Uranium-tolerant fungus screening method based on high-throughput sequencing

Abstract

The invention provides a uranium-tolerant fungus screening method based on high-throughput sequencing, which comprises the following steps: constructing different uranium concentration gradients, planting test plants in soil with the different uranium concentration gradients, collecting rhizosphere soil of the test plants, extracting genomic DNA of the rhizosphere soil, selecting universal primers ITS5-1737F and ITSR-2043R of fungus ITS1 variable regions, and sequencing on a machine, according to the method, raw Reads obtained through sequencing are filtered, removed, denoised and spliced to obtain processed data, the data are screened at the genus level and the species level respectively, and therefore the uranium-tolerant fungi are screened out, the uranium-tolerant fungi can be screened out from the uranium-contaminated soil environment condition, and due to the adaptive characteristic of the uranium-tolerant fungi to the soil environment, the uranium-tolerant fungi can be screened out from the uranium-contaminated soil environment. Compared with a non-indigenous strain, the screened indigenous uranium-tolerant strain can better adapt to a radioactive uranium polluted environment, so that the tolerance is possibly stronger and more stable; in addition, the uranium element has the chemical property of heavy metal and also has radioactivity, so that compared with a heavy metal stress-resistant strain, the fungus screened by the method has a possibly wider application range. The method can provide a basis for ecological quality evaluation and restoration of uranium-polluted soil, and provides a guiding effect for later uranium pollution restoration work.

Description

Technical Field

[0001] The invention relates to the technical field of soil microorganism separation, and in particular to a method for screening uranium-resistant fungi based on high-throughput sequencing. Background Art

[0002] Microorganisms are the most active part of soil organic components and ecosystems, and are the main components of soil decomposition systems. They play a dominant role in soil nutrient transformation cycles, system stability and anti-interference ability, and soil sustainable development. They are considered to be the most sensitive biological indicators of soil quality. In recent years, the relationship between soil heavy metal pollution and soil microbial communities has been a hot issue in the ecological evaluation of soil heavy metal pollution.

[0003] Uranium is a natural radioactive nuclide. Uranium tailings contain 1% to 5% uranium and more than 98% radium and its decay daughters in the original ore. About 1 / 3 of the radioactive nuclides are long-lived, such as 238 U. 232 Th, 226 Ra, 210 Pb, etc. enter the surrounding soil under the scouring and weathering of rainwater or mountain torrents, forming a large area of ​​uranium-contaminated soil, and even endangering human health through the food chain.

[0004] In recent years, the use of plant-microorganism combined remediation of uranium-contaminated soil has been considered a green and feasible remediation technology. For plants, some soil microorganisms can improve their nutritional status, enhance their tolerance to adversity, reduce the occurrence of pests and diseases, and promote plant growth and development. For example, mycorrhizal fungi. Arbuscular mycorrhizalfungi (AMF), as a widely distributed soil fungus, can establish a mutually beneficial symbiotic relationship with more than 80% of plant roots on land. Once a symbiotic relationship is established, AMF can greatly improve the host plant's absorption of a variety of mineral nutrients, especially the difficult-to-mobile phosphorus element in the soil. In addition, AMF has also been shown to significantly improve the host plant's tolerance to drought, salt stress, heavy metals, extreme temperatures (high and low temperatures), low pH, aluminum toxicity, and organic pollutants.

[0005] However, most of the current research focuses on the effects of adding exogenous heavy metals such as mercury, cadmium, lead, and copper on the functions of microorganisms in the soil. Research on the effects of exogenous uranium stress on microbial communities in contaminated soils in uranium mining areas is still in its infancy. Soil microorganisms are easily affected by environmental conditions, especially soil physical and chemical properties. For example, soil pH has been proven to be one of the decisive factors driving soil microbial communities. Stress conditions are also ubiquitous in terrestrial ecosystems, and adverse environmental conditions can not only inhibit the population size of beneficial soil microorganisms, but also affect their functions, making it impossible to fully utilize the potential of soil microbial resources. Therefore, directly excavating fungi that can tolerate uranium stress from their original habitats is of great significance for the screening of microorganisms in plant-microorganism combined remediation technology. Summary of the invention

[0006] The invention discloses a uranium-resistant fungus screening method based on high-throughput sequencing, aiming to solve the technical problems existing in the prior art.

