CN113755606A - Preparation method of high-accuracy functional gene chip for ocean carbon sink - Google Patents
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Abstract
The invention relates to the technical field of ocean carbon sink, in particular to a preparation method of a high-accuracy functional gene chip for ocean carbon sink. The method comprises the following steps: step 1: and constructing a carbon sink functional gene database. Step 2: and acquiring a key functional gene data set in the carbon sink process of the sea area. And step 3: oligonucleotide probes were designed. And 4, step 4: designing and synthesizing functional gene chip. According to the invention, the marine carbon sink key function gene family is more comprehensively covered by large data mining of the metagenome, so that the problem of false negative caused by lack of related gene reference is solved. The invention distinguishes common homologous genes and alleviates the problem of false positive caused by the mixing of homologous gene families in the existing common gene database. In the process of exploring ocean carbon sink, the data result with high accuracy and strong reliability is obtained by an economic and effective method based on the ocean metagenome data set.
Description
Technical Field
The invention relates to the technical field of ocean carbon sequestration, in particular to a preparation method of a high-accuracy functional gene chip for monitoring the carbon sequestration process of ocean micro organisms.
Background
Since the industrial revolution, the great emission of carbon dioxide by human activities, resulting in climate change, has become the largest global environmental problem, seriously threatening the sustainable development of human society. As one of the largest carbon-emitting countries in the world, China firstly puts forward a carbon neutralization schedule, but the China is still in a highly economic growth stage at the present stage and is still in a state of developing countries, so that the 'emission reduction' and 'sink increase' are required to be performed in parallel when the China achieves the carbon neutralization target. In 2019, the climate change meeting of 25 th united nations brings the 'ocean issue' into the meeting agenda for the first time, aims to improve the cognitive ability of each country on the relation between the ocean and the climate change and encourages each country to bring the ocean into a way for coping with the climate change. As a marine country, China has excellent marine natural conditions and excellent space resources, and has the characteristics of long coastline, abundant coastline resources, wide territorial area and the like. Wherein, the total length of the coastline in China exceeds 32600 kilometers, and the coastline longitudinally spans a plurality of climatic zones such as tropical zones, subtropical zones and temperate zones, the territorial area is about 300 ten thousand square kilometers, and the territorial area is about 31.25 percent of the territorial area. The existing research proves that the ocean carbon reservoir is 20 times of the land carbon reservoir and 50 times of the atmospheric carbon reservoir, the ocean is regarded as the important 'regulator' of global climate change as the biggest carbon reservoir on the earth, and the ocean can absorb CO discharged into the atmosphere230% of the total carbon sink potential, the ocean carbon sink chain has huge potential, so that the ocean carbon sink chain key process is analyzed, the ocean negative emission potential is excavated, and the method is one of important ways for assisting China to achieve the aim of carbon neutralization.
Research has been focused for many years primarily on the key marine carbon storage mechanism, Biological Pumps (BP), which indicates that inorganic carbon is converted to organic carbon by photosynthesis, but research accumulation over the last three decades has shown that Particulate Organic Carbon (POC) results in which only about 0.1% of the primary productivity reaches subsea burial, while most POC is converted to CO2And returning to the atmosphere again. In fact, the Organic Carbon in the ocean is mainly present in the form of Dissolved Organic Carbon (DOC), whereas about 95% of the DOC is present as biologically unavailable inert Dissolved Organic Carbon (Recalcitra)nt dispersed Organic Carbon, RDOC). The marine micro biological carbon pump (MCP) proposed by scientists in our country indicates that the marine microorganisms convert organic carbon from an available active state into an unavailable inert state RDOC through physiological ecological and biogeochemical processes, so that the organic carbon can be sequestered on the seabed for thousands of years. Once proposed, MCPs have gained widespread interest and acceptance by international associates, and are known as "behind-the-scenes pushers of huge carbon libraries". MCP emphasizes that microorganisms are main contributors to formation of RDOC, although micro organisms are small, biomass is extremely large, biodiversity is high, and researches on large-scale marine ecosystem experiment systems prove that the microbial carbon pump has extremely high efficiency and can convert active DOC generated by phytoplankton into compounds which are very similar to RDOC in deep sea in less than one year. However, due to the complexity of the marine ecosystem and the carbon sink forming process and the limitation of the existing knowledge system, the difficulty of the research and development of the marine carbon sink checking technology is determined, so far, the key group and the functional gene of the key process of the marine carbon sink chain driven by the micro organisms are not determined internationally, and the carbon fingerprint gene condition of the typical sea area in China is not clear.
