CN114015577B - Construction method of synechococcus strain with cadmium ion stress resistance - Google Patents
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Abstract
The invention provides a construction method of a synechococcus strain with cadmium ion stress resistance. The invention comprises the following steps: 1) Extracting RNA from the kelp, synthesizing cDNA, and amplifying by using a primer 1F and a primer 1R and PCR to obtain a kelp GST gene with a nucleotide sequence shown as SEQ ID No. 1; 2) Cutting, recovering and purifying the giant knotweed GST gene, and connecting the giant knotweed GST gene with a synechococcus protein expression vector to construct the synechococcus expression vector containing the giant knotweed GST gene; 3) And (3) culturing the Synechococcus cultivated to the logarithmic phase, centrifuging, collecting algae cells, re-suspending, adding a Synechococcus expression vector containing a giant kelp GST gene, and culturing to obtain the Synechococcus strain with cadmium ion stress resistance. The invention utilizes the giant alga GST gene to obtain the synechococcus strain with cadmium ion stress resistance, and the synechococcus strain can normally grow under the condition of cadmium ion stress and can normally carry out photosynthesis, thereby improving the growth performance under the condition of cadmium ion stress.
Description
Technical Field
The invention relates to the technical field of algae genetic engineering, in particular to a construction method of a synechococcus strain with cadmium ion stress resistance.
Background
Synechococcus (Synechococcus) is one of the most prominent representative groups of marine cyanobacteria, and was found in 1979 to be a class of ultra-miniature (0.5-2 μm) pellet-shaped photoautotrophic prokaryotes. The Sycochococcus is widely distributed in the ocean all over the world, is huge in quantity, particularly in the coastal waters, and is abundant in the tropical and temperate ocean, and the abundance in the ocean is usually 10 3 -10 5 And each mL. Synechococcus is the dominant component in ultra-micro plankton. Synechococcus is one of the main participants in the global carbon cycle and the main contributor to primary productivity, and Synechococcus and Prochlorococcus (the closely related species of Synechococcus) in the earth's ocean are absorbed about 1×10 per year from the atmosphere 10 T carbon corresponds to two-thirds of the total amount of carbon dioxide in the ocean fixed atmosphere. In addition, the Synechococcus circulates rapidly in the sea micro-food net, has high energy conversion efficiency, and is one of the most important food sources for micro zooplankton.
Cadmium induces ROS (reactive oxygen species) that cause lipid peroxidation, disturbing the intracellular ion balance. Cadmium is difficult to degrade in organisms, can be transmitted along with a food net, and threatens the safety of ocean foods and the health of human beings. Typically, the concentration of cadmium ions in sediment and open sea water is low, but some offshore and estuary areas can become very high in cadmium emissions due to leakage or man-made emissions. Cadmium has no known metabolic function in macroalgae; cadmium, however, has been shown to be a cofactor for diatom carbonic anhydrases. In higher plants, cadmium ions interfere with growth, photosynthesis, ion and water transport and reduce enzyme activity through their reaction with thiol groups.
Because the Synechococcus plays an important role in the marine ecosystem, the method has important significance in researching the ecological environment of the Synechococcus. Heavy metals in the ecological environment, particularly the aquatic environment, have an important influence on the growth of Synechococcus. At present, the photosynthesis of the existing Synechococcus can be reduced under the condition of cadmium ion stress to influence the normal growth.
Disclosure of Invention
The invention aims to provide a construction method of a synechococcus strain with cadmium ion stress resistance, and aims to solve the problem that photosynthesis of synechococcus is reduced under the condition of cadmium ion stress to influence normal growth in the prior art.
