CN111518729A - High-flux separation culture method for crop root system microbiome - Google Patents
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Abstract
The invention discloses a high-flux separation culture method for crop root system microbiome, which comprises the following steps: s1 gradient dilution separation culture of crop root bacteria; s2 extracting DNA of the cultured bacteria; s3 carrying out two-step PCR on the DNA by dividing the hole, taking 799F and 1193R as primers in the first step, taking 799F-2 containing a hole barcode and 1193R-2 containing a plate barcode as primers in the second step, and amplifying a 16S variable region V5-V7 to obtain a bacterial identification library; s4 sequencing analysis; s5 construction of a crop root microbiome. The method can separate and culture a large amount of bacteria in a short time with less manpower, quickly identify the types of the bacteria through a 'one-key' sequence analysis process, can assemble the crop root system microorganism groups, and provides technical support for obtaining more crop root system microorganism resources.
Description
Technical Field
The invention relates to a high-flux separation culture method for crop root system microbiome in the field of biotechnology.
Background
During the evolution of plants, the transition from aquatic to terrestrial life forms root tissue that helps plants to obtain nutrients and water from the soil. A large number of microorganisms of various types inhabit in soil, are selected and enriched by roots of plants to form a root system microorganism group, and participate in important physiological processes of growth and development, nutrition absorption, disease resistance, stress resistance and the like of the plants along with the whole growth cycle of the plants. The interaction between plants and root microorganism groups is closely related to important agricultural problems such as high-efficiency utilization of crop nutrition, continuous cropping, crop rotation and the like.
Plant and root system microbiome interactions involving plant molecular biology (microscopic) and soil environment factors (macroscopic) are an extremely complex process. Although high throughput sequencing technology can effectively detect the composition of root microbiome, the functional studies on the interaction between root microbiome and host plants are still rare. Due to the limitation of microbial culture technology, a large amount of microbes symbiotic with plants cannot be purified and cultured, and only the descriptive work of sequencing technology is relied on, so that the research on the functions of the microbes in the plants and the molecular mechanism of the interaction process of the plants and root system microbiome is greatly restricted.
In 1982, the Colwell laboratory proposed the concept of viable but non-culturable microorganisms, indicating that there are a large number of microorganisms in nature that have not been isolated and cultured in the laboratory. Most microorganisms in soil are in a viable state, but due to medium and technical limitations, artificially isolated bacteria are not too large as 1% -5% of the bacteria present in soil. Since the growing environment of bacteria in nature is often oligotrophic, researchers have proposed a dilution culture method in which the concentration of an environmental sample is constantly diluted, and when the total number of microbial populations in the sample is diluted to a certain concentration, oligotrophic microorganisms present mainly can be prevented from being disturbed by the inhibition, competitive action, and the like of a few dominant microorganisms, thereby greatly increasing the possibility that the majority of oligotrophic microorganisms in soil are cultured.In 2002, Connon et al proposed a high throughput culture method based on a dilution culture method, which mainly utilized a low nutrient medium by diluting the sample to be treated to about 10%3After one/mL, high throughput culture using 48 well cell culture plates of small volume combined with flow cytometry detection to isolate and purify the cultured microorganisms resulted in a number of previously uncultured, more diverse bacteria. Although the method greatly improves the throughput of microorganism isolation culture, a great deal of manpower and time are required in the later identification process, and the method cannot be popularized in any laboratory until now.
The root system microbiome contains thousands of bacteria, the bacteria isolated and cultured at present only account for about 1%, and the factors limiting the establishment of the root system microbiome resources comprise three aspects: 1 isolation culture method, the common monoclonal culture method is to pick a large number of monoclones on the solid culture medium, isolate and culture each monoclone, on a culture plate, when the propagation speed of a dominant bacterium is obviously superior to other bacteria, it is likely that the monoclones picked by the whole plate are all a kind of bacterium, in order to obtain more bacterium species, the separation amount of the plate needs to be enlarged to increase the possibility of more bacterium separation, this method consumes a large amount of manpower, material resources and time; 2, the traditional Sanger sequencing identification method is only suitable for identifying pure bacteria because bacteria in mixed bacteria cannot be distinguished one by one. In addition, the Sanger identification method has the defects of large workload, easy pollution and loss of low-abundance bacteria and the like, so that the Sanger identification method is not suitable for identification of large-batch samples; and 3, sequence analysis, wherein after a bacterial sequence is obtained, professional biological information personnel are required to analyze the sequence, researchers who do not learn the system can only rely on a detection company to feed back a sequence comparison result, autonomous operation cannot be performed, and a large amount of expenditure is still consumed while the sequencing result is not mastered.
Disclosure of Invention
In order to solve the technical problem of using less manpower and carrying out separation culture on a large amount of crop root bacteria in a short time, the invention adopts a gradient dilution method to establish a high-flux separation culture system of bacteria, after a large amount of bacteria are obtained, a two-step PCR method of adding barcode is adopted to build all the separated bacteria into a library, and Hiseq sequencing is combined for identification, so that the high-flux rapid and efficient detection can be met, even if mixed bacteria exist in a hole, the mixed bacteria can be detected, all the steps of analyzing a sequence are integrated into a data packet, and optimization and simplification are carried out, so that a non-professional student telecommunication analyst can also analyze the sequence.
