CN116334266B - Marine streptomycete secondary metabolite gene identification and screening method - Google Patents
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Abstract
The present invention relates to the field of biotechnology. In particular to a method for identifying and screening marine streptomycete secondary metabolite genes. Which comprises the following steps: the method comprises the steps of selecting a marine area to collect strains of marine streptomyces, analyzing the strains of the marine streptomyces, and evaluating the potential of the marine streptomyces; extracting DNA (deoxyribonucleic acid) from the marine streptomycete potential of the strain based on the evaluation result of the marine streptomycete potential of the strain, and sequencing according to the DNA of the strain; the invention designs the primer to amplify the target secondary metabolite gene, improves the identification accuracy, adopts the heterologous expression method in the identification and screening processes, can efficiently screen out the target secondary metabolite, effectively avoids the interference and misjudgment of other metabolites on the identification, improves the specificity of the screening result, and can amplify the target gene in a shorter time by the PCR amplification technology, thereby screening out the novel secondary metabolite more quickly and accurately.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for identifying and screening marine streptomycete secondary metabolite genes.
Background
Streptomyces belongs to the precursor of the penicillin era, and fermentation metabolites and derivatives thereof have wide application in the fields of medicine and agriculture. Hundreds of secondary metabolite genes and their encoded enzymes have been identified from Streptomyces, both of which have important catalytic activities for methylation, deacylation, acylation, hydroxylation, etc. However, in the course of studying the genes of secondary metabolites in Streptomyces, heterogeneous data, such as the presence of a large number of non-coding regions and gene overlaps in the genome of Streptomyces, and the diversity of the secondary metabolite composition of Streptomyces, the complexity and variation of the gene structure and expression, are disadvantageous for the accuracy of gene identification and screening, and therefore, a method for identifying and screening genes of secondary metabolites of Streptomyces oceanicus has been proposed.
Disclosure of Invention
The invention aims to provide a method for identifying and screening marine streptomyces secondary metabolite genes, which aims to solve the problems in the background technology.
To achieve the above object, there is provided a method for identifying and screening a marine Streptomyces secondary metabolite gene, comprising the steps of:
s1, selecting a marine area to collect strains of marine streptomyces, analyzing the strains of the marine streptomyces, and evaluating the potential of the marine streptomyces;
s2, extracting DNA from the marine streptomycete potential of the strain based on the evaluation result of S1, and sequencing the DNA according to the strain;
s3, performing PCR amplification on DNA of the strain serving as a template, and performing predictive analysis on the strain, so as to select a gene family matched with the strain to identify the strain;
s4, comparing and analyzing the PCR product combined with the DNA sequencing data of the strain, and judging that the DNA sequence is complete according to the analysis result;
s5, selecting and searching a corresponding vector according to DNA sequence data of the strain, selecting matched host bacteria, and transforming the vector into the host bacteria for heterologous expression;
s6, separating and purifying the metabolite in the S5 heterologous expression process, and evaluating the target secondary metabolite so as to judge the comprehensive characteristic data of the metabolite.
As a further improvement of the technical scheme, the step of evaluating the potential of the S1 marine streptomycete is as follows:
s1.1, selecting a marine area according to the type of a strain required;
s1.2, collecting strains in the ocean area, detecting and analyzing the strains, and selecting the strains with the production potential of secondary metabolites according to the judgment and analysis results.
As a further improvement of the technical scheme, the step of sequencing the DNA of the strain by S2 comprises the following steps:
s2.1, extracting DNA information of the strain selected according to the S1.2;
s2.2, interrupting the DNA information extracted in the step S2.1, selecting an aptamer for library construction, analyzing and sequencing the library construction, and obtaining the sequence data of the strain according to a sequencing result.
As a further improvement of the present technical scheme, the step of performing PCR amplification of S3 is as follows:
s3.1, collecting and analyzing secondary metabolite data of the strain according to the DNA information extracted in the S2.1, and evaluating by combining the analysis data with the existing network sequence information, so that a primer is arranged in the secondary metabolite;
s3.2, combining the DNA information extracted by the S2.1 with the primer set by the S3.1 for PCR amplification, thereby obtaining a PCR product.
