A method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments

Abstract

The present invention relates to the field of environmental assessment technology, and in particular to a method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments, comprising the following steps: collecting and preserving offshore sediment samples; extracting and testing DNA from offshore sediment samples; fragmenting the DNA of offshore sediment samples that have passed the quality test and constructing a PE library; performing metagenomic sequencing on the offshore sediment samples, completing assembly and non-redundant gene set construction; determining key genes and proteins in the carbon fixation pathway and determining their numbers in the KEGG database; and obtaining carbon fixation pathways and autotrophic microbial population information in a specific environment by analyzing and comparing the gene abundance of corresponding key proteins in different pathways in different environments. The present invention can indirectly reflect the material metabolic pathways in the environment by identifying key and characteristic proteins in the environment, clarify the importance of the role played by different pathways, and obtain information on the types of microorganisms that travel different pathways.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental assessment, and in particular to a method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments. Background Art

[0002] Seagrass beds are an important component of coastal ecosystems. Their growth, reproduction and death processes play an important role in the carbon cycle. They also have ecological and environmental functions such as regulating water quality, capturing freshwater nutrients, and providing habitats for organisms.

[0003] Seagrass beds account for less than 0.2% of the ocean area, but contribute up to 10% of global ocean carbon storage. The huge carbon sequestration potential of seagrass bed ecosystems mainly comes from their relatively high net primary productivity and slow decomposition of organic matter.

[0004] The existing research on carbon fixation in seagrass beds is mainly focused on a single carbon fixation pathway, the Calvin cycle. Information such as the microbial diversity and community structure of the cycle is obtained through cloning library technology. This method obtains a very limited number of microbial species and ignores some autotrophic microbial groups with relatively low abundance. Seagrass bed sediments are an anaerobic or anoxic environment. The carbon fixation function in this environment may not be the most commonly studied Calvin cycle (mainly under aerobic conditions), but there may be more chemoautotrophic microbial species and anaerobic carbon fixation pathways. Therefore, how to analyze the most important carbon fixation pathway in this environment and discover carbon-fixing microorganisms that may not be abundant in the environment can provide guidance for accurately studying the carbon cycle process in seagrass beds and improving the carbon fixation potential of seagrass beds.

[0005] High-throughput sequencing technology has been widely used in the study of microbial diversity and community structure in specific environments. Through gene sequencing technology, nucleic acid and protein sequences in the environment are obtained. Then, by comparing the obtained sequences with existing data in the database and using similarities to annotate species, all species diversity information in the environment can be obtained.

[0006] Since carbon-fixing autotrophic microorganisms have weak species specificity and are widely distributed, it is impossible to identify which autotrophic microorganisms have carbon-fixing functions by microbial species. Therefore, the identification of carbon-fixing autotrophic organisms is completed through genes with specific functions (such as genes that specifically perform carbon fixation functions). That is, genes with specific functions are used as molecular markers to identify microorganisms that can perform corresponding functions.

[0007] For the study of specific functional genes, the traditional method is to use clone library technology, but clone library technology is cumbersome and the amount of data obtained is much lower than that of high-throughput sequencing. Performing hundreds of clones in one experiment consumes a huge amount of work.

[0008] Therefore, how to use high-throughput sequencing technology to analyze the diversity of microorganisms that perform carbon fixation functions in offshore sediments and determine the main carbon fixation pathways is a technical problem that technicians in this field urgently need to solve. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments, which can more comprehensively and accurately identify the carbon fixation pathways and autotrophic microbial species in environmental samples.

[0010] The present invention provides a method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments, comprising the following steps:

[0011] S1. Collection and preservation of offshore sediment samples;

[0012] S2. DNA extraction and quality testing of offshore sediment samples;

[0013] S3, fragmenting the DNA of offshore sediment samples that have passed the quality inspection and constructing a PE library;

[0014] S4. Perform metagenomic sequencing on offshore sediment samples and complete assembly and construction of non-redundant gene sets;

[0015] S5. Identify the key genes and proteins in the carbon fixation pathway and determine their KEGG numbers;

[0016] S6. By analyzing and comparing the gene abundance of corresponding proteins in different pathways in different environments, we can obtain information about the most important carbon fixation pathways in a specific environment and the microbial species in different pathways;

[0017] There is no limitation on the order of steps S2 and S5.