The invention relates to a high-flux separation culture method of a crop root system microorganism group, which comprises the following steps:
s1, grinding crop root systems under aseptic conditions to obtain crop root system homogenate, diluting the crop root system homogenate by adopting a gradient dilution method to obtain a plurality of dilution samples, and culturing each dilution sample on N pieces of 96-pore plates until the number of turbid pores of all dilution samples is not increased any more; turbid holes obtained by culturing the dilution samples with the proportion of the number of the turbid holes to the total number of the dilution samples inoculated by the corresponding dilution samples being 30-50%, wherein the liquid in each turbid hole contains bacteria, and the liquid in each turbid hole is named as cultured bacteria; n is a natural number more than or equal to 2;
s2, extracting the genome DNA of each cultured bacterium under the aseptic condition;
s3, under the aseptic condition, taking the genome DNA of each cultured bacterium as a template, taking 799F and 1193R as primers, respectively carrying out PCR amplification on 16S rRNA gene V5-V7 fragments, and the PCR is called as first-step PCR; performing PCR amplification on a 16S rRNA gene V5-V7 fragment by using the obtained first-step PCR product as a template and 799F-2 and 1193R-2 as primers, wherein the PCR is called as second-step PCR; collecting the second-step PCR products of each of the cultured bacteria and mixing to obtain a mixture of second-step PCR products of all the cultured bacteria, which is called a cultured bacteria identification library;
799F is a single-stranded DNA with a nucleotide sequence of sequence 1, 1193R is a single-stranded DNA with a nucleotide sequence of sequence 2, and 799F-2 and 1193R-2 are primers selected from the following A or B:
A. the 799F-2 is 96 single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on the 799F, and the 1193R-2 is M single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of M-plate barcode on the 1193R;
B. 799F-2 is M single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of M plate barcode on 799F, and 1193R-2 is 96 single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on 1193R;
the 96-well barcode is a marker for marking 96 wells on one 96-well plate, the nucleotide sequences of the 96-well barcode are different, the M-plate barcode is a marker for marking the N96-well plate, the nucleotide sequences of the M-plate barcode are different; m is a natural number which is more than or equal to N;
s4, sequencing the cultured bacterium identification library, comparing a sequence obtained by sequencing with a 16SrRNA gene V5-V7 fragment column in an RDP database to obtain crop root system bacteria OTU, and storing the cultured bacteria corresponding to the crop root system bacteria OTU;
and S5, obtaining the proportion of the cultured bacteria belonging to different phyla and/or high abundance genera according to the bacteria OTU to which all the cultured bacteria belong under the aseptic condition, and recovering and mixing the stored cultured bacteria according to the proportion to obtain the crop root system microorganism group.
Bacteria OTU (Operational taxonomy unit) is considered to belong to the same bacteria in phylogenetic research or population genetics research, wherein the similarity of clustering is more than 97% after comparing the same mark set for a certain taxonomy unit (strain, species, genus, grouping and the like) with a 16S rRNA gene sequence in an RDP database (http:// rdp.cme.msu.edu /) for facilitating analysis.
The microbiology researches symbiotic or pathological microbial ecological groups on animals and plants. Microbiomes include bacteria, archaea, protozoa, fungi, and viruses.
In the above method, N may specifically be 25 to 60, and in the embodiment of the present invention, N is 45.
In the above method, the crop plant may be a monocotyledonous plant (e.g., rice) or a dicotyledonous plant (e.g., Arabidopsis thaliana).
In the above method, the culture in S1 is performed at room temperature.
In the method, in the S1, the crop root homogenate is diluted by a 1/10 XTSB liquid culture medium by adopting a gradient dilution method; the 1/10 XTSB liquid culture medium is liquid with the pH value of 7.3 +/-0.2 and consists of a solute and a solvent, the solvent is water, and the concentration of the solute is 17.0g/L of tryptone, 3.0g/L of soybean papain digest, 5.0g/L of sodium chloride, 2.5g/L of dipotassium hydrogen phosphate and 2.5g/L of glucose.
In the above method, in S4, sequencing was performed using Hiseq2500 platform.
In the above method, in S4, the crop root system bacteria OTU is obtained by performing quality filtration and sample splitting on the sequence according to the hole barcode and the plate barcode.
In the above method, in S4, the specific steps of sequencing analysis are as follows: sequencing the constructed bacteria identification library on a Hiseq2500 platform, wherein each sequence comprises a plate barcode, a pore barcode and a 16S rRNA gene V5-V7 sequence; performing quality filtration and sample splitting on the sequence according to the hole barcode and the plate barcode, removing chimera and low abundance sequence (reading count <8) by using UNOISE algorithm, and defining bacterium OTU (denoising mode, equivalent to 100% similarity); comparing the sequences of the 16S rRNA genes V5-V7 of the cultured bacteria with the sequences of the 16S rRNA genes V5-V7 of the known bacteria OTU in an RDP (http:// RDP. cme. msu. edu /) database, classifying the bacteria OTU by adopting an SINTAX algorithm, and considering that the bacteria OTU belongs to the same OTU if the similarity with the sequences of the 16S rRNA genes V5-V7 of the known bacteria OTU is more than a threshold (97 percent), wherein the cultured bacteria can be cultured; visualization of the dendrogram by GraPhlAn 0.9.7 shows the number and species cultivable in the cultured bacteria.
In the above method, the microbiome is a bacterium.
The invention also provides a separation and culture method of the crop root system microbial component and/or application of the crop root system microbial component in preparation of products. The product contains one or more microorganisms or crop root system microorganism groups obtained by the crop root system microorganism group separation culture method, or contains the crop root system microorganism groups; the product can be a microbial inoculum, or a microbial ecological agent containing the microbial inoculum, or a biological fertilizer containing the microbial inoculum or the microbial ecological agent.
The product described above may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the product, the tomato root microbiome or/and metabolites of the tomato root microbiome may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and filtrate. The preparation formulation of the product can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules. According to the requirement, the product can also be added with surfactant (such as Tween 20, Tween 80, etc.), binder, stabilizer (such as antioxidant), pH regulator, etc.