In order to solve the technical problems, the technical scheme of the invention is realized as follows:
the invention discloses a construction method of a synechococcus strain with cadmium ion stress resistance, which comprises the following steps:
1) Extracting RNA from giant kelp to synthesize cDNA,
using primer 1F:5'-CCGGAATTCATGGCTCCCGTATT-3'
And primer 1R:5'-CGGGGTACCCTAGGCCTTGGATG-3' the number of the individual pieces of the plastic,
PCR amplification to obtain giant kelp GST gene with nucleotide sequence shown as SEQ ID No. 1;
2) Cutting, recovering and purifying the giant knotweed GST gene, and connecting the giant knotweed GST gene with a synechococcus protein expression vector to construct the synechococcus expression vector containing the giant knotweed GST gene;
3) And (3) culturing the Synechococcus cultivated to the logarithmic phase, centrifuging, collecting algae cells, re-suspending, adding a Synechococcus expression vector containing a giant kelp GST gene, and culturing to obtain the Synechococcus strain with cadmium ion stress resistance.
The invention utilizes the connection of the giant alga GST gene and the synechococcus protein expression vector to construct the synechococcus expression vector containing the giant alga GST gene, and the synechococcus expression vector is added into synechococcus cells, and after culturing, the synechococcus strain with cadmium ion stress resistance is obtained; the giant alga GST gene can be efficiently expressed in the Synechococcus, the growth rate of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is obviously increased compared with that of a wild Synechococcus strain under the condition of cadmium ion stress, and the variation of chlorophyll fluorescence parameter Fv/Fm of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is less obvious than that of the Synechococcus strain under the condition of no stress; therefore, the synechococcus strain with the cadmium ion stress resistance can still grow normally under the condition of cadmium ion stress, and photosynthesis can be performed normally, so that the growth performance under the condition of cadmium ion stress is improved.
As a preferred embodiment, the amino acid sequence encoded by the giant alga GST gene is shown as SEQ ID No. 2. The giant alga GST gene is a giant alga glutathione S-transferase gene, glutathione S-transferase (GST) is ubiquitous in all organisms and is a multifunctional protein coded by a large gene family, and the giant alga GST gene can be efficiently expressed in the Synechococcus, so that the growth performance under the condition of cadmium ion stress is improved. The nucleotide sequence of the giant alga GST gene contains 606bp nucleotide, the sequence is the coding sequence of the GST gene, and the coding sequence can code protein with 201 amino acids shown in a sequence table SEQ ID No. 2.
As a preferred embodiment, the synechococcus protein expression vector is pSyn_6. In the invention, the giant alga GST gene is connected with the synechococcus protein expression vector to construct the synechococcus expression vector containing the giant alga GST gene, and the synechococcus expression vector is transformed into E.coli DH5 alpha competent cells. pSyn_6 is the name of a vector, and the synechococcus protein expression vector is a commercial vector, and has the advantages of wide sources, convenient material acquisition, low cost and easy obtainment.
As a preferred embodiment, the PCR amplification conditions are: pre-denaturation at 94 ℃ for 30s; denaturation at 98℃for 10s, annealing at 62℃for 30s, extension at 72℃for 1min, total amplification for 30 cycles, extension at 72℃for 2min. According to GST gene sequences obtained from the kelp transcriptome sequences, enzyme cutting sites contained in the GST gene sequences are screened out through OMIGA software, and specific primers, namely a primer 1F and a primer 1R, of the GST gene sequences containing the proper enzyme cutting sites are designed through Primer5.0 software, and the primer 1F and the primer 1R are directly used by a finished product manufactured by a primer company after the design is finished. The invention effectively controls the amplification conditions and realizes the effective amplification of the giant kelp GST gene.
As a preferred embodiment, the step of resuspension is: centrifuging the algae cells, removing supernatant, adding BG11 liquid culture medium, and re-suspending; again centrifuged, the supernatant removed, BG11 broth added and resuspended. The invention adopts a conventional resuspension method, so that the operation is simple and the realization is convenient; the volume of the BG11 liquid medium used for the first time of resuspension is 10 times of that of the BG11 liquid medium used for the second time of resuspension, the first time of deep resuspension and the second time of repeated resuspension are carried out, and therefore the resuspension effect is improved.