[0007] The present invention adopts the following technical solutions:

[0008] A method for screening uranium-resistant fungi based on high-throughput sequencing comprises: collecting initial soil samples; constructing a uranium concentration gradient, spraying uranyl nitrate solutions of different concentrations in the soil for equilibrium for 5-7 weeks; planting test plants in the soil with the constructed uranium concentration gradient, and collecting rhizosphere soil; extracting genomic DNA from the collected rhizosphere soil, using the soil genomic DNA as a template, and using primers ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and primers ITS2R-2043R

[0009] (5′-GCTGCGTTCTTCATCGATGC-3′) was used to amplify the genome, build a library and perform high-throughput sequencing on the machine; after splicing, quality control, filtering and denoising of the data off the machine, Anova variance analysis was performed on the genus level treatment group to screen out the genera with significant differences between groups; at the species level, Metastats software was used to perform T test on the species abundance data between groups to obtain the p value, and the p value was corrected to obtain the q value; species with differences at the species level were screened out according to the p value or q value, thereby screening out uranium-resistant fungi.

[0010] Preferably, collecting the initial soil sample includes: collecting soil 0-20 cm above the ground.

[0011] Preferably, the uranium concentration gradient is constructed by respectively converting the concentration of 0 mg·kg -1 , 50mg·kg -1 , 100mg·kg -1 The uranyl nitrate solution was sprayed on the soil and allowed to equilibrate for 5-7 weeks.

[0012] Preferably, the test plant is peanut.

[0013] Preferably, extracting the collected rhizosphere soil genomic DNA includes: extracting soil genomic DNA using a DP812 kit, detecting the concentration of the DNA using an enzyme reader, performing detection and amplification based on the detected DNA concentration and amplification region, and detecting the amplified PCR product using agarose gel electrophoresis.

[0014] Preferably, the PCR amplification system is Pusion Hot start flex 2X Master Mix 12.5 μl, Forward Primer 2.5 μl, Reverse Primer 2.5 μl, Template DNA 50 ng, ddH 2 O to 25μl; PCR amplification reaction conditions are: 95℃ pre-denaturation for 5 minutes; then perform cyclic reaction according to the following parameters: 95℃ denaturation for 30 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 40 seconds; cycle 25 times; after the cycle reaction is completed, keep at 72℃ for 7 minutes, and store the reaction product at 4℃.

[0015] Preferably, the Raw Reads obtained by sequencing are filtered using Trimmomatic v0.33 software; then the primer sequences are identified and removed using cutadapt1.9.1 software to obtain Clean Reads that do not contain primer sequences; the dada2 method in QIIME22020.6 is used for denoising, double-end sequence splicing, and chimera sequences are removed to obtain processed data.

[0016] Preferably, at the genus level, genera with significant differences among groups are screened out, and the genera with extremely significant differences (p < 0.01) are Mortierella, Archaeorhizomyces, Psathyrella, Cephalotrichum, Tausonia, and Thermoascus.

[0017] Preferably, at the species level, the species that are tolerant to uranium stress and whose abundance increases significantly are Archaeorhizomyces_borealis, Briansuttonomyces_eucalypti, Cyphellophora_sessilis, and Thermoascus_aurantiacus.