The crop root system microbial component separation culture method can be used for separating and culturing a large amount of bacteria in a short time with less manpower, and rapidly identifying the types of the bacteria through a one-key sequence analysis process, thereby providing technical support for obtaining more crop root system microbial resources.
Drawings
FIG. 1 is a flow chart of the high-throughput isolation and culture method of crop root system microbiome of the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.
In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
Example 1 high-throughput isolation culture method for rice root system microbiome
This example was carried out according to the flow chart (FIG. 1) of the method for high-throughput isolation and culture of crop root microbiome of the present invention.
In Beijing in 2016, the example was carried out on rice, and the variety used was Nipponbare (belonging to japonica rice).
S1, grinding the rice root system under aseptic conditions to obtain crop root system homogenate, diluting the crop root system homogenate by adopting a gradient dilution method to obtain a plurality of dilution samples, and culturing each dilution sample on 45 bacterial culture plates with 96 holes until the turbid hole number of all the dilution samples is not increased any more; and (3) turbid wells obtained by culturing the diluted samples with the ratio of the number of the turbid wells to the total number of the wells inoculated with the corresponding diluted samples being 30-50%, wherein the liquid in each turbid well contains bacteria, and the liquid in each turbid well is named as cultured bacteria. The method comprises the following specific steps:
s1.1, sampling and cleaning: collecting 3 rice plants growing to 7-8 weeks, cutting off a section of root system which is about 10cm away from the root system at the root-tuber junction; washing with sterile water until the soil blocks are washed clean; the washing was performed 3 times with 1 XPBS buffer, each time at 180rpm for 15 min. Sucking water on the surface of the root system by using sterile filter paper, and removing dead roots, impurities and the like by using tweezers; the roots of the 3 seedlings are cut into small sections with the diameter of 1-2mm by scissors, and the small sections of the root systems of the 3 seedlings are mixed together and evenly mixed.
Preparation of mother liquor 10 × PBS buffer solution NaCl 76g, Na2HPO425g，NaH2PO44.68g, add sterile water to 1L, sterilize at 121 ℃ for 15min, adjust pH to 7 with 1mM NaOH.
The stock solution was diluted 10-fold with 10 XPBS buffer to obtain 1 XPBS buffer.
S1.2 extracting bacteria: 0.02g of the mixed root system tissue obtained in S1.1 was weighed into a sterile 1.5ml centrifuge tube, and 200. mu.l of 10mM MgCl which was sterile filtered was added2Grinding root system tissue into pulp by using a sterilizing grinding rod; pipette all homogenates into 25mL 10mM MgCl2And (4) uniformly mixing the solution, and standing for 15min to obtain the crop root homogenate.
S1.3 gradient dilution: the crop root homogenate of step S1.2 was diluted 600-fold, 2000-fold, 6000-fold, and 18000-fold with 1/10 × TSB broth, respectively, to give 4 dilution samples.
The 1/10 XTSB liquid culture medium is liquid with the pH value of 7.3 +/-0.2 and consists of a solute and a solvent, the solvent is water, and the concentration of the solute is 17.0g/L of tryptone, 3.0g/L of soybean papain digest, 5.0g/L of sodium chloride, 2.5g/L of dipotassium hydrogen phosphate and 2.5g/L of glucose.
S1.4 bacterial culture: adding 4 dilution samples into a 96-well bacterial culture plate respectively, wherein each dilution sample is 160 mu l per well, each dilution sample is cultured on 45 96-well plates, and each plate is placed at room temperature (25 ℃) for standing culture after being sealed; daily observations were made until the number of turbid wells for all dilutions of the sample no longer increased, taking approximately 16-20 days.
Turbidity in the wells indicates bacterial growth. The proportion of turbidity in the wells is in Poisson distribution with the appearance of single bacteria, and when the turbidity in the wells is 30-50%, the probability that each well is single bacteria is the highest. The proportion of the number of the holes left turbid to the total number of the holes inoculated by the corresponding dilution sample is 30-50%, and the obtained turbid holes are cultured by the dilution sample: after culturing the dilution sample with the dilution multiple of 2000 times, in 4320 wells in total of 45 96-well bacterial culture plates, 1821 of the wells is turbid, indicating that bacteria grow, and accounts for 42.15% of the total number of wells inoculated by the dilution sample, all turbid wells of the dilution sample after culturing are left, the liquid in each turbid well contains bacteria, and the liquid in each turbid well is named as cultured bacteria.
Taking out a part of the bacterial liquid for culturing bacteria in each hole for bacteria identification, adding 80% (v/v) glycerol into the rest bacterial liquid according to the volume ratio of 1:1, and storing at-80 ℃ for later use.
S2 extracting genomic DNA of each of the cultured bacteria under aseptic conditions.
Extracting DNA from part of bacterial liquid of the cultured bacteria in each hole, and storing the rest bacterial liquid at-80 ℃ for later use. And (3) extracting DNA: and uniformly mixing, cracking and extracting the bacterial liquid of each cultured bacterium to obtain template DNA. The method comprises the following specific steps:
s2.1 adding 10 mul of buffer solution I into each hole of a sterile 96-hole PCR plate, respectively and uniformly mixing bacterial liquid of each cultured bacterium in the original 96-hole cell culture plate, sucking 6 mul of buffer solution I into each hole, and adding the buffer solution I into the 96-hole PCR plate at the corresponding position.
Buffer I: solutes were 25mM NaOH and 0.2mM EDTA, and solvent was water, pH 12.
S2.2, placing the 96-hole PCR plate in a PCR instrument, and carrying out high-temperature thallus lysis in an alkaline environment, wherein the program is set to 95 ℃ for 30 min.
S2.3 adding 10 mul of buffer solution II into each hole, mixing uniformly and centrifuging to obtain the genome DNA of each cultured bacterium.