As a preferred embodiment, the step of culturing is: firstly, placing algae cells added with a Synechococcus expression vector containing a giant alga GST gene in a constant temperature water bath at 34 ℃ for standing for 4 hours; then, the algae liquid is coated on BG11 solid culture medium, and is cultivated by illumination at 27 ℃ for 8 days, so that single spots grow out; finally, the single spots are inoculated into BG11 liquid medium for culture. The invention adopts the conventional culture method, firstly stands in a constant-temperature water bath, then carries out illumination culture, and finally carries out inoculation culture, and the culture method is simple and easy to master.
As a preferred embodiment, in the culture process, the BG11 solid medium is a BG11 solid medium containing 10. Mu.g/mL spectinomycin, and the BG11 liquid medium is a BG11 liquid medium containing 10. Mu.g/mL spectinomycin. The BG11 solid culture medium adopted in the culture process is the same as the BG11 liquid culture medium, and is convenient to select.
As a preferred embodiment, the centrifugation is carried out at room temperature and 14000 rpm. The invention has mild centrifugation condition and convenient operation, and usually the centrifugation is carried out for 1-3min.
Compared with the prior art, the invention has the beneficial effects that: the invention utilizes the connection of the giant alga GST gene and the synechococcus protein expression vector to construct the synechococcus expression vector containing the giant alga GST gene, and the synechococcus expression vector is added into synechococcus cells, and after culturing, the synechococcus strain with cadmium ion stress resistance is obtained; the giant alga GST gene can be efficiently expressed in the Synechococcus, the growth rate of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is obviously increased compared with that of a wild Synechococcus strain under the condition of cadmium ion stress, and the variation of chlorophyll fluorescence parameter Fv/Fm of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is less obvious than that of the Synechococcus strain under the condition of no stress; therefore, the synechococcus strain with the cadmium ion stress resistance can still grow normally under the condition of cadmium ion stress, and photosynthesis can be performed normally, so that the growth performance under the condition of cadmium ion stress is improved.
Drawings
FIG. 1 is a diagram showing the construction of a giant knotweed glutathione S-transferase gene expression vector according to an embodiment of the present invention;
FIG. 2 is a graph showing the result of agarose gel electrophoresis test of the amplified product of transgenic Synechococcus in experiment 1 of the present invention;
FIG. 3 is a graph showing the trend of the growth rate of different Synechococcus in experiment 2 according to the present invention;
FIG. 4 is a graph showing the variation trend of the chlorophyll fluorescence parameters Fv/Fm of different Synechococcus in experiment 2 under different conditions;
in the figure: -wild synechococcus plants; ■ -a synechococcus strain with cadmium ion stress resistance; a-absence of cadmium ion stress; b-cadmium ion stress.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with specific embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention discloses a construction method of a synechococcus strain with cadmium ion stress resistance, which comprises the following steps:
1) Extracting RNA from giant kelp to synthesize cDNA,
using primer 1F:5'-CCGGAATTCATGGCTCCCGTATT-3'
And primer 1R:5'-CGGGGTACCCTAGGCCTTGGATG-3' the number of the individual pieces of the plastic,
PCR amplification to obtain giant kelp GST gene with nucleotide sequence shown as SEQ ID No. 1;
2) Cutting, recovering and purifying the giant knotweed GST gene, and connecting the giant knotweed GST gene with a synechococcus protein expression vector to construct the synechococcus expression vector containing the giant knotweed GST gene;
3) And (3) culturing the Synechococcus cultivated to the logarithmic phase, centrifuging, collecting algae cells, re-suspending, adding a Synechococcus expression vector containing a giant kelp GST gene, and culturing to obtain the Synechococcus strain with cadmium ion stress resistance.
Preferably, the amino acid sequence of the giant alga GST gene is shown as SEQ ID No. 2.
Preferably, the synechococcus protein expression vector is pSyn_6.
