CN118126880A - Method for separating and preserving endophytic bacteria of plants without host background interference for high-throughput screening - Google Patents
Method for separating and preserving endophytic bacteria of plants without host background interference for high-throughput screening Download PDFInfo
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Abstract
The invention discloses a method for separating and preserving endophytic bacteria of plants without host background interference for high-throughput screening, which comprises the following steps: s1, plant pretreatment; s2, obtaining an endophytic bacteria culture; s3, determining the optimal culture concentration of the endophyte culture; s4, separating and culturing endophytes; s5, endophyte screening; s6, purifying and recovering a plant endophytic bacterium 16S full-length gene amplicon; s7, high-throughput sequencing and biological information preservation based on a Nanopore platform. The beneficial effects of the invention are as follows: the rapid identification of the endophyte of the plant is carried out by adopting a high-flux culture method and combining a three-generation full-length high-flux sequencing technology, compared with the existing non-culture method, the method has the advantages that the endophyte can be preserved after the sequencing is finished, and the detection result is more complete; the novel identification and preservation method for the plant culturable endophyte can selectively cultivate the plant endophyte in a targeted manner, and greatly improves the screening and preservation efficiency of the plant endophyte.
Description
Technical Field
The invention relates to the technical field of biological preservation, in particular to a method for separating and preserving endophytic bacteria of plants without host background interference, which is used for high-throughput screening.
Background
Plant bacterial diseases can invade a host through the epidermis under rhizosphere soil or wind and rain conditions, and also invade the host through wounds and the like, pathogenic bacteria can remain in disease residues, seeds, soil and the like for overwintering, and the host can be harmed when meeting proper conditions.
The existing bacterial disease detection method mainly comprises the methods of washing microscopic examination, moisture preservation culture, pathogenicity determination, PCR detection and the like. Although the methods can carry out qualitative detection on pathogenic microorganisms, the traditional methods are limited by the technology, and cannot detect all pathogenic microorganisms in complex samples at one time and cannot carry out high-throughput screening and strain preservation. Therefore, in order to perform early prevention and comprehensive screening of pathogenic microorganisms, the technology of high-throughput detection, screening and preservation of plant endophytic microorganisms has been widely applied to the fields of plant disease detection and the like in recent years, has the advantages of rapidness, reliability, high resolution, good repeatability and the like, and provides a repeatable quantitative research method for research of plant diseases. By means of high throughput sequencing, we can conduct a comprehensive study of interactions of endophytic bacteria in plants. The plant endophytic bacteria are important components in a plant micro-ecological system, form interdependence relationship with plants in a long-term co-evolution process, and the generated active substances have great application prospects in the field of plant disease resistance and other production. However, due to the high homology of plant chloroplasts and mitochondria with endophytic bacteria genes, even if high-throughput sequencing is adopted for related research, the research of plant endophytic bacteria is at a basic research level due to the interference of the genome of a plant host, and most detection methods can only carry out high-throughput sequencing at present, and microorganisms to be detected cannot be stored subsequently. Therefore, the invention designs a high-flux detection, screening and preservation integrated method, and provides a rapid and effective method for researching the plant culturable endophytes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for separating and preserving plant endophyte without host background interference for high-throughput screening, can overcome the technical limitations of culture separation, plant plastid/mitochondrial genome interference and the like in the traditional research method of the plant endophyte, and improves the screening and preserving efficiency of the plant endophyte.