And (2) buffer solution II: 40mM Tris HCl, pH 7.5.
S3, under the aseptic condition, taking the genome DNA of each cultured bacterium as a template, taking 799F and 1193R as primers, respectively carrying out PCR amplification on 16S rRNA gene V5-V7 fragments, and the PCR is called as first-step PCR; performing PCR amplification on a 16S rRNA gene V5-V7 fragment by using the obtained first-step PCR product as a template and 799F-2 and 1193R-2 as primers, wherein the PCR is called as second-step PCR; collecting the second-step PCR products of each of the cultured bacteria and mixing to obtain a mixture of second-step PCR products of all the cultured bacteria, which is called a cultured bacteria identification library;
799F is a single-stranded DNA with a nucleotide sequence of sequence 1, 1193R is a single-stranded DNA with a nucleotide sequence of sequence 2, and 799F-2 and 1193R-2 are primers selected from the following A or B:
A. the 799F-2 is 96 single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on the 799F, and the 1193R-2 is M single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of M-plate barcode on the 1193R;
B. 799F-2 is M single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of M plate barcode on 799F, and 1193R-2 is 96 single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on 1193R;
the 96-well barcode is a marker for marking 96 wells on one 96-well plate, the nucleotide sequences of the 96-well barcode are different, the M-plate barcode is a marker for marking the N96-well plate, the nucleotide sequences of the M-plate barcode are different; the M is the number of sheets of 96-well plates inoculated with the dilution sample left at S1 or more. In this example, the primer was selected from A, the number of 96-well plates was 45, and M was 96.
The method comprises the following specific steps:
s3.1 first step PCR
Performing PCR amplification by using the genome DNA of each cultured bacterium as a template, 799F as a forward primer and 1193R as a reverse primer; setting known bacterium liquid Escherichia coli DH5 alpha (Tiangen Biochemical technology Co., Ltd.) as positive control of a template, and setting Nuclease-Free Water (Nuclease-Free Water) as negative control of the template; performing agarose gel electrophoresis detection on the product, wherein the position of a positive control band is correct, and a negative control band is absent, which indicates that the first round of PCR amplification is qualified;
30 μ L of PCR amplification System consisting of 3 μ L of template DNA, 0.15 μ L of HS-Taq DNA polymerase (TAKARA), and 3 μ L of 10 × PCR Buffer (Mg)2+Plus), 2.4. mu.L dNTPs, 0.3. mu.L forward primer 799F and 0.3. mu.L reverse primer 1193R (Life Technologies), 20.85. mu.L Nuclear-Free Water.
799F：
(sequence 1 in a sequence table); wherein K is T or G.
1193R：
(sequence 2 in the sequence table).
PCR conditions were as follows: the DNA was initially denatured at 94 ℃ for 2 minutes followed by 30 cycles, each cycle denatured at 94 ℃ for 30 seconds, annealed at 55 ℃ for 30 seconds, extended at 72 ℃ for 1 minute, and finally extended at 72 ℃ for 5 minutes.
S3.2 second step PCR
Diluting the PCR amplification products of the first step of each cultured bacterium by 40 times respectively, using the diluted PCR amplification products for the second step of PCR, and carrying out PCR amplification on 16S rRNA genes V5-V7 by using 799F-2 and 1193R-2 as primers; diluting the PCR products of the positive control and the negative control of the first round by 40 times respectively to be used as templates of the positive control and the negative control of the second round; performing agarose gel electrophoresis detection on the product, wherein the position of a positive control band is correct, and a negative control band is absent, which indicates that the second round of PCR amplification is qualified;
30. mu.l of PCR amplification System, 3. mu.l of the diluted first-step amplification product, 0.15. mu.L of HS-Taq DNA polymerase (TAKARA), 3. mu.L of 10 × PCRbuffer (Mg)2+Plus), 2.4. mu.L dNTPs, 0.6. mu.L forward primer 799F-2 and 0.6. mu.L reverse primer 1193R-2, 20.25. mu.L nucleic-Free Water.
The forward primer 799F-2 is 96 single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of 96-well Barcode on 799F, and the structure of the forward primer is P5-Read 1-well Barcode-799F from the 5 'end to the 3' end: the sequence of P5 is 5-AATGATACGGCGACCACCGAGATCT3', the sequence of Read1 is
The well Barcode is determined by the specific position on the plate of each well of the cultured bacteria, and the sequence of 799F is
(K is T or G), and the specific forward primer 799F-2 is selected from 96 kinds of 799F-2 in Table 1 according to the position of the well on a 96-well plate.
The reverse primer 1193R-2 is connected with the 1193RM single-stranded DNAs (deoxyribonucleic acids) obtained from a sequence required by Illumina sequencing and one of M plate barcodes are 1193R with a sequence required by Illumina sequencing and containing plate barcodes, and the structure of the single-stranded DNAs is sequentially P7-Index-Read 2-plate Barcode-1193R from the 5 'end to the 3' end: the sequence of P7 is
The sequence of Index is
Or
The sequence of Read2 is
The plate Barcode is determined specifically on which plate the bacteria are cultured per well, and the sequence of 1193R is
The specific reverse primer 1193R-2 sequence was selected from 96 of 1193R-2 in Table 1 depending on which plate the well was in.
TABLE 1 second round PCR primer sequences (from 5 'to 3')
PCR conditions were as follows: the DNA was initially denatured at 94 ℃ for 2 minutes followed by 25 cycles, each cycle denatured at 94 ℃ for 30 seconds, annealed at 55 ℃ for 30 seconds, extended at 72 ℃ for 1 minute, and finally extended at 72 ℃ for 5 minutes.