As a further improvement of the technical scheme, the step of identifying the strain by selecting the gene family matched with the strain by S3 is as follows:
s3.3, carrying out feature extraction on secondary metabolites of the strain, and selecting corresponding gene families in a network according to feature extraction data;
s3.4, comparing and analyzing the selected gene family and the data of the secondary metabolite, obtaining a genome annotation result of the secondary metabolite according to the analysis result, and screening and identifying the genome annotation result.
As a further improvement of the technical scheme, the S4 pair judgment DNA sequence complete analysis steps are as follows:
s4.1, combining the PCR product obtained in the step S3.2 with the sequence data of the strain obtained in the step S2.2 for comparison and analysis;
s4.2, judging the sequence data accuracy of the strain according to the analysis result obtained in the S4.1, and performing secondary comparison if mutation exists.
As a further improvement of the technical scheme, the step of performing secondary comparison by the S4.2 is as follows:
s4.2.1, analyzing mutation data in the comparison process;
s4.2.2, determining to perform secondary sequencing comparison on the strain sequence data according to the analysis result of S4.2.1.
As a further improvement of the technical scheme, the step of transforming the S5 binding vector into host bacteria for heterologous expression is as follows:
s5.1, collecting sequence data of secondary metabolites of the strain, and selecting corresponding carrier data according to analysis of the sequence data;
s5.2, combining sequence data of the secondary metabolite with corresponding vector data, selecting corresponding host bacteria according to the strain data, and putting the combined data into the host bacteria for heterologous expression, so that the strain gene activity is obtained.
As a further improvement of the technical scheme, the step of S6 judging the comprehensive characteristic data of the strain is as follows:
s6.1, collecting and analyzing process data of the heterologous expression of S5.2, selecting the most obvious metabolite in the process data according to an analysis result, and separating and purifying the metabolite by NMR;
s6.2, judging and analyzing according to the separation and purification results of the metabolite, and obtaining the type, structure and characteristics of the metabolite according to the analysis results.
Compared with the prior art, the invention has the beneficial effects that:
in the method for identifying and screening the marine streptomyces secondary metabolite genes, the primers are designed to amplify target secondary metabolite genes, so that the identification accuracy is improved, and the target secondary metabolite can be efficiently screened out by adopting a heterologous expression method in the identification and screening processes, so that the interference and misjudgment of other metabolites on identification are effectively avoided, the specificity of screening results is improved, and the target genes can be amplified in a short time by a PCR amplification technology, so that novel secondary metabolites can be screened out more quickly and accurately.
Drawings
FIG. 1 is an overall flow diagram of the present invention;
FIG. 2 is a block flow diagram of the invention for evaluating the potential of Streptomyces maritimus;
FIG. 3 is a block diagram of the flow of DNA sequencing according to the strain of the invention;
FIG. 4 is a block flow diagram of the identification of a strain selected to match the strain of the present invention;
FIG. 5 is a block diagram showing the process of judging the integrity of DNA sequence according to the present invention;
FIG. 6 is a block diagram of a process for heterologous expression of a binding vector of the invention transformed into a host bacterium;
FIG. 7 is a block diagram of the process of determining the comprehensive characterization data of the metabolites according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIGS. 1-7, the present embodiment is directed to a method for identifying and screening genes of secondary metabolites of Streptomyces oceanicus, comprising the steps of:
s1, selecting a marine area to collect strains of marine streptomyces, analyzing the strains of the marine streptomyces, and evaluating the potential of the marine streptomyces;
the step of evaluating the potential of S1 marine streptomycete is as follows:
s1.1, selecting a marine area according to the type of a strain required; selecting an appropriate sea area, such as deep sea sediment, seafloor spa, plankton, etc., to collect a sample of potential Streptomyces oceanicus;
s1.2, collecting strains in the ocean area, detecting and analyzing the strains, and selecting the strains with the production potential of secondary metabolites according to the judgment and analysis results. The selection method comprises the following steps of
Collecting a strain sample: accurate sampling was performed using a sterile tool. The sample may be a mineral surface, coral, seaweed, marine debris, or other marine organisms, etc. Samples were processed and prepared to be spread evenly on sterile plates containing medium for culture. Colonies were transplanted to new plates containing medium by a screener and cultured.
Detection of basic biological characteristics: the strain is identified and analyzed for basic biological characteristics through various detection in aspects of morphology, physiology, biochemistry and the like.
Primary metabolite screening: culturing the collected strain, and performing preliminary metabolite screening by adopting a biological fermentation technology or other methods, so as to screen out the strain with higher yield of metabolites.
