CN114381502B

CN114381502B - Plant rhizosphere azotobacter diversity detection primer group, kit and method

Info

Publication number: CN114381502B
Application number: CN202111683745.9A
Authority: CN
Inventors: 邓晔; 颜程良; 王尚; 沈文丽; 何晴
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2023-12-29
Anticipated expiration: 2041-12-24
Also published as: CN114381502A

Abstract

The application provides a primer group, a kit and a method for detecting diversity of plant rhizosphere azotobacter. The method comprises the following steps: pretreating the collected plant sample to obtain bacterial suspension; mixing the bacterial suspension calculated by the cell density, an emulsifying agent and a fusion PCR primer group, and connecting and amplifying the nitrogen fixation functional gene and the gene of the marker species information in a single cell to obtain a fusion product; extracting the fusion product, performing agarose gel electrophoresis, and performing gel cutting purification to obtain a purified fusion product; taking the purified fusion product as a template, mixing with a nested PCR primer group, and reacting to obtain a nested PCR product; and preparing a library by using the nested PCR product, sequencing, and analyzing the diversity of the plant rhizosphere azotobacter according to the sequencing result. The functional genes of the microorganisms can be connected with the genes for marking species information at the single cell level, and the relationship between the functional genes of the microorganisms and the species evolution can be analyzed according to the sequencing result. The operation is simple and the cost is low.

Description

Plant rhizosphere azotobacter diversity detection primer group, kit and method

Technical Field

The application relates to the technical field of environmental biology, in particular to a primer group, a kit and a method for detecting diversity of nitrogen-fixing bacteria of plant rhizosphere.

Background

The nitrogen element circulation is a key bio-geochemical process in the ecological circle, is a main nutrient substance for limiting plant growth in the terrestrial ecosystem, and most of nitrogen in the atmosphere is N in an inert form although the nitrogen content is rich ₂ Plants cannot be directly utilized. Nitrogen is therefore the most common limiting nutrient for plant growth. The conversion of inert nitrogen into a form that can be absorbed by plants is mediated by soil microorganisms, and many bacteria are able to convert atmospheric nitrogen into ammonia through biological nitrogen fixation processes. Therefore, it is important to understand the living state of the microorganism group (such as nitrogen-fixing bacteria) having the important function in the environment.

The method for detecting and evaluating the diversity condition of nitrogen-fixing bacteria in natural habitat has a problem that it is difficult to accurately indicate the diversity condition of nitrogen-fixing bacteria. Therefore, a new method for detecting the diversity of nitrogen-fixing bacteria is needed.

Disclosure of Invention

In view of the above, the present application aims to provide a primer set, a kit and a method for detecting the diversity of nitrogen-fixing bacteria in plant rhizosphere.

Based on the above objects, the present application provides a primer set for detecting diversity of nitrogen-fixing bacteria of plant rhizosphere, comprising: fusion PCR primer group:

primer pair for amplifying nitrogen fixation functional genes: the nucleotide sequence of the F1 primer is shown as SEQ ID NO. 1; the nucleotide sequence of the R1-F2' primer is shown as SEQ ID NO. 3;

primer pairs for amplifying marker species information genes: the nucleotide sequence of the R1-F2' primer is shown as SEQ ID NO. 3; the nucleotide sequence of the R2 primer is shown as SEQ ID NO. 2.

In some of these embodiments, further comprising a nested PCR primer pair:

the nucleotide sequence of the F3 primer is shown as SEQ ID NO. 4; the nucleotide sequence of the R3 primer is shown as SEQ ID NO. 5.

In some embodiments, a primer set as described in any one of the preceding claims and a common auxiliary agent are included.

In some of these embodiments, the kit comprises a fusion PCR kit and a nested PCR kit; the fusion PCR kit comprises a fusion PCR primer and a common auxiliary agent; the nested PCR kit comprises nested PCR primers and common auxiliary agents.

The embodiment of the application also provides a method for detecting the diversity of the nitrogen-fixing bacteria of the plant rhizosphere, which adopts the primer as set forth in any one of the preceding claims or the primer in the kit as set forth in any one of the preceding claims, and comprises the following steps:

pretreating the collected plant sample to obtain bacterial suspension;

mixing the bacterial suspension calculated by the cell density, an emulsifying agent and a fusion PCR primer group, and connecting and amplifying the nitrogen fixation functional gene and the gene of the marker species information in a single cell to obtain a fusion product;

extracting the fusion product, performing agarose gel electrophoresis, and performing gel cutting purification to obtain a purified fusion product;

taking the purified fusion product as a template, mixing with a nested PCR primer group, and reacting to obtain a nested PCR product;

and preparing a library by using the nested PCR product, sequencing, and analyzing the diversity of the plant rhizosphere azotobacter according to the sequencing result.

In some of these embodiments, the nitrogen fixation functional gene is the nifH gene and the gene that marks species information is the 16SrRNA gene.