The aim of the invention is realized by the following technical scheme: a method for the isolation and preservation of endophytic bacteria of plants without host background interference for high throughput screening, comprising the steps of:
S1, pretreatment of plants: taking 0.5-1g of fresh plant root sample, ultrasonic cleaning, soaking and sterilizing in a super clean bench for 1-3 minutes by using a disinfectant, washing with sterile water once, soaking for 20-40 seconds by using 70-75% ethanol solution by volume percent, and then washing with sterile water for 3-5 times to thoroughly remove the disinfectant on the surface of the sample;
s2, obtaining endophyte culture: grinding the pretreated plant sample in an ice bath to be homogenized, adding 20-30ml of 10mM magnesium chloride solution to re-suspend the plant sample in the ice bath to be homogenized, standing at room temperature for 10-20 minutes, extracting a plurality of samples I for determining the optimal concentration of endophyte culture and a plurality of samples II for separating and preserving, carrying out gradient dilution of different concentrations on the samples I, and transferring the diluted plurality of samples I into a sterile porous culture plate for culturing for 30-60 minutes to obtain collected samples under different concentrations;
S3, determining the optimal culture concentration of the endophyte culture: the most positive Kong Kongshu samples were selected as the best samples according to the following formula:
Wherein N neg is the number of negative holes of the multi-hole culture plate in the optimal sample; n total is the total number of wells of the multi-well plate in the optimal sample; v partition is the single-hole culture volume of the multi-hole culture plate in the optimal sample, and the optimal culture concentration C1 of the endophytic bacteria culture is calculated;
S4, separating and culturing endophytes: diluting the sample II to the optimal culture concentration C1, taking the diluted sample II into a porous culture plate, performing static culture at room temperature after film coating, observing the number of opaque holes in the culture process, stopping culture when the number of opaque holes is 30-40%, obtaining a culture, and performing an endophyte screening process;
S5, endophyte screening, which comprises the following substeps:
S501, turbidity screening: sucking the culture in the step S4 into a multi-hole ELISA plate, then scanning an OD value, judging positive if the OD value is larger than 0.01, and selecting a positive culture for subsequent identification;
S502, screening gene copy numbers: sucking 0.5-1 μl of the positive culture in the step S501 to a porous PCR plate, detecting the gene copy number of the culture by using a Biorad CFX96 fluorescent quantitative qPCR instrument, judging that a sample with Ct value less than 35 is positive, and selecting the positive culture for subsequent identification;
S503.PCR and fluorescence screening: the positive culture of step S502 was aspirated at 0.5-1. Mu.l into a multi-well PCR plate, and PCR and fluorescence screening were performed with the following primers and probes added:
upstream primer AMF 5'-AGAGTTTGATCATGGCTCAG-3'
Downstream primer AMR 5'-CAGGCCTAACACATGCAAGTC-3'
Probe AMP 5'-HEX-ZEN (int) -CAGGCCTAACACATGCAAGTC-Iowa Black FQ-3'
After PCR is completed, setting fluorescence excitation wavelength to 400-660nm by using Tecan M200, detecting emission wavelength to 530-730nm, judging that cultures with blank culture medium as a reference and a background value larger than 5 times of the reference are negative, judging that the rest cultures are positive, and selecting positive cultures for subsequent identification;
the PCR and fluorescence screening method can be one or more screening methods of nucleic acid specific dye binding fluorescence screening based on a PCR method, nucleic acid non-specific dye binding fluorescence screening based on a PCR method, taqman probe method screening based on a PCR method, molecular beacon fluorescence screening based on a MGB probe, amplifluor probe fluorescence screening based on a LUX probe fluorescence screening based on a QZyme probe fluorescence screening based on a LocNA probe fluorescence screening based on a Scorpion probe fluorescence screening based on a PCR and a fluorescent enzyme labelling instrument screening. The fluorescent screen may be one or a combination of a single wavelength fluorescent screen, or a multi-wavelength fluorescent screen, or a fluorescent screen based on a PCR method. The PCR may be one or more of a general PCR, or a fluorescent quantitative PCR, or a digital PCR.
