A method for regulating the expression of exogenous genes

Abstract

The present invention provides a method for regulating the expression of exogenous genes, and relates to the field of genetic engineering technology. The method of the present invention is as follows: the ±1 nucleosome sequence and the NFR sequence in the transcription start region of the gene are used as the transcription regulatory sequence of the exogenous gene, and the expression level of the exogenous gene is increased or decreased by any of the following methods: (1) selecting a transcription regulatory sequence containing NFR sequences of different lengths; or (2) increasing or shortening the length of the NFR sequence for the selected transcription regulatory sequence. The present invention uses the ±1 nucleosome sequence in the transcription region of the Saccharomyces cerevisiae gene and the NFR sequence between them as the transcription regulatory sequence of the exogenous gene, and regulates the level of transgenic expression by selecting and adjusting the length of the NFR sequence, thereby providing a new design idea for regulating the expression of exogenous genes.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for regulating the expression of exogenous genes. Background Art

[0002] Gene expression regulation is a hot topic that people have always paid attention to. Theoretical research on gene expression regulation has been widely used in transgenic engineering. In transgenic engineering, most studies are based on one-dimensional sequence information of gene transcription regions to explore the relationship between regulatory elements in the sequence and the expression level of exogenous genes, with the aim of increasing the expression level of exogenous genes as much as possible. However, in eukaryotes, transgenic technology still faces challenges in the expression regulation of transgenics. Therefore, exploring a regulatable transgenic expression system is a current research difficulty. Studies have shown that increasing promoter strength can increase the expression level of transgenics. Due to the complexity of gene expression regulation in eukaryotic organisms, not all strong promoters can achieve the highest protein expression and activity. Increasing the number of copies of the inserted gene can increase the expression level of the gene, but adding more gene copies cannot increase the expression level of the transgenic. Given that the usage frequencies of synonymous codons in protein coding sequences vary in Escherichia coli, yeast, animals and plants, transgenic codon optimization is one of the common methods to increase the expression level of proteins. However, synonymous codon optimization may change the structure and stability of proteins, causing changes in the function and activity of proteins. In addition, studies have shown that the expression level of transgenes can be increased by adding enhancers, but they must be used in combination with strong promoters, and in some cases, the addition of enhancers may affect the gene expression of the host.

[0003] In eukaryotes, chromosomes exist in the form of nucleosome arrays. Nucleosomes are composed of a 147bp DNA fragment and a histone octamer containing 2 copies of H2A, H2B, H3 and H4. DNA is assembled into a "beaded" linear nucleosome array. The position of nucleosomes on the DNA sequence can regulate the interaction between DNA binding proteins and DNA, thereby regulating gene transcription and expression. In the gene transcription start region, there are two well-positioned nucleosomes upstream and downstream of the transcription start site, called -1 nucleosome and +1 nucleosome, respectively. Between the ±1 nucleosomes is a nucleosome-free region (NFR) of variable length. Studies have found that NFRs are commonly found in the gene transcription regions of yeast, plants, fruit flies, nematodes and humans. The position of the -1 nucleosome upstream of the NFR is highly variable, while the position of the +1 nucleosome downstream of the NFR is relatively stable. NFR is an important functional element for the initiation of gene transcription. It is rich in transcription factor (TFs) binding sites, providing an entry point for protein complexes involved in gene transcription to enter chromatin and initiate gene transcription by binding to transcription factors. The binding efficiency of transcription factors and NFRs determines the transcription efficiency of genes.

[0004] It can be seen that there are certain limitations to explore the expression regulation of transgenics only from the perspective of one-dimensional DNA sequences. Exploring the expression regulation of transgenics at the level of nucleosome arrays is indispensable for improving the theory of gene expression regulation. At present, there are no reports on the study of regulating the expression of exogenous genes through NFR sequences. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for regulating the expression of exogenous genes, using the ±1 nucleosome sequence in the transcription start region of the Saccharomyces cerevisiae gene and the NFR sequence therebetween as the transcription regulatory sequence of the exogenous gene, and by selecting and adjusting the length of the NFR sequence in the transcription regulatory sequence, the expression level of the exogenous gene can be increased or decreased.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for regulating the expression of an exogenous gene, the method comprising the following steps: using the ±1 nucleosome sequence and the NFR sequence in the transcription start region of the gene as the transcription regulatory sequence of the exogenous gene, and increasing or decreasing the expression level of the exogenous gene by any of the following methods: (1) selecting a transcription regulatory sequence containing NFR sequences of different lengths; or (2) increasing or shortening the length of the NFR sequence for the selected transcription regulatory sequence.

[0008] Preferably, the transcriptional regulatory sequence is derived from one or more of the transcriptional regulatory regions of Saccharomyces cerevisiae genes YBL032W, YBL025W, YBL071W-A, YAL030W, YAR008W and YAR027W.

