CN112143772A - Bioassay method for evaluating detoxification effect of microorganisms on lead ions in environment - Google Patents

Abstract

The invention relates to a bioassay method for evaluating the detoxification effect of microorganisms on lead ions in the environment. Detoxification effect, the detoxification effect of the test organisms in the lead-containing soil environment is characterized by the test method for the growth of N. benthamiana seedlings in the lead-containing soil environment, which can intuitively reflect the adsorption effect of different test microorganisms on the lead ions in the environment, so that the The biological detoxification ability of different strains was characterized. The lead ion fixation ability of the tested strain is evaluated by the biological assay method provided by the present invention, which is closer to the bioremediation of the real heavy metal lead-contaminated environment. At the same time, the present invention is also applicable to other metal ion-tolerant strains except lead. Relevant research on biological detoxification has far-reaching significance for the realization of in situ remediation of heavy metal-contaminated environments.

Description

Bioassay method for assessing microbial detoxification of lead ions in the environment

technical field

The invention relates to a bioassay method for evaluating the detoxification effect of microorganisms on lead ions (Pb ²⁺ ) in the environment. To characterize the immobilization effect of the tested microorganisms on Pb ²⁺ in the environment.

Background technique

Humans have discovered the presence of the heavy metal lead (Pb) as early as around 3000 BC, and it is also used to make batteries, cables and ammunition, and sometimes as a gasoline additive. Some lead compounds are also used as raw materials for synthetic rubber, glass, pigments and other products. Metal lead also has excellent corrosion resistance and acid and alkali resistance, so it is widely used in the manufacture of chemical and metallurgical equipment and other industries. The rapid development of the national economy has increased the demand for lead in my country's industry. Today, my country's lead consumption has surpassed that of the United States, ranking first in the world. At the same time, lead, as one of the five major heavy metals in soil pollution in my country, has caused more and more serious environmental pollution problems. According to statistics, about 1.3104hm ² of arable land in China is polluted by metallic lead, resulting in a substantial reduction in grain yield of 1.0107 tons. Moreover, in recent years, some edible crops have often been detected with excessive levels of heavy metals such as lead. Lead has been considered by the World Health Organization (WHO) to be the most serious environmental poison for children, and infants and young children are mainly exposed to lead through soil media. Currently accepted technologies for decontamination of lead-contaminated soil require burial or excavation of contaminated soil, but these treatment procedures are often ineffective in removing Pb (and other metals) or reducing its bioavailability. Furthermore, replacing the original contaminated soil with clean soil after excavation requires clean soil and a repository of contaminated soil, which is highly destructive and unsustainable. Therefore, in situ remediation technology has become a research hotspot to solve heavy metal pollution.

At present, the use of microbial adsorbents to achieve the adsorption of Pb ²⁺ has the advantages of high removal efficiency, low price, simple operation, easy cultivation of strains, and little impact on the environment. Engineering means to fix specific proteins on the outer membrane of cells to achieve the purpose of Pb ²⁺ being bound and removed outside the cell, and it has been widely used in the field of heavy metal adsorption and removal. However, most of the existing studies are limited to laboratory shake flask culture, and the tested strains have not been applied to the in situ remediation of lead-containing environments, and before practical application, there is also a lack of characterization mechanisms for the remediation effect of the test microorganisms immobilizing heavy metal ions . Therefore, at this stage, it is necessary to establish a bioassay method that can operate in a laboratory environment to simulate the Pb ²⁺ contamination state in the actual environment, and put specific microorganisms that have adsorption effect on Pb ²⁺ into a lead-containing environment to achieve free. The fixation and detoxification of Pb ²⁺ , and the characterization of the microbial detoxification effect by bioassay, so as to provide a research basis for the in-situ remediation of lead-contaminated wastewater or soil.

Tobacco plants are often used as laboratory model plants for research due to their advantages of small genome, short culture period and obvious plant growth characteristics. Under natural conditions, tobacco plants can germinate in a water environment when the temperature and humidity are moderate, and can maintain a good growth situation after being transplanted into a soil environment. However, when there is a certain concentration of free Pb ²⁺ in the tobacco growth environment, it is easy for plants to absorb and utilize it, so that both seed germination and plant growth are affected by the toxicity of free lead. yellow necrosis and other characteristics. Theoretically, when specific microorganisms that have adsorption effect on Pb ²⁺ are added to the lead-containing environment, Pb ²⁺ can be adsorbed by the microorganism strains and converted from free state to bound state, realizing the immobilization of free state Pb ²⁺ and reducing Pb 2+ The bioavailability of ²⁺ makes it difficult to be absorbed by tobacco plants, so as to achieve the effect of biological detoxification.