S6, purifying and recovering the plant endophytic bacteria 16S full-length gene amplicon, namely carrying out PCR amplification on the positive culture in the step S503, detecting a reaction product after the PCR is finished by using 1-2% agarose gel electrophoresis, cutting gel by using a QIAquick gel recovery kit to recover a target strip with the fragment size of 1400-1600bp, and eluting by using TE buffer solution to recover the target DNA fragment;
S7, high-throughput sequencing and biological information preservation based on a Nanopore platform: constructing a gene amplicon library, sequencing a target DNA fragment based on the gene amplicon library, wherein a sequencing platform is Nanopore GridIon, sequencing lower machine data is in a fast5 format, converting electric signal data recorded in a fast5 file into sequence and quality information recorded in a fastq format, and preserving.
The disinfectant is sodium hypochlorite solution with 5% of available chlorine concentration.
The sample I was subjected to 200-fold, 600-fold, 2000-fold, 6000-fold, 18000-fold and 54000-fold gradient dilution with 10% TSB medium in the step S2.
The 10% TSB culture medium comprises one or more of triazines, imidazolones, phenylureas and diphenyl ether compounds.
The sterile multi-well culture plate is a 96-well culture plate, and the addition volume of each well of the sterile multi-well culture plate is 160 mu l.
In the step S502, a qPCR premix, a 16SU515F/U806R primer pair and water are added into the culture and mixed uniformly before the gene copy number detection. The detection of the gene copy number is carried out by the prior means, the mixing proportion of the qPCR premix, the 16SU515F/U806R primer pair and water is carried out according to the prior proportion, and the details are not repeated.
The 16S U515F/U806R primer sequence is as follows: U515F:5'-GTGCCAGCMGCCGCGG-3'/U806R 5'-GGACTACHVGGGTWTCTAAT-3'.
The gene amplicon library was constructed using the Genomic-DNA-by-Ligation Kit.
In the step S6, the PCR premix, the 16S 8F/1492R primer pair and water are added into the positive culture and uniformly mixed for PCR amplification.
In the step S501, a Tecan M200 microplate reader is adopted to scan OD value at 600nm of visible light.
Therefore, in order to obtain the optimal sample of the endophytic bacteria, the application avoids pollution of the existing means to the plant genome, adopts sample II to carry out multiple groups of dilution and observe the positive hole number of the culture plate to determine the optimal dilution concentration (cultivation concentration) of the endophytic bacteria, overcomes the technical limitations of culture separation, plant plastid/mitochondrial genome interference and the like in the traditional research method of the endophytic bacteria, adopts multiple groups of screening methods to determine relatively stable endophytic bacteria information, and brings about the precision and accuracy which cannot be achieved by other conventional methods to the research of the endophytic bacteria.
The beneficial effects of the application are as follows: 1. the application adopts the high-flux culture method and combines the three-generation full-length high-flux sequencing technology to rapidly identify the endophyte, compared with the existing non-culture method, the method can preserve the endophyte after the sequencing is completed, and the detection result is more complete; 2. the application provides a novel method for identifying and preserving plant culturable endophyte, which can selectively culture plant endophyte in a targeted manner.
Detailed Description
The technical scheme of the present invention is described in further detail below with reference to examples, but the scope of the present invention is not limited to the following.
Example 1
A method for the isolation and preservation of endophytic bacteria of plants without host background interference for high throughput screening, comprising the steps of:
S1, pretreatment of plants: taking 0.8g of fresh plant root samples (cowpea leaves), carrying out ultrasonic cleaning, soaking and sterilizing in a super clean bench for 2 minutes by using a disinfectant, washing with sterile water once, soaking for 30 seconds by using an ethanol solution with the volume percentage of 72%, and then washing with sterile water for 5 times to thoroughly remove the disinfectant on the surfaces of the samples;
S2, obtaining endophyte culture: grinding the pretreated plant sample in an ice bath to be homogenized, adding 25ml of 10mM magnesium chloride solution to re-suspend the plant sample in the ice bath to be homogenized, standing at room temperature for 15 minutes, extracting a plurality of samples I for determining the optimal concentration of endophyte culture and a plurality of samples II for separating and preserving the plant sample, carrying out gradient dilution of different concentrations on the samples I, and transferring the diluted plurality of samples I into a sterile porous culture plate for culturing for 40 minutes to obtain collected samples under different concentrations;
In the step S2, 10% TSB culture medium (containing 0.05% -chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) is used for carrying out gradient dilution on the sample I by 200 times, 600 times, 2000 times, 6000 times, 18000 times and 54000 times.