[0009] More preferably, the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAL030W is as described in SEQ ID NO: 1; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAR008W is as described in SEQ ID NO: 2; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAR027W is as described in SEQ ID NO: 3; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL071W-A is as described in SEQ ID NO: 4; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL032W is as described in SEQ ID NO: 5; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL025W is as described in SEQ ID NO: 6.

[0010] Preferably, the NFR sequence length > 150 bp is a long NFR sequence, called an L-NFR sequence; the NFR sequence length < 150 bp is a short NFR sequence, called an S-NFR sequence.

[0011] Preferably, the method for increasing the length of the NFR sequence comprises the following steps: taking a 30-80 bp DNA fragment in the middle and upper reaches of the NFR sequence, and serially connecting it to the original NFR sequence to obtain an increased NFR sequence; the method for shortening the length of the NFR sequence comprises the following steps: cutting off a 30-80 bp DNA fragment in the middle and upper reaches of the NFR sequence, and splicing the NFR sequences at both ends together to form a shortened NFR sequence.

[0012] More preferably, the range of the increased NFR sequence length x is 150≤x≤220 bp; the range of the shortened NFR sequence length x is 100≤x<150 bp.

[0013] Preferably, the coding sequence of the exogenous gene is linked to the 3' end of the transcription regulatory sequence.

[0014] Preferably, the expression cassette of the exogenous gene consists of a -1 nucleosome sequence, an NFR sequence, a +1 nucleosome sequence, the exogenous gene and a CYC1 terminator.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The TSS of most genes of Saccharomyces cerevisiae is located downstream of NFR or in the +1 nucleosome, and the ±1 nucleosome and the NFR sequence between them have an important influence on the regulation of gene expression. The binding efficiency of TF and NFR sequence is closely related to the length of NFR sequence. The present invention provides a method for regulating the expression of exogenous genes, using the ±1 nucleosome sequence of the transcription start region of Saccharomyces cerevisiae gene and the NFR sequence between them as the transcriptional regulatory sequence of the exogenous gene, and taking the GFP exogenous gene as an example to explore the influence of the NFR sequence length on the expression level of the GFP exogenous gene. For the transcriptional regulatory sequence of long NFR (>150bp), the relative mRNA expression amount and protein expression amount of the GFP gene are significantly higher than those of short NFRs (<150bp). When the length of the long NFR sequence is shortened, the relative mRNA expression amount and protein expression level of the GFP gene are significantly reduced. Conversely, when the length of the short NFR sequence is increased, the relative mRNA expression level and protein expression level of the GFP gene are significantly improved. The results of the present invention show that the length of the NFR sequence is a key factor in regulating the expression of exogenous genes. Increasing the length of the NFR sequence can increase the expression level of the exogenous gene. Conversely, shortening the length of the NFR sequence can reduce the expression level of the exogenous gene. The present invention verifies the influence of the length of the NFR sequence on the expression level of the exogenous gene at the experimental level, explores a new regulatable exogenous gene expression method, and provides a new design idea for regulating the expression of exogenous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The NFR length distribution of the transcription start regions of 1258 Saccharomyces cerevisiae genes;

[0018] Figure 2 Figure 2 is the relationship between NFR length and relative mRNA expression level; Figure A shows the relative mRNA expression level of 1258 Saccharomyces cerevisiae genes, with the two groups of NFR lengths bounded by 150 bp; Figure B shows the two groups of NFR lengths bounded by 120 bp; Figure C shows the RT-qPCR validation of RNA-seq data, with the two groups of NFR lengths bounded by 150 bp, ignoring genes with extremely low FPKM values ​​(FPKM<1), and randomly selecting 37 long NFR genes and 49 short NFR genes, FPKM represents the number of fragments per kilobase of transcript per million mapped reads;

[0019] Figure 3The structure diagram of the transcriptional regulatory sequences of six Saccharomyces cerevisiae genes; TSS is the transcription start site, -1N represents -1 nucleosome, +1N represents +1 nucleosome, NFR represents the DNA sequence between ±1 nucleosome positions, ATG is the start codon, and the yellow area represents the partial coding sequence of the yeast gene; YAL030W, YAR008W and YAR027W are short NFR sequences (<150bp), YBL071W-A, YBL032W and YBL025W are long NFR sequences (>150bp);

[0020] Figure 4 The structure diagrams of three NFR+ and three NFR- transcriptional regulatory sequences; in the transcriptional regulatory sequences of YAL030W, YAR008W and YAR027W genes (named NFR+), the shorter NFR increased by 80bp; in the transcriptional regulatory sequences of YBL071W-A, YBL032W and YBL025W genes (named NFR-), the longer NFR shortened by 80bp; the oblique line box indicates the sequence fragment inserted in the NFR region; the cross line box indicates the sequence fragment cut out in the NFR region;

[0021] Figure 5 It is the structure diagram of the exogenous gene;

[0022] Figure 6 Construction process for pYES-AUR1-NFR-GFP plasmid;