To sum up, in order to visually characterize the actual detoxification effect of the tested microorganisms on Pb ²⁺ , as a preference, the present invention establishes a bioassay method with Nicotiana benthamiana as the characterization plant. and the growth situation of tobacco plants in lead-containing soil, which intuitively reflects the immobilization effect of the tested microbial strains on free Pb ²⁺ , and confirms the biological detoxification effect of the tested microbial strains on free Pb ²⁺ in the environment. The research on in-situ remediation of tested microorganisms into Pb ²⁺ polluted environment provides the basis and basis.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a biological assay method for characterizing the detoxification effect of tested microbial strains on free Pb ²⁺ in the environment, as shown in Figure 1, the method takes N. benthamiana as the observation object, and the The germination rate of seeds in lead-containing microbial cell suspensions characterizes the detoxification effect of the test microorganisms in the lead-containing water environment, and the test organisms in the lead-containing soil environment are characterized by the assay method for the growth of N. benthamiana seedlings in the lead-containing soil environment The detoxification effect, thus intuitively reflecting the ability of microorganisms to fix Pb ²⁺ in the environment.

For realizing the above-mentioned object effect, the present invention provides following technical scheme:

The steps to characterize the germination rate of N. benthamiana seeds in lead-containing microbial cell suspensions are as follows:

(1) Preparation of the test microorganism bacterial suspension: According to the test microorganism's requirements for growth conditions, select the corresponding medium and protein inducer, and wash with double distilled water (ddH ₂ O) after inducing culture for 24 hours to make OD ₆₀₀ = 3 cell suspensions;

(2) Pb ²⁺ addition: add 500-2000 μM of Pb ²⁺ to each test microbial cell suspension and the same volume of distilled water respectively and mix well to make each test microbial lead-containing cell suspension and microorganism-free Pb ²⁺ aqueous solution;

(3) seed arrangement: place the pre-sterilized and cut filter paper on the bottom of the sterile petri dish, and evenly arrange the precisely counted N. benthamiana seeds on the filter paper;

(4) Tobacco germination: add the test microorganism lead-containing cell suspension prepared in step (2) and the Pb ²⁺ aqueous solution without microorganisms to the petri dish in step (3), so that the liquid level is just covered seeds, then incubate the dish under white light at 25-28°C; preferably for 3 days;

(5) Calculation of germination rate: when the seed coat rupture is visible, it is considered that the seed has sprouted, and the germination rate of the seed is calculated;

Preferably, the tobacco seeds accurately counted in the step (3) are 50-100;

The tested microbial strains used for Pb ²⁺ adsorption in the step (1) include wild-type host strain E. coli BL21 (DE3) and three surface display engineering strains BL21 (pR-FL) and BL21 (pR691-FL) and BL21 (pD-FL); the nucleotide sequence of the recombinant plasmid pR-FL in BL21 (pR-FL) is as SEQ ID No. 1; the nucleotide sequence of the recombinant plasmid pR691-FL in BL21 (pR691-FL) is as SEQ ID No.2; the nucleotide sequence of the recombinant plasmid pD-FL in BL21 (pD-FL) is as SEQ ID No.3;

Among them, BL21(pR-FL), BL21(pR691-FL) and BL21(pD-FL) are according to the Chinese patent application number 2020102466711 filed by Tianjin University on 2020-3-31, and the name of the invention is "for specific adsorption of heavy metals" The surface display engineering bacteria of lead and its construction method and application are constructed by the construction method of the specific lead binding protein surface display engineering bacteria disclosed in ". The nucleotide sequence of the recombinant plasmid pR-FL in BL21(pR-FL) is shown in SEQ ID No.1, and the nucleotide sequence of the recombinant plasmid pR691-FL in BL21(pR691-FL) is shown in SEQ ID No.2. The nucleotide sequence of the recombinant plasmid pD-FL in BL21(pD-FL) is shown in SEQ ID No.3.

