CN107828907B - Application of Physarum polycephalum Ppgst gene as biomarker - Google Patents
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to an application of a poverty vesiculosus Ppgst gene as a biomarker, namely an application of the poverty vesiculosus Ppgst gene as a biomarker in detection of anatase type nano titanium dioxide in a dark environment. The poecilomyces multiceps Ppgst gene can indicate the stress action of anatase type nano titanium dioxide under the condition of no illumination, and the transcription level of the gene has positive correlation in a certain range with the concentration of the anatase type nano titanium dioxide. In addition, the transcription level of the gene can also indicate the stress of anatase type nanometer titanium dioxide with different grain diameters. The results show that Ppgst can indicate that the anatase type nano titanium dioxide is polluted in the environment without illumination, namely the gene has potential application prospect in the aspect of environmental pollution monitoring.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an application of a Physarum polycephalum Ppgst gene as a biomarker.
Background
Physarum polycephalum (Physarum polycephalum) is an environment-sensitive eukaryotic slime mold, and can sense various external environmental stimuli such as chemical substances, gravity, light, magnetic field, electric field and the like. Glutathione transferase (GST) is a typical detoxification enzyme, which can relieve the toxic action of various organic or inorganic substances on organisms, the antioxidant property of the GST plays an important role in the detoxification process of the organisms, GST has an important significance on the life activities of environmental organisms, the gene encoding GST protein is Glutathione transferase gene (Ppgst), Ppgst genes of a plurality of environmental organisms are separated and identified, however, Ppgst genes of Phyllostachys polycephala are not separated.
Nano titanium dioxide (nTiO)2) Has special physical and chemical properties, especially anatase type nTiO2Often added to a variety of industrial and commercial products. Under the condition of illumination, nTiO2Can generate a large amount of oxidation free radicals to oxidize various biological macromolecules, resulting in oxidative damage of cells, nTiO2May be leaked and discharged to the environment through production and transportation links, endanger environmental organisms and destroy ecological balance. Therefore, it is very necessary to develop nTiO2Environmental indicators, indicating nTiO in the environment2And (4) pollution.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides application of a Physarum polycephalum Ppgst gene as a biomarker, and aims to solve the problem of nTiO in the existing environment2The pollution detection method is limited.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a Physarum polycephalum Ppgst gene serving as a biomarker in detection of anatase type nano titanium dioxide in a dark environment.
The invention discovers that the poecilomyces multiceps Ppgst gene can indicate the stress action of anatase type nano titanium dioxide under the condition of no illumination, and the transcription level of the gene is in positive correlation with the concentration of the anatase type nano titanium dioxide within a certain range. In addition, the transcription level of the gene can also indicate the stress of anatase type nanometer titanium dioxide with different grain diameters. The results all show that Ppgst can indicate the environmental pollution of the anatase type nano titanium dioxide under the condition of no illumination, namely the gene has potential application prospect in the aspect of environmental pollution monitoring.
Drawings
FIG. 1 is a graph showing the effect of anatase-type nano-titanium dioxide on the growth of Podocarpus multiceps in dark conditions in example 4 of the present invention, wherein the dotted line shows the initial weight of Podocarpus multiceps protoplasm cells, the stress time is 3 days, and the stress experiment is repeated three times;
FIG. 2 is a graph showing the relationship between the transcription level of the Ppgst gene of Physarum polycephalum and the stress concentration of anatase nano-titanium dioxide under dark conditions in example 4 of the present invention;
FIG. 3 is the transcription response reaction of the Ppgst gene of Physarum polycephalum in example 4 to anatase type nanometer titanium dioxide with different particle sizes under dark conditions; a: the relationship between the transcription level of the Ppgst gene and the stress of 60nm anatase nano titanium dioxide (different concentrations), B: the relationship between the transcription level of the Ppgst gene and the stress of 80nm anatase nano titanium dioxide (different concentrations).
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides application of a Physarum polycephalum Ppgst gene serving as a biomarker in detection of anatase type nano titanium dioxide in a dark environment.