Detailed metabolite analysis: further metabolite analysis is performed on strains with potential metabolites to determine the type, nature and specific biosynthetic pathways of the metabolites.
S2, extracting DNA from the marine streptomycete potential of the strain based on the evaluation result of S1, and sequencing the DNA according to the strain;
the step of sequencing the DNA of the strain by the S2 is as follows:
s2.1, extracting DNA information of the strain selected according to the S1.2;
s2.2, interrupting the DNA information extracted in the S2.1, selecting an aptamer, carrying out library construction, analyzing and sequencing the library construction, wherein the library construction method comprises the following steps:
strain pretreatment and genome DNA extraction: the strain is first grown to a suitable growth phase (usually the logarithmic growth phase) and appropriate colonies are collected. Extracting genome DNA by using genome extraction kit such as konjak fungus genome DNA extraction kit.
DNA quality control: the purity and quality of the extracted DNA are confirmed to meet the research requirements, such as detecting the A260/280 ratio and the A260/230 ratio of the DNA, and judging whether the purity and the quality are polluted or not.
Library construction: after the quality of the extracted genome DNA meets the requirements, breaking the genome DNA and selecting the proper size by adopting a library construction method of an Illumina platform. And selecting proper connection aptamer for library construction, and performing library quality inspection.
Sequencing: and carrying out high-throughput sequencing on the constructed library to obtain sequence data of the genome of the strain.
Data preprocessing: quality control is performed on the sequencing data, for example, low quality, low alignment, data with inserted or deleted bases is removed and filtered and corrected to ensure high quality input data for subsequent analysis, and the strain sequence data is obtained according to the sequencing result.
S3, performing PCR amplification on DNA of the strain serving as a template, and performing predictive analysis on the strain, so as to select a gene family matched with the strain to identify the strain;
the step of PCR amplification of S3 is as follows:
s3.1, collecting and analyzing secondary metabolite data of the strain according to the DNA information extracted in the S2.1, and evaluating by combining the analysis data with the existing network sequence information, so that a primer is arranged in the secondary metabolite;
a conserved region or a fragment of known sequence of the secondary metabolite gene of interest was identified, and a pair of primers was designed in this region to amplify a fragment of about 0.5kb in length. The primers positioned at different positions can effectively avoid amplifying non-target sites, and ensure the specificity and accuracy of amplified products. The Primer design can be performed by using various existing Primer design software, such as Primer3, oligo6 and the like;
s3.2, combining the DNA information extracted by the S2.1 with the primer set by the S3.1 for PCR amplification, thereby obtaining a PCR product. The PCR system and conditions may be varied depending on the primer selected and may be adjusted according to the specific conditions. It is generally recommended to split the PCR system into two parts: the first stage is 95 ℃ for 2min, and the second stage is 25,30 or 35 cycles: 94 ℃ for 30s,55 ℃ for 30s and 72 ℃ for 1min. The final extension was 72 ℃ for 7min.
The step of identifying the strain by selecting the gene family matched with the strain in S3 is as follows:
s3.3, carrying out feature extraction on secondary metabolites of the strain, and selecting corresponding gene families in a network according to feature extraction data;
s3.4, comparing and analyzing the selected gene family and the data of the secondary metabolite, obtaining a genome annotation result of the secondary metabolite according to the analysis result, and screening and identifying the genome annotation result. The following are possible methods:
polyketide synthase gene family screening: polyketide synthase (PolyketideSynthase, PKS) is a key enzyme for the synthesis of polyketides and polyester-based secondary metabolites. Searching for positive predictive results for a family of PKS genes from the genome annotation results may use software tools such as antisMASH, etc.
Non-polyketide synthase gene family screening: non-polyketide synthases (NonribosomalPeptideSynthetase, NRPS) are commonly used to synthesize secondary metabolites such as linear and cyclic peptides, glycopeptides, and the like. Likewise, searching for NRPS gene families from genomic predictions may be accomplished using software such as anti-SMASH.
Polyketide synthase gene family screening: polyketide synthases (T1 PKS) are similar to polyketide synthases and are used to synthesize polyketide secondary metabolites, but differ greatly in structure and function. The T1PKS gene family was searched from the genomic prediction.
Other gene family screening: based on the biological characteristics of the different classes of secondary metabolites, other potential gene families can be further screened for annotation results and identified.