The microbial culture medium corresponding to the four tested strains in the present invention is all LB medium, and its composition is (calculated in 1 L of medium): 10 g NaCl, 10 g tryptone and 5 g yeast powder, and the medium is adjusted after all dissolving pH to 7.2～7.4, and autoclave at 121℃ for 20min. The protein inducers corresponding to the four tested strains were all isopropyl-β-D-thiogalactoside (IPTG).

The preparation of the test microorganism cell suspension in the described step (1), the steps are as follows:

1) Strain activation: pick a single colony of the tested strain from the plate and inoculate it into 3-5ml of LB medium, and place it at 37°C and cultivate it in a shaker at 200-220rpm overnight;

2) Transfer: transfer the activated microbial strain to a shake flask containing LB liquid medium according to the inoculation ratio of 1%;

3) Protein induction: when the OD ₆₀₀ of the bacterial solution reaches 0.6-0.8, add 0.2-0.5mM IPTG inducer to each shake flask, and place it in a constant temperature shaker at 22-26°C and 200-220rpm to induce protein expression;

4) Cell collection: after 24 hours of induction culture, centrifuge at 5000-8000rpm for 8-10 minutes, wash with sterile double-distilled water (ddH ₂ O), preferably three times; resuspend the cell pellet in ddH ₂ O, A cell suspension of _OD600 =3 was made.

Through all the above steps, comparing the germination rate of tobacco seeds in the lead-containing aqueous solution with or without microbial cells can intuitively reflect the detoxification effect of the tested microorganisms on Pb ²⁺ in the aqueous solution.

The present invention also provides an assay method for the growth of N. benthamiana plants in lead-containing soil. The tested strains used are the same as those in step 1, and the steps are as follows:

(1) Tobacco seed germination: put sterile gauze at the bottom of the petri dish, preferably three layers of sterile gauze; N. benthamiana seeds are evenly arranged on the gauze, spray once every day with ddH ₂ O, fully wet the seeds and the gauze, Germinate at room temperature for 25-30 days;

(2) Preparation of the tested microorganism bacterial suspension: according to the requirements of each tested microorganism for growth conditions, select the corresponding medium and protein inducer, and prepare a cell suspension with OD ₆₀₀ =3 after inducing and culturing for 24 hours;

(3) Preparation of lead-containing soil: firstly add 500-2000 μM of Pb ²⁺ to the test microbial cell suspension and the same volume of distilled water in step (2) and mix well to prepare the test microbial lead-containing cell suspension and the Pb ²⁺ aqueous solution without microorganisms; then mix the pre-sterilized plant growth nutrient soil with the above-mentioned lead-containing solution at a ratio of 1:1 (v/w), fully wet the soil, and make lead-contaminated soil. nutrient soil for plant growth;

(4) Tobacco plant cultivation: the N. benthamiana seedling of step (1) is transplanted in the nutrient soil of step (3), is placed in a sunny greenhouse, and is watered once every two days and cultivated continuously for 20-50 days;

(5) Weighing of the plants: The plants were taken out from the pots, the soil at the roots was cleaned, and the biomass of the plants was weighed.

In the above step (3), the plant growth nutrient soil is sterilized by high temperature and high pressure steam, and the condition is 121°C for 20 minutes, and then placed in a 55-65°C oven for 24 hours to fully dry the soil.

Through the above steps, comparing the growth trend and plant biomass of tobacco plants in the lead-containing soil treated with microorganisms and the lead-containing soil without microorganisms can intuitively reflect the detoxification effect of the tested microorganisms on Pb ²⁺ in the soil.

The advantages of the invention are as follows: a biological assay method based on N. benthamiana plants is established, and the assay method is closer to the real heavy metal pollution environment than the shake flask experiment. As shown in Figure 1, both the germination rate of the seeds in the lead-containing aqueous solution can reflect the immobilization effect of the tested microbial strains on Pb ²⁺ in the water environment, and the growth situation of the plants in the lead-containing soil can reflect the affected bacteria. The detoxification effect of the test microbial strains on Pb ²⁺ in the soil environment can more intuitively characterize and evaluate the actual remediation effect of the free Pb ²⁺ in the fixed environment of the test microbial strains. profound meaning.