In the embodiment of the invention, the poecilomyces polycephalus Ppgst gene can indicate the stress action of anatase type nano titanium dioxide under the condition of no illumination, and the transcription level of the gene is in positive correlation with the concentration of the anatase type nano titanium dioxide within a certain range. In addition, the transcription level of the gene can also indicate the stress of anatase type nanometer titanium dioxide with different grain diameters. The results all show that Ppgst can indicate the environmental pollution of the anatase type nano titanium dioxide under the condition of no illumination, namely the gene has potential application prospect in the aspect of environmental pollution monitoring.
In the embodiment of the invention, in order to prove that the Ppgst gene of the acinetobacter polycephalus indicates the stress action of the anatase type nano titanium dioxide, the following researches are carried out: firstly, anatase type nanometer titanium dioxide with different concentrations stresses the acinetobacter polycephalus, and the full-length cDNA sequence of the Ppgst gene is cloned by an RACE technology. Then, a primer is designed according to the sequence, the transcription level of Ppgst is detected by Real-Time PCR, and the transcription response condition of the Ppgst gene to different concentrations of anatase type nano titanium dioxide is analyzed. Finally, P.polycephalum is stressed by anatase nano titanium dioxide with different particle sizes and different concentrations respectively, the transcription level of Ppgst is detected, and the response condition of the gene to the anatase nano titanium dioxide with different particle sizes is analyzed.
Furthermore, the nucleotide sequence of the above-mentioned Physarum polycephalum Ppgst gene is shown in SEQ ID NO. 1, or the nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO. 1. The nucleic acid molecules provided in the examples of the present invention may be sequences that are different from the nucleotide sequences disclosed in the examples of the present invention but encode the same protein due to the complementary sequence of the nucleotide sequences or due to the degeneracy of the genetic code.
Furthermore, the amino acid sequence of the protein expressed by the gene Ppgst of the Physarum polycephalum is shown in SEQ ID NO. 2, or the amino acid sequence shown in SEQ ID NO. 2 is subjected to deletion, insertion or substitution to obtain the amino acid sequence with the same function.
SEQ ID NO:1：
Wherein the content of the first and second substances,
is a start codon for the gene encoding the polypeptide,
the nucleotide sequence between the two is a stop codon and codes the amino acid sequence shown in SEQ ID NO. 2.
SEQ ID NO:2：
MSKPRLSYFGGRGLAEPIRLLLKDAKVEYEEVNLGTAANGVFPEAFLALKASGVLDYGSVPYWEENGLNLVQSFAISRHLARKHGYNGENEAEAAKIDSVVEGTRDLVMAIRKVLTVPAEQKAQTWQEISKVDIPKWFGFFEALLKKSGTGFFVGKKASLADIYVIAYTEEIRCLDKSALASFPLLSAHLDAMFARPNLAAYAADPNAIPSFARLWHKNNNNK。
Further, when the anatase type nano titanium dioxide is detected in a dark environment, the concentration of the anatase type nano titanium dioxide in a sample is 9-15 mg/ml. Within the concentration range, the transcription level of the Ppgst gene and the stress concentration of the anatase nano titanium dioxide are in positive correlation, so that the anatase nano titanium dioxide concentration in a sample to be detected can be deduced more accurately according to the expression level of the Ppgst gene.
Further, when the anatase type nano titanium dioxide is detected in a dark environment, the particle size of the anatase type nano titanium dioxide is 60-80 nm.
Further, the step of detecting the anatase type nano titanium dioxide in the dark environment comprises the following steps:
s01: carrying out stress treatment on the acinetobacter polycephalus in a dark environment on a sample to be detected containing anatase type nano titanium dioxide;
s02: detecting the expression level of Ppgst gene in the stress-treated Physarum polycephalum;
s03: and deducing the concentration of the anatase type nano titanium dioxide in the sample to be detected according to the expression level of the Ppgst gene.
In a preferred embodiment, the time period for the stress treatment is 3-5 days. More effectively, the method for detecting the transcription level of the Ppgst gene in the stress-treated Physarum polycephalum is reverse transcription fluorescence quantitative PCR.