Preferably, the conditions of the PCR amplification are: pre-denaturation at 94 ℃ for 30s; denaturation at 98℃for 10s, annealing at 62℃for 30s, extension at 72℃for 1min, total amplification for 30 cycles, extension at 72℃for 2min.
Preferably, the step of resuspension is: centrifuging the algae cells, removing supernatant, adding BG11 liquid culture medium, and re-suspending; again centrifuged, the supernatant removed, BG11 broth added and resuspended.
Preferably, the step of culturing is: firstly, placing algae cells added with a Synechococcus expression vector containing a giant alga GST gene in a constant temperature water bath at 34 ℃ for standing for 4 hours; then, the algae liquid is coated on BG11 solid culture medium, and is cultivated by illumination at 27 ℃ for 8 days, so that single spots grow out; finally, the single spots are inoculated into BG11 liquid medium for culture.
Preferably, in the culture process, the BG11 solid medium is a BG11 solid medium containing 10 mug/mL of spectinomycin, and the BG11 liquid medium is a BG11 liquid medium containing 10 mug/mL of spectinomycin.
Preferably, the centrifugation is performed at room temperature and 14000 rpm.
Example 1
The invention discloses a construction method of a synechococcus strain with cadmium ion stress resistance, which comprises the following steps:
cloning of S1 kelp GST Gene
The total kelp RNA extraction kit is Omega Bio-Tek (U.S. Co., ltd. (http:// www.omegabiotek.com /), and the reverse transcription kit is Ai Kerui bioengineering Co., ltd. (http:// agbio.com cn) and various restriction enzymes are purchasedFrom Neon England Biotechnology (U.S. Co., ltd. (http// www.neb.com /), T 4 DNA Ligase was purchased from Dalianbao bioengineering Co., ltd (http:// takara. Com. Cn).
About 0.1g of fresh kelp was weighed and subjected to extraction of total RNA, and the specific method was performed by referring to the procedure of the above-mentioned Omega Bio-Tek company plant sample extraction instruction, and cDNA synthesis was performed on the extracted total RNA (i.e., cDNA was reverse transcribed using RNA as a template), and the specific method was performed by referring to the procedure of the agbio company instruction. The sequence of the giant kelp GST gene is amplified by a conventional PCR method under the guidance of a primer 1F and a primer 1R by taking the first strand of the synthesized cDNA as a template. The amplification conditions were: pre-denaturation at 94 ℃ for 30s; denaturation at 98℃for 10s, annealing at 62℃for 30s, extension at 72℃for 1min, total amplification for 30 cycles, extension at 72℃for 2min. After the reaction is finished, 2% agarose gel electrophoresis detection is carried out on the PCR amplification product, and a DNA fragment of about 700bp is recovered and purified and sequenced to obtain the required full length of the kelp GST gene.
Primer 1F:5' -CCGGAATTCATGGCTCCCGTATT-3' (underlined sequence is restriction endonuclease EcoRI recognition site and protecting base);
primer 1R:5' -CGGGGTACCCTAGGCCTTGGATG-3' (the underlined sequence is the recognition site for restriction endonuclease KpnI and a protecting base);
and screening enzyme cutting sites contained in the GST gene sequence through OMIGA software according to the GST gene sequence obtained by the kelp transcriptome sequence, and designing specific primers, namely a primer 1F and a primer 1R, of the GST gene sequence containing the proper enzyme cutting sites through Primer5.0 software.
Sequencing results show that the DNA fragment of about 700bp contains a 606bp nucleotide sequence shown in a sequence table SEQ ID No. 1, the sequence is a coding sequence of a GST gene, the coding sequence can code a protein consisting of 201 amino acids shown in a sequence table SEQ ID No. 2, and the protein is named GST.
Construction of S2 GST Gene Synechococcus expression vector
Plasmid miniprep kit was purchased from Tiangen Biochemical technology (Beijing) Co., ltd (http:// www.tiangen.com /).