Description of drawings

Figure 1 is a schematic diagram of the bioassay method for the detoxification of lead ions in the environment by the tested microorganisms

Figure 2. Micrographs of germination of N. benthamiana seeds in lead-containing aqueous solutions treated with different tested strains

Fig. 3 Growth status of N. benthamiana plants in lead-containing soil treated with different tested strains

Detailed ways

Below in conjunction with accompanying drawing and embodiment, the present invention will be further described:

When free Pb ²⁺ was not contained in the environment, the four tested microorganisms in the present invention did not have any promotion or adverse effect on tobacco seed germination and plant growth.

Example 1 Preparation of Test Microorganism Cell Suspension for Pb ²⁺ Adsorption

(1) Strain activation: Pick a single colony of the test strains BL21, BL21(pR-FL), BL21( ^pR691 -FL) and BL21(pD-FL) for Pb adsorption from the plate and inoculate it into 5ml containing kanamycin-resistant solution in LB medium, wherein the wild-type control strain BL21 did not add resistance to the medium during activation. Incubate the shaker tube at 37°C, 220rpm in a shaker overnight;

(2) Transfer: transfer to a shake flask containing 100 mL of anti-anti-liquid medium according to the inoculation ratio of 1%;

(3) Protein induction: when the OD ₆₀₀ of the bacterial solution reaches 0.6 to 0.8, add IPTG with a final concentration of 0.5 mM to each shake flask, and place the shake flask in a constant temperature shaker at 22°C and 220 rpm to induce protein expression;

(4) Cell collection: After 24 hours of induction culture, centrifuge at 8000 rpm for 8 minutes, wash three times with sterile double-distilled water, and resuspend the cell pellet in ddH ₂ O to prepare a cell suspension with OD ₆₀₀ =3.

Through the above steps, all tested strains BL21(pR-FL), BL21(pR691-FL), BL21(pD-FL) and BL21 were obtained cell suspensions with _OD600 =3 for subsequent bioassay studies.

Wherein, the LB medium components used for strain culture in the present invention are (in 1 L medium): 10 g NaCl, 10 g tryptone and 5 g yeast powder, all of which are dissolved and the pH of the medium is adjusted to 7.2-7.4. And autoclaved at 121°C for 20min.

Embodiment 2 Germination of Nicotiana benthamiana seeds in lead-containing aqueous solution

(1) Preparation of tested microorganism bacterial suspension: According to the steps provided in Example 1, BL21, BL21 (pR-FL), BL21 (pR691-FL) and BL21 (pD-FL) strains with OD ₆₀₀ =3 were prepared respectively cell suspension;

(2) Pb ²⁺ addition: respectively take 20 mL of the test microorganism cell suspension and the same volume of distilled water, add 2000 μM of Pb ²⁺ to them and mix them evenly to make the test microorganism lead-containing cell suspension and microorganism-free cell suspension. Pb ²⁺ aqueous solution;

(3) seed arrangement: place the pre-sterilized and cut filter paper on the bottom of the sterile petri dish, and evenly arrange 100 precisely counted N. benthamiana seeds on the filter paper;

(4) Tobacco germination: add the test microorganism lead-containing cell suspension prepared in step (2) and the Pb ²⁺ aqueous solution without microorganisms to the petri dish in step (3), so that the liquid level is just covered seeds and incubate the petri dishes at 28°C for 3 days under white light irradiation;

(5) Calculation of germination rate: when the seed coat rupture is visible, it is considered that the seed has sprouted, the germination rate of the seed is calculated, and the germination state is observed under an optical microscope;

(6) Statistical analysis of seed germination rate using SPSS software.

The results showed that the germination rate of N. benthamiana in a 2000 μM Pb ²⁺ aqueous solution without any microbial strains reached 18%. pD-FL) and the wild-type starting strain BL21 of the control group were treated with lead-containing aqueous solution and the germination rates are shown in Table 1. It can be seen that the ability of the wild-type starting strain BL21 to immobilize Pb ²⁺ is very small, but the germination rate of tobacco seeds treated with the three engineered strains is about 1.5 times higher than that of the seeds treated with BL21. In addition, it can be seen from Fig. 2 that the average shoot length of the tobacco seeds treated with engineered bacteria was more than 8 times that of BL21 treatment, indicating that the three outer membrane display engineering bacteria played an important role in reducing the toxicity of Pb ²⁺ . By comparing the detoxification effects of the three tested engineered bacteria, it was found that the germination rate of the seeds treated with the PbrR691 engineered bacteria solution was relatively the highest, reaching 55%, followed by PbrR, and the lowest PbrD, 49%. The above results show that the three tested lead-binding protein surface display engineering bacteria in the present invention can effectively adsorb Pb ²⁺ in lead-containing aqueous solution, thereby effectively protecting seeds from the toxicity of heavy metal ions.