The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.
Example 1
Cultivation of strains and post-stress treatment
The Physarum polycephalum M3CVII (ATCC 204388) was deposited in this laboratory. After the bacterial plasmogen cells of the multi-headed blepharipoda are cultured by a liquid culture medium to the end of a logarithmic growth phase, 0.1g of the bacterial plasmogen cells of the multi-headed blepharipoda are inoculated to a solid culture medium for 5 days, and then the bacterial plasmogen cells are divided into 0.1g of cells and are respectively inoculated to the solid culture medium containing anatase type nano titanium dioxide. At the end of each stress treatment, the protoplasm cells were detached from the medium and weighed.
Example 2
Extraction of Total RNA and cloning of Ppgst Gene
According to the results of subsequent example 4, the Physarum polycephalum was stressed with 15mg/mL of anatase-type nano-titanium dioxide, and total RNA of the Physarum polycephalum was extracted after 3 days.
And designing a primer according to the known sequence of the Ppgst gene, and cloning the full-length cDNA by utilizing a RACE technology. Extracting total RNA of the stress-treated Physarum polycephalum with an RNA extraction Kit RNeasy Plant Mini Kit (QIAGEN, Germany), and amplifying with 5' -Full RACE Kit (Takara, Japan) and a primer pair (F1: 5'-TGTTGTCGTTCGCC-3'; F1: 5'-GTCAAGGATAGCAGCACCATCAGCGT-3'); the 5' terminal sequence of clone Ppgst was determined using the DreamTaq kit (Fermentas, USA) and primer set (F2: 5'-TCACTTGTTCGCTCCCGTGTTTCAT-3'; R2: 5'-GTTTTCCCAGTCACGAC-3'). Finally, the extracted cDNA sequences were sequenced and aligned using clustal X2 software. The signal peptide, transmembrane domain and secondary domain of the cDNA-encoded protein were analyzed using online software signalp4.1, TMHMM and GOR4, respectively.
The results show that: the cDNA contains a 672bp development reading frame, the start codon and the stop codon of the reading frame are respectively positioned at 76-78 bp and 748-750 bp, and the development reading frame of the cDNA codes a polypeptide (Genbank No. MF568068) containing 223 amino acid residues. The polypeptide has a molecular weight of about 24.62kDa and an isoelectric point of about 8.52. The gene encodes a polypeptide having two typical GST domains: GST _ N _ Sigma _ like domain and GST _ C _ family domain, which do not have transmembrane domain and signal peptide, is an intracellular GST. In addition, the cDNA contains a polyadenylation signal (AATAA) and a polyadenylic tail. The analysis result of the transmembrane structure shows that the polypeptide does not have a transmembrane domain and belongs to an intracellular protein. The secondary structure analysis results showed that the polypeptide contained 52.47% alpha helix, 10.31% extended chain and 37.22% random coil. The secondary structure analysis result also shows that the N end of the polypeptide is provided with a beta alpha beta alpha motif (amino acid residues 5 to 84), and the C end is provided with a plurality of almost continuous alpha spiral structures (amino acid residues 91 to 204).
Example 3
Method for detecting Ppgst gene transcription level
Total RNA was extracted according to the method of example 2, followed by degradation of the genomic residue using EasyScript One-Step gDNA Removal Kit (TransGen biotech, Beijing, China), cDNA Synthesis using cDNA Synthesis SuperMix Kit (TransGen biotech, Beijing, China), primer Synthesis (F3: 5'-TGAAGAAGTCAATCTCGGAACGGCTG-3'; R3:5'-CAAGGTGTCGGCTA ATGGCAAAGGA-3'), and detection of the transcription level of Ppgst gene using SYBR Premix ExTaqIITi RNase H Plus Kit (TaKaRa, Tokyo) Kit and 7300Real Time PCR instrument (BBI), selection of GAPDH gene as internal reference, and 2-Step DNA Removal Kit (TransGen Biotech, Beijing, China), and detection of the transcription level of Ppgst gene using 2-△△CtAnd analyzing the detection data. Finally, the colony weight of the Phycomyces polycephalus and the transcription level of the Ppgst gene were counted using the statistical software JMP 5.0(SAS Institute, Cary, NC, USA).
