGTCAAGATATCCACATGTGTTTTTAGTAAACAAATTTTGGGACCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAACATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTGCATGATATTAAATAGCTTGGCAGCAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATATGTAGCTTTCGACATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAGCTAGCGAATTCTCTCTTGGGGCTGAAGTGAAATTTAAAAAAGTCGCTTGAGGCTCAGCCGGAATTATAAAACATCACCTGAGTCTTGAGAGCGCTTTCACTCACCTGAGGCTCAGCTGAAATTTCAAAAAGTCACTTGAGCCCAGAAGGAGTGTTTCACCCCCTGAGGCTATAACGTTCGTTATTTTAATACCTAAATAAACAAAAATATATGGTACAGGAACGCGAGGCAACGCGCCGATACAGGGTCAATGGGTACACGAGAGGGTGACACTAGGCGTAGAAAGTCATTAGTATAAAATACAGTGGTATATAGTAGATATTTAGTTTGTTTTCCTTTTCTTTTTCTCCAAACGATATCAGACATTTGTCTGATAATGAAGCATTATCAGACAAATGTCTGATATCGTTTTTCAATAATAATATACATCATCACAAAACAAACAAACATAGCATCGCAAGCCCCATCATGCCACCACCGTCCGCTGTGATCGCAACTCATGTTTCCGGCGGTATTCTGCAATGAATTGGAGAACCTCGTCTGAGATAATTCCATGCCATTGTTCGAACAACTGGAGGCTAGGATGAGCTGAGAAGGATTGAGCGACCAAGCGCGGACTTGACGGTGGGCTGAGTGGTGGGCTACCAAGGCTGTTACCCTCCTCTTCAAGTAGCTCCTCGCGAGATAAAGGTTTATTAGAAGGATCCTTCAAAACATATATTTCACTGCCCAATGGGGCTTCCTTGTAAAAACCTGATATAAAGGCAAATACACGGTCATCTACAGTCACACTACCATGACTGTAGTGTGATCTAGCCACTGCACTTTGAATTTCACGGTCCCCAGCCCAATTGCCCAGTGAGGAGACATATTTTCCCTTCTCAGATCTCGATAAAAAGGTCGCCATTAAATGTCTTCCAAAATGAGACTTCGGGCCGTTCCAGATCTTGAAGACTGGTTCATCGACATGCTGGGTAAGAAACCTAGAGAACGTTCTGGCCAATGACTCTGGTAAAAACTGATGAGTTTGATTAGTTGGTCTATTACTGGATACTGTTTTTTCAATAGGCGAGCAAACACGCAAATAGTCGTATAATGATATCAGAAGATCGCAATCACCATTCACAGGATAGAAGTTAACGTACCGTTCAGTTCTGCTTTTCGTTTCTGTCACAGTAGCACGCACAATTGGGCCCAGAAATGAATTGTTGTAGATCTCAAAAGTCCTTGGATCTAGATTCTTCAGATCGCTGTATCTGCAGCAATTTCCAACAGCTCCCAGAAGTAGCAATCGGTATTCCGCTCGTTTTGTAGTGGTTACGCAGGACTGATCGAAGAAGCAGGCAATCCTGGAGACAATCTTCCAAATATCTTTTTCTTTTGACAGAATATTAGTGAATTGTAATCCAACCATAGAAGCATCGTATTTATGTGTTTCCTCGTAGCGATCAAACAAGGAAACTTCTTGATTTTTAAATGGGCTAACAACAACCTTGTAAGAAGGCAATGCTGATTCGATATCCTTTTGCAGAGACTCTGTCTTTCTTAGTCTAACAGTGAATTTGATAATTTTGTCATCCTTATCAAAAGACAGAGATTTGCCAATTGCGCTCTTGTAAGAGCGGTAGGTATTGATTTTCATCTCGCGTCGGATAGATAGCGACTGCATTGTCAAGATAGAGAATAGGGACGCCAGCTTATTTCTAATTTCTTTCGCATTTATATTAAAGGTGTCAGATTCCAGAATTTCATTAATTTCATTAGCACACTGATGAGGTGTGAGGTGAGCAGCCTCCGCAAAGGTAGACATAGGGGCATTGGTTGGAGGCCTTTGAGGTACCACTAGAGTGCTGCAAACATAGCACCGTTCGAGACTTTAAAATCTTCAGTTTTAAAATTATGAAAAAAAACATCGTCCTGAGTTGAAACGGTCGTTTCAACCTCCGTGTACAGAAAGATACATAGCATATGGCAAGCTGCACGCAGCGTAAACATGCCGGACAACTGTCATTTCGTCAGATCAGTTGATCTACTCTCTGTGATACTGCTTCGTTTGTCCACGGAGGTCGGACTAACTCTCACCACGCTTCCACGGCATTCGAAAGAACTAATATTGTATCATTGTACATATGAGGAACACGCAGTTGAACTGAGCAAACCAGGACTCAGGAAAGCAGGAGGTAAGTGCTCGCTTTTCGTGGATCCAGAGGAACGTGAAAATTCGCCTTCTCCTCCTATACCGCCGTATCAGATATCAGAGATGCCCCTTCATGAACTTCTCGAGTCAGGCAATGCTAAATTGGTTCCAAATCCCGAGTTTGATCTAACTGATCCAGACGACTTTCATAAGTGTTTCTCGGTCACCTATTCAGCATTATCTTTAATGGTACCATATCTGCCCAGAGCTGCTCTAAAGGCTGCTCGAGTGTTTTGTAAAGATCATTCAATATTAACAACGGATATGCTTGATTTGAATTATCTTGAAGAGCTAATTGAGTTCTCAAAGGAAACTGTGAACAAAATCCCAGCTAGAATCCCTATAGAGGACATGCTTCTCGAGCGGGGATATGTGCTACCATGGGTTCATGGTGGTACAGTGAAGGGAGGAAAGCTACTGACCCCCAACGATTGATTCTTTACCGAATCATTGCATAATTCATTGCATAATTCATTGCAGAATACCGCCGGAAACATGAGTTGCGATCACAGCGGACGGTGGTGGCATGATGGGGCTTGCGATGCTATGTTTGTTTGTTTTGTGATGATGTATATTATTATTGAAAAACGATATCAGACATTTGTCTGATAATGCTTCATTATCAGACAAATGTCTGATATCGTTTGGAGAAAAAGAAAAGGAAAACAAACTAAATATCTACTATATACCACTGTATTTTATACTAATGACTTTCTACGCCTAGTGTCACCCTCTCGTGTACCCATTGACCCTGTATCGGCGCGTTGCCTCGCGTTCCTGTACCATATATTTTTGTTTATTTAGGTATTAAAATTTACTTTCCTCATACAAATATTAAATTCACCAAACTTCTCAAAAACTAATTATTCGTAGTTACAAACTCTATTTTACAATCACGTTTATTCAACCATTCTACATCCAATAACCAAAATGCCCATGTACCTCTCAGCGAAGTCCAACGGTACTGTCCAATATTCTCATTAAATAGTCTTTCATCTATATATCAGAAGGTAATTATAATTAGAGATTTCGAATCATTACCGTGCCGATTCGCACGCTGCAACGGCATGCATCACTAATGAAAAGCATACGACGCCTGCGTCTGACATGCACTCATTCTGAAGAAGATTCTGGGCGCGTTTCGTTCTCGTTTTCCTCTGTATATTGTACTCTGGTGGACAATTTGAACATAACGTCTTTCACCTCGCCATTCTCAATAATGGGTTCCAATTCTATCCAGGTAGCGGTTAATTGACGGTGCTTAAGCCGTATGCTCACTCTAACGCTACCGTTGTCCAAACAACGGACCCCTTTGTGACGGGTGTAAGACCCATCATGAAGTAAAACATCTCTAACGGTATGGAAAAGAGTGGTACGGTCAAGTTTCCTGGCACGAGTCAATTTTCCCTCTTCGTGTAGATCAGAGGCTATATACATGCCGAGGTATTCGATCACTCTACGATGACGGTCTGTTAGCTCAACAACTTCTTCTAAATGCTCCATAACCGTAACGTAAGAAGCATAACTGTCAATACTGAAGTCATCCCAGTTTATTGGTGCTCCTGTTGAACAGTCATCCACTATATGTTCGAATAGCCCAGGATCACGAGGAGGTCCTACAAACGGATACGGTACAGTCTTCTTTTTATAGTCTGCAAATTCTAGAATAGCATTTTTTATCCAATAGTGTCGAATCGTCCTAGCCGTTCTACCGATAAAGGATCCAATGTGATTATTAGCTCCACTACACGATATGTTAAGTTTGATCGATGTCTTGTTAACAAACGCTAAACTCAAGTTCGGCATTTCCAACAGCGAGAAGAAATCATCAATTCCATCGGCTATCTCTTGATAAGTCATTAGATCATATACCTTCTCGGGATGTCGTTGAGTTACTTTATGACTAGAAATCTTCAGGTTATCATCAACGTAATTGTTCTCCAATAGCTCTGGAGAGGGACATAACAATACTTTGATTTTTTCCATGGCCTGGACTTGTTTCCGTAGGAAATACTTGTTCTTTTGTAGACGTTCCATGATGAGTTTGTATACCTCTGCTGGAGATATCCATTCTAGATCTTTGATATAAGTTTGGTATGGTAAAGAGTTGATTTTGTAGGACACGTAAATCTGCGCTAGATAAGTACATTGTGCAAATGCCTCTGGTACTTCGTAAGACCCATGCTGCGTAATTATAGTATTATTGAGTGGATCATAAGCGTTGTACTCGTTTTTGAATTTAAAACTGTCTAATAAGGCCCTGTAAATCTCTCTGACTTGTTGTACACCTTTCTGCTCTTCGGGACTGAGATCGGATAGCAATGGAGCAGCAGTTTCTGAGCTTTCTGATGGGGCTGACATGGCAGATGCCTATTCAATGCTGCCTTTTGTTTGGGAGGTTATGAAATGCATCTGTTTACATTGTATGTAATACCCTTACTAGGCAATGTTATAAGCAAAAATCCTTTGATCACATGGAATATCACTTTATACGTGTTGAAATATGCAAAAAAACAGTCCCCCTGAGCTCAGGGGGTGGTTTACGCTTTTGAGGCTCAGCAGCGCGGAAGCTCCACCCCGGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACGAATAGCCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTCCAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAAGGGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCAGTAAATCGGAAGGGTAAACGGATGCCCCCATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGGGCTAGGGCGGTGGGAAGTGTAGGGGTCACGCTGGGCGTAACCACCACACCCGCCGCGCTTAATGGGGCGCTACAGGGCGCGT。
2.2 Promoter PCR
Amplification primers were designed for the Spg4 promoter obtained in example 1, as shown by P22-F and P22-R in Table 4. Using the K.marxinus CJ3113 genome as a template, the Spg4 promoter was amplified using the above primer pairs P22-F and P22-R according to the PCR system and PCR program shown in the following Table, to obtain an Spg4 promoter amplification product.
Meanwhile, amplification primers were designed for the Inu promoter, as shown in P in Table 4 INU -F and P INU -R. Using the K.marxinus CJ3113 genome as a template, the primer set P was used INU -F and P INU And R, amplifying the Inu gene according to a PCR system and a PCR program of the following table to obtain an Inu gene amplification product.
Table 5: PCR System of promoter Gene (50. Mu.L)
Table 6: PCR program for promoter Gene
And the PCR product is cleaned and recovered after the correctness of the PCR product is verified by agarose gel electrophoresis.