S3, determining the optimal culture concentration of the endophyte culture: the most positive Kong Kongshu samples were selected as the best samples according to the following formula:
Wherein N neg is the number of negative holes of the multi-hole culture plate in the optimal sample; n total is the total number of wells of the multi-well plate in the optimal sample; v partition is the single-hole culture volume of the multi-hole culture plate in the optimal sample, and the optimal culture concentration C1 of the endophytic bacteria culture is calculated;
In this example, the number of positive wells for single-cell isolation was the greatest with 96 wells, and the proportion of final positive wells was about 36.8%, with at most 35 wells positive, N total＝96,Nneg = 96-35 = 61, and c1 = 2.86cp/ml.
S4, separating and culturing endophytes: diluting the sample II to the optimal culture concentration C1, taking the diluted sample II into a porous culture plate, performing static culture at room temperature after film coating, observing the number of opaque holes in the culture process, stopping culture when the number of opaque holes is 35%, obtaining a culture, and performing an endophyte screening process;
S5, endophyte screening, which comprises the following substeps:
S501, turbidity screening: sucking the culture in the step S4 into a multi-hole ELISA plate, then scanning an OD value, judging positive if the OD value is larger than 0.01, and selecting a positive culture for subsequent identification;
S502, screening gene copy numbers: sucking 0.5 μl of the positive culture in the step S501 into a multi-hole PCR plate, detecting gene copy number of the culture by using a Biorad CFX96 fluorescent quantitative qPCR instrument, judging that a sample with Ct value less than 35 is positive, and selecting the positive culture for subsequent identification; before gene copy number detection, qPCR premix, 16S U515F/U806R primer pair and water are added into the culture and mixed uniformly. Wherein, 16S U515F/U806R primer sequence is: U515F:5'-GTGCCAGCMGCCGCGG-3'/U806R 5'-GGACTACHVGGGTWTCTAAT-3'. The reaction procedure was 1min at 95℃for 1 cycle; the fluorescence reading is carried out at 98 ℃ for 5s,55 ℃ for 30s and 68-72 ℃ for 30s and 40 cycles at the end of each cycle, and the qPCR premix is BlasTaq TM X2 qPCR Mastermix (EvaGreen is the fluorescent specific dye).
S503.PCR and fluorescence screening: pipette 0.5. Mu.l of the positive culture of step S502 into a multi-well PCR plate, add the following primers and probes for PCR and fluorescence detection:
upstream primer AMF 5'-AGAGTTTGATCATGGCTCAG-3'
Downstream primer AMR 5'-CAGGCCTAACACATGCAAGTC-3'
Probe AMP 5'-HEX-ZEN (int) -CAGGCCTAACACATGCAAGTC-Iowa Black FQ-3'
The reaction procedure was 1min at 95℃for 1 cycle; at 98℃for 5s, at 60℃for 30s,40 cycles, each cycle was completed and fluorescence reading was performed with qPCR premix BlasTaq TM X PCR Master mix. Setting fluorescence excitation wavelength 535nm and emission wavelength 556nm for detection by using Tecan M200 after PCR, judging that cultures with a blank culture medium as a reference and a background value greater than 5 times of the reference are negative, and selecting positive cultures for subsequent identification, wherein the rest cultures are positive;
S6, purifying and recovering a plant endophytic bacterium 16S full-length gene amplicon, namely carrying out PCR amplification on the positive culture in the step S503, wherein a MegaFi TM Pro Fidelity 2X PCR Master mix (G886) is used as a PCR premix, and the reaction condition is 1min at 95 ℃ for 1 cycle; 98 ℃ for 5s,55 ℃ for 30s,68-72 ℃ for 30s,25 cycles; 5min at 72 ℃ for 1 cycle; the PCR is completed under the constant temperature PCR circulation condition of 25 ℃, the reaction product after the PCR is detected by 1% agarose gel electrophoresis, the detection condition is 5V/cm for 20min, the QIAquick gel recovery kit is used for cutting gel to recover target bands with the fragment size of 1500bp, and TE buffer is used for eluting and recovering target DNA fragments.