Example 4
Anatase type nano titanium dioxide stress polycephalon bacterium suffering from different concentrations and different particle sizes
In order to identify the Ppgst gene capable of indicating the stress of the anatase type nano titanium dioxide, different concentrations of the anatase type nano titanium dioxide are firstly used for stressing the acinetobacter polycephalus in the experiment so as to determine the stress concentration of the anatase type nano titanium dioxide used in the follow-up research.
In the research, four anatase type nanometer titanium dioxide with different concentrations (5mg/mL, 9mg/mL, 15mg/mL and 18mg/mL) are used for stressing the poverty vesiculosus protoplasm mass cells in a dark environment, total RNA of the poverty vesiculosus protoplasm mass cells is extracted on the 3 rd day, and the transcription response condition of the Ppgst gene is detected by using a real-time quantitative PCR technology, wherein the result is shown in figure 1. The data results from fig. 1 show that: the anatase type nano titanium dioxide of 5mg/mL and 9mg/mL does not obviously inhibit the growth of the acinetobacter polycephalus, while the anatase type nano titanium dioxide of 15mg/mL and 18mg/mL can obviously inhibit the growth of the acinetobacter polycephalus, wherein the concentration of 15mg/mL is close to the semi-lethal concentration of the anatase type nano titanium dioxide on the acinetobacter polycephalus, and therefore the concentration is selected as the stress concentration for subsequent research.
In the research, four anatase type nano titanium dioxide with different concentrations (9mg/mL, 12mg/mL, 15mg/mL and 18mg/mL) are used for stressing the poverty vesiculosus protoplasm cells in a dark environment, total RNA of the poverty vesiculosus protoplasm cells is extracted on the 3 rd day, and the transcription response condition of the Ppgst gene is detected by using a real-time quantitative PCR technology, wherein the result is shown in FIG. 2. The data results from fig. 2 show that: the transcription level of the Ppgst gene is positively correlated with the stress concentration of the anatase type nano titanium dioxide within the range of 9-15mg/mL, and within the concentration range, the higher the stress concentration of the anatase type nano titanium dioxide is, the higher the transcription level of the gene is.
Finally, the research stresses the poverty acinus protoplasm cells in dark environment with two anatase type nanometer titanium dioxide with different particle sizes (60nm and 80nm), extracts total RNA on day 3, and detects the transcription response condition of the Ppgst gene by using a real-time quantitative PCR technology, and the result is shown in figure 3. FIG. 3A is a graph of: the relationship between the transcription level of Ppgst gene and 60nm anatase type nano titanium dioxide stress, and FIG. 3B is: the relationship between the transcription level of the Ppgst gene and the stress of 80nm anatase type nano titanium dioxide. As can be seen from the data in FIG. 3, the transcription level of Ppgst gene is positively correlated with the stress concentration (9-15mg/mL) of the anatase nano titanium dioxide with different particle sizes, which indicates that the transcription level of Ppgst gene can reflect the stress action of the anatase nano titanium dioxide with different particle sizes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Shenzhen university
<120> application of Physarum polycephalum Ppgst gene as biomarker
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 774
<212> DNA
<213> Physarum polycephalum (Physarumpolycephalum)