[0023] Figure 7 It is the restriction analysis of the recombinant plasmid; Figure A shows the pYES-AUR plasmid was digested with SnaBI / HpaI to obtain the AUR1 fragment; Figure B shows the pYES-AUR1-NFR-GFP plasmid was digested with SacI / SphI to obtain the NFR-GFP fragment; M represents DL5000 DNA marker, from top to bottom: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

[0024] Figure 8 For the detection of exogenous gene GFP in recombinant Saccharomyces cerevisiae. N is the negative control, the gene PCR amplification band using the genomic DNA of Saccharomyces cerevisiae BY4741 as the template, 1-6 are the gene PCR amplification bands using the genomic DNA of 6 transformants as the template, M represents DL2000 DNA marker, from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

[0025] Fig. 9Analysis of GFP exogenous gene expression levels in L-NFR and S-NFR groups; Figure A is the relative mRNA expression of GFP gene in L-NFR and S-NFR groups detected by RT-qPCR; Figure B is the quantitative detection of GFP protein expression levels in L-NFR and S-NFR groups by ELISA kit; Figure C is the expression of GFP protein in L-NFR and S-NFR groups by western blotting using anti-GFP antibody; In Figures A and B, the mean ± SEM data are from three NFRs, and each NFR group has three replicates. Independent t-test (two-tailed) was used, **p<0.01, ***p<0.001;

[0026] Fig.10 Analysis of GFP exogenous gene expression levels in NFR-group and NFR+group; Figure A is the relative mRNA expression of GFP gene in NFR-group and NFR+group detected by RT-qPCR; Figure B is the quantitative detection of GFP protein expression level in NFR-group and NFR+group by ELISA kit; Figure C is the expression of GFP protein in NFR-group and NFR+group detected by western blotting using anti-GFP antibody. In Figures A and B, the mean ± SEM data are from three NFRs, and each NFR group has three replicates. Independent t-test (two-tailed) was used, **p<0.01, ***p<0.001;

[0027] Fig.11 Comparative analysis of the expression level of GFP exogenous gene. Figure A shows the relative mRNA expression of GFP gene under L-NFR and NFR- regulation detected by RT-qPCR; Figure B shows the GFP protein expression level under L-NFR and NFR- regulation detected by ELISA kit; Figure C shows the expression of GFP protein under L-NFR and NFR- regulation detected by western blotting using anti-GFP antibody; Figure D shows the relative mRNA expression of GFP gene under S-NFR and NFR+ regulation detected by RT-qPCR; Figure E shows the GFP protein expression level under S-NFR and NFR+ regulation detected by ELISA kit; Figure F shows the expression of GFP protein under S-NFR and NFR+ regulation detected by western blotting using anti-GFP antibody; In Figures A, B, D and E, the mean ± SEM of 3 replicates for each pair of NFR was taken. Paired t test (two-tailed) was used, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0028] The present invention provides a method for regulating the expression of an exogenous gene, the method comprising the following steps: using the ±1 nucleosome sequence and the NFR sequence between the transcription start region of a saccharomyces cerevisiae gene as a transcription regulatory sequence, and regulating the expression level of the exogenous gene by any of the following methods: (1) selecting a transcription regulatory sequence containing NFR sequences of different lengths; or (2) increasing or shortening the length of the NFR sequence for the selected transcription regulatory sequence; the regulation includes increasing the expression level of the exogenous gene or decreasing the expression level of the exogenous gene. The present invention achieves the purpose of regulating the expression of the exogenous gene by selecting and adjusting the length of the NFR sequence in the transcription regulatory sequence. In the present invention, the central position of the ±1 nucleosome in the transcription start region of the gene is derived from the single base pair precision positioning data of the nucleosome of saccharomyces cerevisiae given by Brogaard et al. (Brogaard K, Xi L, Wang JP, et al. Amap of nucleosome positions in yeast at base-pair resolution. Nature, 2012, 486 (7404): 496-501). The transcriptional regulatory sequence of each gene was taken from the Saccharomyces Genome Database (SGD, https: / / www.yeastgenome.org / ), version number R64. In the present invention, the Saccharomyces cerevisiae strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) was purchased from Shanghai Weidi Biotechnology Co., Ltd. and used as the host for Saccharomyces cerevisiae transformation experiments.

[0029] In the present invention, the NFR sequence length>150bp is a long NFR sequence, called L-NFR sequence; the NFR sequence length<150bp is a short NFR sequence, called S-NFR sequence. The present invention obtained the MNase-seq data of the BY4741 strain of Saccharomyces cerevisiae, and obtained the ±1 nucleosome position of the gene transcription start region. The present invention took the nucleosome sequence length as 150bp. Based on RNA-seq data, the relative mRNA expression levels of 1258 Saccharomyces cerevisiae genes were obtained and analyzed. The Saccharomyces cerevisiae genes were divided into two groups, long NFR and short NFR, and the relationship between the difference in mRNA relative expression levels and NFR length was tested.