In some of these embodiments, the emulsifier comprises the following volume fractions: 4% UMIL EM90 oil, 0.05% Triton X-100 and 95.95% mineral oil.

In some embodiments, the preparing a library using nested PCR products further comprises: and (3) performing agarose gel electrophoresis on the nested PCR product, and performing gel cutting purification to obtain a purified nested PCR product.

In some of these embodiments, the extracting the fusion product specifically comprises:

collecting the fusion product and performing first centrifugation to remove the upper oil phase;

adding water saturated diethyl ether and performing a second centrifugation treatment to remove an upper liquid phase;

adding water saturated ethyl acetate, and performing third centrifugation to remove an upper liquid phase;

the resultant was placed in a fume hood and air dried.

In some of these embodiments, the cell density calculation specifically comprises:

preparing a fluorescent reagent;

diluting the ATP standard solution to different concentrations, and preparing a standard curve;

fluorescent reagents were added to the bacterial suspension, RLU values were recorded and converted to ATP concentration, and the cell density of the bacterial suspension was calculated.

From the above, the primer group, the kit and the method for detecting the diversity of the nitrogen-fixing bacteria of the plant rhizosphere provided by the application can obtain bacterial suspension by preprocessing the collected plant samples; mixing the bacterial suspension subjected to cell density calculation, an emulsifying agent and a fusion PCR primer group, and connecting and amplifying the nitrogen fixation functional gene and the gene of the marker species information in a single cell to obtain a fusion product; extracting the fusion product, performing agarose gel electrophoresis, and performing gel cutting purification to obtain a purified fusion product; taking the purified fusion product as a template, mixing with a nested PCR primer group, and reacting to obtain a nested PCR product; and preparing a library by using the nested PCR product, sequencing, and analyzing the diversity of the plant rhizosphere azotobacter according to the sequencing result. The functional genes of the functional microorganisms can be connected with the genes of the marker species information on a single cell level, so that the diversity analysis of the functional microorganism groups is realized, the relation between the functional genes of the microorganisms and the species evolution is analyzed according to the sequencing result, and the specificity of functional bacterial community detection in the fields of environment, agriculture and the like is improved. The method has the advantages of simple operation, low cost, higher flux, more definite amplified objects, capability of generating a large amount of effective data and the like.

Drawings

In order to more clearly illustrate the technical solutions of the present application or related art, the drawings that are required to be used in the description of the embodiments or related art will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort to those of ordinary skill in the art.

FIG. 1 is a schematic diagram of fusion PCR and nested PCR;

FIG. 2 is a schematic flow chart of a method for detecting diversity of nitrogen-fixing bacteria of plant rhizosphere according to an embodiment of the present application;

FIG. 3 is a schematic flow chart of example 6;

FIG. 4 is an electrophoretogram after fusion PCR reaction of example 6;

FIG. 5 is an electrophoresis chart after the nested PCR reaction of example 6;

FIG. 6 is a schematic representation of the nested PCR product-generation sequencing results of example 6.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail below with reference to the accompanying drawings.

It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present application should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present application belongs.

Some methods of detecting the diversity of microbial populations (nitrogen-fixing bacteria) rely to a large extent on the isolated culture of microorganisms and assessment of their metabolic capacity. With the development of molecular biology, biological process-critical enzyme genes during various metabolic cycles were selected as biomarkers for assessing the functional diversity status of microorganisms in natural habitats. However, currently, only the functional diversity of nitrogen-fixing bacteria can be analyzed by using a single functional gene, and the species diversity of functional microorganisms cannot be indicated.

Based on this, the embodiment of the application provides that functional sequences are linked with gene fragments of marker species information in a microbial community with complex structure, and amplified and sequenced to determine the species diversity status of specific functional microorganisms. The method can solve the problem that the species diversity of the azotobacter is difficult to accurately indicate in the existing method to a certain extent.

The embodiment of the application provides a plant rhizosphere azotobacter diversity detection primer group, which comprises a fusion PCR primer group:

The fusion PCR has the main function of connecting and amplifying the sequence of the functional gene of the plant rhizosphere azotobacter and the gene of the marker species information to obtain a fusion product of the functional gene and the gene of the marker species information. The principle of fusion PCR is shown in FIG. 1.

In some embodiments, the nitrogen fixation functional gene may be nifH gene and the gene marking species information may be 16S rRNA gene.

Specifically, the primer pairs for amplifying the nitrogen fixation nifH gene are shown in table 1 as follows:

f1 primer: 5'-ATSGCCATCATYTCRCCGGA-3' as shown in SEQ ID NO. 1;

R1-F2' primer: 5 '-ttaccgcgckgctgrcactgcgaxyccsaargcbgactc-3', wherein k=g or T; r=a or G; y=c or T; s=c or G, b= G, T or C, as shown in SEQ ID No. 3.