S3.3 obtaining a culture bacterium identification library
After the second PCR, the amplified products in the other wells except the positive and negative controls in each PCR plate are mixed in equal volume to form gel samples to be cut, each gel sample to be cut consists of 75 mul of second PCR products and 15 mul of Loading Buffer, and agarose gel is used for gel cutting and recovery.
A1.2% agarose gel was prepared, voltage set 126V, and run for about 40-50 min. The amplified fragment (about 500bp) of the 16S rRNA gene and all fragments in a gel 2mm above and below the fragment were excised, and the gel masses were filled into 2mL sterile centrifuge tubes for use
The SV Gel and PCR Clean-up System kit (Promega) was used for Gel cutting recovery and purification with Agencourt AMPure XP beads (Beckman) to obtain DNA samples from each PCR plate. DNA concentration was determined from Quant-iTTMPicoGreenTMThe dsDNA Assay Kit (Invitrogen corporation) was used, and DNA samples of each PCR plate were mixed with the same amount of DNA standard to obtain a culture bacterial identification library.
S4, sequencing the cultured bacteria identification library, comparing the sequence obtained by sequencing with the 16SrRNA gene V5-V7 fragment column in the RDP database to obtain the crop root system bacteria OTU, and storing the cultured bacteria corresponding to the crop root system bacteria OTU. The method comprises the following specific steps:
the cultured bacteria identification library is sequenced on a Hiseq2500 platform to obtain 6756 sequences of rice root bacteria, and each sequence comprises a plate barcode, a pore barcode and a 16S rRNA gene V5-V7 sequence. And (3) performing quality filtering and sample splitting on the sequence according to the hole barcode and the plate barcode, and defining OTU (denoising mode, which is equivalent to 100% similarity) after removing chimera and low-abundance sequence (reading count is less than 8) by using UNOISE algorithm. The sequences of the 16SrRNA genes V5-V7 of the cultured bacteria are compared with the sequences of the 16SrRNA genes V5-V7 of the known bacteria OTU in an RDP (http:// RDP. cme. msu. edu /) database, the bacteria OTU is classified by adopting an SINTAX algorithm, and the bacteria OTU is considered to belong to the same OTU when the similarity with the sequences of the 16SrRNA genes V5-V7 of the known bacteria OTU is more than a threshold value (97 percent), and the cultured bacteria can be cultured. Visualization of the dendrogram by GraPhlAn 0.9.7 shows the number and species culturable in the cultured bacteria, giving a total of 278 non-redundant bacterial OTUs.
According to the result of the identification at S4, a representative strain 522 of the non-redundant bacterium OTU was selected from the cultured bacteria reserved at S1, recovered by continuous plating, and the strain was preserved. The method comprises the following specific steps:
1) and (3) recovering bacteria: according to the result of the identification in the step S4, a representative strain of the non-redundant bacteria OTU is selected from the cultured bacteria preserved and ready for use in S1, streaked and activated on a 1/2TSB solid medium plate, sealed and left at room temperature (25 ℃) for about 3 days, and a single colony is picked and transferred to a new 1/2TSB solid medium plate for purification 2 times;
2) and (3) strain preservation: the bacteria which are suitable for growing in the liquid culture medium and the bacteria which are not suitable for growing in the liquid culture medium are respectively preserved by the following methods a or b:
a, selecting bacteria suitable for growing in a liquid culture medium into an 1/2TSB liquid culture medium, culturing for 5-7 days at 28 ℃ and 220rpm to obtain a bacterial liquid, carrying out low-speed centrifugal concentration, removing a supernatant, leaving 5ml of the bacterial liquid to mix uniformly, sucking 650 mu l of the concentrated bacterial liquid to mix with 80% of sterile glycerol with the same volume, and standing at-80 ℃ for strain preservation;
b, selecting bacteria which are not suitable for growing in the liquid culture medium into a microbank bacteria storage tube, and storing the strains at minus 80 ℃.
Identity with Sanger sequencing: the saved bacterial DNA of each strain was extracted, 16S rRNA gene was amplified with primers 27F and 1492R, and finally reverse Sanger sequencing was performed with primer 1492R.
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-TACGGCTACCTTGTTACGACTT-3’。
The successful sequences were sequenced by intercepting the sequences with amplification primers 799F and 1193R (Life technologies) used in constructing the bacterial identification library and aligning with the sequencing results of S4, resulting in sequences of 498 strains, which are high quality bacterial strains.
S5, obtaining the proportion of the cultured bacteria belonging to different phyla and/or abundant genera according to the bacteria OTU to which all the cultured bacteria belong under aseptic conditions, wherein the species is more than 4 phyla Proteobacteria (Proteobacteria): actinomycetea (actinobacillus): firmicutes (Firmicutes): the ratio of Bacteroidetes (bacteroidides) was 30:10:8:3, with 3 bacteroides in the lesser species: there are 3 species of curvularia (Chloroflexi), 2 species of Acidobacteria (Acidobacteria), 1 species of Verrucomicrobia (Verrucomicrobia), according to Proteobacteria (Proteobacteria): actinomycetea (actinobacillus): firmicutes (Firmicutes): bacteroidetes (bacteroidides): closterobacterium (Chloroflexi): acidobacterium phylum (Acidobacterium): the ratio of Verrucomicrobia (Verrucomicrobia) is 30:10:8:3:1:1:1, the preserved cultured bacteria are recovered and mixed to obtain the rice root system microorganism group.