[0030] In the present invention, the method for selecting a transcriptional regulatory sequence comprises the following steps: in a saccharomyces cerevisiae gene, NFR sequences of different lengths can be selected as transcriptional regulatory sequences of exogenous genes. In the present invention, 6 transcriptional regulatory sequences are selected to be derived from one or more of the transcriptional regulatory regions of saccharomyces cerevisiae genes YBL032W, YBL025W, YBL071W-A, YAL030W, YAR008W and YAR027W. In the selected 6 transcriptional regulatory sequences, the NFR lengths are 218bp, 214bp, 208bp, 134bp, 123bp and 95bp, respectively, and the selected NFR length ranges between 80-220bp. The first 3 are L-NFR transcriptional regulatory sequences, and the last 3 are S-NFR transcriptional regulatory sequences. In the present invention, the expression difference of the exogenous gene GFP under the regulation of the L-NFR group and the S-NFR group is explored.

[0031] In the present invention, the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAL030W is as shown in SEQ ID NO: 1; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAR008W is as shown in SEQ ID NO: 2; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YAR027W is as shown in SEQ ID NO: 3; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL071W-A is as shown in SEQ ID NO: 4; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL032W is as shown in SEQ ID NO: 5; the transcription regulatory sequence of the Saccharomyces cerevisiae gene YBL025W is as shown in SEQ ID NO: 6.

[0032] In the present invention, the method for increasing the length of the NFR sequence comprises the following steps: taking a 30-80 bp DNA fragment in the middle and upper reaches of the NFR sequence, and serially connecting it to the original NFR sequence to obtain an increased NFR sequence; the method for shortening the length of the NFR sequence comprises the following steps: cutting off a 30-80 bp DNA fragment in the middle and upper reaches of the NFR sequence, and splicing the NFR sequences at both ends together to form a shortened NFR sequence. In a specific embodiment of the present invention, for the S-NFR group, a 30-80 bp DNA fragment is taken from the middle and upper reaches of the NFR sequence and is serially connected to the original NFR sequence to obtain an increased NFR+ sequence; as an practicable manner, the length of the increased NFR sequence DNA fragment is 30bp, 40bp, 50bp, 60bp, 70bp, or 80bp; the method for shortening the length of the NFR sequence comprises the following steps: for the L-NFR group, a 30-80 bp DNA fragment is cut off from the middle and upper reaches of the NFR sequence, and the NFR sequences at both ends are spliced ​​together to form a shortened NFR- sequence; as an practicable manner, the length of the shortened NFR sequence DNA fragment is 30bp, 40bp, 50bp, 60bp, 70bp, or 80bp.

[0033] In the present invention, the range of the length x of the NFR sequence after the increase is preferably 150≤x≤220bp; the range of the length x of the NFR sequence after the shortening is preferably 90≤x<150bp. NFR sequences that are too long or too short will cause changes in the gene transcription regulation pattern, and the critical point of the NFR length needs further experimental verification.

[0034] In the present invention, the coding sequence of the exogenous gene is connected to the 3' end of the transcriptional regulatory sequence. In the present invention, the coding sequence of the green fluorescent protein (GFP) gene is selected as the exogenous gene, and the GFP gene coding sequence information is derived from the NCBI nucleic acid database (https: / / www.ncbi.nlm.nih.gov / , AccessionNumber: KF040397).

[0035] In the present invention, the expression cassette of the exogenous gene consists of -1 nucleosome sequence, NFR sequence, +1 nucleosome sequence, exogenous gene and CYC1 terminator. In a specific embodiment of the present invention, when the coding sequence of GFP gene is used as the exogenous gene, the expression cassette consists of -1 nucleosome sequence, NFR sequence, +1 nucleosome sequence, GFP gene and CYC1 terminator. In the present invention, the CYC1-ter sequence on the Saccharomyces cerevisiae expression vector pYES2 is selected as a unified termination sequence to eliminate the influence of different 3'UTR sequences on transgenic expression.

[0036] In a specific embodiment of the present invention, the pYES2 plasmid was used as the backbone, and the 2μori fragment was replaced with the AUR1 gene to construct a single copy integration expression vector with AUR1 as the integration site. The full length of the AUR1 gene is 2904 bp, and the amplified AUR1 gene sequence is 100% consistent with the nucleotide sequence of the AUR1 gene in GenBank (GenBank Accession No. Z28004.1).