China's carbon market as the second market of global quota trading, compared with ' ocean carbon sink ', the ' forestry carbon sink ' has been developed more mature, including the measurement and detection of forest carbon sink amount. The forestry carbon sink enters a carbon trading market and has been successfully traded for a plurality of times, and the ecological benefit value of the forestry carbon sink is gradually realized. For the development of the ocean carbon sink, research on the measurement and detection of the blue carbon sink in the coastal zone is started at present, and mainly relates to mangrove, seaweed beds and the like, but the evaluation standards, the checking standards and the like are still insufficient, and the current stage is still in a blank state. The marine ecosystem is complex, DOC metabolic activities generated by different micro organism representative groups are different, and DOCs with different relative activities are converted into different types of RDOC. At present, the gene chip is one of effective and promising means for exploring the organic carbon metabolism characteristics of marine micro organisms, and the gene chip only contains partial key genes which are interested by researchers, so that the subsequent analysis is simpler, time-saving, economical and effective. With the development of Next Generation Sequencing (NGS), the data volume obtained by sequencing increases exponentially, and the simplicity and effectiveness of subsequent data analysis become one of the important points of attention of people. The traditional Polymerase Chain Reaction (PCR) has high requirements on primer selection and amplification, the primer design is very difficult only for functional genes which are interested by researchers, especially for samples in complex environments, the accuracy of the final amplification result is difficult to grasp, the repeatability of the experiment is low, and by combining the factors, the gene chip shows the specific advantages in the aspects of researching the complex environments such as oceans and analyzing the key process of the carbon sink Chain. The existing gene chip mainly comprises a high-flux qPCR gene chip and a functional gene chip, wherein the high-flux qPCR gene chip cannot link a microbial community structure with the functions of an ecological system of the microbial community structure, the functional gene chip can link species and functions and is more suitable for researching samples from complex environments, but the functional gene chip specially aiming at the ocean carbon sink process is not developed up to now.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a preparation method of a high-accuracy functional gene chip for ocean carbon sink.
The technical scheme of the invention is as follows:
a preparation method of a high-accuracy functional gene chip for ocean carbon sink comprises the following steps:
step 1: and constructing a carbon sink functional gene database.
Step 2: and acquiring a key functional gene data set in the carbon sink process of the sea area.
And step 3: oligonucleotide probes were designed.
And 4, step 4: synthesizing functional gene chip.
Further, the specific steps of step 1 are:
1) obtaining basic nucleotide and protein sequences of a micro-organism key gene family involved in the ocean carbon sink process, and incorporating partial sequences with 30% sequence similarity as a threshold into a seed database through sequence alignment.
2) Fusing the seed database in the step 1) with a public database, carrying out sequence comparison on the seed database and a public source database, obtaining a homologous sequence belonging to a target genome by using parameters of E value 1E-5 and global sequence similarity of 30%, extracting a sequence of which a comparison result meets a threshold value, incorporating the sequence into the seed database, and removing redundancy of the combined database to form a carbon sink functional gene database.
Further, the specific steps of step 2 are: carrying out voyage survey and on-site sampling on the sea area in China to obtain sea water and sediment samples with different depths, carrying out sea water filter membrane filtration on the sea water samples, extracting DNA of the filter membrane and the sediment samples to obtain total DNA of a microbial community, carrying out metagenome sequencing to obtain a sea metagenome data set, and comparing the sea metagenome data set with a carbon sink function gene database to obtain a key function gene data set in the carbon sink process of the sea area.
