CN107603893B - Porphyra tenera with high resistance to cadmium as well as extraction method and application thereof - Google Patents

Abstract

The invention provides a rhodosporidium having high resistance to cadmium, an extraction method and application thereof, the bacterial strain is rhodosporidium, is registered and preserved in China general microbiological culture Collection center in 12.09.2017, with the preservation number of CGMCC No.14159, and the ITS sequence is shown in the sequence table SEQ.ID.NO: 1 is shown. The strain has a cadmium adsorption function, can adsorb cadmium in soil, and reduces the absorption of plants on cadmium, thereby promoting the growth of plants.

Description

A kind of uricaria with high resistance to cadmium and extraction method and application

technical field

The invention belongs to the technical field of screening of cadmium-resistant strains, and in particular relates to a purple sp. with high resistance to cadmium and an extraction method and application.

Background technique

Official documents show that the polluted area of our cultivated land exceeds 19.4% of the total cultivated land, and cadmium is the most widely polluted pollutant, accounting for about 7% of the total cultivated land. At the same time, under the background of "all rivers are polluted", cadmium pollution cannot be ignored in various water bodies including natural rivers, lakes, urban sewage rivers and industrial sewage seepage pits. The ecological restoration of cadmium in cultivated land and industrial sewage has become a worldwide problem. Taking the pollution of cultivated land in China as an example, the pollution is mostly non-point source pollution and difficult to remove in time. The fallow restoration faces great economic pressure, which makes the conventional treatment strategy of removing heavy metals face many obstacles. The safe utilization system of polluted farmland can realize the production of safe agricultural products without removing or slowly removing heavy metals, so it has become one of the hotspots in the treatment of heavy metal pollution in farmland.

Microbial fixatives based on cadmium-resistant microorganisms have important application prospects in the safe utilization system of cadmium-contaminated farmland. It utilizes the metabolic ability of cadmium-resistant microorganisms, reduces the effectiveness of heavy metal cadmium in soil in situ, and reduces the absorption of heavy metal cadmium by crops, thereby realizing the production of safe agricultural products. The microbial fixative is simple to apply, has no secondary pollution and is low in cost, and is expected to replace traditional chemical fixatives. Therefore, how to obtain a strain with high resistance to cadmium is an urgent problem to be solved.

SUMMARY OF THE INVENTION

In view of the above-mentioned problems in the prior art, the present invention provides a strain of Pseudomonas with high resistance to cadmium and an extraction method and application. has great application potential.

For realizing the above-mentioned purpose, the technical scheme that the present invention solves its technical problem adopts is:

A kind of Purpureo cillium sp. with high resistance to cadmium. It is registered and deposited by the General Microbiology Center of the Species Depository Management Committee, and the deposit number is CGMCC No. 14159, and its ITS sequence is shown in the sequence table SEQ.ID.NO:1.

The extraction method of above-mentioned bacterial strain, comprises the following steps:

Select cultivated soil, then add _CdCl2 solution with a concentration of 15-20mM to the soil, leave it at room temperature for 20-23 days, then mix the soil, randomly select soil from it, and perform a 10-fold gradient dilution with water, take each dilution The resulting soil suspension was evenly spread in Martin's medium containing 15-20mM CdCl ₂ , and cultured at 26-28°C, and then the single colony with the highest tolerance was isolated by the plate coating method, and the single colony was inoculated. Cultured in Martin's medium without CdCl ₂ to obtain strain ZYZX-YZ1.

Wherein, the Martin medium contains the following components by weight: 1% glucose, 0.5% peptone, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ·7H ₂ O, 1.5% agar, 0.001% red Bengal, and the rest are distilled water.

The purple bacteria ZYZX-YZ1 obtained by the above extraction is used in the restoration of heavy metal cadmium-contaminated soil or water bodies or other cadmium-contaminated environments, and the purple bacteria ZYZX-YZ1 can also be used to prepare a cadmium adsorbent.

The invention provides a cadmium-resistant purpura, an extraction method and application, and has the following beneficial effects:

(1) The present invention extracts a strain resistant to heavy metal cadmium from soil, and the tolerance of the extracted strain to heavy metal cadmium can reach up to 100 mM in a liquid medium, showing strong resistance to cadmium. The source of the strain is easy to obtain, the separation and purification method is simple and convenient, and the cost is low.

(2) The extracted Pseudomonas ZYZX-YZ1 has the function of cadmium adsorption, which can adsorb cadmium in the soil and promote the growth of plants.

Description of drawings

Figure 1 shows the growth of strain ZYZX-YZ1 in Martin's medium plate.

Figure 2 is a morphological diagram of the hyphae and conidia stained with Lactophenol Gossypium blue under the light microscope.

Figure 3 shows the results of PCR amplification of strain 18S rDNA and ITS.

Figure 4 is a phylogenetic analysis diagram based on ITS sequences.