[0018] The invention provides a uranium-resistant fungus screening method based on high-throughput sequencing. Different uranium concentration gradients are constructed, and test plants are planted in soils with different uranium concentration gradients, rhizosphere soils of the test plants are collected, genomic DNA of the rhizosphere soils is extracted, universal primers ITS5-1737F and ITS2R-2043R of fungal ITS1 variable regions are selected for on-machine sequencing, Raw Reads obtained by sequencing are filtered, removed, denoised, and spliced ​​to obtain processed data, and the data are screened at the genus level and the species level, so as to screen out uranium-resistant fungi. The method can screen out uranium-resistant fungi from uranium-contaminated soil environmental conditions. Due to the adaptability of the fungi to the soil environment, the screened indigenous uranium-resistant strains can better adapt to the radioactive uranium contaminated environment than non-indigenous strains, and thus may have stronger tolerance and more stability. In addition, since the uranium element has radioactivity in addition to the chemical properties of heavy metals, the fungi screened out by the method have a wider range of applications than strains resistant to heavy metal stress, can provide a basis for ecological quality assessment and restoration of uranium-contaminated soil, and provide guidance for later uranium pollution restoration work. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the dilution curve of soil fungi treated with uranium provided by an embodiment of the present invention;

[0020] Figure 2 Shown is a histogram of variance analysis between genus levels provided by an embodiment of the present invention;

[0021] Figure 3 It shows the difference in species abundance between groups at the species level provided by the embodiments of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. In the description of the present invention, it should be noted that the term "or" is usually used in the sense of including "and / or", unless the content clearly indicates otherwise.

[0023] Example

[0024] Screening method for uranium-resistant fungal strains based on high-throughput sequencing:

[0025] 1. Determination of the physical and chemical properties of the test soil: Soil was collected from farmland around the Shaoguan mining area in Guangdong Province, and the soil was collected from 0-20 cm above the ground. The initial soil sample was collected before treatment, and the physical and chemical properties of the soil were determined to be: pH 5.28, water content 17.97%, total nitrogen 1.64 g kg -1 , total phosphorus 0.36g·kg -1, total potassium 28.23g·kg -1 、Organic matter 44.16g·kg -1 , water-soluble salt 0.3g·kg -1 、Uranium content 3mg·kg -1 , which is fertile farmland soil.

[0026] 2. Uranium concentration gradient setting: set 3 different uranium concentration gradients, and change uranyl nitrate (UO 2 (NO 3 ) 2 6H 2 O) solution was sprayed on the soil to form different uranium concentration gradients, namely: 0 mg·kg -1 , 50mg·kg -1 , 100mg·kg -1 After 6 weeks of equilibrium, soils with different uranium concentration gradients were placed in flower pots, with 4.0 kg of soil in each pot. Three parallel samples were set up in each group. Peanuts were selected as test plants, with three plants planted in each pot. In the early stage of cultivation, water was applied once every evening.

[0027] 3. Four months after planting, the peanut roots were dug up and rhizosphere soil samples were collected using the “shaking root method”: first, the loose soil attached to the surface of the peanut roots was shaken off, and then the soil within 0 to 1 mm of the roots was brushed with a brush, i.e., the rhizosphere soil. After removing visible roots, gravel and other impurities, the soil was stored in a -80°C refrigerator for soil fungal sequencing;

[0028] 4. Use DP812 soil genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract nucleic acid. Take 1 μl of nucleic acid, add 1X dsDNAHS Working Solution, use synergy HTX (manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.) to detect the concentration of nucleic acid, and use the fully automatic ultra-high throughput sequencing platform (Bai Ling 1000) according to the detected nucleic acid concentration and amplification area. ) Amplification detection: The integrity of the amplified PCR product was detected by 1.8% agarose gel electrophoresis (Beijing Bomei Fuxin Technology Co., Ltd.).

[0029] The universal primers ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2R-2043R (5′-GCTGCGTTCTTCATCGATGC-3′) of the variable region of fungal ITS1 were selected for amplification. The PCR amplification system was: Pusion Hotstart flex2X Master Mix 12.5 μl, Forward Primer 2.5 μl, Reverse Primer 2.5 μl, Template DNA 50 ng, and ddH 2 O to 25μl. The reaction conditions for PCR amplification were: 95℃ pre-denaturation for 5 minutes; then the cycle reaction was performed according to the following parameters: 95℃ denaturation for 30 seconds, 50℃ renaturation for 30 seconds, 72℃ extension for 40 seconds; 25 cycles; after the cycle reaction, the reaction was kept at 72℃ for 7 minutes, and the reaction product was stored at 4℃. The purified PCR product was recovered by gel excision using the Monarch DNA gel recovery kit (Beijing Hongyue Innovation Technology Co., Ltd.).