[0018] As a preferred embodiment of the present technical solution, in step S1, the collected offshore sediment samples are divided into two groups, one group is air-dried at room temperature for determination of physical and chemical parameters; the other group is stored at -20°C for DNA extraction and metagenomic sequencing.

[0019] As a preferred embodiment of the present technical solution, in step S2, the quality detection of the offshore sediment sample DNA includes detection of DNA concentration and purity;

[0020] Preferably, the concentration of DNA in each sample is ≥2 ng / μL, and the amount of DNA in each sample is greater than 0.25 μg.

[0021] As a preferred embodiment of the present technical solution, step S3 specifically includes: connecting the DNA fragments and the adapter, screening and removing the adapter self-ligated fragments, enriching the DNA template using PCR, recovering the PCR products, and obtaining a PE library;

[0022] Preferably, the length of the DNA fragment is 380-420 bp.

[0023] As a preferred embodiment of the present technical solution, in step S4, after the offshore sediment sample is subjected to metagenomic sequencing, quality control is performed to remove adapters and reads with a length less than 50 bp and an average base quality value less than 20.

[0024] As a preferred embodiment of the present technical solution, in step S4, during the splicing and assembly, the sequences meeting the quality requirements are spliced ​​and assembled using splicing software, and overlapping groups ≥300 bp are screened as splicing and assembly results.

[0025] As a preferred embodiment of the present technical solution, in step S4, when constructing the non-redundant gene set, first, open reading frames are predicted for the overlapping groups, genes with nucleic acid lengths greater than or equal to 100 bp are selected, and they are translated into amino acid sequences; the gene sequences predicted for all samples are clustered, and the longest gene in each category is taken as the representative sequence to construct a non-redundant gene set.

[0026] As a preferred embodiment of the present technical solution, in step S5, the carbon fixation pathway includes CBB cycle, rTCA cycle, rAcCoA pathway, 3HP cycle, 3HP / 4HB cycle and DC / HB cycle;

[0027] Among them, the CBB cycle is numbered M00165 in KEGG, its key gene and protein are Ribulose-bisphosphate carboxylase (encoding gene rbcL), and the protein is numbered K01601 in KEGG;

[0028] The rTCA cycle is numbered M00173 in KEGG, and its key gene and protein are ATP citrate lyase (encoding gene aclA), and the protein is numbered K15230 in KEGG;

[0029] The rAcCoA pathway is numbered M00377 in KEGG, and its key gene and protein are 5-methyltetrahydrofolate corrinoid / iron sulfur protein methyltransferase (encoding gene acsE), and the protein is numbered K15023 in KEGG.

[0030] The 3HP cycle is numbered M00376 in KEGG, and its key gene and protein are 2-methylfumaryl-CoAisomerase (encoding gene mct), and the protein is numbered K14470 in KEGG;

[0031] The 3HP / 4HB cycle is numbered M00375 in KEGG, and its key gene and protein are 3-hydroxypropionyl-coenzyme A synthetase, which is numbered K15018 in KEGG.

[0032] The DC / HB cycle is numbered M00374 in KEGG, and its key gene and protein are 4-hydroxybutyrate---CoA ligase (encoding gene 4hbl), and the protein is numbered K14467 in KEGG.

[0033] As a preferred embodiment of the present technical solution, step S6 specifically includes comparing the amino acid sequence of the non-redundant gene set with the KEGG database to obtain the KEGG function corresponding to the gene; using the gene abundance to obtain the abundance of the corresponding functional category, and obtaining the carbon fixation pathway in a specific environment and the microbial species information of different pathways. If this characteristic protein exists in a certain sample, it is considered that the environment contains this carbon fixation pathway. The more genes containing this protein, the greater the role of this pathway in this environment.

[0034] As a preferred embodiment of this technical solution, the physical and chemical parameters include sediment water content, sediment pH, sediment organic matter content and sediment nitrogen and phosphorus content.