Results and advantages
In this example, 6756 strains of rice rhizobacteria were co-isolated and cultured, wherein 278 species of bacteria with the only 16S rRNA account for about 60% of the species of rice rhizobacteria, 498 high-quality rice rhizobacteria strains were prepared, which provide a good resource foundation for further research on the functions of crop rhizobacteria, and any one or a combination of several of the strains could be obtained to produce a microbial inoculum, or a microbial ecological preparation containing the microbial inoculum, or a biofertilizer product containing the microbial inoculum or the microbial ecological preparation. Furthermore, the rice microbial group can be prepared according to the proportion of different phyla and/or high abundance genera to which the cultured bacteria belong, the composition height of the rice microbial group is close to that of the in-situ microbial group, and the obtained rice microbial group can be used for preparing a microbial inoculum, a microbial ecological agent containing the microbial inoculum, or a biological fertilizer product containing the microbial inoculum or the microbial ecological agent.
Example 2 Arabidopsis thaliana root system microbiome high-flux isolation culture method
This example was carried out according to the flow chart (FIG. 1) of the method for high-throughput isolation and culture of crop root microbiome of the present invention.
The example was carried out in Beijing in 2017 on Arabidopsis thaliana grown in the greenhouse, the variety Columbia (Columbia).
S1, grinding the root system of Arabidopsis thaliana to obtain crop root homogenate under an aseptic condition, diluting the crop root homogenate by adopting a gradient dilution method to obtain a plurality of dilution samples, and culturing each dilution sample on 45 bacterial culture plates with 96 holes until the number of turbid holes of all the dilution samples is not increased any more; and (3) turbid wells obtained by culturing the diluted samples with the ratio of the number of the turbid wells to the total number of the wells inoculated with the corresponding diluted samples being 30-50%, wherein the liquid in each turbid well contains bacteria, and the liquid in each turbid well is named as cultured bacteria. The method comprises the following specific steps:
s1.1, sampling and cleaning: collecting all root systems of 3 arabidopsis thaliana which grows to 6-7 weeks; washing with sterile water until the soil blocks are washed clean; the washing was performed 3 times with 1 XPBS buffer, each time at 180rpm for 15 min. Sucking water on the surface of the root system by using sterile filter paper, and removing dead roots, impurities and the like by using tweezers; the roots of the 3 seedlings are cut into small sections with the diameter of 1-2mm by scissors, and the small sections of the root systems of the 3 seedlings are mixed together and evenly mixed.
Preparation of mother liquor 10 × PBS buffer solution NaCl 76g, Na2HPO425g，NaH2PO44.68g, add sterile water to 1L, sterilize at 121 ℃ for 15min, adjust pH to 7 with 1mM NaOH.
The stock solution was diluted 10-fold with 10 XPBS buffer to obtain 1 XPBS buffer.
S1.2 extracting bacteria: 0.02g of the mixed root system tissue obtained in S1.1 was weighed into a sterile 1.5ml centrifuge tube, and 200. mu.l of 10mM MgCl which was sterile filtered was added2Grinding root system tissue into pulp by using a sterilizing grinding rod; pipette all homogenates into 25mL 10mM MgCl2And (4) uniformly mixing the solution, and standing for 15min to obtain the crop root homogenate.
S1.3 gradient dilution: the crop root homogenate from step S1.2 was diluted 600-fold, 2000-fold, and 6000-fold with 1/10 × TSB broth, respectively, to give 3 dilution samples.
The 1/10 XTSB liquid culture medium is liquid with the pH value of 7.3 +/-0.2 and consists of a solute and a solvent, the solvent is water, and the concentration of the solute is 17.0g/L of tryptone, 3.0g/L of soybean papain digest, 5.0g/L of sodium chloride, 2.5g/L of dipotassium hydrogen phosphate and 2.5g/L of glucose.
S1.4 bacterial culture: adding 3 dilution samples into a 96-well bacterial culture plate respectively, wherein each dilution sample is 160 mu l per well, each dilution sample is cultured on 45 96-well plates, and each plate is placed at room temperature (25 ℃) for standing culture after being sealed; daily observations were made until the number of turbid wells for all dilutions of the sample no longer increased, taking approximately 16-20 days.
Turbidity in the wells indicates bacterial growth. The proportion of turbidity in the wells is in Poisson distribution with the appearance of single bacteria, and when the turbidity in the wells is 30-50%, the probability that each well is single bacteria is the highest. The proportion of the number of the holes left turbid to the total number of the holes inoculated by the corresponding dilution sample is 30-50%, and the obtained turbid holes are cultured by the dilution sample: after culturing the dilution sample with the dilution multiple of 6000 times, 45 96-well bacterial culture plates account for 4320 wells, the turbidity in 1598 wells indicates that bacteria grow and accounts for 37 percent of the total number of wells inoculated by the dilution sample, all the turbid wells after culturing the dilution sample are left, the liquid in each turbid well contains bacteria, and the liquid in each turbid well is named as cultured bacteria.
Taking out a part of the bacterial liquid for culturing bacteria in each hole for bacteria identification, adding 80% (v/v) glycerol into the rest bacterial liquid according to the volume ratio of 1:1, and storing at-80 ℃ for later use.
S2 extracting genomic DNA of each of the cultured bacteria under aseptic conditions.
Extracting DNA from part of bacterial liquid of the cultured bacteria in each hole, and storing the rest bacterial liquid at-80 ℃ for later use. And (3) extracting DNA: and uniformly mixing, cracking and extracting the bacterial liquid of each cultured bacterium to obtain template DNA. The method comprises the following specific steps:
s2.1 adding 10 mul of buffer solution I into each hole of a sterile 96-hole PCR plate, respectively and uniformly mixing bacterial liquid of each cultured bacterium in the original 96-hole cell culture plate, sucking 6 mul of buffer solution I into each hole, and adding the buffer solution I into the 96-hole PCR plate at the corresponding position.
Buffer I: solutes were 25mM NaOH and 0.2mM EDTA, and solvent was water, pH 12.