S7, high-throughput sequencing and biological information preservation based on a Nanopore platform: constructing a gene amplicon library, sequencing a target DNA fragment based on the gene amplicon library, wherein a sequencing platform is Nanopore GridIon, a sequencing mode is R10.4.1, sequencing machine data is fast5 format, and Guppy software is used for converting electric signal data recorded in a fast5 file into sequence and quality information recorded in a fastq format and preserving the sequence and quality information. The test process comprises the following steps: a. distinguishing the samples according to Barcode; b. the species classification information was partitioned using the Silva database. The gene amplicon library was constructed using the Genomic-DNA-by-Ligation Kit, specifically using the Ligation sequencing amplicons V (SQK-LSK 114) Kit.
Sequencing data corresponds to the wells in the multi-well plate in example 1, if the sample is detected to be single bacteria (more than or equal to 95% of single species) by sequencing, 800 μl of culture medium of the sample well is sucked, 800 μl of 80% sterile glycerol is added, and the mixture is frozen in a refrigerator at-80 ℃ after uniform mixing, liquid nitrogen or dry ice.
Example 2
A method for the isolation and preservation of endophytic bacteria of plants without host background interference for high throughput screening, comprising the steps of:
S1, pretreatment of plants: taking 0.5g of fresh plant root sample, ultrasonic cleaning, soaking and sterilizing in a super clean bench with disinfectant for 1 minute, washing with sterile water once, soaking in 75% ethanol solution for 20 seconds, and washing with sterile water for 3 times to thoroughly remove the disinfectant on the surface of the sample;
S2, obtaining endophyte culture: grinding the pretreated plant sample in an ice bath to be homogenized, adding 20ml of 10mM magnesium chloride solution to re-suspend the plant sample in the ice bath to be homogenized, standing the plant sample at room temperature for 10 minutes, extracting a plurality of samples I for determining the optimal concentration of endophyte culture and a plurality of samples II for separating and preserving the endophyte culture, carrying out gradient dilution on the samples I with different concentrations, and transferring the diluted plurality of samples I into a sterile porous culture plate for culturing for 30 minutes to obtain collected samples with different concentrations;
S3, determining the optimal culture concentration of the endophyte culture: the most positive Kong Kongshu samples were selected as the best samples according to the following formula:
Wherein N neg is the number of negative holes of the multi-hole culture plate in the optimal sample; n total is the total number of wells of the multi-well plate in the optimal sample; v partition is the single-hole culture volume of the multi-hole culture plate in the optimal sample, and the optimal culture concentration C1 of the endophytic bacteria culture is calculated;
s4, separating and culturing endophytes: diluting the sample II to the optimal culture concentration C1, taking the diluted sample II into a porous culture plate, performing static culture at room temperature after film coating, observing the number of opaque holes in the culture process, stopping culture when the number of opaque holes is 30%, obtaining a culture, and performing an endophyte screening process;
S5, endophyte screening, which comprises the following substeps:
S501, turbidity screening: sucking the culture in the step S4 into a multi-hole ELISA plate, then scanning an OD value, judging positive if the OD value is larger than 0.01, and selecting a positive culture for subsequent identification;
S502, screening gene copy numbers: sucking 0.5 μl of the positive culture in the step S501 into a multi-hole PCR plate, detecting gene copy number of the culture by using a Biorad CFX96 fluorescent quantitative qPCR instrument, judging that a sample with Ct value less than 35 is positive, and selecting the positive culture for subsequent identification;
s503.PCR and fluorescence screening: pipette 0.5. Mu.l of the positive culture of step S502 into a multi-well PCR plate, add the following primers and probes for PCR and fluorescence detection:
upstream primer AMF 5'-AGAGTTTGATCATGGCTCAG-3'