The primer pairs for amplifying the 16SrRNA gene are shown in Table 1:

R1-F2' primer: 5 '-ttaccgcgckgctgrcactgcgaxyccsaargcbgactc-3', wherein k=g or T; r=a or G; y=c or T; s=c or G, b=g or T or C, as shown in SEQ ID No. 3;

r2 primer: 5'-GGTTACCTTGTTACGACTT-3', which is shown in SEQ ID NO. 2.

The primer can be used for specifically detecting microorganisms with the nitrogen fixation function from a large number of microorganism groups, and can also obtain the fragment sequence of the functional gene nifH for evolution analysis.

TABLE 1 primer sequences

In some embodiments, further comprising a nested PCR primer set: the nucleotide sequence of the F3 primer is shown as SEQ ID NO. 4; the nucleotide sequence of the R3 primer is shown as SEQ ID NO. 5. That is, as shown in table 1, the nested PCR primers were:

f3 primer: 5'-GGCATNGCRAANCCVCCRCANAC-3' as shown in SEQ ID NO. 4;

r3 primer: 5'-GGACTACNVGGGTWTCTAAT-3' as shown in SEQ ID NO. 5; wherein v=g, C or a; n= A, T, C or G; r=a or G; w=a or T.

The primer can well enrich fusion products derived from the same single cell and is used for gene sequencing analysis and the like. The principle of nested PCR is shown in FIG. 1.

The fusion PCR primer and the nested PCR primer can specifically detect functional microorganisms with nitrogen fixation functional genes from a large number of microorganism groups, and can acquire fragment sequences of the functional genes for analyzing species diversity of the nitrogen fixation microorganisms.

Based on the same inventive concept, the embodiment of the application also provides an application of the primer group in preparation of a kit for detecting the diversity of the nitrogen-fixing bacteria of the rhizosphere of plants.

In some embodiments, the kit comprises a fusion PCR kit and a nested PCR kit.

Based on the same inventive concept, the embodiment of the application also provides a kit for detecting the diversity of the nitrogen-fixing bacteria of the plant rhizosphere, which comprises the primer group and the common auxiliary agent.

In some embodiments, the kit comprises a fusion PCR kit and a nested PCR kit; the fusion PCR kit comprises a fusion PCR primer and a common auxiliary agent; the nested PCR kit comprises nested PCR primers and common auxiliary agents.

In some embodiments, the fusion PCR kit may specifically include: fusion PCR primer set, 2 XHI-FI PCR Mix, BSA, tween-20 and sterile water as described previously.

In some embodiments, as in Table 2, the concentrations of the F1 primer, the R1-F2' primer, and the R2 primer in the fusion PCR kit may be 10. Mu.M, 1. Mu.M, and 10. Mu.M, respectively.

TABLE 2 fusion PCR reaction System

In some embodiments, the ratio of F1 primer, R1-F2' primer, R2 primer, 2 XHI-FI PCR Mix, BSA, and Tween-20 double may be 10. Mu.L, 1. Mu.L, 10. Mu.L, 50. Mu.L, 2. Mu.L, and 2. Mu.L, respectively.

In some embodiments, the nested PCR kit may specifically include: nested PCR primer set, 2 XHI-FI PCR Mix and sterile water as described previously.

In some embodiments, as in table 3, the concentrations of the F3 primer and the R3 primer in the nested PCR kit may be 10 μm and 10 μm, respectively.

TABLE 3 nested PCR reaction System

In some embodiments, the ratio of F3 primer, R3 primer, and 2 XHI-FI PCR Mix may be 4. Mu.L, and 50. Mu.L, respectively.

Based on the same inventive concept, referring to fig. 2, the embodiment of the present application further provides a method for detecting diversity of nitrogen-fixing bacteria in rhizosphere of plants, including:

s100, preprocessing an acquired plant sample to obtain bacterial suspension;

s200, mixing the bacterial suspension subjected to cell density calculation, an emulsifying agent and a fusion PCR primer group, and connecting and amplifying a nitrogen fixation functional gene and a gene of marker species information in a single cell to obtain a fusion product;

s300, extracting the fusion product, performing agarose gel electrophoresis, and performing gel cutting and purification to obtain a purified fusion product;

s400, mixing the purified fusion product serving as a template with a nested PCR primer set, and reacting to obtain a nested PCR product;

s500, preparing a library by using the nested PCR product, sequencing, and analyzing the diversity of the plant rhizosphere azotobacter according to the sequencing result.