[0035] Compared with the existing technology, the method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments of the present invention has the following advantages:

[0036] The present invention provides a method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments. The method mainly comprises: performing metagenomic sequencing and species annotation on the sample tested, and simultaneously determining the key and characteristic proteins of six carbon fixation pathways and their numbered KOs in the KEGG database. Through analysis of the abundance of different KOs and species annotation, the most important carbon fixation pathways in a specific environment and information on the microbial species of different pathways are obtained. That is, if the corresponding encoded protein is found in the environmental sample, it is considered that this environment has this carbon fixation metabolic pathway. The abundance of microorganisms with six different metabolic pathways is used to determine which pathway plays the most important role in carbon fixation. The microbial diversity and community structure of the corresponding pathways can also be analyzed. Compared with traditional clone library technology, this method can simultaneously study six different carbon fixation pathways in offshore areas using data obtained by the same method. The data can be compared horizontally to assess the importance of different pathways in a specific environment. Traditional clone library technology can only be used to analyze the importance of a single pathway, which cannot be compared horizontally. In addition, the microbial diversity obtained by traditional clone library technology is relatively low in abundance, and even with a large workload, only information on a few hundred microbial species can be obtained. However, metagenomic methods based on high-throughput sequencing can obtain information on thousands of species, with a throughput several orders of magnitude higher, and can more comprehensively and accurately reflect the information on carbon-fixing autotrophic microbial populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 The geographical location of the site is used in the embodiment of the present invention, where TEH represents Swan Lake, LDW represents Lidao Bay, and SGW represents Sanggou Bay;

[0039] Figure 2 is the abundance of key proteins in different carbon fixation pathways in the examples of the present invention;

[0040] Figure 3 This is a histogram of the autotrophic community structure (phylum level) of the present invention that uses the CBB cycle;

[0041] Figure 4 CCA analysis of the relationship between the autotrophic community structure and environmental factors of the CBB cycle of the present invention

[0042] Figure 5 This is a histogram of the community structure (phylum level) of autotrophic organisms that operate the rAcCoA pathway of the present invention;

[0043] Figure 6 RDA analysis of the relationship between the autotrophic community structure and environmental factors of the rAcCoA pathway of the present invention;

[0044] Figure 7 The histogram of the autotrophic community structure (class level) of the present invention using the 3HP cycle;

[0045] Figure 8 This is the CCA analysis of the relationship between the autotrophic community structure and environmental factors of the 3HP cycle of the present invention. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Example

[0050] S1. Collection and preservation of offshore sediment samples

[0051] First, sampling was carried out from three representative offshore sites in Weihai, which are located in Swan Lake, Lidao Bay and Sanggou Bay respectively. Three replicates were taken from each sampling site in sediments with and without giant algae seaweed, for a total of 18 samples, which were named TEH_S1, TEH_S2, TEH_S3, TEH_C1, TEH_C2, TEH_C3, LDW_S1, LDW_S2, LDW_S3, LDW_C1, LDW_C2, LDW_C3, SGW_S1, SGW_S2, SGW_S3, SGW_C1, SGW_C2, SGW_C3. The suffix S represents samples with seaweed, and the suffix C represents sediment controls without seaweed. The sampling sites are as follows: Figure 1 shown.

[0052] The sample collection point was located 0-15 cm from the sediment surface. The collected nearshore sediment samples were divided into two groups. One group was air-dried at room temperature for the determination of physical and chemical parameters (sediment moisture content, sediment pH, sediment organic matter content, and sediment nitrogen and phosphorus content); the other group was stored at -20°C for DNA extraction and metagenomic sequencing.

[0053] S2. DNA extraction and quality testing of offshore sediment samples

[0054] Genomic DNA was extracted from the samples using Soil DNA Kit (Omega Bio-tek, USA) was used. DNA concentration and purity were then determined using a micro-fluorescence analyzer (TBS-380) and a NanoDrop 200 spectrophotometer, respectively. DNA sample concentrations ≥ 2 ng / μL and sample volumes greater than 0.25 μg were sufficient for subsequent PE library construction.

[0055] S3. Fragment the DNA of offshore sediment samples that have passed the quality inspection and construct a PE library

[0056] The DNA was fragmented using an automatic focusing acoustic genome shearer (Gene Corporation, China), and fragments of approximately 400 bp were screened for the construction of a PE library.