S2.2, placing the 96-hole PCR plate in a PCR instrument, and carrying out high-temperature thallus lysis in an alkaline environment, wherein the program is set to 95 ℃ for 30 min.
S2.3 adding 10 mul of buffer solution II into each hole, mixing uniformly and centrifuging to obtain the genome DNA of each cultured bacterium.
And (2) buffer solution II: 40mM Tris HCl, pH 7.5.
S3, under the aseptic condition, taking the genome DNA of each cultured bacterium as a template, taking 799F and 1193R as primers, respectively carrying out PCR amplification on 16S rRNA gene V5-V7 fragments, and the PCR is called as first-step PCR; performing PCR amplification on a 16S rRNA gene V5-V7 fragment by using the obtained first-step PCR product as a template and 799F-2 and 1193R-2 as primers, wherein the PCR is called as second-step PCR; collecting the second-step PCR products of each of the cultured bacteria and mixing to obtain a mixture of second-step PCR products of all the cultured bacteria, which is called a cultured bacteria identification library;
799F is a single-stranded DNA with a nucleotide sequence of sequence 1, 1193R is a single-stranded DNA with a nucleotide sequence of sequence 2, and 799F-2 and 1193R-2 are primers selected from the following A or B:
A. the 799F-2 is 96 single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on the 799F, and the 1193R-2 is M single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of M-plate barcode on the 1193R;
B. 799F-2 is M single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of M plate barcode on 799F, and 1193R-2 is 96 single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on 1193R;
the 96-well barcode is a marker for marking 96 wells on one 96-well plate, the nucleotide sequences of the 96-well barcode are different, the M-plate barcode is a marker for marking the N96-well plate, the nucleotide sequences of the M-plate barcode are different; the M is the number of sheets of 96-well plates inoculated with the dilution sample left at S1 or more. In this example, the primer was selected from A, the number of 96-well plates was 45, and M was 96. The procedure of S3 in example 1 was followed to obtain a library of cultured bacteria.
S4, sequencing the cultured bacteria identification library, comparing the sequence obtained by sequencing with the 16SrRNA gene V5-V7 fragment column in the RDP database to obtain the crop root system bacteria OTU, and storing the cultured bacteria corresponding to the crop root system bacteria OTU. The method comprises the following specific steps:
the cultured bacteria identification library is sequenced on a Hiseq2500 platform to obtain sequences of 5185 strains of arabidopsis root bacteria, and each sequence comprises a plate barcode, a pore barcode and a 16S rRNA gene V5-V7 sequence. And (3) performing quality filtering and sample splitting on the sequence according to the hole barcode and the plate barcode, and defining OTU (denoising mode, which is equivalent to 100% similarity) after removing chimera and low-abundance sequence (reading count is less than 8) by using UNOISE algorithm. The sequences of the 16SrRNA genes V5-V7 of the cultured bacteria are compared with the sequences of the 16SrRNA genes V5-V7 of the known bacteria OTU in an RDP (http:// RDP. cme. msu. edu /) database, the bacteria OTU is classified by adopting an SINTAX algorithm, and the bacteria OTU is considered to belong to the same OTU when the similarity with the sequences of the 16SrRNA genes V5-V7 of the known bacteria OTU is more than a threshold value (97 percent), and the cultured bacteria can be cultured. Visualization of the dendrogram by GraPhlAn 0.9.7 showed the number and species culturable in the cultured bacteria, resulting in a total of 238 non-redundant bacterial OTUs.
According to the result of the identification at S4, 445 representative strains of non-redundant bacteria OTU were selected from the cultured bacteria reserved for storage at S1, and recovered by continuous plating to store the strains. The method comprises the following specific steps:
1) and (3) recovering bacteria: according to the result of the identification in the step S4, a representative strain of the non-redundant bacteria OTU is selected from the cultured bacteria preserved and ready for use in S1, streaked and activated on a 1/2TSB solid medium plate, sealed and left at room temperature (25 ℃) for about 3 days, and a single colony is picked and transferred to a new 1/2TSB solid medium plate for purification 2 times;
2) and (3) strain preservation: the bacteria which are suitable for growing in the liquid culture medium and the bacteria which are not suitable for growing in the liquid culture medium are respectively preserved by the following methods a or b:
a, selecting bacteria suitable for growing in a liquid culture medium into an 1/2TSB liquid culture medium, culturing for 5-7 days at 28 ℃ and 220rpm to obtain a bacterial liquid, carrying out low-speed centrifugal concentration, removing a supernatant, leaving 5ml of the bacterial liquid to mix uniformly, sucking 650 mu l of the concentrated bacterial liquid to mix with 80% of sterile glycerol with the same volume, and standing at-80 ℃ for strain preservation;
b, selecting bacteria which are not suitable for growing in the liquid culture medium into a microbank bacteria storage tube, and storing the strains at minus 80 ℃.
Identity with Sanger sequencing: the saved bacterial DNA of each strain was extracted, 16S rRNA gene was amplified with primers 27F and 1492R, and finally reverse Sanger sequencing was performed with primer 1492R.
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-TACGGCTACCTTGTTACGACTT-3’。
The successful sequences were sequenced by intercepting the sequences with amplification primers 799F and 1193R (Life technologies) used in constructing the bacterial identification library and aligning with the sequencing results of S4, resulting in 412 strains with high quality bacterial strains.
S5, under aseptic conditions, obtaining the proportion of the cultured bacteria belonging to different phyla and/or abundant genera as Proteobacteria (Proteobacteria) according to the bacteria OTU to which all the cultured bacteria belong: actinomycetes door
(Actinobacteria): firmicutes (Firmicutes): and (3) recovering the preserved cultured bacteria according to the proportion, and mixing to obtain the arabidopsis root system microorganism group.