Downstream primer AMR 5'-CAGGCCTAACACATGCAAGTC-3'
Probe AMP 5'-HEX-ZEN (int) -CAGGCCTAACACATGCAAGTC-Iowa Black FQ-3'
After PCR is finished, setting fluorescence excitation wavelength to 400nm and emission wavelength to 530nm by using Tecan M200, detecting, judging that cultures with blank culture medium as a reference and a background value larger than 5 times of the reference are negative, judging that the rest cultures are positive, and selecting positive cultures for subsequent identification;
S6, purifying and recovering the plant endophytic bacteria 16S full-length gene amplicon, namely carrying out PCR amplification on the positive culture in the step S503, detecting a reaction product after the PCR is finished by using 1% agarose gel electrophoresis, cutting gel to recover a target strip with the fragment size of 1400bp by using a QIAquick gel recovery kit, and eluting and recovering the target DNA fragment by using TE buffer solution;
S7, high-throughput sequencing and biological information preservation based on a Nanopore platform: constructing a gene amplicon library, sequencing a target DNA fragment based on the gene amplicon library, wherein a sequencing platform is Nanopore GridIon, sequencing lower machine data is in a fast5 format, converting electric signal data recorded in a fast5 file into sequence and quality information recorded in a fastq format, and preserving.
Example 3
A method for the isolation and preservation of endophytic bacteria of plants without host background interference for high throughput screening, comprising the steps of:
S1, pretreatment of plants: taking 1g of fresh plant root sample, ultrasonic cleaning, soaking and sterilizing in a super clean bench for 3 minutes by using a disinfectant, washing with sterile water once, soaking for 40 seconds by using 70% ethanol solution, and washing with sterile water for 5 times to thoroughly remove the disinfectant on the surface of the sample;
S2, obtaining endophyte culture: grinding the pretreated plant sample in an ice bath to be homogenized, adding 30ml of 10mM magnesium chloride solution to re-suspend the plant sample in the ice bath to be homogenized, standing at room temperature for 20 minutes, extracting a plurality of samples I for determining the optimal concentration of endophyte culture and a plurality of samples II for separating and preserving the plant sample, carrying out gradient dilution of different concentrations on the samples I, and transferring the diluted plurality of samples I into a sterile porous culture plate for culturing for 60 minutes to obtain collected samples under different concentrations;
S3, determining the optimal culture concentration of the endophyte culture: the most positive Kong Kongshu samples were selected as the best samples according to the following formula:
Wherein N neg is the number of negative holes of the multi-hole culture plate in the optimal sample; n total is the total number of wells of the multi-well plate in the optimal sample; v partition is the single-hole culture volume of the multi-hole culture plate in the optimal sample, and the optimal culture concentration C1 of the endophytic bacteria culture is calculated;
S4, separating and culturing endophytes: diluting the sample II to the optimal culture concentration C1, taking the diluted sample II into a porous culture plate, performing static culture at room temperature after film coating, observing the number of opaque holes in the culture process, stopping culture when the number of opaque holes is 40%, obtaining a culture, and performing an endophyte screening process;
S5, endophyte screening, which comprises the following substeps:
S501, turbidity screening: sucking the culture in the step S4 into a multi-hole ELISA plate, then scanning an OD value, judging positive if the OD value is larger than 0.01, and selecting a positive culture for subsequent identification;
S502, screening gene copy numbers: sucking 1 μl of the positive culture in the step S501 into a multi-hole PCR plate, detecting gene copy number of the culture by using a Biorad CFX96 fluorescent quantitative qPCR instrument, judging that a sample with Ct value less than 35 is positive, and selecting the positive culture for subsequent identification;
