CN108004252A - Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled - Google Patents
Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled Download PDFInfo
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- CN108004252A CN108004252A CN201711231277.5A CN201711231277A CN108004252A CN 108004252 A CN108004252 A CN 108004252A CN 201711231277 A CN201711231277 A CN 201711231277A CN 108004252 A CN108004252 A CN 108004252A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/405—Assays involving biological materials from specific organisms or of a specific nature from algae
Abstract
The invention discloses Chlamydomonas reinhardtiig3280.t2Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, belongs to biological technical field and field of environment pollution control.The present invention is by by VIII fragments of aph of the resistant gene containing paromomycin（SEQ ID NO:3）The Chlamydomonas reinhardtii mutation algae strain significantly sensitive to cadmium is obtained in radom insertion chlamydomonas, further identification finds that the cadmium sensitiveness of mutation algae strain is due to that VIII fragments of aph are inserted intog3280.t2Gene（CDS sequences such as SEQ ID NO:Shown in 1）In caused by.By reducing Chlamydomonas reinhardtiig3280.t2The expression of gene, makeg3280.t2Gene, which inactivates or lacks the gene, can reduce the cadmium tolerance of Chlamydomonas reinhardtii；The cadmium responsive type Chlamydomonas reinhardtii algae strain obtained by genetic engineering means may be used on carrying out by biological means in water body environment among the monitoring and detection of cadmium pollution, and then new biomaterial is provided for sewage detection.
Description
Technical field
The present invention relates to biological technical field and field of environment pollution control, and in particular to Chlamydomonas reinhardtii g3280.t2 genes
Application in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled.
Background technology
Since 21 century, with the development of science and technology, cadmium is widely used in life：Normal cell, smoke shell are glimmering
Light powder, pigment, alloy manufacture etc..The compound of cadmium is also once widely used in manufacture stabilizer for plastics, video mapping pipe fluorescent powder etc..
A large amount of industrial wastewaters, the discharge of discarded object, makes cadmium ion enter soil, air, or even food.Heavy metal cadmium is toxic, is not
Human essential elements, cadmium is entered by food, water, air and accumulated in vivo in human body, therefore can be to human body, animals and plants
The health care belt of body carrys out hidden danger.Cadmium can to respiratory tract produce stimulation, long-term exposure can cause anosmia, gum macula lutea or gradually into
Yellow circle, cadmium compound are not easy by intestinal absorption, but can be lodged in liver through breathing by body absorption or kidney damages, especially with
It is the most obvious to kidney damage, osteoporosis and softening are can also result in, long-term suction cadmium can produce chronic Zhong Du ﹐ and cause kidney to damage
Though Hai ﹐ are mainly shown as time of the category Zheng Chang ﹐ more of the filtering function containing a large amount of low molecular weight protein Zhi ﹐ glomerulus but renal tubule in urine
Receive function but Jian Tui ﹐ and urinate cadmium discharge increase.So the water pollution gone out therewith, air pollution etc. are urgently to be resolved hurrily, add
Strong environmental monitoring should not be underestimated.The method of Monitoring of Cadmium includes in environment：Atomic fluorescence spectrometry, atomic absorption spectrophotometry,
Double-current total spectrophotometry, anodic stripping voltammetry and oscilloscopic polarography etc., are also progressively becoming one using biological means monitoring
The new pollution prevention measure of kind.
Chlamydomonas reinhardtii is as unicellular eukaryote, and condition of culture is simple, and growth cycle is short, and photosynthetic efficiency is high, with dynamic plant
Thing is compared, it is easier to obtains mutant, environmental suitability is strong, is a kind of preferably pattern life of water pollution monitoring and improvement
Thing., can not only be into present invention finds Chlamydomonas reinhardtii g3280.t2 genes and its function of encoding proteins regulation and control cadmium tolerance
One step is used to study chlamydomonas heavy metal cadmium metabolic regulation network, and heavy metal cadmium based theoretical is managed for biology ruling by law, by
The mutant algae strain (KY1) that g3280.t2 gene mutations are formed is even more to have significant sensitiveness to caddy, may be used on water
Among the monitoring and detection for carrying out cadmium pollution in body environment by biological means, and then new biological material is provided for waste water control
Material.