Further, in step 3, the oligonucleotide probes are synthesized from sequence-specific probes for a single sequence and group-specific probes for multiple sequences, and the sequence-specific probes and the group-specific probes are subjected to conditional screening to obtain final probes.
Further, the conditions for screening the single sequence specific probes are: sequence specific probes against a single sequence between the non-target sequence: the degree of similarity with the sequence is not higher than 90-95%, the similarity is not higher than 20 continuous bases, and the Gibbs free energy is not lower than-32 kcal/mol.
Further, the screening conditions for the group-specific probes were: between group-specific probes for multiple sequences and non-target sequences: the sequence similarity is not higher than 90-95%, no more than 20 continuous bases are the same, and the Gibbs free energy is not lower than-32 kcal/mol; while among the specific probes belonging to the same group: the sequence similarity is higher than 96% -98%, more than 35 continuous bases are consistent, and Gibbs free energy is not higher than-60 kcal/mol.
Further, the specific steps of step 4 are: selecting a certain amount of carbon sink functional gene probes, arranging the probes on a chip according to a certain coordinate position at a probe theoretical hybridization temperature of 42-45 ℃, hybridizing a plurality of labeled target nucleotide sequences with the probes on the specific sites of the MCPCHIP, and preparing the synthesis functional gene chip MCPCHIP by in-situ synthesis of the probes or attaching the probes to the surface of a solid phase by using an array robot.
The invention achieves the following beneficial effects:
according to the invention, the marine carbon sink key function gene family is more comprehensively covered by large data mining of the metagenome, so that the problem of false negative caused by lack of related gene reference is solved.
The invention distinguishes common homologous genes and alleviates the problem of false positive caused by the mixing of homologous gene families in the existing common gene database.
In the process of exploring ocean carbon sink, the data result with high accuracy and strong reliability is obtained by an economic and effective method based on the ocean metagenome data set.
Drawings
FIG. 1 is a flow chart of the preparation method of a high-accuracy functional gene chip for ocean carbon sink of the present invention.
Detailed Description
To facilitate an understanding of the present invention by those skilled in the art, specific embodiments thereof are described below with reference to the accompanying drawings.
The invention provides a preparation method of a functional gene chip for analyzing ocean carbon sink with high accuracy based on an ocean metagenome dataset, and the flow of the preparation method refers to fig. 1, and the preparation method specifically comprises the following steps:
step 1: construction of high-accuracy carbon sink functional gene database
By genome big data mining, a micro organism key gene family participating in the ocean carbon sink process is determined, and corresponding nucleotide and protein sequences are obtained. The carbon sink functional gene database comprises a plurality of sub-processes related to the carbon sink process, such as carbon fixation, carbon emission, organic matter synthesis, organic matter degradation, organic matter conversion and the like, and functional genes closely related to the carbon sink process mediated by marine microorganisms in the existing functional gene chip system are integrated. The final coverage carbon gene family will be greater than 2,500, containing more than 100,000 gene sequences. In order to make the technical scheme of the invention clearer, the step is further detailed.
(a) By genome big data mining, a micro-organism key gene family which is definitely involved in the ocean carbon sink process is obtained, basic nucleotide and protein sequences are obtained, then, the obtained nucleotide and protein sequences are screened and filtered by using a sequence alignment tool (such as DIAMOND and USEARCH), and partial sequences with the sequence similarity of 30 percent as a threshold value are brought into a seed database.
(b) Fusing a seed database with a public database (such as arcOG, COG, KEGG, eggNOG, GenBank RefSeq and the like), and comparing the sequence of the seed database with a public source database, wherein the use parameter is an E value of 1E-5, the global sequence similarity is 30% so as to obtain a homologous sequence belonging to a target genome, so that the problem of high false positive commonly existing in the existing database is solved. And extracting a sequence of which the comparison result meets a threshold value, bringing the sequence into a seed database, and removing redundancy of the combined database to form a carbon sink functional gene database MCPBase.