And (3) carrying out double digestion on the DNA fragment containing the nucleotide shown in the sequence table SEQ ID No. 1 and the polcoculine protein expression vector pSyn_6 which are obtained by PCR amplification in the S1 by adopting EcoRI and KpnI, recovering and purifying enzyme digestion products, connecting, and constructing the polcoculine expression vector of the giant alga GST after enzyme digestion and identification of the connection products, and converting the polcoculine expression vector into E.coli DH5 alpha competent cells. Extracting plasmids through a plasmid small extraction kit, and carrying out sequencing identification to ensure that the kelp GST gene is connected to a synechococcus protein expression vector pSyn_6; the construction diagram of the Synechococcus expression vector of the giant alga GST is shown in figure 1.
S3 obtaining transgenic Synechococcus
The S2 constructed Synechococcus expression vector containing the Cytomentosa GST gene is transformed into Synechococcus, and the specific method is as follows:
culturing Synechococcus in logarithmic phase, centrifuging at 14000rpm at room temperature for 3min, and collecting 1.5mL of algae cells; centrifuging, removing supernatant, adding 1mL of BG11 liquid medium, and re-suspending; centrifuging at 14000rpm at room temperature for 1min, removing supernatant, adding 100 μl of BG11 liquid culture medium, and re-suspending; then 100ng of the S2-constructed Synechococcus expression vector containing the giant kelp GST gene is added, a centrifuge tube (EP tube) is gently stirred, the EP tube is added with a black cover, and the mixture is subjected to constant temperature water bath at 34 ℃ and is kept stand for 4 hours.
The EP tube was removed, rubbed with 70% ethanol, 100. Mu.L of the algae solution was spread on BG11 solid medium containing 10. Mu.g/mL spectinomycin, and the culture was carried out at 27℃under light irradiation for 8 days to grow single spots, thereby obtaining transformants. Inoculating the single spot into BG11 liquid culture medium containing 10 mug/mL spectinomycin, and culturing to obtain the Synechococcus strain with cadmium ion stress resistance.
Experiment 1 PCR identification of transgenic Synechococcus
Extracting genome DNA from the Synechococcus strain with cadmium ion stress resistance obtained in the first embodiment, performing PCR amplification by using 2F and 2R primers, and performing 2% agarose gel electrophoresis detection on the PCR amplification product; as can be seen from FIG. 2, GST gene of kelp was amplified in Synechococcus; therefore, the transformation is successful, and the high-efficiency expression of the giant alga GST gene in the Synechococcus is realized.
Primer 2F:5'-ATGGCTCCCGTATTCAACTA-3';
primer 2R:5'-CTAGGCCTTGGATGCGTAG-3'.
The wild Synechococcus plant growing to logarithmic phase and the Synechococcus plant with cadmium ion stress resistance obtained in example one were diluted to OD with BG11 medium 750 The value of the solution is 0.01, the solution containing cadmium ions is added to ensure that the concentration of the cadmium ions in each sample is 0.2mg/L, and blank controls are respectively added. The above sample was placed in a constant temperature incubator at 100. Mu. Mol/(m) 2 S) culturing at 25.+ -. 2 ℃ under illumination intensity, and measuring OD of the sample every 2 days 750 Values were measured for 12 days and the chlorophyll fluorescence parameters Fv/Fm of the samples were determined on day 10.
As can be seen from FIG. 3, the growth rate of the Synechococcus strain with the stress resistance to cadmium ions under the condition of cadmium ion stress is obviously increased compared with that of a wild Synechococcus strain under the condition of cadmium ion stress, which shows that the Synechococcus strain with the stress resistance to cadmium ions has a faster growth rate under the condition of cadmium ion stress.