S4, comparing and analyzing the PCR product combined with the DNA sequencing data of the strain, and judging that the DNA sequence is complete according to the analysis result;
the S4 is used for completely analyzing the DNA sequence, and comprises the following steps:
s4.1, combining the PCR product obtained in the step S3.2 with the sequence data of the strain obtained in the step S2.2 for comparison and analysis; after the PCR product is obtained, it is subjected to sequencing analysis and comparison. The alignment method includes BLAST and Clustal common alignment tools. The sequence is checked for accuracy and integrity.
S4.2, judging the sequence data accuracy of the strain according to the analysis result obtained in the S4.1, and performing secondary comparison if mutation exists.
The step of performing secondary comparison in S4.2 is as follows:
s4.2.1, analyzing mutation data in the comparison process;
s4.2.2, determining to perform secondary sequencing comparison on the strain sequence data according to the analysis result of S4.2.1. The method comprises the following steps:
sequencing analysis: after the PCR product is subjected to high-throughput sequencing of the Illumina platform, different software tools can be used for cutting sequencing data, removing low-quality sequences, performing error correction, removing joints and other pretreatment processes. The processed high quality sequencing data can be used for annotation and alignment analysis.
And (3) comparison: the correctness and relativity of the sequence are further verified by comparing the sequencing data after the alignment treatment with sequences in a database of known sequences (such as NCBI and the like). The operation of the referenceable alignment may be performed using a computing tool such as Bowtie, BWA or BLAST, etc.
The secondary comparison method comprises the following steps: further alignment of default alignment results generated by conventional alignment algorithms (e.g., BLAST or BWA) helps to further improve alignment accuracy. By using software tools such as SAMtools, GATK or Picard, etc. to rank the data, eliminating repeated reads and generating analysis results such as sequence depth, GC content and variation based on information such as base quality, etc., potential secondary metabolite genes can be more accurately identified and screened.
S5, selecting and searching a corresponding vector according to DNA sequence data of the strain, selecting matched host bacteria, and transforming the vector into the host bacteria for heterologous expression;
the step of transforming the S5 binding vector into host bacteria for heterologous expression is as follows:
s5.1, collecting sequence data of secondary metabolites of the strain, and selecting corresponding carrier data according to analysis of the sequence data;
s5.2, combining sequence data of the secondary metabolite with corresponding vector data, selecting corresponding host bacteria according to the strain data, and putting the combined data into the host bacteria for heterologous expression, so that the strain gene activity is obtained. If it is desired to optimize the expression of the target gene, a specific vector, for example, a vector containing a strong promoter or reporter gene may be selected, and the expression amount and activity of the target gene may be improved.
S6, separating and purifying the metabolite in the S5 heterologous expression process, and evaluating the target secondary metabolite so as to judge the comprehensive characteristic data of the metabolite.
The step of S6 judging the comprehensive characteristic data of the strain is as follows:
s6.1, collecting and analyzing process data of the heterologous expression of S5.2, selecting the most obvious metabolite in the process data according to an analysis result, and separating and purifying the metabolite by NMR; the usual hydrogen nuclear magnetic resonance spectrum formula data include the following parameters:
chemical shift (chemical shift): the displacement of the hydrogen nuclei in the sample relative to the reference compound is indicated generally by ppm (partspermillion). Chemical shift can be expressed by the formula:
；
wherein the resonance frequency of the hydrogen nuclei in the sample is the resonance frequency of the hydrogen nuclei in the reference compound.
Wherein the method comprises the steps ofFor the resonance frequency of the hydrogen nuclei in the sample, < >>Is the resonance frequency of the hydrogen nuclei in the reference compound.
Seed number (couplingconstate) J: indicating the interaction between two adjacent hydrogen nuclei, typically expressed in Hz. The seed number can be calculated by the double t-peak occurring between the two hydrogen nuclei of the oxoalkane.
Seed coupling mode (couplingpattern): the manner of interaction between adjacent two hydrogen nuclei in the resulting spectral line is described. The seed-coupled mode may be a singlet t-peak, a doublet t-peak, a triplet Q-peak, a quartet, or the like. Seed chemical shift (coupling shift) dj: the difference in magnetic field between two adjacent hydrogen nuclei is represented and is determined by the coupling constant J. Seed chemical shift can be expressed by the formula:
；
wherein the method comprises the steps ofIs the resonant frequency.