[0037] In a specific embodiment of the present invention, all sequences are inserted into the pYES2 plasmid to obtain the plasmid pYES-AUR1-NFR-GFP. The plasmids are digested with two pairs of restriction endonucleases, namely, the pYES-AUR plasmid is digested with SnaBI / HpaI to obtain the AUR1 fragment; the pYES-AUR1-NFR-GFP plasmid is digested with SacI / SphI to obtain the NFR-GFP fragment. The results confirmed that the AUR1 and NFR-GFP fragments were integrated into the plasmid pYES2. The Ura3 gene on the pYES2 plasmid is an auxotrophic selection marker. Through the AUR1 gene, the expression cassette of the GFP gene and the Ura3 gene are integrated into the 11th chromosome of Saccharomyces cerevisiae BY4741, specifically: the expression vector pYESA-NFR-GFP is linearized using the restriction endonuclease EcoR I. Referring to the LiAc method described by Gietz and Schiestl, the linearized DNA fragment pYESA-NFR-GFP was transformed into Saccharomyces cerevisiae BY4741 cells using Yeast Transformation System 2 (Takara, Dalian). The linearized DNA fragment was integrated into chromosome XI of Saccharomyces cerevisiae by homologous recombination with the AUR1 gene as the homologous arm, and then the transformants were selected on SD-Ura agar medium.

[0038] In the present invention, a yeast genomic DNA extraction kit (Takara, Dalian) was used to extract the genomic DNA of Saccharomyces cerevisiae BY4741 and recombinant Saccharomyces cerevisiae. The PCR method was used to detect and determine whether the exogenous gene was integrated into the chromosome XI in the Saccharomyces cerevisiae genome. The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 15s, 60°C annealing for 30s, 72°C extension for 50s, 30 cycles; 72°C extension for 5 minutes. The primers used for PCR amplification are shown in Table 1. The expression products were then subjected to quantitative PCR (qPCR), Western Blot analysis, and GFP protein quantitative detection.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The primers, transcriptional regulatory sequences and GFP gene sequences used in the following examples were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. The Ultra One Step cloning kit was purchased from Novozymes.

[0041] Example 1 Relationship between NFR length and gene expression in Saccharomyces cerevisiae

[0042] The center position of the ±1 nucleosome in the gene transcription start region is derived from the single base pair precision positioning data of the Saccharomyces cerevisiae nucleosome given by Brogaard et al. The transcriptional regulatory sequence of each gene is taken from the Saccharomyces Genome Database (SGD, https: / / www.yeastgenome.org / ), version number R64. The length of the ±1 nucleosome sequence in the present invention is 150 bp.

[0043] The present invention first examined the relationship between NFR length and gene expression level of Saccharomyces cerevisiae. The present invention obtained MNase-seq data of Saccharomyces cerevisiae BY4741 strain and analyzed the nucleosome position of gene transcription start region. Based on MNase-seq sequencing data, the present invention obtained and analyzed NFRs of 1258 genes, and the NFR length ranged from 80bp to 370bp. Among them, 80% of NFR lengths were less than 150bp, and 20% of NFR lengths were greater than 150bp. The length distribution of NFR sequences of 1258 genes is as follows: Figure 1 shown.

[0044] Based on RNA-seq data, the present invention obtained and analyzed the relative mRNA expression levels of 1258 Saccharomyces cerevisiae genes, and expressed the gene expression levels using FPKM values. Compared with genes with NFR lengths less than 150 bp, the relative mRNA expression of genes with NFR lengths greater than 150 bp was approximately 30% higher ( Figure 2 A) The relative mRNA expression of genes with NFR length greater than 120 bp is about 70% higher than that of genes with NFR length less than 120 bp ( Figure 2 B). After removing genes with extremely low FPKM values ​​(FPKM<1), 37 long NFR genes (≥150bp) and 49 short NFR genes (<150bp) were randomly selected. The relative mRNA expression levels of the two groups of genes were detected by RT-qPCR. The results showed that the relative mRNA expression of long NFR genes was 15 times that of short NFR genes (P<0.0001; Figure 2 C) Therefore, these data indicate that the length of NFRs in S. cerevisiae is positively correlated with gene expression levels.

[0045] Example 2 Construction of exogenous gene transcription regulatory sequence

[0046] The ±1 nucleosome sequence of the transcription region of the yeast saccharomyces cerevisiae gene and the NFR sequence between them are selected as the transcription regulatory sequence of the exogenous gene. In the yeast saccharomyces cerevisiae gene, NFR sequences of different lengths can be selected as the transcription regulatory sequence of the exogenous gene. The present invention selects 6 transcription regulatory sequences, of which 3 long NFR sequences are derived from the transcription regulatory regions in the yeast saccharomyces cerevisiae genes YBL032W, YBL025W and YBL071W-A, and the NFR lengths are 218bp, 214bp and 208bp respectively, which are called L-NFR transcription regulatory sequences. 3 short NFR sequences are derived from the transcription regulatory regions in the yeast saccharomyces cerevisiae genes YAL030W, YAR008W and YAR027W, and the NFR lengths are 134bp, 123bp and 95bp respectively, which are called S-NFR transcription regulatory sequences. The length of the L-NFR sequence is between 200 and 220bp, and the length of the S-NFR sequence is between 90 and 150bp. The translation start site ATG of the regulatory sequences of YBL032W, YBL025W, YAL030W, YAR008W and YBL071W-A genes is located in the +1 nucleosome, and the translation start site ATG of the YAR027W gene is located downstream of the +1 nucleosome. The information of the six transcriptional regulatory sequences is shown in Table 1. The structures of the six transcriptional regulatory sequences are shown in Figure 3 shown.

[0047] The present invention marks the position of the TSS site as 0, and all other positions are referenced to the TSS. The length of the ±1 nucleosome sequence is set to 150 bp, and the sequence from 75 bp upstream of the center of the -1 nucleosome to 75 bp downstream of the center of the +1 nucleosome is used as the transcriptional regulatory sequence of the exogenous gene.

[0048] Table 1 Structural information of transcriptional regulatory regions of Saccharomyces cerevisiae genes

[0049]

[0050] Example 3 NFR+ and NFR- sequence construction

[0051] Based on the original 3 L-NFR and 3 S-NFR transcriptional regulatory sequences, the length of the NFR sequence was changed. For the L-NFR sequence, an 80 bp DNA fragment was cut near its 5' end to make the NFR sequence length less than 150 bp, which was marked as NFR-. For the S-NFR sequence, an 80 bp DNA fragment was taken near its 5' end and repeated in series to make its length greater than 150 bp, which was marked as NFR+. The structure of the adjusted transcriptional regulatory sequence is shown in Figure 4 The length of its NFR sequence is shown in Table 1. Figure 4As shown, in the NFR sequence of YBL071W-A, a DNA fragment with a length of 80 bp was cut off from the upstream of the -47 bp site, and the NFR sequences at both ends were spliced ​​together to form an NFR-sequence with a length of 128 bp; in the NFR sequence of YBL032W, a DNA fragment with a length of 80 bp was cut off from the upstream of the -72 bp site, and the NFR sequences at both ends were spliced ​​together to form an NFR-sequence with a length of 138 bp; in the NFR sequence of YBL025W, a DNA fragment with a length of 80 bp was cut off from the upstream of the -134 bp site, and the NFR sequences at both ends were spliced ​​together to form an NFR-sequence with a length of 134 bp. In the NFR sequence of YAL030W, a DNA fragment with a length of 80bp between -46bp and -125bp was taken and inserted into -126bp to form a NFR+ sequence with a length of 214bp; in the NFR sequence of YAR008W, a DNA fragment with a length of 80bp between -36bp and -115bp was taken and inserted into -116bp to form a NFR+ sequence with a length of 203bp; in the NFR sequence of YAR027W, a DNA fragment with a length of 80bp between -19bp and -98bp was taken and inserted into -99bp to form a NFR+ sequence with a length of 175bp. Finally, 3 NFR- and 3 NFR+ transcriptional regulatory sequences were obtained.

[0052] Example 4 Exogenous gene sequence design

[0053] The coding sequence of the green fluorescent protein gene was selected as the exogenous gene. The coding sequence of the exogenous gene was connected to the 3' end of the transcriptional regulatory sequence. In order to ensure that the reading frame of the exogenous gene was not destroyed, the partial coding sequence of the selected yeast gene was a multiple of 3. The CYC1-ter sequence on the Saccharomyces cerevisiae expression vector pYES2 was selected as a unified termination sequence to eliminate the influence of different 3'UTR sequences on the expression of the exogenous gene. The complete exogenous gene structure is shown in Figure 5 .

[0054] Example 5 Construction of single copy integration expression vector

[0055] The GFP gene and AUR1 gene were connected to the pYES2 vector using a cloning kit. The steps are as follows: AUR1 gene was amplified from Saccharomyces cerevisiae genomic DNA using AUR1-F / R primers (Table 2). The amplified product was connected to the pMD18-T plasmid, sequenced and verified. Using VF / R primers, reverse PCR amplification was performed by Prime STAR HSPCR (Takara, Dalian, China) to knock out the 2μori fragment on the pYES2 plasmid to obtain the linearized vector pYES2. The AUR1 gene was connected to the linearized pYES2 vector using seamless cloning to replace the 2μori fragment and complete the construction of a single copy integration expression vector. Then, the GFP target gene and transcriptional regulatory sequence were introduced into the multiple cloning site upstream of the CYC1 terminator of the pYES2 plasmid to complete the construction of the homologous recombination expression plasmid pYES2-AUR1-NFR-GFP ( Figure 6 ). The plasmid was digested with two pairs of restriction endonucleases to obtain AUR1 and NFR-GFP fragments. The results confirmed that AUR1 and NFR-GFP fragments were integrated into plasmid pYES2 ( Figure 7 ).