Results and advantages
In this example, 5185 arabidopsis root bacteria are co-isolated and cultured, wherein the bacteria comprise 238 kinds of bacteria with the only 16S rRNA, which account for about 58% of the species of arabidopsis root bacteria, and 412 high-quality arabidopsis root bacteria strains are prepared, so that a good resource basis is provided for further research on the functions of crop root microorganisms, and any one or more of the obtained strains can be used to produce a microbial inoculum, or a microbial ecological preparation containing the microbial inoculum, or a biological fertilizer product containing the microbial inoculum or the microbial ecological preparation. Furthermore, the arabidopsis thaliana microbial group can be prepared according to the proportion of different phyla and/or high abundance genera to which the cultured bacteria belong, the composition height of the arabidopsis thaliana microbial group is close to that of the in-situ microbial group, and the obtained arabidopsis thaliana microbial group can be used for preparing a microbial inoculum, or a microbial ecological agent containing the microbial inoculum, or a biological fertilizer product containing the microbial inoculum or the microbial ecological agent.
The embodiment of the invention aims at the rice root system microbiome and the arabidopsis root system microbiome, and other crop root system microbiomes can also adopt the same method to carry out high-flux separation culture and identification on the root system microbiome.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
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<120> high flux separation culture method for crop root system microbiome
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Claims (10)
1. A high flux separation culture method of crop root system microbiome is characterized in that: the method comprises the following steps:
s1, grinding crop root systems under aseptic conditions to obtain crop root system homogenate, diluting the crop root system homogenate by adopting a gradient dilution method to obtain a plurality of dilution samples, and culturing each dilution sample on N pieces of 96-pore plates until the number of turbid pores of all dilution samples is not increased any more; turbid holes obtained by culturing the dilution samples with the proportion of the number of the turbid holes to the total number of the dilution samples inoculated by the corresponding dilution samples being 30-50%, wherein the liquid in each turbid hole contains bacteria, and the liquid in each turbid hole is named as cultured bacteria; n is a natural number more than or equal to 2;
s2, extracting the genome DNA of each cultured bacterium under the aseptic condition;
s3, under the aseptic condition, taking the genome DNA of each cultured bacterium as a template, taking 799F and 1193R as primers, respectively carrying out PCR amplification on 16S rRNA gene V5-V7 fragments, and the PCR is called as first-step PCR; performing PCR amplification on a 16S rRNA gene V5-V7 fragment by using the obtained first-step PCR product as a template and 799F-2 and 1193R-2 as primers, wherein the PCR is called as second-step PCR; collecting the second-step PCR products of each of the cultured bacteria and mixing to obtain a mixture of second-step PCR products of all the cultured bacteria, which is called a cultured bacteria identification library;
799F is a single-stranded DNA with a nucleotide sequence of sequence 1, 1193R is a single-stranded DNA with a nucleotide sequence of sequence 2, and 799F-2 and 1193R-2 are primers selected from the following A or B:
A. the 799F-2 is 96 single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on the 799F, and the 1193R-2 is M single-stranded DNAs obtained by connecting a sequence required for Illumina sequencing and one of M-plate barcode on the 1193R;
B. 799F-2 is M single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of M plate barcode on 799F, and 1193R-2 is 96 single-stranded DNA obtained by connecting a sequence required for Illumina sequencing and one of 96-well barcode on 1193R;
the 96-well barcode is a marker for marking 96 wells on one 96-well plate, the nucleotide sequences of the 96-well barcode are different, the M-plate barcode is a marker for marking the N96-well plate, the nucleotide sequences of the M-plate barcode are different; m is a natural number which is more than or equal to N;
s4, sequencing the cultured bacteria identification library, comparing a sequence obtained by sequencing with a 16S rRNA gene V5-V7 fragment column in an RDP database to obtain crop root bacteria OTU, and storing the cultured bacteria corresponding to the crop root bacteria OTU;
and S5, obtaining the proportion of the cultured bacteria belonging to different phyla and/or high abundance genera according to the bacteria OTU to which all the cultured bacteria belong under the aseptic condition, and recovering and mixing the stored cultured bacteria according to the proportion to obtain the crop root system microorganism group.
2. The method of claim 1, wherein: the N is a natural number of 25-60, specifically 45.
3. The method of claim 2, wherein: the crop is monocotyledon or dicotyledon.
4. The method according to any one of claims 1-3, wherein: in S1, the culture was performed at room temperature.
5. The method according to any one of claims 1-4, wherein: in the S1, diluting the crop root homogenate by a 1/10 XTSB liquid culture medium by adopting a gradient dilution method; the 1/10 XTSB liquid culture medium is liquid with the pH value of 7.3 +/-0.2 and consists of a solute and a solvent, the solvent is water, and the concentration of the solute is 17.0g/L of tryptone, 3.0g/L of soybean papain digest, 5.0g/L of sodium chloride, 2.5g/L of dipotassium hydrogen phosphate and 2.5g/L of glucose.
6. The method according to any one of claims 1-5, wherein: in S4, sequencing was performed using the Hiseq2500 platform.
7. The method according to any one of claims 1-6, wherein: and in the S4, performing quality filtration and sample splitting on the sequence according to the hole barcode and the plate barcode to obtain the crop root system bacteria OTU.
8. The method according to any one of claims 1-7, wherein: the microbiome is a bacterium.
9. A crop root microbiome obtainable by the method of any one of claims 1 to 8.
10. Use of the method of any one of claims 1 to 8, and/or the crop plant root microbiome of claim 9 in the manufacture of a product; the method is characterized in that: the product contains a microorganism or a plurality of microorganisms or a crop root system microbiome obtained by the method of any one of claims 1 to 8, or contains a crop root system microbiome as defined in claim 9.
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Application publication date: 20200811 |