According to the method for detecting the diversity of the nitrogen-fixing bacteria in the rhizosphere of the plants, the acquired plant samples are preprocessed to obtain bacterial suspension; mixing the bacterial suspension subjected to cell density calculation, an emulsifying agent and a fusion PCR primer group, and connecting and amplifying the nitrogen fixation functional gene and the gene of the marker species information in a single cell to obtain a fusion product; extracting the fusion product, performing agarose gel electrophoresis, and performing gel cutting purification to obtain a purified fusion product; taking the purified fusion product as a template, mixing with a nested PCR primer group, and reacting to obtain a nested PCR product; and preparing a library by using the nested PCR product, sequencing, and analyzing the diversity of the plant rhizosphere azotobacter according to the sequencing result. The functional genes of the functional microorganisms can be connected with the genes of the marker species information on a single cell level, so that the diversity analysis of the functional microorganism groups is realized, the relation between the functional genes of the microorganisms and the species evolution is analyzed according to the sequencing result, and the specificity of functional bacterial community detection in the fields of environment, agriculture and the like is improved. The method has the advantages of low operation cost, higher flux, more definite amplified objects, capability of generating a large amount of effective data and the like.

In some embodiments, in step S100, the preprocessing may specifically include: suspending the collected plant root sample in sterile water, mixing uniformly, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain bacterial suspension.

In some embodiments, in step S200, the cell density is calculated, specifically, by using ATP fluorescent microbiological detection. Specifically, the method comprises the following steps:

preparing a fluorescent reagent;

fluorescent reagents were added to the bacterial suspension, RLU values were recorded and converted to ATP concentration, and the cell density of the bacterial suspension was calculated. The cell density is calculated by an ATP fluorescent microorganism detection method, so that the accurate cell density can be obtained, and a subsequent fusion PCR reaction system is better promoted.

In some embodiments, the emulsifier may be a UMIL EM90 emulsified oil. After the emulsified oil is mixed with the bacterial suspension, the liquid drops can wrap single cells after dispersion treatment. Namely, single cell separation of the bacterial suspension is realized, and single cells are obtained.

In some embodiments, the UMIL EM90 emulsified oil may include the following components in volume fractions: 4% umil em90 oil; 0.05% Triton X-100; the balance of mineral oil. That is, the emulsifier comprises the following substances in volume fraction: 4% UMIL EM90 oil, 0.05% Triton X-100 and 95.95% mineral oil. The UMIL EM90 emulsified oil with the components can enable the liquid drops to have better high-temperature-resistant stability, so that single cells wrapped by the liquid drops can stably react at a high temperature of PCR and are favorable for later extraction and separation.

In some embodiments, the volume ratio of the fusion PCR reaction system obtained after mixing the bacterial suspension with the fusion PCR primer set to the aforementioned UMIL EM90 emulsified oil may be 1:6.

in some embodiments, the nitrogen fixation functional gene may be a nifH gene and the marker species information gene may be a 16Sr RNA gene. The gene combination is selected to fuse the most conserved gene coding nitrogen fixation enzyme ferritin contained in the nitrogen fixation microorganism with the gene marking species information, so that the functional gene is related to the species evolution relationship, and the diversity condition of the nitrogen fixation bacteria is accurately indicated.

In some embodiments, the fusion PCR primer set comprises:

f1 primer: 5'-ATSGCCATCATYTCRCCGGA-3' as shown in SEQ ID NO. 1;

the primer of R1-F2' is 5' -TTACCGCGGCKGCTGRCACTGCGAYCCSAARGCBGA CTC-3', and is shown as SEQ ID NO. 3; wherein k=g or T; r=a or G; y=c or T; s=c or G, b= G, T or C;

r2 primer: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO. 2. The primer can be used for specifically detecting microorganisms with the nitrogen fixation function from a large number of microorganism groups, and can also obtain the fragment sequence of the functional gene nifH for evolution analysis.

Specifically, the primer pairs for amplifying the nitrogen fixation nifH gene are respectively:

f1 primer: 5'-ATSGCCATCATYTCRCCGGA-3' as shown in SEQ ID NO. 1;

R1-F2' primer: 5 '-ttaccgcgckgctgrcactgcgaxyccsaargcbgactc-3', wherein k=g or T; r=a or G; y=c or T; s=c or G, b= G, T or C as shown in SEQ ID No. 3;

the primer pairs for amplifying the 16SrRNA gene are respectively as follows:

r2 primer: 5'-GGTTACCTTGTTACGACTT-3', which is shown in SEQ ID NO. 2.

In some embodiments, in step S300, the extraction may specifically be performed with diethyl ether and ethyl acetate. The UMIL EM90 emulsifying system can be destroyed by the extraction to extract the single cells coated by the polymer.

In some embodiments, the extracting the fusion product specifically comprises:

s310, collecting the fusion product and performing first centrifugation to remove an upper oil phase;

s320, adding water saturated diethyl ether, and performing second centrifugation to remove an upper liquid phase;

s330, adding water saturated ethyl acetate, and performing third centrifugation to remove an upper liquid phase;

and S340, placing the obtained product in a fume hood for air drying.

Wherein the duration of the first centrifugation may be greater than the second centrifugation and the third centrifugation. The duration of the second centrifugation process and the third centrifugation process may be the same. The rotational speed of the first centrifugal process, the rotational speed of the second centrifugal process, and the rotational speed of the third centrifugal process may be the same.