[0030] The purified PCR product was recovered by gel excision using the Monarch DNA gel recovery kit (Beijing Hongyue Innovation Technology Co., Ltd.), and the genome was amplified using adapter + universal primers; the library was then constructed using P5 / P7 + index + adapter, and the constructed library was sequenced using Illumina Novaseq 6000.

[0031] 5. Use Trimmomatic v0.33 software to filter the Raw Reads obtained by sequencing; then use cutadapt 1.9.1 software to identify and remove primer sequences to obtain Clean Reads that do not contain primer sequences; use the dada2 method in QIIME22020.6 to denoise, splice double-end sequences and remove chimera sequences to obtain the final valid data;

[0032] After connecting to a universal adapter suitable for high-throughput sequencing, the first round of PCR amplification is performed to obtain a first-round PCR product with universal adapters at both ends; using the first-round PCR product as a template, a second round of PCR amplification is performed using universal primers with sequencing adapters and indexes at the 5' end of the PCR product to obtain a second-round product with sequencing adapters and index sequences at both ends;

[0033] After the second-round PCR amplification products are purified, high-throughput sequencing is performed, and the Raw Reads obtained from the high-throughput sequencing are processed and converted into Clean Reads;

[0034] The sequences in Clean Reads were compared with the corresponding sequences of fungal ITS1 rRNA with clear annotation information. The sequences under these conditions were screened out by setting the similarity of the comparison to be greater than or equal to 97% and the coverage to be greater than 99.6% and less than 100.4%. The screened sequences were compared with the fungal reference sequences with the highest similarity in NCBI by one-to-one Blast alignment to obtain the final valid data for annotation. Table 1 is a statistical table of the sample sequencing data processing results.

[0035] 6. At the genus level, the three treatment groups (uranium concentration 0 mg kg -1 、Uranium concentration 0mg·kg -1 、Uranium concentration 0mg·kg -1 ) data were subjected to Anova analysis of variance, and the genera with significant differences among the groups were screened out (p < 0.05);

[0036] 7. At the species level, Metastats software was used to conduct T-tests on species abundance data between groups and to perform pairwise analysis between groups to screen out species that caused differences in the composition of samples between the two groups.

[0037] Table 1 Statistics of sample sequencing data processing results

[0038]

[0039]

[0040] Figure 1 The dilution curve of soil fungi treated with uranium provided by the embodiment of the present invention is shown; Figure 1 As shown in the figure, the dilution curves of the 9 treatment groups are within a certain range. As the number of sequencing increases, if the curve shows a sharp rise, it means that a large number of species are discovered in the community; when the curve tends to be flat, it means that the species in this environment will not increase significantly with the increase in the number of sequencing, indicating that the sample size is sufficient to cover all fungi, and the sequencing results can better represent the actual situation of the microorganisms in the samples.

[0041] Figure 2 : is a histogram of variance analysis between genus levels provided by an embodiment of the present invention; Figure 2 As shown in the figure, at the genus level, there were 6 genera with extremely significant differences among the groups (**p<0.01), namely Mortierella, Archaeorhizomyces, Psathyrella, Cephalotrichum, Tausonia, and Thermoascus. The genera whose abundance increased significantly after uranium treatment were screened out, namely Psathyrella and Thermoascus.

[0042] Figure 3 The figure shows the species abundance differences between species level groups provided by the embodiment of the present invention, such as Figure 3 As shown, at the species level, with U0 (uranium concentration 0mg·kg -1 ) compared to U50 (uranium concentration 50 mg kg -1 ) treatment, the species with significantly increased abundance included Psathyrella lutensis, Coniochaeta lignicola, unclassified_Talaromyces, unclassified Saccharomyces, Cladosporium funiculosum, unclassified Glomeraceae, Neocucurbitaria irregularis, Funneliformis mosseae, and U100 (uranium concentration 100 mg kg -1 ) treatment, the species with significantly increased abundance included Psathyrella lutensis, Archaeorhizomyces_borealis, Alternaria alstroemeriae, and Cladosporium funiculum.