As can be seen from fig. 4, the chlorophyll fluorescence parameter Fv/Fm of the wild synechococcus plant under the cadmium ion stress is obviously reduced compared with that of the wild synechococcus plant under the condition of no cadmium ion stress, and the wild synechococcus plant almost dies; therefore, wild Synechococcus plants cannot normally perform photosynthesis under cadmium ion stress conditions. However, the chlorophyll fluorescence parameter Fv/Fm of the chlorella strain with the cadmium ion stress resistance obtained by the invention is slightly reduced and the change is not obvious compared with the chlorophyll fluorescence parameter Fv/Fm of the chlorella strain without the cadmium ion stress; therefore, the synechococcus strain with the cadmium ion stress resistance can also normally perform photosynthesis under the condition of cadmium ion stress.
Therefore, compared with the prior art, the invention has the beneficial effects that: the invention utilizes the connection of the giant alga GST gene and the synechococcus protein expression vector to construct the synechococcus expression vector containing the giant alga GST gene, and the synechococcus expression vector is added into synechococcus cells, and after culturing, the synechococcus strain with cadmium ion stress resistance is obtained; the giant alga GST gene can be efficiently expressed in the Synechococcus, the growth rate of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is obviously increased compared with that of a wild Synechococcus strain under the condition of cadmium ion stress, and the variation of chlorophyll fluorescence parameter Fv/Fm of the Synechococcus strain with the cadmium ion stress resistance under the condition of cadmium ion stress is less obvious than that of the Synechococcus strain under the condition of no stress; therefore, the synechococcus strain with the cadmium ion stress resistance can still grow normally under the condition of cadmium ion stress, and photosynthesis can be performed normally, so that the growth performance under the condition of cadmium ion stress is improved.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence list
<110> Qingdao university of science and technology; yellow sea aquatic institute of China aquatic science institute
<120> construction method of Synechococcus strain having cadmium ion stress resistance
<130> 2021
<160> 6
<170> PatentIn version 3.5
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<211> 606
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atggctcccg tattcaacta cttcggaatc cccgcccgtg gcgaggcaac ccgcgtcgct 60
ctggccgtcg ccggcgttga cttcgaggac aagcgcatgt ccttcgaaga gttcggtgcc 120
tccgagttca agactctgcc cgtctaccag atggacggaa ccgactacac ccagtcgacc 180
gcccttttgc gatacgccgg taagctcggc ggcacgtacc cggaaagccc tctcgccgcc 240
ctcaaggtgg acgagatcgt gatgatcgcg gaggacgtgt tcatcaacct cttctccacc 300
atgggagaga aggacgaggc caagaagttg cacaagtgcc agactctgat ggccggcaaa 360
gtgaaggagc tgctggagga catcgccagg aaggtggaat ccaacaagag ctcggccttc 420
tgcgtcgggg actgcctcac catcgccgac atcacgattc acgccgtgtt cgccacagtg 480
caggctggct tcctggctgg catccccaag accatggtgg aggacatctg tccatccctc 540
aaggcggtcg acaacgccgt catggcacac ccgaaggtga aggcgtacta cgcatccaag 600
gcctag 606
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Met Ala Pro Val Phe Asn Tyr Phe Gly Ile Pro Ala Arg Gly Glu Ala
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Thr Arg Val Ala Leu Ala Val Ala Gly Val Asp Phe Glu Asp Lys Arg
20 25 30
Met Ser Phe Glu Glu Phe Gly Ala Ser Glu Phe Lys Thr Leu Pro Val
35 40 45
Tyr Gln Met Asp Gly Thr Asp Tyr Thr Gln Ser Thr Ala Leu Leu Arg
50 55 60
Tyr Ala Gly Lys Leu Gly Gly Thr Tyr Pro Glu Ser Pro Leu Ala Ala
65 70 75 80
Leu Lys Val Asp Glu Ile Val Met Ile Ala Glu Asp Val Phe Ile Asn
85 90 95
Leu Phe Ser Thr Met Gly Glu Lys Asp Glu Ala Lys Lys Leu His Lys
100 105 110