Figure 5 shows the results of changes in the diameter of the mycelium of the strain under the stress of 16 mM CdCl ₂ .

Figure 6 shows the dry weight results of mycelia after culturing for 3 days under different cadmium concentrations.

Figure 7 shows the results of cadmium removal rate after culturing for 3 days under different cadmium concentrations.

Figure 8 shows the effect of strains on tobacco cadmium tolerance results.

Figure 9 shows the effect of strains on the accumulation of cadmium in tobacco.

Detailed ways

Example 1 Isolation of strains

The soil samples for this experiment were collected at the Luancheng Experimental Station of the Chinese Academy of Sciences. The geographical location is 37°53'N and 114°41'E. It belongs to the warm temperate semi-humid monsoon climate. . The annual average temperature is 12.2°C, and the annual average precipitation is 530mm. The cultivated soil soils were collected from 3 points for mixed sample treatment.

5mL of 16mM CdCl ₂ solution was added to each 50mL centrifuge tube containing 20g of soil samples in advance, and placed at room temperature for 21 days. Take 5g of the treated soil sample to make a soil suspension, and make 10-fold ratio dilution according to 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , and 10 ^-5 . 200 μL of the diluted soil suspension was evenly spread on Martin medium (1% glucose, 0.5% peptone, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ·7H ₂ O, 1.5% MgSO ₄ . agar, 0.001% red Bengal), and cultured in an incubator at 28°C.

The screened cadmium-resistant microorganisms were streaked in Martin's medium containing 16 mM CdCl ₂ and cultured in an incubator at 28°C. Repeat the streak two to three times until a single colony grows in the medium. A single colony was inoculated into a petri dish without CdCl ₂ , and the cultured strain was preserved as a parent seed.

Example 2 Morphological identification

1. Colony morphology observation

The strains were inserted into Martin's medium plates, and placed at 28°C for continuous cultivation for 12 days, and the morphological characteristics of the colonies were observed. The results are shown in Figure 1.

As shown in Figure 1, the colony has bacterial rings, the mycelium is white, the texture is velvety, the conidia structure is produced in large quantities, and the color of the conidia surface is light pink gray; there is no exudate on the surface.

2. Microscopic observation

Pick out a few colonies from the Martin's medium plate with a dissecting needle and place it on a glass slide with a drop of lactate phenol cotton blue staining solution in the middle, and then use two dissecting needles to disperse the colonies as much as possible and mix them with lactic phenol. Mix the cotton blue staining solution, add a cover glass, press gently, and dry the water around the cover glass with absorbent paper. Observe the morphology of hyphae and conidia under a microscope. The results are shown in Figure 2.

It can be seen from Figure 2 that the cell wall of the vegetative mycelium is smooth, the conidiophore is smooth, and stands upright on the vegetative mycelium; the sporophore has several whorled branches, and the top becomes tapered; the conidia is oval, chained from top to bottom. arranged in shape.

3. Molecular biological identification

The genomic DNA of fungal strains was extracted by conventional methods, and 18S rDNA and ITS were amplified by the designed primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the 18S rDNA and ITS products were subjected to 1.5% Agarose gel electrophoresis, the electrophoresis results are shown in Figure 3. The amplified product was recovered and purified by gel and then ligated with the vector pMD19T vector, and the positive recombinant plasmid was subjected to Sanger sequencing to determine the ITS sequence.

The ITS sequence is shown in the sequence table SEQ.ID.NO: 1, and the specific sequence is:

GAACCTGCGGAGGGATCATTACCGAGTTATACAACTCCCAAACCCACTGTGAACCTTACCTCAGTTGCCTCGGCGGGAACGCCCCGGCCGCCTGCCCCCGCGCCGGCGCCGGACCCAGGCGCCCGCCGCAGGGACCCCAAACTCTCTTGCATTACGCCCAGCGGGCGGAATTTCTTCTCTGAGTTGCACAAGCAAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGAGCCCCCCCCGGGGGCCTCGGTGTTGGGGGACGGCACACCAGCCGCCCCCGAAATGCAGTGGCGACCCCGCCGCAGCCTCCCCTGCGTAGTAGCACACACCTCGCACCGGAGCGCGGAGGCGGTCACGCCGTAAAACGCCCAACTTTCTTAGAGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGG

The ITS sequences were input into the online BLAST software for sequence alignment, and several registered ITS sequences with the highest identity and similarity were selected for phylogenetic tree construction. The results are shown in Figure 4.

After ITS identification, the strain was preliminarily confirmed as Purpureocillium sp strain ZYZX-YZ1.

Example 3 Cadmium tolerance and cadmium adsorption experiments

Since the test strain was selected from 16 mM CdCl ₂ pretreated soil and purified in Martin medium plates with 16 mM CdCl ₂ concentration, the strain could grow well under 16 mM CdCl ₂ stress.