The content of the invention
The present invention primary and foremost purpose in provide Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled
Application.Based on the function of g3280.t2 gene regulation Chlamydomonas reinhardtii cadmium tolerances, the present invention also aims to provide Rhein
Application of the chlamydomonas g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is reduced, there is provided the acquisition of Chlamydomonas reinhardtii cadmium responsive type algae strain
Application of method and the strain of cadmium responsive type algae etc..
The purpose of the present invention is achieved through the following technical solutions：
The present invention is notable to cadmium by having been obtained in the VIII fragment radom insertion chlamydomonas of aph by the resistant gene containing paromomycin
Sensitive Chlamydomonas reinhardtii mutation algae strain, further identification find that the cadmium sensitiveness of mutation algae strain is due to that VIII fragments of aph are inserted into
Caused by the 5th extron of g3280.t2 genes.Gene of the g3280.t2 genes in JGI Chlamydomonas reinhardtii gene databases is compiled
Number it is Cre03.g160200.t1.2, positioned at No. 3 chromosome of Chlamydomonas reinhardtii, its CDS sequence such as SEQ ID NO:Shown in 1.
Based on above-mentioned discovery, an object of the present invention is to provide Chlamydomonas reinhardtii g3280.t2 genes in regulation and control Chlamydomonas reinhardtii
Application in cadmium tolerance.
The nucleotides sequence of the g3280.t2 genes is classified as one kind in following sequences：
(1) such as SEQ ID NO are included:The nucleotide sequence of 894bp shown in 1；
(2) comprising coding SEQ ID NO:The nucleotide sequence of protein shown in 2；
(3) include and SEQ ID NO:Nucleotide sequence shown in 1 is with more than 90% homology and coding identical function
The nucleotide sequence of protein.
The encoding proteins of the g3280.t2 genes are one kind in following albumen：
(1) such as SEQ ID NO are included:Amino acid sequence shown in 2；
(2) include by SEQ ID NO:Substitution of the amino acid sequence shown in 2 through several amino acid, missing or increase etc.
What is changed and derive has and SEQ ID NO:The identical active derived protein of albumen shown in 2.
The second object of the present invention, which is to provide above-mentioned Chlamydomonas reinhardtii g3280.t2 genes, is reducing Chlamydomonas reinhardtii cadmium tolerance
In application.By reducing the expression of Chlamydomonas reinhardtii g3280.t2 genes, g3280.t2 genes is inactivated or lack g3280.t2
Gene is so as to reduce tolerance of the Chlamydomonas reinhardtii to cadmium.
The third object of the present invention is to provide reduces the tolerance of Chlamydomonas reinhardtii cadmium using above-mentioned Chlamydomonas reinhardtii g3280.t2 genes
The method of property.The method is by reducing the expression of Chlamydomonas reinhardtii g3280.t2 genes, g3280.t2 genes is inactivated or is lacked
G3280.t2 genes are lost to reduce tolerance of the Chlamydomonas reinhardtii to cadmium.
The fourth object of the present invention is to provide a kind of Chlamydomonas reinhardtii cadmium responsive type algae strain.Responsive type algae strain be without
Under the g3280.t2 gene mutations strain of g3280.t2 gene activities or g3280.t2 gene-deleted strains, or g3280.t2 gene expressions
The g3280.t2 gene expression defect strains of drop.
The fifth object of the present invention is to provide a kind of preparation method of Chlamydomonas reinhardtii responsive type algae strain.The preparation method bag
Include：Chlamydomonas reinhardtii g3280.t2 gene mutation strains are obtained by the method for insertion mutation or rite-directed mutagenesis, pass through structure
Crispr carriers and then acquisition Chlamydomonas reinhardtii g3280.t2 gene-deleted strains, Rhein is obtained by building RNAi interference vectors
Chlamydomonas g3280.t2 gene expression defect strains.