Table 1 Effects of Pb ²⁺ on the germination rate of N. benthamiana seeds

^* Indicates a significant difference between the germination rates of the treated seeds of the wild-type starting strain BL21 and the PbrR/PbrR691/PbrD surface display engineered bacteria (P<0.001, F test); ^** indicates the wild-type host strain BL21 and PbrR/PbrR691/ There was a significant difference between the germination shoot lengths of the treated seeds with PbrD surface display engineering bacteria (P<0.001, F test).

Example 3 Growth of N. benthamiana plants in lead-containing soil

(1) Tobacco seed germination: put three layers of sterile gauze at the bottom of the petri dish, evenly arrange the N. benthamiana seeds on the gauze, spray once a day with ddH ₂ O, fully wet the seeds and the gauze, and place them at room temperature 30 days of germination;

(2) Preparation of test microorganism bacterial suspension: According to the steps provided in Example 1, BL21, BL21 (pR-FL), BL21 (pR691-FL) and BL21 (pD-FL) strains with OD ₆₀₀ =3 were prepared respectively cell suspension;

(3) Preparation of lead-containing soil: respectively take 20 mL of the test microbial cell suspension prepared in step (2) and the same volume of distilled water, add 500 μM of Pb ²⁺ to it and mix well to prepare the test microbial lead-containing cells Suspension and Pb ²⁺ aqueous solution without microorganisms; then the plant growth nutrient soil sterilized by high pressure steam sterilization in advance and the above Pb ²⁺ solution are mixed at a ratio of 1:1 (v/w) to make The Pb ²⁺ concentration in the soil reaches 500μmol.kg ^-1 to make lead-contaminated nutrient soil for plant growth;

(4) Tobacco plant cultivation: the tobacco seedlings in the step (1) are transplanted into the nutrient soil that the step (3) has handled, placed in a sunny greenhouse, watered once every two days and cultivated continuously for 20 days, observe plant growth condition;

The results are shown in Figure 3. After 20 days of transplanting, the tobacco seedlings transplanted in the lead-containing soil that did not contain any microbial strains withered and died within 7 days, but the four tested strains treated the soil. The transplanted tobacco seedlings could grow normally. First, it was confirmed that the four tested strains had a certain fixed effect on lead ions in the soil; The growth of tobacco in the pot is relatively good, slightly better than BL21 (pD-FL), and the tobacco growth in the soil treated by BL21 is the worst, indicating that the three tested lead-binding protein surface display engineering bacteria in the present invention will be displayed within 20 days. It can effectively alleviate the toxic effect of Pb ²⁺ in soil on plants, while the detoxification effect of wild-type strain BL21 on Pb ²⁺ in soil is relatively weak.

Example 4 Growth of N. benthamiana plants in lead-containing soil

(1) Tobacco seed germination: put three layers of sterile gauze at the bottom of the petri dish, evenly arrange the N. benthamiana seeds on the gauze, spray once a day with ddH ₂ O, fully wet the seeds and the gauze, and place them at room temperature 30 days of germination;

(2) Preparation of test microorganism bacterial suspension: According to the steps provided in Example 1, BL21, BL21 (pR-FL), BL21 (pR691-FL) and BL21 (pD-FL) strains with OD ₆₀₀ =3 were prepared respectively cell suspension;

(3) Preparation of lead-containing soil: respectively take 20 mL of the test microbial cell suspension prepared in step (2) and the same volume of distilled water, add 500 μM of Pb ²⁺ to it and mix well to prepare the test microbial lead-containing cells Suspension and Pb ²⁺ aqueous solution without microorganisms; then the plant growth nutrient soil sterilized by high pressure steam sterilization in advance and the above Pb ²⁺ solution are mixed at a ratio of 1:1 (v/w) to make The Pb ²⁺ concentration in the soil reaches 500μmol.kg ^-1 to make lead-contaminated nutrient soil for plant growth;

(4) Tobacco Plant Cultivation: the tobacco seedling in step (1) is transplanted in the nutrient soil that step (3) has handled, is placed in the greenhouse with sufficient sunlight, and is watered once every two days and cultivated continuously for 50 days;

(5) Plant weighing: observe the growth status of the plants, after 50 days, the plants are taken out from the pots, the soil at the roots is cleaned, and the plant biomass is weighed.