A hole was made on the edge of the colony in good growth state with a diameter of 0.7cm, and the solid medium block containing mycelium was inoculated into a Martin medium plate with a concentration of _16mM CdCl2, and the diameter of the mycelium was recorded every day. See Figure 5.

It can be seen from Figure 5 that the diameter of hyphae in _both cadmium-free and cadmium-containing media showed a gradually increasing trend. It is not very large, indicating that the strain has a strong tolerance to cadmium.

Punch holes on the edge of the colony in good growth state with a diameter of 0.7cm, and inoculate the solid medium block containing mycelium from the punched holes in 100mL of Martin's liquid medium with different concentrations of CdCl ₂ for 3 days, and then collect the bacteria by centrifugation. The dry weight of the bacteria was weighed, and the results were shown in Figure 6. The cadmium content was measured in the liquid medium after collecting the mycelium, and the results are shown in Figure 7.

It can be seen from Figure 6 and Figure 7 that under a certain cadmium concentration, the mycelium has a certain adsorption effect on cadmium. When the cadmium concentration is 0.5mM, the dry weight of the mycelium is the heaviest, and when the cadmium concentration is 0.05mM The adsorption rate is the largest, and when the cadmium concentration exceeds a certain value, the growth of mycelium will be inhibited.

Example 4 Pot experiment

In this experiment, tobacco (Nicotiana benthamiana) was selected for pot experiment. The pots are 10cm long, 10cm wide and 10cm high; each pot is planted with one tobacco tree.

Two-week-old tobacco plants with good and consistent growth conditions were selected and divided into two groups, one group was not inoculated with fungi, and the other group was inoculated with fungi (1g fresh weight mycelium per 50g dry weight soil). From the day of inoculation, 30 mL of cadmium solutions with different concentrations were poured into each pot every 5 days for 1 month to observe the growth of tobacco. The results are shown in Figure 8.

It can be seen from Figure 8 that the tobacco plants inoculated with the strain ZYZX-YZ1 can still grow when the cadmium concentration is 400 mg/kg, and the leaves are normal green, but the tobacco plants not inoculated with the strain ZYZX-YZ1 become dry at this concentration. death, indicating that the strain repaired soil containing cadmium and increased the tolerance of tobacco to cadmium.

The roots and aerial parts of tobacco were collected respectively, dried and digested with nitric acid, and then the content of cadmium in the samples was determined by flame atomic absorption method. The results are shown in Figure 9.

It can be seen from Figure 9 that when the cadmium concentration was 100 mg/kg and 200 mg/kg, respectively, the cadmium accumulation in the roots of the tobacco plants inoculated with the strain ZYZX-YZ1 decreased by 52.3% and 44.1%, respectively, and the aerial parts also decreased to varying degrees; When the concentration was 400 mg/kg, the cadmium accumulation in the roots and aerial parts of the tobacco plants not inoculated with the strain ZYZX-YZ1 was much higher than that of the tobacco plants with the strain ZYZX-YZ1. The test results further proved that the strain ZYZX-YZ1 can remediate soil containing cadmium, thereby reducing the absorption of cadmium by tobacco.

sequence listing

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> A kind of Lavus sp. with high resistance to cadmium and its extraction method and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 587

<212> DNA

<213> Artificial Sequence

<400> 1

gaacctgcgg agggatcatt accgagttat acaactccca aacccactgt gaaccttacc 60

tcagttgcct cggcgggaac gccccggccg cctgcccccg cgccggcgcc ggacccaggc 120

gcccgccgca gggaccccaa actctcttgc attacgccca gcgggcggaa tttcttctct 180

gagttgcaca agcaaaaaca aatgaatcaa aactttcaac aacggatctc ttggttctgg 240

catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc 300

atcgaatctt tgaacgcaca ttgcgcccgc cagcattctg gcgggcatgc ctgttcgagc 360

gtcatttcaa ccctcgagcc ccccccgggg gcctcggtgt tgggggacgg cacaccagcc 420

gccccccgaaa tgcagtggcg accccgccgc agcctcccct gcgtagtagc acacacctcg 480

caccggagcg cggaggcggt cacgccgtaa aacgcccaac tttcttagag ttgacctcgg 540

atcaggtagg aatacccgct gaacttaagc atatcaataa gcggagg 587

Claims (3)

1. A rhodosporidium (purpurocillium sp.) with high resistance to cadmium is characterized in that the rhodosporidium is named ZYZYZX-YZ 1, the rhodosporidium is registered and preserved in the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms in 2017, 09 and 12 months, the preservation number is CGMCC No.14159, and the ITS sequence is shown in the sequence table SEQ.ID No: 1 is shown.

2. Use of the cadmium-resistant Porphyromonas purpurea of claim 1 in remediation of soil or water contaminated with cadmium.

3. Use of the purple cell bacterium having high resistance to cadmium according to claim 1 for preparing a cadmium adsorbent.

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