[0057] The library was constructed using the DNA library construction kit NEXTflex™ Rapid DNA-Seq (Bioo Scientific, USA). The specific process involved first ligating the DNA fragments to the adapters, then removing the adapter-ligated fragments using magnetic bead screening, and then enriching the DNA templates by PCR. Finally, the PCR products were recovered using magnetic beads to obtain the final PE library.

[0058] S4. Perform metagenomic sequencing on offshore sediment samples and complete assembly and non-redundant gene set construction

[0059] Metagenomic sequencing was performed at Shanghai Meiji Biopharmaceutical Technology Co., Ltd. using the Illumina NovaSeq (Illumina, USA) sequencing platform. Data quality control was performed using fastp software (version 0.20.0), removing adapters and reads less than 50 bp in length and with an average base quality score less than 20. Sequences meeting quality requirements were then assembled using the MEGAHIT assembly software, with contigs ≥300 bp selected as the final assembly. Open reading frames (ORFs) were then predicted for these contigs using MetaGene. Genes with a nucleic acid length of 100 bp or longer were selected and translated into amino acid sequences. The predicted gene sequences for all samples were clustered using CD-HIT (90% similarity and 90% coverage), with the longest gene in each cluster selected as the representative sequence to construct a non-redundant gene set. High-quality reads from each sample were aligned to the non-redundant gene set using SOAPaligne software (95% similarity), and gene abundance in the corresponding sample was calculated.

[0060] S5. Identify the key genes and proteins of the six carbon fixation pathways and determine their KEGG numbers

[0061] CBB cycle, the pathway is numbered M00165 in KEGG, the key gene and protein in this pathway is Ribulose-bisphosphate carboxylase (encoding gene rbcL), and the protein KEGG number is K01601;

[0062] rTCA cycle, which is numbered M00173 in KEGG. The key gene and protein in this pathway is ATP citratelyase (encoding gene aclA), and the protein KEGG number is K15230;

[0063] rAcCoA pathway, which is numbered M00377 in KEGG. The key gene and protein in this pathway are 5-methyltetrahydrofolate corrinoid / iron sulfur protein methyltransferase (encoding gene acsE), and the protein KEGG number is K15023;

[0064] 3HP cycle, the pathway is numbered M00376 in KEGG, the key gene and protein of the pathway is 2-methylfumaryl-CoA isomerase (encoding gene mct), and the protein KEGG number is K14470;

[0065] 3HP / 4HB cycle, the pathway is numbered M00375 in KEGG, and the key gene and protein in this pathway is 3-hydroxypropionyl-coenzyme A dehydratase, with the protein KEGG number K15019;

[0066] DC / HB cycle, this pathway is numbered M00374 in KEGG, the key genes and proteins in this pathway are 4-hydroxybutyrate---CoA ligase (encoding gene 4hbl), and the protein KEGG number is K14467.

[0067] S6. By analyzing and comparing the gene abundance of corresponding proteins in different pathways in different environments, we can obtain the most important carbon fixation pathways in specific environments and the information on the microbial species in different pathways.

[0068] After identifying the key characteristic proteins of the six autotrophic carbon fixation pathways in the KEGG database, the amino acid sequences of the non-redundant gene set were aligned with the KEGG database using Diamond software (BLASTP alignment parameters were set to an expected e-value of 1e-5) to obtain the KEGG functions corresponding to the genes. The abundance of the corresponding functional category was calculated by summing the gene abundances corresponding to the KO and module.

[0069] Depend on Figure 2 It can be seen that the CBB cycle is the most important carbon fixation pathway in the sampled environment, followed by the rAcCoA carbon fixation pathway. These two pathways are absolutely dominant in Sanggou Bay and Swan Lake areas. Although the other three carbon fixation pathways also exist in Sanggou Bay and Swan Lake areas, their role is very small. Lidao Bay is different from the other two. The CBB cycle and 3HP cycle are the most important carbon fixation pathways. The DC / HB cycle was not detected at all sites.