s503.PCR and fluorescence screening: 1 μl of the positive culture of step S502 was aspirated into a multi-well PCR plate, and the following primers and probes were added for PCR and fluorescence detection:
upstream primer AMF 5'-AGAGTTTGATCATGGCTCAG-3'
Downstream primer AMR 5'-CAGGCCTAACACATGCAAGTC-3'
Probe AMP 5'-HEX-ZEN (int) -CAGGCCTAACACATGCAAGTC-Iowa Black FQ-3'
Setting fluorescence excitation wavelength of 660nm and emission wavelength of 730nm for detection by using Tecan M200 after PCR, judging that cultures with a blank culture medium as a reference and a background value greater than 5 times of the reference are negative, judging that the rest cultures are positive, and selecting positive cultures for subsequent identification;
s6, purifying and recovering the plant endophytic bacteria 16S full-length gene amplicon, namely carrying out PCR amplification on the positive culture in the step S503, detecting a reaction product after the PCR is finished by using 2% agarose gel electrophoresis, cutting gel to recover a target strip with the fragment size of 1600bp by using a QIAquick gel recovery kit, and eluting and recovering the target DNA fragment by using TE buffer solution;
S7, high-throughput sequencing and biological information preservation based on a Nanopore platform: constructing a gene amplicon library, sequencing a target DNA fragment based on the gene amplicon library, wherein a sequencing platform is Nanopore GridIon, sequencing lower machine data is in a fast5 format, converting electric signal data recorded in a fast5 file into sequence and quality information recorded in a fastq format, and preserving.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (10)
1. A method for the isolation and preservation of endophytic bacteria of plants without host background interference for high throughput screening, comprising the steps of:
S1, pretreatment of plants: taking 0.5-1g of fresh plant root sample, ultrasonic cleaning, soaking and sterilizing in a super clean bench for 1-3 minutes by using a disinfectant, washing with sterile water once, soaking for 20-40 seconds by using 70-75% ethanol solution by volume percent, and then washing with sterile water for 3-5 times to thoroughly remove the disinfectant on the surface of the sample;
s2, obtaining endophyte culture: grinding the pretreated plant sample in an ice bath to be homogenized, adding 20-30ml of 10mM magnesium chloride solution to re-suspend the plant sample in the ice bath to be homogenized, standing at room temperature for 10-20 minutes, extracting a plurality of samples I for determining the optimal concentration of endophyte culture and a plurality of samples II for separating and preserving, carrying out gradient dilution of different concentrations on the samples I, and transferring the diluted plurality of samples I into a sterile porous culture plate for culturing for 30-60 minutes to obtain collected samples under different concentrations;
S3, determining the optimal culture concentration of the endophyte culture: the most positive Kong Kongshu samples were selected as the best samples according to the following formula:
Wherein N neg is the number of negative holes of the multi-hole culture plate in the optimal sample; n total is the total number of wells of the multi-well plate in the optimal sample; v partition is the single-hole culture volume of the multi-hole culture plate in the optimal sample, and the optimal culture concentration C1 of the endophytic bacteria culture is calculated;
S4, separating and culturing endophytes: diluting the sample II to the optimal culture concentration C1, taking the diluted sample II into a porous culture plate, performing static culture at room temperature after film coating, observing the number of opaque holes in the culture process, stopping culture when the number of opaque holes is 30-40%, obtaining a culture, and performing an endophyte screening process;
S5, endophyte screening, which comprises the following substeps:
S501, turbidity screening: sucking the culture in the step S4 into a multi-hole ELISA plate, then scanning an OD value, judging positive if the OD value is larger than 0.01, and selecting a positive culture for subsequent identification;
S502, screening gene copy numbers: sucking 0.5-1 μl of the positive culture in the step S501 to a porous PCR plate, detecting the gene copy number of the culture by using a Biorad CFX96 fluorescent quantitative qPCR instrument, judging that a sample with Ct value less than 35 is positive, and selecting the positive culture for subsequent identification;