In some embodiments, step S320 and step S330 may be repeated twice, respectively, to better wash the fusion product.

In some embodiments, in step S400, the residual diethyl ether and ethyl acetate can be volatilized as clean as possible by an air drying process.

In some embodiments, purification may be performed specifically by a gel purification kit to avoid the effect of non-specific amplification of other interval fragments on subsequent reactions.

In some embodiments, in step S400, the nested PCR primers are:

f3 primer: 5'-GGCATNGCRAANCCVCCRCANAC-3' as shown in SEQ ID NO. 4;

r3 primer: 5'-GGACTACNVGGGTWTCTAAT-3' as shown in SEQ ID NO. 5; wherein v=g, C or a; n= A, T, C or G; r=a or G; w=a or T. The primer can well enrich fusion products derived from the same single cell and is used for gene sequencing analysis and the like. In some embodiments, after step S400, before step S500, the method may further comprise performing agarose gel electrophoresis on the nested PCR product, and performing gel cutting purification to obtain a purified nested PCR product. So as to improve the purity of the nested PCR product, and better carry out subsequent sequencing and the like.

According to the primer group, the kit and the method for detecting the diversity of the nitrogen-fixing bacteria of the plant rhizosphere, provided by the embodiment of the application, bacterial suspension is prepared from a plant root sample, the cell density is calculated by adopting an ATP fluorescent microorganism detection method, the liquid drop is wrapped by single cells by emulsifying UMIL EM90 emulsified oil, fusion PCR is performed by using a fusion PCR primer group consisting of an F1 primer, an R1-F2' primer and an R2 primer, nifH genes and 16S rRNA genes are connected, fusion products derived from the same single cells are enriched by matching with the F3 primer and the R3 primer, and effective analysis of the diversity of the nitrogen-fixing bacteria of the plant rhizosphere is realized by constructing a gene library, performing gene sequencing analysis and the like.

According to the method, an emulsifier UMIL EM90 is utilized to separate a microbial community in a plant root sample environment into single microbial cells wrapped by oil drops, then nifH genes with nitrogen fixation function and amplified fragments of 16SrRNA genes with marker species information are connected through parallel fusion and nested polymerase chain reaction amplification (PCR), so that the types and community structures of the microorganisms with nitrogen fixation function are detected in the plant root sample, meanwhile, the relationship between functional genes and species evolution of the nitrogen fixation microorganisms is analyzed through comparison of the nitrogen fixation gene fragments and the base sequence fragments of 16S rRNA, and the specificity of functional bacterial community detection in the fields of environment, agriculture and the like is improved.

TABLE 4 comparison of the inventive process with the analogous process

Referring to table 4, the method of the present application, cell sorting stage, is simple and easy to implement by emulsification, paired separation and tandem PCR methods and has low consumables compared to fluorescence flow-based cell sorting (FACS-activated cell sorting) and microfluidic droplet methods (microfluidic) based on fluorescence signals. Compared with genome Multiple Displacement Amplification (MDA), the amplification target of the invention is more definite, can generate a large amount of effective data and can represent and reflect the characteristics of the whole functional community. Meanwhile, the defects of low coverage of the metagenome technology to the group, high cost of single sample, large data acquisition amount, high requirement on hardware, redundant analysis process and the like can be overcome. The method can identify the specific functional microorganism group from millions of microorganism groups at one time, and overcomes the defects that the effective data size of acquiring the whole genome sequence is low, the operation technology of micro-fluidic is relied on, the experiment is difficult, the reagent consumable is expensive and the like, which are not targeted in the traditional single-cell sequencing.

The application provides possibility for discovering new assumed azotobacter on the basis of revealing known azotobacter functional flora. Potential applications include identification of functional community members, tracking of horizontal gene transfer networks, and mapping of ecological interactions between microbial cells. Can be applied to the fields of biotechnology industrial production, ecological microorganism research, agriculture and plant microorganism, and molecular genetics and evolution. In terms of the field of biotechnology production, the method is used for developing new species of azotobacter and provides a technical means for industries such as grasslands. The rhizosphere azotobacter can help promote plant growth, increase the nutrient content and enzyme activity of soil, and improve the microbial diversity of soil. For scientific research in the field of ecological microorganisms, the method of the invention can be used for precisely revealing microorganisms with specific nitrogen fixation and generation functions in the environment, so as to explain scientific problems in various ecological fields, such as ecological species and functions, and the like, and perfect a geobiochemical circulation model, and the like, thereby strongly promoting ecological research progress.

The method of the invention not only provides species information, but also provides functional gene sequence information when determining nitrogen fixation functional microorganisms. Thus, not only can new nitrogen fixation functional species be revealed, but also related research hot spots in biological evolution, such as evolution of nitrogen fixation functional genes in inheritance, gene level transfer of nitrogen fixation microorganisms in evolution and the like, can be revealed through co-evolution of species evolution and nitrogen fixation functional genes.