Step 2: obtaining key functional gene data set of carbon sink process in typical sea area of China
Carrying out voyage investigation and on-site sampling on a typical sea area (such as a Bohai sea shellfish algae culture area, a yellow sea enteromorpha outbreak area, a south sea coral reef area and other sea areas) in China to obtain sea water and sediment samples at different depths, filtering the sea water sample by using a filter membrane, extracting DNA of the filter membrane and the sediment to obtain total DNA of a microbial community, carrying out metagenome sequencing to obtain a marine metagenome data set, and comparing the marine metagenome data set with a carbon sink function gene database MCPPase to obtain a key function gene data set in the carbon sink process of the typical sea area in China.
And step 3: design of oligonucleotide probes
Because the sample has the problems of high complexity, high functional gene sequence homology and the like, the invention designs two specific probe nucleotide sequences, namely a sequence specific probe aiming at a single sequence and a group specific probe aiming at a plurality of sequences. The final synthesized oligonucleotide probe is about 50 mers in length.
Based on a probe design tool and an algorithm (such as Commoligo), simultaneously taking conditions such as sequence similarity, allowed continuous base length, Gibbs free energy and the like into consideration, and taking 0.1 as a threshold value of global matching, sequencing the oligonucleotide probes to be selected so as to select an optimal object as a final probe. In the present invention, the screening criteria for oligonucleotide probes include the following conditions:
(a) sequence specific probes against a single sequence between the non-target sequence: the degree of similarity with the sequence is not higher than 90-95%, the similarity is not higher than 20 continuous bases, and the Gibbs free energy is not lower than-32 kcal/mol.
(b) Between group-specific probes for multiple sequences and non-target sequences: the sequence similarity is not higher than 90-95%, no more than 20 continuous bases are the same, and the Gibbs free energy is not lower than-32 kcal/mol; while among the specific probes belonging to the same group: the sequence similarity is higher than 96% -98%, more than 35 continuous bases are consistent, and Gibbs free energy is not higher than-60 kcal/mol.
And 4, step 4: chip synthesis
Selecting a certain amount of carbon sink functional gene probes, arranging the probes on a chip according to a certain coordinate position at a probe theoretical hybridization temperature of 42-45 ℃, hybridizing a plurality of labeled target nucleotide sequences with the probes on the specific sites of the MCPCHIP, and preparing the synthesis functional gene chip MCPCHIP by in-situ synthesis of the probes or attaching the probes to the surface of a solid phase by using an array robot.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (7)
1. A preparation method of a high-accuracy functional gene chip for ocean carbon sink is characterized by comprising the following steps:
step 1: constructing a carbon sink functional gene database;
step 2: acquiring a key functional gene data set in the carbon sequestration process of the sea area;
and step 3: designing an oligonucleotide probe;
and 4, step 4: synthesizing functional gene chip.
2. The method for preparing the high-accuracy functional gene chip for the ocean carbon sink according to claim 1, wherein the specific steps of the step 1 are as follows:
1) acquiring basic nucleotide and protein sequences of a key gene family of the micro organisms participating in the ocean carbon sink process, combining high-accuracy gene annotation information, and bringing partial sequences with sequence similarity of 30% serving as a threshold value into a seed database through sequence comparison;
2) fusing the seed database in the step 1) with a public database, carrying out sequence comparison on the seed database and a public source database, obtaining a homologous sequence belonging to a target functional gene by using the parameters of 1E-5 and the global sequence similarity of 30%, extracting a sequence of which the comparison result meets a threshold value, incorporating the sequence into the seed database, and removing redundancy of the combined database to form a carbon sink functional gene database.
3. The method for preparing the high-accuracy functional gene chip for the ocean carbon sink according to claim 1, wherein the specific steps of the step 2 are as follows: carrying out voyage survey and on-site sampling on the sea area in China to obtain sea water and sediment samples with different depths, carrying out filter membrane filtration on the sea water samples, extracting DNA of the filter membrane and the sediment samples to obtain total DNA of a microbial community, carrying out metagenome sequencing to obtain a sea metagenome data set, and comparing the sea metagenome data set with a carbon sink function gene database to obtain a key function gene data set in the carbon sink process of the sea area.