[0070] The present invention further analyzed the community structure of autotrophic organisms in the main carbon fixation pathways of sediment samples, specifically by constructing gene sets using the KEGG numbers of the key proteins of each carbon fixation pathway. Therefore, three gene sets were constructed, named K01601, K15023, and K14470. Each gene set was then annotated with species, and the amino acid sequences of the non-redundant gene sets were compared with the NR database using Diamond software (the BLASTP comparison parameter set the expected value e-value to 1e-5), and the species annotations were obtained through the taxonomic information database corresponding to the NR library. The abundance of the species was then calculated using the sum of the gene abundances corresponding to the species, and the composition ratio of different species at the phylum level was calculated. The relationship between environmental factors (nitrogen, phosphorus, pH, organic matter, etc.) and different autotrophic community structures was analyzed by redundancy analysis RDA / CCA. Among them, the community structure of autotrophic organisms that carry out the CBB cycle is as follows: Figure 3 , and its relationship with environmental factors such as Figure 4 As shown: The structure of the autotrophic community that carries out the rAcCoA pathway is as follows Figure 5 Its relationship with environmental factors is shown in Figure 6 As shown: the structure of the autotrophic community that carries out 3HP cycle and Figure 7 The relationship with environmental factors is shown in Figure 8 shown.

[0071] Figure 3 This is a histogram of the community structure (phylum level) of autotrophic organisms that run the CBB cycle. This figure does not show the Proteobacteria, which has the highest content. Proteobacteria has the highest abundance in all samples and is absolutely dominant, with a minimum of about 70% and a maximum of 85%. Figure 3 It can be seen that the autotrophic community structure of Lidao Bay LDW is significantly different from that of Sanggou Bay and Swan Lake. This may be because the seagrass beds in Lidao Bay are closer to the village and are more seriously polluted by life than the other two sites. This conclusion can also be seen from Figure 4 The results (arrows represent environmental factors; longer lines indicate greater influence; points represent samples; smaller distances from the sample point to the environmental factor projection to the arrow point indicate a greater impact) show that nitrogen and phosphorus content have the most significant impact on the community structure of the CBB cycle. Samples from Lidao Bay are significantly more affected by nitrogen and phosphorus than those from Sanggou Bay and Swan Lake. Autotrophic biodiversity in Lidao Bay is also lower than in other areas, suggesting that the detrimental effects of organic pollution on the carbon sequestration capacity of seagrass beds warrant attention.

[0072] Figure 5This is a bar graph of the community structure (phylum level) of autotrophic organisms that travel the rAcCoA pathway. This graph does not show the Proteobacteria, which has the highest abundance. Proteobacteria has the highest abundance in all samples and is absolutely dominant. Figure 2 It can be seen that this pathway mainly exists in the seagrass beds of Sanggou Bay and Swan Lake, and Chloroflexi is the most abundant species except Proteobacteria. Figure 6 It can be seen that N, P and organic matter content have the most significant effects on the community structure of the rAcCoA pathway, organic matter has a particularly significant impact on Sanggou Bay, and nitrogen content has the most significant impact on Swan Lake.

[0073] Figure 7 The histogram of the autotrophic community structure (class level) of the 3HP cycle shows that α-Proteobacteriahe has the highest abundance in the samples of Lidao Bay, while SGW and THE play a lower role in this pathway ( Figure 2 ).Depend on Figure 8 It can be seen that N, P and pH have the most significant effects on the community structure of the 3HP cycle, and P has the greatest impact on the Lidao Bay samples.