[0043] Cladosporium_funiculosum, unclassified_Pseudeurotium, unclassified Naganishia, Briansuttonomyces_eucalypti, Cyphellophora sessilis, Thermoascus aurantiacus, Aquadiscula_appendiculata.

[0044] There are four fungal species that have a certain tolerance to uranium stress, namely Archaeorhizomyces_borealis, Briansuttonomyces_eucalypti, Cyphellophora_sessilis, and Thermoascus aurantiacus.

[0045] Thermoascus_aurantiacus.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for screening uranium-resistant fungi based on high-throughput sequencing, characterized in that: include: Collect initial soil samples; Construct a uranium concentration gradient by spraying different concentrations of uranyl nitrate solution on the soil to equilibrate for 5-7 weeks; Test plants were grown in soils with constructed uranium concentration gradients, and rhizosphere soil was collected; The collected rhizosphere soil genomic DNA was extracted and the soil genomic DNA was used as a template with primers ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and primers ITS2R-2043R (5′-GCTGCGTTCTTCATCGATGC-3′) to amplify the genome, build a library and perform high-throughput sequencing; After splicing, quality control, filtering, and denoising the data, Anova analysis of variance was performed on the genus level treatment groups to screen out the genera with significant differences between the groups; At the species level, Metastats software was used to perform a T test on the species abundance data between groups to obtain the p-value, and the p-value was corrected to obtain the q-value; species with differences at the species level were screened out based on the p-value or q-value, thereby screening out uranium-resistant fungi.

2. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: Collecting the initial soil sample includes: collecting soil 0-20 cm from the ground.

3. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: The uranium concentration gradient is constructed by: -1 , 50mg·kg -1 , 100mg·kg -1 The uranyl nitrate solution was sprayed on the soil and allowed to equilibrate for 5-7 weeks.

4. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: The test plant was peanut.

5. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: Extracting and collecting rhizosphere soil genomic DNA includes: extracting soil genomic DNA using a DP812 kit, detecting the concentration of DNA using an enzyme reader, performing detection and amplification based on the detected DNA concentration and amplification region, and detecting the amplified PCR product using agarose gel electrophoresis.

6. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 5, characterized in that: The PCR amplification system was Pusion Hot start flex 2X Master Mix 12.5 μl, Forward Primer 2.5 μl, Reverse Primer 2.5 μl, Template DNA 50 ng, and ddH2O to 25 μl; The amplification reaction conditions of PCR were as follows: pre-denaturation at 95°C for 5 minutes; then the cycle reaction was performed according to the following parameters: denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 40 seconds; the cycle was repeated 25 times; after the cycle reaction was completed, the reaction product was kept at 72°C for 7 minutes and stored at 4°C.

7. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: Trimmomatic v0.33 software was used to filter the Raw Reads obtained by sequencing; then cutadapt 1.9.1 software was used to identify and remove primer sequences to obtain Clean Reads that did not contain primer sequences; the dada2 method in QIIME22020.6 was used for denoising, double-end sequence splicing, and chimera sequences were removed to obtain processed data.

8. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: At the genus level, the genera with significant differences among the groups were screened out. The genera with extremely significant differences (p < 0.01) were Mortierella, Archaeorhizomyces, Psathyrella, Cephalotrichum, Tausonia, and Thermoascus.

9. The method for screening uranium-resistant fungi based on high-throughput sequencing according to claim 1, characterized in that: At the species level, the species that were tolerant to uranium stress and whose abundance increased significantly were Archaeorhizomyces_borealis, Briansuttonomyces_eucalypti, Cyphellophora_sessilis, and Thermoascus_aurantiacus.

Images