4. The method for preparing the high-accuracy functional gene chip for the ocean carbon sink according to claim 1, wherein the method comprises the following steps: the oligonucleotide probes in step 3 are synthesized from sequence-specific probes for a single sequence and group-specific probes for multiple sequences, which are screened by conditions to obtain the final probes.
5. The method for preparing a high-accuracy functional gene chip for ocean carbon sink according to claim 4, wherein the screening conditions of the single-sequence specific probe are as follows: sequence specific probes against a single sequence between the non-target sequence: the degree of similarity with the sequence is not higher than 90-95%, the similarity is not higher than 20 continuous bases, and the Gibbs free energy is not lower than-32 kcal/mol.
6. The method for preparing a high-accuracy functional gene chip for ocean carbon sink according to claim 4, wherein the screening conditions of the group-specific probes are as follows: between group-specific probes for multiple sequences and non-target sequences: the sequence similarity is not higher than 90-95%, no more than 20 continuous bases are the same, and the Gibbs free energy is not lower than-32 kcal/mol; while among the specific probes belonging to the same group: the sequence similarity is higher than 96% -98%, more than 35 continuous bases are consistent, and Gibbs free energy is not higher than-60 kcal/mol.
7. The method for preparing the high-accuracy functional gene chip for the ocean carbon sink according to claim 1, wherein the specific steps of the step 4 are as follows: selecting a certain amount of carbon sink functional gene probes, arranging the probes on a chip according to a certain coordinate position at a theoretical hybridization temperature of 42-45 ℃, and manufacturing the functional gene chip MCPCHIp by in-situ synthesis of the probes or attaching the probes to the surface of a solid phase by using an array robot.
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| CN115779809A (en) * | 2022-10-10 | 2023-03-14 | 山东大学 | Biological microcapsule for realizing multi-pump cooperation and application |
| CN115779809B (en) * | 2022-10-10 | 2024-05-14 | 山东大学 | Biological microcapsule for realizing multi-pump cooperation and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240418A1 (en) * | 2002-05-03 | 2006-10-26 | Diggans James C | Canine gene microarrays |
| CN105401221A (en) * | 2015-12-29 | 2016-03-16 | 中国科学院天津工业生物技术研究所 | Gene chip for detecting bacteroid community in marine environment and application thereof |
| CN110592185A (en) * | 2018-12-25 | 2019-12-20 | 首都医科大学附属北京安贞医院 | Method for designing hypercholesteremia virulence gene screening probe and gene chip thereof |
| CN112111592A (en) * | 2020-09-22 | 2020-12-22 | 中南大学 | Functional gene chip for identifying medicinal plant species and/or screening potential medicinal components and preparation and use methods thereof |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240418A1 (en) * | 2002-05-03 | 2006-10-26 | Diggans James C | Canine gene microarrays |
| CN105401221A (en) * | 2015-12-29 | 2016-03-16 | 中国科学院天津工业生物技术研究所 | Gene chip for detecting bacteroid community in marine environment and application thereof |
| CN110592185A (en) * | 2018-12-25 | 2019-12-20 | 首都医科大学附属北京安贞医院 | Method for designing hypercholesteremia virulence gene screening probe and gene chip thereof |
| CN112111592A (en) * | 2020-09-22 | 2020-12-22 | 中南大学 | Functional gene chip for identifying medicinal plant species and/or screening potential medicinal components and preparation and use methods thereof |
Non-Patent Citations (1)
| Title |
|---|
| 焦念志等: "实施海洋负排放践行碳中和战略", 中国科学:地球科学, 11 March 2021 (2021-03-11), pages 638 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115779809A (en) * | 2022-10-10 | 2023-03-14 | 山东大学 | Biological microcapsule for realizing multi-pump cooperation and application |
| CN115779809B (en) * | 2022-10-10 | 2024-05-14 | 山东大学 | Biological microcapsule for realizing multi-pump cooperation and application thereof |
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