The technical scheme of the invention is further described below with reference to the specific embodiments.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 screening and preparation of primers for nifH Gene

The performance of the primer determines the quality of the detection effect of the kit. The application screens 30 pairs of primers against nifH genes, and finally screens a pair of optimal nifH gene primers. This example is intended to illustrate the screening procedure of primer pairs for nifH genes.

Primer design: primer pairs for amplifying nifH gene were designed for nifH gene nucleic acid sequences, and the results are shown in Table 5.

And (3) test design: the nifH gene PCR primers with obvious amplification effect are screened from 30 pairs of primers in table 5 by pre-experiment. And performing nested PCR reaction by taking the nifH gene PCR product as a template, and determining nested PCR primers and nifH gene end fusion primers through the amplification effect of the PCR reaction.

TABLE 5 primer sequences for amplifying nifH Gene

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TABLE 6 functional Gene end PCR reaction System

TABLE 7 functional Gene end PCR reaction conditions

The test method comprises the following steps: the reaction system was prepared as shown in Table 6, and the reaction conditions were as shown in Table 7, and the PCR amplification was performed, followed by running gel on agarose gel, and the amplification effect was observed.

Test results: the primer amplification effect is shown in Table 8, and the conditions of agarose gel electrophoresis of common and nested PCR reactions at the functional gene end are combined to determine the nifH gene end fusion primer F1 (5 '-ATSGCCATCATYTCRCCGGA-3') and nifH gene end nested primer F3 (5 '-GGCATNGCRAANCCVCCRCANAC-3').

TABLE 8 primer amplification Effect

Primer pair numbering	1	2	3	4	5	6	7	8	9	10
											Amplification Effect	+	+	-	-	-	-	++	++	++	-
Primer pair numbering	11	12	13	14	15	16	17	18	19	20
											Amplification Effect	+++	+++	-	-	-	-	-	-	-	-
Primer pair numbering	21	22	23	24	25	26	27	28	29	30
											Amplification Effect	-	-	+	+	-	-	-	-	+	+

+++: with unique and bright bands

++: with unique but darker bands

+: having bands of interest but comprising non-specific amplification

-: non-strip belt

Example 2 screening and preparation of primers for 16S rRNA Gene

The performance of the primer determines the quality of the detection effect of the kit. The present application uses 16S universal amplification primers F2, R2 and R3 for 16S rRNA gene selection.

As in table 9.

TABLE 9 16S rRNA Gene primer sequences

Primer name	Sequence information (5 '-3')	Sequence numbering
			F2	GTGYCAGCMGCCGCGGTAA	SEQ ID NO:19
R2	GGTTACCTTGTTACGACTT	SEQ ID NO:2
			R3	GGACTACNVGGGTWTCTAAT	SEQ ID NO:5

And (3) test design: for the 16S rRNA gene, a 16S universal amplification primer pair F2 and R2 is adopted, a 16S rRNA gene PCR product is used as a template for nested PCR reaction, and the amplification effect is observed through agarose gel electrophoresis.

The test method comprises the following steps: the reaction system was prepared as shown in Table 10, and the reaction conditions were as shown in Table 11, and the PCR amplification was performed, followed by running gel on agarose gel, and the amplification effect was observed.

TABLE 10 16S rRNA gene end PCR reaction system

TABLE 11 16S rRNA Gene end PCR reaction conditions

Test results: the primer amplification effect is shown in Table 12, and the PCR amplification effect of the 16S rRNA gene primer is obvious, so that the 16S rRNA gene end fusion primer R2 (5'-GGTTACCTTGTTACGACTT-3') and the 16S rRNA gene end nest primer R3 (5 '-GGACTACNVGGGTWTCTAAT-3') are determined.

TABLE 12 primer amplification Effect

Primer pair	F2、R2	F2、R3
			Amplification Effect	+++	+++

+++: with unique and bright bands

++: with unique but darker bands

+: having bands of interest but comprising non-specific amplification

-: non-strip belt

Example 3 preparation of primers for bypass

The primers R1 for amplifying nifH gene determined in example 1 and the primers for amplifying 16S rRNA gene determined in example 2 are spliced to obtain the bypass primer R1-F2', and the bypass primer can connect functional gene nifH gene and phylogenetic gene 16S rRNA gene together in the fusion PCR reaction process.

