Microflora specific function gene diversity analysis primer pairs and analysis method
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A technology for diversity analysis and microbial community, applied in the field of microbial community-specific functional gene diversity analysis primer pairs and analysis
Active Publication Date: 2020-06-02
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 functional genes and primers thereof for the diversity analysis of specific functional genes of microbial communities, the specific circumstances are as follows:
[0070] A pair of primers for amplifying the nifH gene of a nitrogen-fixing microbial community, the specific sequence of which is:
[0071] Primer F (nifH): AAAGGYGGWATCGGYAARTCCACCAC (SEQ ID NO. 1)
[0072] Primer R (nifH): TTGTTSGCSGCRTACATSGCCATCAT (SEQ ID NO. 2)
[0073] A pair of primers for amplifying the nirS gene of a denitrifying microbial community, the specific sequence of which is:
[0074] Primer F (Cd3a): GTSAACGTSAAGGARACSGG (SEQ ID NO.3)
[0075] Primer R (R3cd): GASTTCGGRTGSGTCTTGA (SEQ ID NO.4), a primer pair for amplifying the nirK gene of denitrifying microbial communities, the specific sequence is:
[0076] Primer F (nirK1aCu): ATCATGGTSCTGCCGCG (SEQ ID NO.5)
[0077] Primer R (nirKR3Cu): GCCTCGATCAGRTTGTGGTT (SEQ ID NO. 6)
[0078] Wherein, in th...
Embodiment 2
[0118] Take a soil sample, extract DNA, and add three kinds of corresponding functional gene primers to amplify the corresponding functional gene. 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 (10μΜ) 1.5μL Primer R (10μΜ) 1.5μL KOD enzyme 1μL template 1-5μL (ie 100ng) h 2 o
Make up 50μL
[0122] The amplification program is: 94°C, 2min; 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 for PCR product purification, after purification, use Qubit3.0 to quantify. Amplification results see figure 1 , image 3 and Figure 5 , PCR amplified bands showed that the specificity of the corresponding functional gene primer amplification was ...
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Abstract
The invention belongs to the field of biological information analysis and in particular relates to microflora specific function gene diversity analysis primer pairs and an analysis method. The invention discloses three microflora specific function gene diversity analysis primer pairs and a method for obtaining corresponding function gene segments by using the three primer pairs. The invention further discloses a microflora specific functional gene diversity analysis method and application of the method in analyzing diversity of three microflora specific functional genes. The microflora specific function gene diversity analysis primer pairs provided by the invention have remarkable amplification advantages, the microflora specific function gene diversity analysis method provided by the invention has a comprehensively integral analysis process, that is, for microbial populations with specific functions, in microflorae, biological information contents with complex function genes can be explored, and microbial diversity of function florae can be dug.
Description
technical field [0001] The invention belongs to the field of biological information analysis, and in particular relates to a pair of primers for analyzing the diversity of specific functional genes of microbial communities and an analysis method. Background technique [0002] Microorganisms commonly exist 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. Microbial community research mainly includes the encoding of enzymes related to biogeochemical cycle processe...
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