2.3 cleavage ligation and transformation
The recombinant vector plasmid pHKD-noP-EGFP obtained in the step 2.1 is subjected to enzyme digestion according to the following table, and then is respectively connected with the Spg4 gene amplification product and the Inu gene amplification product obtained in the step 2.2 in a seamless manner to obtain recombinant vectors pHKD-P22-EGFP and pHKD-P INU -EGFP。
Table 7: enzyme cutting system (50 mu L)
The enzyme digestion system in Table 7 was used for the reaction at 37℃for 30min, and the reaction was recovered.
Table 8: seamless cloning system (20. Mu.L)
The seamless cloning system configuration in Table 8 was followed, and the reaction was carried out at 37℃for 30min, cooled to 4℃or immediately cooled on ice. mu.L of the ligation product was added to DH 5. Alpha. Competent cells, and ice-incubated for 30min. And (5) carrying out heat shock in a water bath at the temperature of 42 ℃ for 45-60 s, rapidly transferring to an ice bath, and standing for 2min. 700. Mu.L of antibiotic-free LB medium was added thereto, and resuscitated at 37℃and 200rpm for 60 minutes. 80. Mu.L of resuscitating culture solution is coated with Amp-resistant LB medium and cultured upside down at 37 ℃ overnight.
2.4 Positive clone screening and plasmid extraction
Positive clones were picked up and cultured in 0.5mL Amp-resistant LB medium at 37℃and 200rpm for 4 to 5 hours, and then subjected to colony verification according to tables 9 and 10. And after the PCR product is verified to be correct by agarose gel electrophoresis, sequencing the Huada gene. The strain with correct sequencing result is subjected to glycerol sterilization and plasmid is extracted.
Wherein the verification primer pair includes P22YZ-F (GAAAGGAAGGGAAGAAAGCGSEQ ID No: 17) and P22YZ-R (CAGTAAATAATTCTTCACCTTTGGAAACSEQ ID No: 18).
Table 9: colony validation PCR system (20. Mu.L)
TABLE 10 colony validation PCR procedure
2.5 Plasmid electrotransfer Kluyveromyces marxianus and verification
10 mu L (not more than 1000 ng) of the constructed plasmid is added into competent cells of Kluyveromyces marxianus (K.marxinus CJ 3113) and the mixture is subjected to ice bath for 2-5 min. Transferring to a precooled electric rotating cup, and selecting Pic mode to perform electric shock. After the end of the shock, 750. Mu.L of 1M sorbitol and 750. Mu.L of YPD medium were added rapidly, and resuscitated at 30℃at 200rpm for 1h. And (5) coating a proper amount of resuscitating bacteria liquid with a Hyg resistant YPD culture medium, and culturing the mixture for 48 hours at the temperature of 30 ℃ in an inverted way.
Positive clones were picked and cultured in 5mL of Hyg resistant YPD medium at 30 ℃/200rpm overnight. mu.L of the culture medium was taken and 100. Mu.L of ddH was added thereto 2 O, removing the supernatant after centrifugation. Adding 50 mu L TE buffer solution, and stopping for 30sec after high fire in a microwave oven for 1 min; medium and high fire for 30sec, stopping for 10s; after 10sec of medium-high fire, colony verification was performed as a verification template according to tables 6 and 7. The PCR product is verified to be correct by agarose gel electrophoresis and then glycerinum bacteria are preserved, and the PCR product is named KM:: P22-EGFP and KM::: P respectively INU -EGFP。
2.6 Shaking flask fermentation
Overnight activated KM:: P22-EGFP and KM:: P were used INU The EGFP and KM original strain bacterial solutions are respectively inoculated into 50mL of non-antibiotic YD culture medium with the inoculum size of 1 percent, and are subjected to shaking flask fermentation at 30 ℃/200rpm for 72 hours. Taking the fermentation liquid for 48h and 72h respectively, centrifuging, taking the supernatant, and verifying the expression result by 4% -20% SDS-PAGE, wherein as shown in the figure 4, the quantity of the EGFP protein expressed by P22 is obviously higher than that of P INU 。
2.7 Determination of fluorescence intensity of supernatant of fermentation broth
Taking 1-2 mL 48h or 72h shake flask fermentation broth, centrifuging at 8000rpm for 1min, taking supernatant for 100 times dilution, taking 8#K.M as blank, and using a QuBit instrument to measure the green fluorescence intensity of 510-580 nm under 470nm blue light excitation, wherein the result is shown in FIG. 5, EGFP fluorescence intensity P22 is obviously higher than P at 48 hours and 72 hours INU 。
EXAMPLE 3P 22 promoter expression beta-lactoglobulin assay
Referring to example 2, pHKD-P22-EGFP and pHKD-P are used INU -an EGFP plasmid, replacing the EGFP gene with a β -lactoglobulin gene (βlg) having the amino acid sequence: LIVTQTMKGLDIQKVAGTWYSLA MAASDISLLDAQSAPLREEVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKAPVMHIRLSFNPTQLEEQCHI (SEQ ID No: 19). Construction of plasmid pHKD-P22- βLG and pHKD-P INU -βLG。
Plasmid pHKD-P22-beta LG and pHKD-P INU Electrotransformation of beta LG into KM competent cells, construction of KM:: P22-beta LG and KM:: P INU βlg strain, validated as shown in figure 6.