S6.2, according to the judgment and analysis of the separation and purification results of the metabolites, the heterologously expressed metabolites are separated and purified by adopting High Performance Liquid Chromatography (HPLC), mass Spectrum (MS) or hydrogen Nuclear Magnetic Resonance (NMR) and other technologies. From which the target secondary metabolite is detected and by comparison, judgment and identification, the type, structure and characteristics of the target metabolite are confirmed. The metabolite species, structure and characteristics are obtained from the analysis results.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments, and that the above-described embodiments and descriptions are only preferred embodiments of the present invention, and are not intended to limit the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A method for identifying and screening marine streptomycete secondary metabolite genes is characterized in that: the method comprises the following steps:
s1, collecting strains of marine streptomyces in a selected marine area, and evaluating the strains with the production potential of secondary metabolites in the marine streptomyces;
s2, extracting DNA of a strain with a secondary metabolite production potential, and sequencing according to the DNA of the strain;
s3, performing PCR amplification by taking DNA of the strain as a template, wherein:
s3.1, collecting and analyzing secondary metabolite data of the strain according to the extracted DNA information, and evaluating by combining the analysis data with the existing network sequence information, so that a primer is arranged in the secondary metabolite;
s3.2, carrying out PCR amplification on the extracted DNA information by combining the primer set in the S3.1, thereby obtaining a PCR product;
s3.3, carrying out feature extraction on secondary metabolites of the strain, and selecting corresponding gene families in a network according to feature extraction data;
s3.4, combining the selected gene family with the data of the secondary metabolite to perform comparison and analysis, obtaining a genome annotation result of the secondary metabolite according to the analysis result, and screening and identifying the genome annotation result;
s4, comparing and analyzing the PCR product combined with the DNA sequencing data of the strain, and judging that the DNA sequence is complete according to the analysis result;
s5, selecting and searching a corresponding vector according to DNA sequence data of secondary metabolites of the strain, selecting matched host bacteria, and transforming the vector into the host bacteria for heterologous expression;
s6, separating and purifying the metabolite in the S5 heterologous expression process, and evaluating the target secondary metabolite so as to judge the comprehensive characteristic data of the metabolite.
2. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 1, wherein: the evaluation steps of the secondary metabolite production potential in S1 marine Streptomyces are as follows:
s1.1, selecting a marine area according to the type of a strain required;
s1.2, collecting strains in the ocean area, detecting and analyzing the strains, and selecting the strains with the production potential of secondary metabolites according to the judgment and analysis results.
3. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 2, wherein: the step of sequencing the DNA of the strain by the S2 is as follows:
s2.1, extracting DNA information of the strain selected according to the S1.2;
s2.2, interrupting the DNA information extracted in the step S2.1, selecting an aptamer for library construction, analyzing and sequencing the library construction, and obtaining the sequence data of the strain according to a sequencing result.
4. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 3, wherein: the S4 is used for completely analyzing the DNA sequence, and comprises the following steps:
s4.1, combining the PCR product obtained in the step S3.2 with the sequence data of the strain obtained in the step S2.2 for comparison and analysis;
s4.2, judging the sequence data accuracy of the strain according to the analysis result obtained in the S4.1, and performing secondary comparison if mutation exists.
5. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 4, wherein: the step of performing secondary comparison in S4.2 is as follows:
s4.2.1, analyzing mutation data in the comparison process;
s4.2.2, determining to perform secondary sequencing comparison on the strain sequence data according to the analysis result of S4.2.1.
6. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 1, wherein: the step of transforming the S5 binding vector into host bacteria for heterologous expression is as follows:
s5.1, collecting sequence data of secondary metabolites of the strain, and selecting corresponding carrier data according to analysis of the sequence data;
s5.2, combining sequence data of the secondary metabolite with corresponding vector data, selecting corresponding host bacteria according to the strain data, and putting the combined data into the host bacteria for heterologous expression, so that the strain gene activity is obtained.
7. The method for identifying and screening marine Streptomyces secondary metabolite genes according to claim 6, wherein: the step of S6 judging the comprehensive characteristic data of the strain is as follows:
s6.1, collecting and analyzing process data of the heterologous expression of S5.2, selecting the most obvious metabolite in the process data according to an analysis result, and separating and purifying the metabolite by NMR;
s6.2, judging and analyzing according to the separation and purification results of the metabolite, and obtaining the type, structure and characteristics of the metabolite according to the analysis results.
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