Cys Gln Thr Leu Met Ala Gly Lys Val Lys Glu Leu Leu Glu Asp Ile
115 120 125
Ala Arg Lys Val Glu Ser Asn Lys Ser Ser Ala Phe Cys Val Gly Asp
130 135 140
Cys Leu Thr Ile Ala Asp Ile Thr Ile His Ala Val Phe Ala Thr Val
145 150 155 160
Gln Ala Gly Phe Leu Ala Gly Ile Pro Lys Thr Met Val Glu Asp Ile
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Claims (8)
1. The construction method of the synechococcus strain with the cadmium ion stress resistance is characterized by comprising the following steps:
1) Extracting RNA from giant kelp to synthesize cDNA,
using primer 1F:5'-CCGGAATTCATGGCTCCCGTATT-3'
And primer 1R:5'-CGGGGTACCCTAGGCCTTGGATG-3' the number of the individual pieces of the plastic,
PCR amplification to obtain giant kelp GST gene with nucleotide sequence shown as SEQ ID No. 1;
2) Cutting, recovering and purifying the giant knotweed GST gene, and connecting the giant knotweed GST gene with a synechococcus protein expression vector to construct the synechococcus expression vector containing the giant knotweed GST gene;
3) And (3) culturing the Synechococcus cultivated to the logarithmic phase, centrifuging, collecting algae cells, re-suspending, adding a Synechococcus expression vector containing a giant kelp GST gene, and culturing to obtain the Synechococcus strain with cadmium ion stress resistance.
2. The construction method of the synechococcus strain with the cadmium ion stress resistance according to claim 1, which is characterized in that:
the coding amino acid sequence of the giant kelp GST gene is shown as SEQ ID No. 2.
3. The construction method of the synechococcus strain with the cadmium ion stress resistance according to claim 1, which is characterized in that:
the synechococcus protein expression vector is pSyn_6.
4. The construction method of the synechococcus strain with the cadmium ion stress resistance according to claim 1, which is characterized in that:
the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 30s; denaturation at 98℃for 10s, annealing at 62℃for 30s, extension at 72℃for 1min, total amplification for 30 cycles, extension at 72℃for 2min.
5. The method for constructing a chlorella strain with cadmium ion stress resistance according to any one of claims 1 to 4, wherein:
the step of resuspension is as follows: centrifuging the algae cells, removing supernatant, adding BG11 liquid culture medium, and re-suspending; again centrifuged, the supernatant removed, BG11 broth added and resuspended.
6. The construction method of the chlorella strain with cadmium ion stress resistance according to claim 5, wherein the construction method comprises the following steps:
the culturing steps are as follows: firstly, placing algae cells added with a Synechococcus expression vector containing a giant alga GST gene in a constant temperature water bath at 34 ℃ for standing for 4 hours; then, the algae liquid is coated on BG11 solid culture medium, and is cultivated by illumination at 27 ℃ for 8 days, so that single spots grow out; finally, the single spots are inoculated into BG11 liquid medium for culture.
7. The construction method of the chlorella strain with cadmium ion stress resistance according to claim 6, wherein the construction method comprises the following steps:
in the culture process, the BG11 solid medium is a BG11 solid medium containing 10 mug/mL of spectinomycin, and the BG11 liquid medium is a BG11 liquid medium containing 10 mug/mL of spectinomycin.
8. The construction method of the chlorella strain with cadmium ion stress resistance according to claim 5, wherein the construction method comprises the following steps:
the centrifugation was performed at room temperature and 14000 rpm.
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| 不同环境胁迫因子对藻类分子生物学特性的影响研究进展;魏静;林莉;潘雄;刘敏;李明;;长江科学院院报;37(04);14-24 * |
| 巨藻中谷胱甘肽S转移酶基因对细长聚球藻PCC7942耐镉性的影响;顾梓鹏 等;渔业科学进展;第44卷(第2期);127-136 * |
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