[0056] The Ura3 gene on the pYES2 plasmid is an auxotrophic selection marker. Through the AUR1 gene, the expression cassette of the GFP gene and the Ura3 gene are integrated into the 11th chromosome of Saccharomyces cerevisiae BY4741, specifically: the expression vector pYESA-NFR-GFP is linearized using the restriction endonuclease EcoR I. Referring to the LiAc method described by Gietz and Schiestl, the linearized DNA fragment pYESA-NFR-GFP is transformed into Saccharomyces cerevisiae BY4741 cells using Yeast Transformation System 2 (Takara, Dalian), and the linearized DNA fragment is integrated into the XIth chromosome of Saccharomyces cerevisiae by homologous recombination with the AUR1 gene as the homologous arm, and then the transformants are screened on SD-Ura agar medium. Among them, the expression cassette of the GFP gene consists of -1 nucleosome sequence, NFR sequence, +1 nucleosome sequence, GFP gene and CYC1 terminator.

[0057] Transformants were screened on SD / -Ura medium, and the presence of the GFP gene in the transformed strain of Saccharomyces cerevisiae was detected by PCR using primers GFP-F / R. Agarose gel electrophoresis showed that a 714 bp DNA fragment was detected in the recombinant strain DNA, and subsequent sequencing of the fragment confirmed that it was consistent with the sequence of the fragment, but was not detected in the control line ( Figure 8 ). Experiments showed that the gene fragment had been integrated into the Saccharomyces cerevisiae genome.

[0058] Table 2 Primer sequences used to construct vectors

[0059]

[0060] Example 6 Analysis of GFP exogenous gene expression under NFR sequence regulation

[0061] According to Example 4, the present invention chemically synthesized three L-NFR and three S-NFR transcriptional regulatory sequences and the GFP gene coding sequence, cloned them into the pYESA vector, and transferred them into Saccharomyces cerevisiae BY4741 cells for expression. Under the regulation of the L-NFR sequence, the relative expression level of the GFP gene mRNA was higher than that of the S-NFR sequence, and the average relative expression level under the regulation of the L-NFR sequence was 2.8 times that of the S-NFR sequence ( Fig. 9 A), the independent t-test results showed that the relative expression levels between the L-NFR group and the S-NFR group were significantly different (p<0.01). The expression of GFP protein was detected using Western blot (WB) and ELISA kits. By WB analysis, a 27kDa protein product was detected in the recombinant Saccharomyces cerevisiae BY4741 cells, which was consistent with the molecular weight of the amino acid sequence of the 714bp GFP gene ( Fig. 9 C). The expression level of GFP protein under the regulation of L-NFR sequence is higher than that of S-NFR sequence. The average expression level of GFP protein under the regulation of L-NFR sequence is 7 times that of S-NFR sequence ( Fig. 9 B), the independent t-test results showed that the difference between the two was extremely significant (p<0.001). The protein expression level was consistent with the relative expression level of mRNA. The results showed that in the transcriptional regulatory sequence of Saccharomyces cerevisiae, the length of the NFR sequence was positively correlated with gene expression.

[0062] According to Example 4, the present invention chemically synthesized 3 NFR- and 3 NFR+ transcriptional regulatory sequences and the GFP gene coding sequence, cloned them into the pYESA vector, and transferred them into Saccharomyces cerevisiae BY4741 cells for expression. The relative expression level of the GFP gene mRNA under the regulation of the NFR+ sequence was significantly higher than that of the NFR- sequence, and the average relative expression level of the GFP gene mRNA under the regulation of the NFR+ sequence was 5.3 times that of the NFR- sequence ( Fig.10 A), the independent t-test results showed that the difference between the NFR- group and the NFR+ group was extremely significant (p<0.001). The expression of GFP protein was detected using Western blot (WB) and ELISA kits. The WB test results showed that ( Fig.10C), at a protein molecular weight of about 27kDa, an obvious antigen-antibody reaction band appeared in the transgenic Saccharomyces cerevisiae protein sample, indicating that GFP was expressed in Saccharomyces cerevisiae. Under the regulation of NFR+ sequence, the expression level of GFP protein was significantly higher than that of NFR-sequence. The average expression level of GFP protein under the regulation of NFR+ sequence was 4 times that of NFR-sequence ( Fig.10 B), the independent t-test results showed that there was a very significant difference between the two (p<0.01). The results of protein level detection were consistent with those of mRNA level. Compared with the original transcriptional regulatory sequence, shortening NFR significantly reduced the expression level of the exogenous gene, and lengthening NFR significantly increased the expression level of the exogenous gene.