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ggttgggcag tagttcataa aacgcaggtc catccccttg gtagctagta aacctagcaa 60
gcataagtat aagcatgagc aagcccaggc tatcgtactt tggaggaagg ggactcgctg 120
agcccatcag gttgctttta aaagatgcca aggtggaata tgaagaagtc aatctcggaa 180
cggctgccaa tggcgtgttt cccgaggctt tcctcgctct taaggcttct ggtgttctag 240
actacggttc tgttccttat tgggaggaga atggattgaa ccttgtgcag tcctttgcca 300
ttagccgaca ccttgccagg aagcacggat ataacgggga gaatgaggca gaagcagcta 360
aaattgactc ggttgtggaa ggcacgagag atttggtgat ggccatccgc aaagtgctta 420
cggttcccgc tgaacaaaag gcacaaacat ggcaagagat ttcaaaggtg gacattccca 480
agtggtttgg cttctttgaa gctcttttga agaaaagtgg taccggattt tttgttggaa 540
agaaggcctc gcttgccgat atctacgtca ttgcttacac agaggagatt cgttgtcttg 600
acaaatcggc cctagcttcc ttccccctcc tctccgccca cctggatgcc atgtttgcgc 660
gcccgaacct tgctgcgtac gctgccgacc ccaacgctat cccctcattt gcacgtttat 720
ggcataaaaa taataataac aaataacatg aaaaaccaaa ataaaaaaaa aaaa 774
<210> 2
<211> 223
<212> PRT
<213> Physarum polycephalum (Physarumpolycephalum)
<400> 2
Met Ser Lys Pro Arg Leu Ser Tyr Phe Gly Gly Arg Gly Leu Ala Glu
1 5 10 15
Pro Ile Arg Leu Leu Leu Lys Asp Ala Lys Val Glu Tyr Glu Glu Val
20 25 30
Asn Leu Gly Thr Ala Ala Asn Gly Val Phe Pro Glu Ala Phe Leu Ala
35 40 45
Leu Lys Ala Ser Gly Val Leu Asp Tyr Gly Ser Val Pro Tyr Trp Glu
50 55 60
Glu Asn Gly Leu Asn Leu Val Gln Ser Phe Ala Ile Ser Arg His Leu
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Ala Arg Lys His Gly Tyr Asn Gly Glu Asn Glu Ala Glu Ala Ala Lys
85 90 95
Ile Asp Ser Val Val Glu Gly Thr Arg Asp Leu Val Met Ala Ile Arg
100 105 110
Lys Val Leu Thr Val Pro Ala Glu Gln Lys Ala Gln Thr Trp Gln Glu
115 120 125
Ile Ser Lys Val Asp Ile Pro Lys Trp Phe Gly Phe Phe Glu Ala Leu
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Leu Lys Lys Ser Gly Thr Gly Phe Phe Val Gly Lys Lys Ala Ser Leu
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Ala Asp Ile Tyr Val Ile Ala Tyr Thr Glu Glu Ile Arg Cys Leu Asp
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Lys Ser Ala Leu Ala Ser Phe Pro Leu Leu Ser Ala His Leu Asp Ala
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Met Phe Ala Arg Pro Asn Leu Ala Ala Tyr Ala Ala Asp Pro Asn Ala
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Claims (5)
1. The application of the reagent for detecting the gene Ppgst of the Physarum polycephalum, which is used for detecting anatase type nano titanium dioxide in a dark environment, is characterized in that the nucleotide sequence of the gene Ppgst of the Physarum polycephalum is shown as SEQ ID NO. 1; the amino acid sequence of the protein expressed by the gene Ppgst of the Physarum polycephalum is shown as SEQ ID NO. 2;
the step of detecting the anatase type nano titanium dioxide in the dark environment comprises the following steps:
carrying out stress treatment on the acinetobacter polycephalus in a dark environment on a sample to be detected containing anatase type nano titanium dioxide;
detecting the expression level of Ppgst gene in the stress-treated Physarum polycephalum;
and deducing the concentration of the anatase type nano titanium dioxide in the sample to be detected according to the expression level of the Ppgst gene.
2. The use as claimed in claim 1, wherein the anatase nano-titania is present in a concentration of 9-15 mg/ml.
3. The use as claimed in claim 1, wherein the anatase nano-titania has a particle size of 60-80 nm.
4. The use of claim 1, wherein the period of stress treatment is 3-5 days.
5. The use according to claim 4, wherein the method for detecting the transcription level of the Ppgst gene in the stress-treated Physarum polycephalum is reverse transcription fluorescence quantitative PCR.
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