Example 4 method for detecting diversity of plant rhizosphere azotobacter

Referring to FIG. 3, the method for detecting the diversity of the nitrogen-fixing bacteria of the rhizosphere of the plant mainly comprises the following steps: (a) Pretreating the collected plant sample to obtain bacterial suspension;

the biological samples used in this example were collected at the Qinghai province-feature pasture resource ecological test station, which included 38 samples of Gramineae and leguminous plants. Shaking off large soil of plant roots, shearing the plant roots by using disinfected scissors, placing the roots in a sterilized 50ml centrifuge tube, adding 30ml PBS (0.02 mol/L, pH=7.0) buffer solution, placing the mixture on a shaking table for shaking for 30min, then performing ultrasonic treatment for 10min, clamping the roots into another 50ml centrifuge tube by using sterile forceps, repeating the previous step, cleaning for three times, mixing cleaning liquid, centrifuging, and freeze-drying the centrifuged soil.

Weigh 0.1g of freeze-dried soil into a 2ml centrifuge tube. Adding 1ml NaCl solution (0.7%), mixing, performing maximum speed vortex for 5min, performing ultrasonic vibration for 1min, performing vortex for 5min again, and centrifuging at a low speed for 30s at a rotating speed of 3000g in a centrifuge, and collecting supernatant to a 2ml centrifuge tube to obtain cell suspension.

(b) Calculating to obtain cell density of the bacterial suspension by adopting an ATP fluorescent microorganism detection method;

melting Bac Titer-Glo ^TM Buffer solution, which is equilibrated at room temperature 48 hours in advance before use, and lyophilized powder Bac Titer-Glo before use ^TM The substrate was equilibrated at room temperature 2 hours in advance. Bac Titer-Glo ^TM Transferring the buffer solution to the freeze-dried powder Bac Titer-Glo ^TM In the substrate bottle, the lyophilized powder enzyme/substrate mixture is dissolved to form a fluorogenic reagent. Mix by gentle shaking, shake or reverse upside down to homogenize the solution. The reagents were equilibrated at room temperature for at least 15 minutes, sub-packaged and stored at-20℃in the dark.

ATP standard (10mM,Promega Corporation) was diluted with sterile water without ATP at various concentrations (0.00005-1 nM) prior to detection and used to make standard curves. The water bath is opened in advance to set the temperature at 38 ℃, the required reagent is packaged into a 1.5ml centrifuge tube, the sample and the reagent are heated for at least 1 minute respectively at 38 ℃, 50 mu L of the reagent is added into 500 mu L of the sample, the sample is immediately placed on an ATP instrument for measurement and counting after being uniformly mixed, each sample is repeatedly measured for 3 times, the interval between the samples is more than 10 seconds for integer time, the RLU is unit, finally, the RLU is converted into the ATP concentration through a standard curve, and the cell density of the bacterial suspension is calculated.

(c) Mixing a bacterial suspension PCR system with an emulsifying agent, and connecting and amplifying nitrogen fixation functional genes and genes of marker species information in each single cell by adopting a fusion PCR method to obtain a fusion product;

the calculated bacterial suspension density was adjusted to contain 100 ten thousand cells per 30 μl (reaction volume for each sample), and a PCR reaction system was prepared according to table 13.

TABLE 13 fusion PCR reaction System

After mixing 100. Mu.L of the PCR reaction system with 600. Mu.L of the emulsified oil UMIL EM90, the droplets were allowed to encapsulate single cells and to take on a milky color after vortexing at 3000rpm for 1 min. The UMIL EM90 emulsified oil for fusion PCR reaction has high temperature stability, so that the liquid drop can stably react at the high temperature of PCR and is favorable for later extraction and separation. The emulsified oil comprises the following components in percentage by volume:

4% umil em90 oil; 0.05% Triton X-100; the rest is mineral oil

50. Mu.L of the reaction mixture was dispensed into PCR vials, each sample was dispensed into about 12 vials, and then subjected to fusion PCR reactions under the conditions shown in Table 14:

TABLE 14 fusion PCR reaction conditions

After the reaction, the fusion product was collected into a 1.5mL centrifuge tube and subjected to the next operation for use.

(d) Extracting the fusion PCR product by using diethyl ether and ethyl acetate, and carrying out agarose gel electrophoresis, and cutting gel to purify DNA;

breaking the ABIL emulsifying system, releasing the gel beads, and extracting the single cells coated by the polymer. The resulting fusion PCR product was centrifuged at 13,000g for 5min at room temperature, and the upper oil phase was discarded. Shaking the water saturated diethyl ether (water-diethyl ether mixture in the same volume) for 30 seconds, occasionally opening the lid to prevent pressure build up. The phase was allowed to stabilize before use and was taken up from the top (diethyl ether) phase. 1mL of water-saturated diethyl ether was added to each sample, gently vortexed, centrifuged at 13,000g for 1min, the upper liquid phase discarded, and the procedure repeated once. The same procedure was followed with water saturated ethyl acetate (same volume of water-ethyl acetate mixture) for 2 washes. After the completion, the mixture is placed in a fume hood and dried for 15min, so that residual diethyl ether and ethyl acetate are volatilized as completely as possible, and 100-150 mu L of the product is collected.