[0063] The present invention uses qPCR method to detect the difference in relative expression level of GFP gene mRNA under the regulation of L-NFR and corresponding NFR-transcriptional regulatory sequence and S-NFR and corresponding NFR+transcriptional regulatory sequence. Western blot (WB) and ELISA kits were used to detect the difference in expression level of GFP protein under the regulation of L-NFR and corresponding NFR-transcriptional regulatory sequence and S-NFR and corresponding NFR+transcriptional regulatory sequence ( Fig.11 Western blot analysis showed that a 27 kDa product was detected in the recombinant Saccharomyces cerevisiae protein sample ( Fig.11 C and 11F), indicating that GFP protein was expressed. In the YBL025W, YBL032W and YBL071W-A transcriptional regulatory sequences, compared with the original L-NFR, under the regulation of NFR-sequence, the relative expression level of GFP gene mRNA and protein expression level were significantly reduced, and the relative expression level of GFP gene mRNA was reduced by about 19, 22 and 9 times, respectively ( Fig.11 A), the protein expression levels decreased by approximately 3, 6, and 4 times, respectively ( Fig.11 B). Paired t-test results showed that the differences in relative mRNA expression and protein expression in the three groups of L-NFR and NFR-pairing were extremely significant. In the YAR008W, YAR027W and YAL030W transcriptional regulatory sequences, compared with the original S-NFR sequence, the relative mRNA expression level and protein expression level of the GFP gene under the regulation of the NFR+ sequence were significantly increased, and the relative mRNA expression level of the GFP gene increased by about 11, 33 and 4 times, respectively ( Fig.11 D), the protein expression levels increased by approximately 6, 15 and 3 times, respectively ( Fig.11E). Paired t-test results showed that the differences in relative mRNA expression and protein expression between the three groups of S-NFR and NFR+ were extremely significant. The results showed that changes in the length of NFR transcriptional regulatory sequences significantly affected the expression of exogenous genes. When the NFR sequence was shortened to less than 150 bp, the expression level of the exogenous gene could be significantly downregulated. When the NFR sequence was increased to more than 150 bp, the expression level of the exogenous gene could be significantly upregulated.

[0064] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A method for regulating the expression of an exogenous gene, characterized in that: The method comprises the following steps: using the ±1 nucleosome sequence and the NFR sequence in the gene transcription start region as the transcription regulatory sequence of the exogenous gene, and increasing or decreasing the expression level of the exogenous gene by selecting the transcription regulatory sequence containing the NFR sequence of different lengths; the transcription regulatory sequence is derived from one or more of the transcription regulatory regions of the yeast Saccharomyces cerevisiae genes YBL032W, YBL025W, YBL071W-A, YAL030W, YAR008W and YAR027W; The lengths of NFRs in the six transcriptional regulatory sequences are 218 bp, 214 bp, 208 bp, 134 bp, 123 bp and 95 bp, respectively; the first three are L-NFR transcriptional regulatory sequences, and the last three are S-NFR transcriptional regulatory sequences; The transcriptional regulatory sequences of the Saccharomyces cerevisiae genes YAL030W, YAR008W, YAR027W, YBL071W-A, YBL032W and YBL025W are nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 6, respectively; The coding sequence of the exogenous gene is connected to the 3' end of the L-NFR transcription regulatory sequence or the 3' end of the S-NFR transcription regulatory sequence.

2. A method for regulating the expression of an exogenous gene, characterized in that: The method comprises the following steps: using the ±1 nucleosome sequence and the NFR sequence in the gene transcription start region as the transcription regulatory sequence of the exogenous gene, and increasing or decreasing the expression level of the exogenous gene by increasing or shortening the length of the NFR sequence in the selected transcription regulatory sequence; Increasing or shortening the length of the NFR sequence means that: on the basis of the three L-NFR transcription regulatory sequences and the three S-NFR transcription regulatory sequences described in claim 1, the position of the TSS site is marked as 0, and the other positions are all referenced to the TSS. In the transcription regulatory sequence of the YBL071W-A gene, a DNA fragment with a length of 80 bp is cut off from the upstream of the -47 bp site, and the NFR sequences at both ends are spliced ​​together to form an NFR-sequence with a length of 128 bp; in the transcription regulatory sequence of the YBL032W gene, a DNA fragment with a length of 80 bp is cut off from the upstream of the -72 bp site, and the NFR sequences at both ends are spliced ​​together to form an NFR-sequence with a length of 138 bp; in the transcription regulatory sequence of the YBL025W gene, a DNA fragment with a length of 80 bp is cut off from the upstream of the -134 bp site. A fragment, the NFR sequences at both ends were spliced ​​together to form a 134bp NFR-sequence; in the transcriptional regulatory sequence of the YAL030W gene, a DNA fragment with a length of 80bp between -46bp and -125bp was taken and inserted into -126bp to form a 214bp NFR+ sequence; in the transcriptional regulatory sequence of the YAR008W gene, a DNA fragment with a length of 80bp between -36bp and -115bp was taken and inserted into -116bp to form a 203bp NFR+ sequence; in the transcriptional regulatory sequence of the YAR027W gene, a DNA fragment with a length of 80bp between -19bp and -98bp was taken and inserted into -99bp to form a 175bp NFR+ sequence; finally, 3 NFR- and 3 NFR+ transcriptional regulatory sequences were obtained; The coding sequence of the exogenous gene is connected to the 3' end of the NFR+ transcription regulatory sequence or the 3' end of the NFR- transcription regulatory sequence.

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