The results are shown in Figure 3. The tobacco plants transplanted in the soil treated with the surface display engineering bacteria containing the lead-binding proteins PbrR, PbrR691 and PbrD all grew well, and the seedlings treated with the wild-type strain BL21 grew poorly, but in the absence of Tobacco seedlings transplanted in lead-containing soil containing any microbial strain withered and died within 7 days, indicating that the three tested lead-binding protein surface-displayed engineered bacteria in the present invention indeed effectively relieved the soil during the 50-day observation period. The toxic effect of Pb ²⁺ on plants in BL21 was extremely weak, while the detoxification effect of wild-type strain BL21 on Pb ²⁺ in soil was extremely weak.

From the quantification results in Table 2, it can be seen that the biomass of tobacco seedlings grown in the soil treated with the PbrR691 surface display engineered strain was relatively the highest after 50 days of culture, reaching 2.67 g, which was 13.35 times that of BL21; PbrR and PbrD display strains treated The biomass of tobacco grown in the treated soil was not much different, 1.49 g and 1.46 g, respectively, which were more than 6 times higher than that of BL21, confirming that the presence of the target protein on the cell surface enhanced the immobilization of Pb ²⁺ in soil by the engineered strain Therefore, the toxicity of heavy metal ions to plants growing in the soil is alleviated. This research is of great significance for heavy metal remediation.

Table 2 Effects of Pb ²⁺ seeds on the growth of N. benthamiana plants

The application test results of the above three groups of examples show that the Pb ²⁺ immobilization ability of the four tested strains is evaluated by the biological assay method provided by the present invention, which is closer to the real bioremediation of the heavy metal lead polluted environment. By comparing the germination rate of N. benthamiana seeds under lead-containing conditions, it can be seen intuitively that the immobilization ability of BL21 in lead-containing aqueous solution is very weak, and the immobilization ability of BL21 (pR691-FL) on Pb ^{2+ is} stronger than that of BL21 (pR691-FL) BL21 (pR-FL), stronger than BL21 (pD-FL); through the experiment of transplanting 30-day-old N. benthamiana seedlings to lead-containing soil, it can be seen intuitively that the tested strain BL21 has a strong effect on the soil environment. The detoxification effect of Pb ²⁺ is also very weak, but the detoxification effect of the other three engineering bacteria is very obvious.

The addition of the two test results can test the biological detoxification ability of different strains in practical application: BL21(pR691-FL)>BL21(pR-FL)>BL21(pD-FL)>>BL21. The three surface display engineering strains with good Pb ²⁺ adsorption have been tested, and they can be further considered for in-situ remediation of heavy metal lead-contaminated environments. At the same time, the invention is also applicable to the related research on the biological detoxification of other metal ion-tolerant strains except lead.

The above content is a further detailed description of the present invention in conjunction with specific embodiments. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and modifications can also be made. These Improvements and modifications should also be considered within the scope of the present invention.

sequence listing

<110> Tianjin University

<120> Bioassay method for assessing microbial detoxification of lead ions in the environment

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ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggtttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100

ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160

acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220

ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280

tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340

cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400

caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460

acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520

gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580

ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgaat 5640

atccagatcg gcgagcttgc caagcgcacc gcatgcccgg tggtgaccat tcgcttctac 5700

gaacaagaag ggctgttgcc gccgccgggc cgcagccggg ggaattttcg cctgtatggc 5760

gaggagcacg tggagcgctt gcagttcatt cgtcactgcc ggtctctgga tatgccgttg 5820

agcgacgtac ggaccttatt gagttaccgg aagcggcccg accaggattg cggtgaagtc 5880

aatatgctct tggatgagca catccgtcag gtcgaatctc ggatcggagc tttgctcgaa 5940

ctgaagcacc atttggtgga actgcgcgaa gcctgttctg gtgccaggcc cgcccaatcg 6000

tgcgggattc tgcagggact gtcggactgc gtgtgtgata cgcgggggac caccgcccat 6060

ccaagcgacc atcatcacca tcaccactag gaattcgagc tccgtcgaca agcttgcggc 6120

cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 6180

agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 6240

acgggtcttg aggggtttttt tgctgaaagg aggaactata tccggat 6287

<210> 2

<211> 6248

<212> DNA

<213> Artificial Sequence

<400> 2

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatcctttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccaggggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gccttttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggtttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100

ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160

acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220

ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280

tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340

cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400

caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460

acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520

gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580

ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgatg 5640

cggatcggtg aactgggcaa gaaggcagat tgcttggtgc agaccgtgcg cttttacgag 5700

tcagaaggct tgctgcccga gcctgcacgt agcgagggca acttcaggct ctatgacgaa 5760

gtccatttgc agcgcttgct gttcatccgc cgctgccggg cgaaggacat gacgctggat 5820

gagatccgtc aactgctgaa cttacgggat cggccagagt tgggctgcgg cgaggtgaac 5880

gcgctggtcg acgctcatat cgcgcaagtg cggaccaaga tgaaggaatt gcgcgccttg 5940

gagcgcgagt taatggatct gcgacgctcc tgcgatagcg cccgaacctc gcgcgagtgc 6000

ggcattctca acagcttggc cgagcccgcc catcatcacc atcaccactg agaattcgag 6060

ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 6120

tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 6180

ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 6240

atccggat 6248

<210> 3

<211> 6575

<212> DNA

<213> Artificial Sequence

<400> 3

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagtttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatcctttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccaggggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gccttttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggtttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100

ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160

acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220

ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280

tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340

cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400

caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460

acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520

gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580

ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgatg 5640

ccagtgtatt tggctgattt gcaacgacgt ctggcgcgag gcgccgccct acaacccaaa 5700

acttgcatga tttcgttggc aggcgacgcg cgacatcggg ggcgaaaacg gcacggaatg 5760

gcggctttca tgcgagggag ccaggggggc ttctggccgg gaagcgacct tcgtcacagc 5820

gcaagtcccg gtcatgctct cggtgtacga tgtgcgggga gaaccgtccg agcgggacga 5880

gacattgtgg aatctcaaag tccatggggg cagctatggg gttttcttgc cgaaagtttc 5940

tcattgcccg tgacgatact ctctggcagc tgccaaccac caaattccag cggatgttgc 6000

gggagccggc caaccactgc ttgtccactt ttgccgggca acgcgcacgc atggccgatg 6060

tggtcgtcga actggtggcc agagagccag tgcgcgtcgt tcgaacgacg ttttccattc 6120

tcacgttcga tgccgaaggt tgcctcgatc caggggcatt cgaaaagcag caatttgcgc 6180

tcgcggagtc ggtcgttgcc cccgtcttcg ccgcatccgt ggatgagagc aagcaacccg 6240

tcgttgacgc gtccgcgcga ttcattgcgc aagggggcca atgggttccg acacgagctt 6300

tggcgcgcgc gatcgatgag gcggcgttgg ggcaacgacg gtgcctacgc ctgtaggcat 6360

catcaccatc accactagga attcgagctc cgtcgacaag cttgcggccg cactcgagca 6420

ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 6480

tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 6540

gggttttttg ctgaaaggag gaactatatc cggat 6575

Claims (8)

1. A bioassay method for evaluating the detoxification of microorganisms to lead ions in an environment; the method is characterized in that the Nicotiana benthamiana is taken as an observation object, and the detoxification effect of the tested microorganism in a lead-containing water environment is represented by the germination rate of Nicotiana benthamiana seeds in a lead-containing microorganism cell suspension; the method for detecting the growth of the Nicotiana benthamiana seedlings in the lead-containing soil is used for representing the detoxification effect of the tested organism in the lead-containing soil environment, so that the Pb in the environment by the microorganism is intuitively reflected²⁺The fixing ability of (a).