Agarose gel with 1% concentration is prepared, the agarose gel is placed in an electrophoresis tank of 1 xTAE buffer solution, the collected product is mixed with loading buffer solution (with the final concentration of 1 x) and then spotted in the gel tank, the voltage of 110V and the current of 300mA are set for 40min, after the agarose gel is finished, the gel running result is observed in an electrophoresis imager, the target fragment length is about 1330bp, the fragment with the length of 1000-2000bp is cut off, and the fragment is purified by a gel purification kit, so that the influence of nonspecific amplification of other interval fragments on subsequent reactions is avoided. This interval band is not apparent or visible because the fusion product is produced in a smaller amount and can be further enriched by the nest reaction in the next step.

(e) Taking the purified fusion product as a template, and performing nested PCR reaction to obtain a nested PCR product;

a nested PCR reaction system was prepared according to Table 15, and Table 16 shows the nested PCR reaction conditions.

TABLE 15 nested PCR reaction System

TABLE 16 nested PCR reaction conditions

The nested PCR products after reaction were also subjected to agarose gel electrophoresis, and as described above, a distinct target fragment was observed in the electrophoresis imager, the target fragment was about 590bp long, and the fragment was subjected to gel cutting recovery and purification, and then sent to generation sequencing. Fusion amplicons derived from the same single cell, including about 300 base sequences from the functional gene nifH gene and about 260 base sequences from 16SrRNA, were obtained.

(f) And preparing a library by using the purified nest type PCR product, sequencing, and analyzing the diversity of the nitrogen-fixing bacteria of the plant rhizosphere according to the sequencing result.

The nested products were prepared into libraries according to the Miseq kit preparation guidelines (Illumina) and sequenced on the Illumina Miseq platform.

Test results: as shown in fig. 4, 5 and 6. See FIG. 4 for an electrophoretogram after fusion PCR reactions. See FIG. 5 for an electrophoresis pattern after nested PCR reactions. Taking the environmental sample DNA as an example, the first generation sequencing result of the nested PCR products is shown in FIG. 6. Wherein the 16S sequence of the marker phylogenetic gene is the first half, the nifH sequence of the marker azotobacter functional gene is the second half, and the middle is connected by a bridging primer.

As can be seen, the method of example 5 can link the 16S rRNA gene fragment with the functional sequence nifH gene fragment and amplify it, and can determine the diversity of the specific nitrogen-fixing bacteria. The sequencing result can be used for investigating the structural diversity of azotobacter community composition, and the co-evolution of genes can be checked by comparing the base sequences of the azotobacter functional genes and the 16S rRNA genes.

Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.

While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of those embodiments will be apparent to those skilled in the art in light of the foregoing description.

The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.

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Claims

1. The plant rhizosphere azotobacter diversity detection primer group is characterized by comprising: fusion PCR primer group and nested PCR primer pair:

primer pairs for amplifying marker species information genes: the nucleotide sequence of the R1-F2' primer is shown as SEQ ID NO. 3; the nucleotide sequence of the R2 primer is shown as SEQ ID NO. 2;

the nucleotide sequence of the F3 primer is shown as SEQ ID NO. 4; the nucleotide sequence of the R3 primer is shown as SEQ ID NO. 5; the nitrogen fixation functional gene is nifH gene, and the gene for marking species information is 16S rRNA gene.

2. The kit for detecting the diversity of the nitrogen-fixing bacteria of the plant rhizosphere is characterized by comprising the primer set and common auxiliary agents as claimed in claim 1.

3. The kit of claim 2, wherein the kit comprises a fusion PCR kit and a nested PCR kit; the fusion PCR kit comprises a fusion PCR primer and a common auxiliary agent; the nested PCR kit comprises nested PCR primers and common auxiliary agents.

4. A method for detecting diversity of nitrogen-fixing bacteria in plant rhizosphere, which is characterized by adopting the primer as claimed in claim 1, and comprising the following steps:

pretreating the collected plant sample to obtain bacterial suspension;

5. The method for detecting the diversity of nitrogen-fixing bacteria of plant rhizosphere according to claim 4, wherein the emulsifier comprises the following substances in volume fraction: 4% UMIL EM90 oil, 0.05% Triton X-100 and 95.95% mineral oil.

6. The method for detecting diversity of nitrogen-fixing bacteria of plant rhizosphere according to claim 4, wherein the method for preparing the library by using the nested PCR product further comprises: and (3) performing agarose gel electrophoresis on the nested PCR product, and performing gel cutting purification to obtain a purified nested PCR product.

7. The method for detecting the diversity of nitrogen-fixing bacteria of plant rhizosphere according to claim 4, wherein the extracting the fusion product specifically comprises:

the resultant was placed in a fume hood and air dried.

8. The method for detecting the diversity of nitrogen-fixing bacteria of plant rhizosphere according to claim 4, wherein the cell density calculation specifically comprises:

preparing a fluorescent reagent;