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Primer pair and analysis method for microbial community-specific functional gene diversity analysis
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A technology of diversity analysis and microbial community, applied in the field of primer pairs and analysis of specific functional gene diversity analysis of microbial community, to achieve the effect of strong specificity and good amplification effect
Active Publication Date: 2022-07-01
GUANGZHOU GENE DENOVO BIOTECH
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[0003] At present, there is no public technology. It only needs to design primers for the conserved region of a certain type of functional gene, amplify and sequence the sequence of a specific functional gene, and then start the analysis of functional gene diversity, aiming at the microbial population with a specific function in the microbial community Carry out microbial diversity analysis of this functional group
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Embodiment 1
[0069] The present invention provides three kinds of functional genes and their primers for analyzing the diversity of specific functional genes of a microbial community, and the specific conditions are as follows:
[0070] A primer pair for amplifying the nifH gene of nitrogen-fixing microbial community, the specific sequence is:
[0071] Primer F(nifH): AAAGGYGGWATCGGYAARTCCACCAC (SEQ ID NO. 1)
[0072] Primer R (nifH): TTGTTSGCSGCRTACATSGCCATCAT (SEQ ID NO. 2)
[0073] A primer pair for amplifying the nirS gene of a denitrifying microbial community, the specific sequence is:
[0074] Primer F (Cd3a): GTSAACGTSAAGGARACSGG (SEQ ID NO. 3)
[0075] Primer R (R3cd): GASTTCGGRTGSGTCTTGA (SEQ ID NO.4) is a primer pair for amplifying the nirK gene of the denitrifying microbial community, the specific sequence is:
[0076] Primer F (nirK1aCu): ATCATGGTSCTGCCGCG (SEQ ID NO. 5)
[0077] Primer R (nirKR3Cu): GCCTCGATCAGRTGTGGTT (SEQ ID NO. 6)
[0078] Wherein, in the sequence tabl...
Embodiment 2
[0118] Take soil samples, extract DNA, and add primers for three corresponding functional genes to amplify the corresponding functional genes. The steps include:
[0119] (1) The first round of amplification:
[0120] The amplification system is:
[0121] KOD Enzyme Amplification System 50μL 10×Buffer KOD 5μL 2mM dNTPs 5μL 25mM MgSO 4
3μL Primer F (10μM) 1.5μL Primer R (10μM) 1.5μL KOD enzyme 1μL Template 1-5μL (ie 100ng) H 2 O
Make up 50 μL
[0122] The amplification program was: 94°C, 2 min; 98°C, 10s; 58-66°C, 30s; 68°C, 30s; 30 cycles; 68°C, 5min.
[0123] (2) The first round of amplification product purification includes: using AMPure XP Beads to purify the PCR product, and quantifying it with Qubit3.0 after purification. Amplification results see figure 1 , image 3 and Figure 5 , PCR amplification bands show that the specificity of the corresponding functional gene primers is good, and...
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Abstract
The invention belongs to the field of biological information analysis, and particularly relates to a primer and an analysis method for analyzing the diversity of specific functional genes of a microbial community. The invention discloses three primer pairs for analyzing specific functional gene diversity of microbial communities, and a method for obtaining corresponding functional gene fragments by using the three primer pairs. The invention also discloses a microbial community specific functional gene diversity analysis method and its application in analyzing the above-mentioned three microbial community specific functional gene diversity. The three microbial community-specific functional gene diversity analysis primer pairs of the present invention have significant amplification advantages, and the microbial community-specific functional gene diversity analysis method of the present invention has a more comprehensive and integrated analysis process, that is, for the specific functional gene diversity in the microbial community. A microbial population with a specific function can explore the more complex biological information content of functional genes and mine the microbial diversity of this functional population.
Description
technical field [0001] The invention belongs to the field of biological information analysis, and in particular relates to a primer pair and an analysis method for analyzing the diversity of specific functional genes of a microbial community. Background technique [0002] Microorganisms are ubiquitous in various environments and play a role in their respective environments. Among them, some microorganisms with special functions have received extensive attention and research due to the importance or particularity of their functions. Such microorganisms are called functional Microorganisms, such as ammonia oxidizing bacteria, sulfur bacteria, nitrifying bacteria, etc. The genes that govern the important functions of these functional bacteria are called functional genes. Functional genes refer to genes that encode enzymes that catalyze specific biological processes. The study of microbial communities mainly includes the encoding of enzymes related to biogeochemical cycling proc...
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