2. The method as claimed in claim 1, wherein the method is used for lead ions (Pb)²⁺) The adsorbed tested microorganism strains comprise wild host bacteria E.coli BL21(DE3) and three surface display engineering bacteria BL21(pR-FL), BL21(pR691-FL) and BL21 (pD-FL); wherein the nucleotide sequence of the recombinant plasmid pR-FL in BL21(pR-FL) is shown in SEQ ID No. 1; the nucleotide sequence of the recombinant plasmid pR691-FL in BL21(pR691-FL) is shown in SEQ ID No. 2; the nucleotide sequence of the recombinant plasmid pD-FL in BL21(pD-FL) is shown in SEQ ID No. 3.

3. The method of claim 1, wherein the germination rate of nicotiana benthamiana seeds in a suspension of lead-containing microbial cells is characterized by the following steps:

(1) preparation of test microbial suspension: selecting corresponding culture medium and protein inducer according to the requirement of the tested microorganism on growth conditions, inducing and culturing for 24h, and adding double distilled water (ddH)₂O) washing to OD₆₀₀A cell suspension of ═ 3;

(2)Pb²⁺adding: separately, 500-2000. mu.M Pb was added to each of the microbial cell suspensions tested and the distilled water of the same volume²⁺And mixing uniformly to prepare lead-containing cell suspension and Pb without microorganisms of each tested microorganism²⁺An aqueous solution;

(3) seed arrangement: placing the pre-sterilized and cut filter paper at the bottom of an aseptic culture dish, and uniformly distributing the accurately counted Nicotiana benthamiana seeds on the filter paper;

(4) germinating tobacco: mixing the tested microorganism prepared in the step (2) with a lead-containing cell suspension and Pb without microorganisms²⁺Respectively adding the aqueous solutions into the culture dishes obtained in the step (3) to enable the liquid level to just cover the seeds, and then incubating the culture dishes at 25-28 ℃ under the irradiation of white light;

(5) and (3) calculating the germination rate: when the seed coat rupture is visible, the seed is considered to have germinated, and the germination rate of the seed is calculated.

4. The method as set forth in claim 2, wherein the tobacco seeds are accurately counted in the step (3) in 50-100 pieces.

5. The method of claim 1, wherein the assay for the growth of nicotiana benthamiana seedlings in lead-containing soil is characterized by the following steps:

(1) germinating tobacco seeds: placing sterile gauze at the bottom of the culture dish, uniformly distributing the Nicotiana benthamiana seeds on the gauze, and using ddH every day₂Spraying O, fully wetting seeds and gauze, and germinating at room temperature for 25-30 days;

(2) test microbial suspensionsPreparation: selecting corresponding culture medium and protein inducer according to the requirement of each tested microorganism on growth conditions, and preparing OD after induction culture for 24h₆₀₀A cell suspension of ═ 3;

(3) preparing lead-containing soil: adding 500-2000 mu M Pb to the cell suspension of the test microorganism in the step (2) and distilled water of the same volume²⁺And mixing uniformly to prepare the tested microorganism lead-containing cell suspension and the Pb without the microorganism²⁺An aqueous solution; then uniformly mixing the pre-sterilized plant growth nutrient soil and the lead-containing solution in a ratio of 1: 1(v/w), and fully wetting the soil to prepare the lead-polluted plant growth nutrient soil;

(4) tobacco plant culture: transplanting the seedling of the Nicotiana benthamiana in the step (1) into the nutrient soil in the step (3), placing the seedling in a greenhouse with sufficient sunlight, and watering once every two days to continuously culture for 20-50 days;

(5) plant weighing: the plants were taken out of the pot, the soil from the roots was cleaned and the biomass of the plants was weighed.

6. The method as claimed in claim 3 or 5, wherein in the step (3), the plant growth nutrient soil is sterilized by high temperature and high pressure steam under the condition of 121 ℃ for 20min, and then is placed in an oven at 55-65 ℃ for 24h to fully dry the soil.

7. The method according to claim 3 or 5, wherein the culture medium for the microorganisms corresponding to the four test strains is LB medium, and the composition of the culture medium is 1L: and (3) dissolving 10g of NaCl, 10g of tryptone and 5g of yeast powder, adjusting the pH value of the culture medium to 7.2-7.4, and sterilizing for 20min by high-pressure steam at 121 ℃.

8. The protein inducers corresponding to the four tested strains are isopropyl-beta-D-thiogalactoside (IPTG).

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