[0074] In summary, the analysis of different carbon fixation pathways through the KO numbers of key proteins in the KEGG database revealed that the CBB cycle is the main carbon fixation pathway in the seagrass bed sediments in Weihai. Due to the serious pollution from domestic life, the carbon fixation autotrophic biodiversity of the seagrass beds in Lidao Bay has decreased, and the main carbon fixation pathways are also different from those in Sanggou Bay and Swan Lake. Therefore, the present invention can indirectly reflect the material metabolic pathways in a specific environment by identifying key and characteristic proteins in the environment, and clarify the importance of the roles played by different pathways by comparing the relative abundance of proteins; and by annotating the proteins with species, obtain information on the types of microorganisms that travel in different pathways, thereby completing a complete analysis of the carbon fixation pathways and microbial community structure of the autotrophic microorganisms in the seagrass bed sediments, providing theoretical guidance for improving the carbon sequestration capacity of offshore areas.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing autotrophic microbial flora and carbon fixation pathways in offshore sediments, characterized in that: The following steps are involved: S1. Collection and preservation of offshore sediment samples; S2. DNA extraction and quality testing of offshore sediment samples; S3, fragmenting the DNA of offshore sediment samples that have passed the quality inspection and constructing a PE library; S4. Perform metagenomic sequencing on offshore sediment samples and complete assembly and construction of non-redundant gene sets; S5. Identify the key genes and proteins in the carbon fixation pathway and determine their KEGG numbers; S6. By analyzing and comparing the gene abundance of corresponding proteins in different pathways in different environments, information about carbon fixation pathways in specific environments and the microbial species in different pathways can be obtained; Among them, steps S2 and S5 are not limited in order; In step S5, the carbon fixation pathway includes CBB cycle, rTCA cycle, rAcCoA pathway, 3HP cycle, 3HP / 4HB cycle and DC / HB cycle; Among them, the CBB cycle is numbered M00165 in KEGG, and its key gene and protein are Ribulose-bisphosphate carboxylase, and the protein is numbered K01601 in KEGG; The rTCA cycle is numbered M00173 in KEGG, and its key gene and protein are ATP citrate lyase, and the protein is numbered K15230 in KEGG; The rAcCoA pathway is numbered M00377 in KEGG, and its key gene and protein are 5-methyltetrahydrofolate corrinoid / iron sulfur protein methyltransferase, and the protein is numbered K15023 in KEGG; The 3HP cycle is numbered M00376 in KEGG, and its key gene and protein are 2-methylfumaryl-CoAisomerase, and the protein is numbered K14470 in KEGG; The 3HP / 4HB cycle is numbered M00375 in KEGG, and its key gene and protein are 3-hydroxypropionyl-coenzyme A synthetase, which is numbered K15018 in KEGG. The DC / HB cycle is numbered M00374 in KEGG, and its key gene and protein are 4-hydroxybutyrate---CoAligase, and the protein is numbered K14467 in KEGG; Step S6 specifically includes comparing the amino acid sequences of the non-redundant gene set with the KEGG database to obtain the KEGG functions corresponding to the genes; using the gene abundance to obtain the abundance of the corresponding functional category, and obtaining the carbon fixation pathways in a specific environment and the microbial species information of different pathways.

2. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: In step S1, the collected offshore sediment samples are divided into two groups. One group is air-dried at room temperature for determination of physical and chemical parameters; the other group is stored at -20°C for DNA extraction and metagenomic sequencing.

3. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: In step S2, the quality test of the offshore sediment sample DNA includes the test of DNA concentration and purity; The DNA concentration in each sample was ≥2 ng / μL, and the amount of each sample was greater than 0.25 μg.

4. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: Step S3 specifically includes: connecting the DNA fragments and the adapter, screening and removing the adapter self-ligated fragments, enriching the DNA template using PCR, recovering the PCR products, and obtaining a PE library; The length of the DNA fragment is 380-420 bp.

5. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: In step S4, after metagenomic sequencing of offshore sediment samples, quality control is performed to remove adapters and reads with lengths less than 50 bp and average base quality values ​​less than 20.

6. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: In step S4, during the splicing and assembly, the sequences that meet the quality requirements are spliced ​​and assembled using splicing software, and overlapping groups ≥300 bp are screened as splicing and assembly results.

7. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 1, characterized in that: In step S4, when constructing the non-redundant gene set, first, open reading frames are predicted for the overlapping groups, genes with nucleic acid lengths greater than or equal to 100 bp are selected, and they are translated into amino acid sequences; the gene sequences predicted for all samples are clustered, and the longest gene in each class is taken as the representative sequence to construct the non-redundant gene set.

8. The method for analyzing the autotrophic microbial flora and carbon fixation pathways in offshore sediments according to claim 2, characterized in that: The physical and chemical parameters include sediment water content, sediment pH, sediment organic matter content, and sediment nitrogen and phosphorus content.

Images