S503.PCR and fluorescence screening: the positive culture of step S502 was aspirated at 0.5-1. Mu.l into a multi-well PCR plate, and PCR and fluorescence detection were performed with the following primers and probes added:
upstream primer AMF 5'-AGAGTTTGATCATGGCTCAG-3'
Downstream primer AMR 5'-CAGGCCTAACACATGCAAGTC-3'
Probe AMP 5'-HEX-ZEN (int) -CAGGCCTAACACATGCAAGTC-Iowa Black FQ-3'
After PCR is completed, setting fluorescence excitation wavelength to 400-660nm by using Tecan M200, detecting emission wavelength to 530-730nm, judging that cultures with blank culture medium as a reference and a background value larger than 5 times of the reference are negative, judging that the rest cultures are positive, and selecting positive cultures for subsequent identification;
S6, purifying and recovering the plant endophytic bacteria 16S full-length gene amplicon, namely carrying out PCR amplification on the positive culture in the step S503, detecting a reaction product after the PCR is finished by using 1-2% agarose gel electrophoresis, cutting gel by using a QIAquick gel recovery kit to recover a target strip with the fragment size of 1400-1600bp, and eluting by using TE buffer solution to recover the target DNA fragment;
S7, high-throughput sequencing and biological information preservation based on a Nanopore platform: constructing a gene amplicon library, sequencing a target DNA fragment based on the gene amplicon library, wherein a sequencing platform is Nanopore GridIon, sequencing lower machine data is in a fast5 format, converting electric signal data recorded in a fast5 file into sequence and quality information recorded in a fastq format, and preserving.
2. A method of isolated preservation of endophytic bacteria of plants without host background interference for high throughput screening according to claim 1, wherein the disinfectant is a sodium hypochlorite solution with an available chlorine concentration of 5%.
3. The method for isolated preservation of endophytic bacteria of plants without host background interference for high throughput screening according to claim 1, wherein the sample I is subjected to a 200-fold, 600-fold, 2000-fold, 6000-fold, 18000-fold, 54000-fold gradient dilution with 10% tsb medium in step S2.
4. A method of isolated preservation of host background interference free endophytic bacteria for high throughput screening according to claim 3, wherein the 10% tsb medium comprises one or more of triazines, imidazolinones, phenylureas, diphenyl ether compounds.
5. A method of isolated preservation of host background interference free endophytic bacteria in plants for high throughput screening according to claim 1, wherein the sterile multi-well plate is a 96 well plate with an add-on volume per well of 160 μl.
6. The method for separating and preserving endophytic bacteria without host background interference for high throughput screening according to claim 1, wherein in the step S502, qPCR premix, 16S U515f/U806R primer pair and water are added to the culture before the gene copy number detection.
7. The method for isolated preservation of endophytic bacteria of plants without host background interference for high throughput screening according to claim 6, wherein the 16s U515f/U806R primer sequences are: U515F:5'-GTGCCAGCMGCCGCGG-3'/U806R 5'-GGACTACHVGGGTWTCTAAT-3'.
8. The method for isolated preservation of host background interference-free endophytic bacteria in plants for high throughput screening according to claim 1, wherein the gene amplicon library is constructed using Genomic-DNA-by-Ligation Kit.
9. The method for isolated preservation of endophytic bacteria of plants without host background interference for high throughput screening according to claim 1, wherein in step S6, PCR premix, 16S 8f/1492R primer pair and water are added to the positive culture and mixed uniformly for PCR amplification.
10. The method for isolated preservation of endophytic bacteria of plants without host background interference for high throughput screening according to claim 1, wherein the OD value scanning is performed with visible light 600nm using a Tecan M200 microplate reader in step S501.
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