Overnight activated KM:: P22-. Beta.LG and KM:: P were used INU The strain liquids of the original strain beta LG and KM are respectively inoculated in 50mL of an antibiotic-free YD culture medium with the inoculum size of 1 percent, and are subjected to shaking flask fermentation at 30 ℃/200rpm for 72 hours. Taking the fermentation liquids for 48h and 72h respectively, centrifuging, taking the supernatant, and verifying the expression result by 4% -20% SDS-PAGE, wherein as shown in FIG. 7, the amount of P22 expressed beta LG protein is obviously higher than that of P INU 。
Claims (12)
1. A fungal-derived promoter element, wherein the nucleotide sequence of said promoter element is set forth in SEQ ID No: 1.
2. A transcriptional expression cassette comprising the promoter element of claim 1, and a coding gene operably linked to the promoter element.
3. A recombinant expression vector comprising the promoter element of claim 1 or the transcriptional expression cassette of claim 2.
4. A recombinant cell comprising the promoter element of claim 1, or the transcriptional expression cassette of claim 2, or the recombinant expression vector of claim 3; the original cell of the recombinant cell is selected from Kluyveromyces marxianusMother and motherKluyveromycesmarxianusKluyveromyces lactisKluyveromyceslactisPichia kluyveriPichiakluyveriOr Pichia pastorisPichiapastoris。
5. Use of at least one of the promoter element of claim 1, the transcription expression cassette of claim 2, the recombinant expression vector of claim 3, the recombinant cell of claim 4 in (a) and/or (b):
(a) Enhancing the transcription level of the encoding gene, or preparing a reagent or a kit for enhancing the transcription level of the encoding gene;
(b) Preparing exogenous protein, or preparing reagent or kit for preparing exogenous protein.
6. The use of claim 5, wherein the exogenous protein comprises a milk protein.
7. The use according to claim 6, wherein the milk proteins comprise at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin or osteopontin.
8. A method of enhancing transcription of a gene encoding a foreign protein, said method comprising operably linking the promoter element of claim 1 to a gene encoding a foreign protein prior to introducing into a host cell.
9. The method of claim 8, wherein the exogenous protein comprises at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin, or osteopontin.
10. The method of claim 8, wherein the host cell is selected from the group consisting of kluyveromyces marxianusKluyveromycesmarxianusKluyveromyces lactisKluyveromyceslactisPichia kluyveriPichiakluyveriOr PichiaYeastPichia pastoris。
11. A method for producing milk protein, comprising culturing the recombinant cell of claim 4 and isolating and collecting milk protein from the fermentation broth; wherein the recombinant cell comprises the promoter element of claim 1 operably linked to a milk protein encoding gene.
12. The method of claim 11, wherein the milk protein comprises at least one of beta-lactoglobulin, alpha-lactalbumin, casein, lactoferrin, or osteopontin.
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