The sixth object of the present invention is to provide monitoring, inspection of the above-mentioned Chlamydomonas reinhardtii cadmium responsive type algae strain in heavy metal cadmium
Application in survey.According to the strain of g3280.t2 gene expression defects, g3280.t2 gene mutations strain or the g3280.t2 of Chlamydomonas reinhardtii
Gene-deleted strain is to the sensitiveness of heavy metal cadmium, the characteristic that can only be grown in the environment containing heavy metal cadmium of low concentration, its
It can be applied in the monitoring and detection of cadmium pollution, be particularly applicable in sewage in the monitoring and detection of cadmium, such as the Rhein clothing
Whether the strain of algae cadmium responsive type algae can exceeded with the cadmium content in monitoring of environmental water body, convenient to detect cadmium pollution degree in environment and rise
To prevention or improve Environmental Cadmium Pollution.The Chlamydomonas reinhardtii cadmium responsive type algae strain is applied to monitor or detects in environment water
The situation of heavy metal cadmium, a kind of new thinking is provided for the detection of Heavy Metals in Waters cadmium and Biochemical method sewage
With new material.Meanwhile it can be further used for studying chlamydomonas heavy metal cadmium metabolic regulation net based on g3280.t2 gene functions
Network, heavy metal cadmium based theoretical is managed for biology ruling by law.
The beneficial effects of the invention are as follows：
(1) present invention discover that g3280.t2 genes and its encoding proteins be a kind of new with heavy metal cadmium metabolic regulation phase
The gene and albumen of pass, have no relevant report at present.
(2) method for the acquisition 0.4mM caddy sensitive mutants that the present invention uses is simple, efficient, can use for reference
In other model organisms.
(3) the 0.4mM caddies sensitive mutant (KY1) that the present invention obtains is due to that VIII fragment forward directions of aph are inserted into
The 5th extron position of the gene order and formed, to 0.4mM caddies show it is very sensitive, be conducive to further grind
Study carefully mechanism of action of the gene in chlamydomonas heavy metal cadmium metabolic regulation.
(4) in the present invention g3280.t2 gene insertion mutation bodies (KY1) that obtain under conditions of caddy containing 0.4mM,
There is significant sensitiveness relative to wild type chlamydomonas, in monitoring and detection that cadmium pollution waters can be further applied.
Brief description of the drawings
Fig. 1 is agarose gel electrophoresis figure after VIII plasmid enzyme restrictions of pJMG-aph.
Fig. 2 is wild type Chlamydomonas reinhardtii 21gr and cadmium responsive type chlamydomonas mutant (KY1) under the conditions of 0.4mM caddies
Grow comparison diagram.
Fig. 3 is cadmium responsive type chlamydomonas mutant (KY1) agarose gel electrophoresis figure after RESDA-PCR.
Fig. 4 is the insertion mutation site schematic diagram of g3280.t2 genes in cadmium responsive type chlamydomonas mutant (KY1).
Embodiment
In order to enable related personnel to be better understood from the present invention, the present invention is carried out with reference to embodiment and attached drawing detailed
Thin description, the method not clearly stated in embodiment is conventional experimental method.
Used medium formula is as follows in following embodiments：
TAP culture medium prescriptions：Tris 1.21g, SolutionI (salting liquid) 12.5mL, Solution II (phosphate)
0.188mL, SolutionIII (trace element) 0.5mL, GAA (glacial acetic acid) 0.5mL, adds each component, is settled in order
500mL.Solid medium then adds 7.5g Agar (agar).
TAgP culture medium prescriptions：Tris 1.21g, SolutionI (salting liquid) 12.5mL, sodium glycero-phosphate (1M)
0.376mL, SolutionIII (trace element) 0.5mL, GAA (glacial acetic acid) 0.5mL, adds each component, is settled in order
500mL.7.5g Agar (agar) are then added to prepare solid medium.
The acquisition of 1 chlamydomonas insertion mutation body of embodiment
(1) picking TAP cultured on solid medium wild type Chlamydomonas reinhardtii algae strain 21gr in good condition is extremely equipped with TAP liquid
In the blowing cylinder of body culture medium, ventilation culture to concentration is 1 × 10 under the conditions of continuous illumination7The sterile of 250mL is forwarded to after/mL
In shaking flask, cell concentration is diluted to as 1 × 10 with liquid TAP6/ mL, cultivates 24h on shaking table under continuous illumination, to final concentration of 4
×106/mL。
(2) by I-HF enzyme digestions pJMG-aph of EcoR, VIII plasmids (Zhangfeng Hu, Yinwen Liang, Wei He,
Junmin Pan*,Cilia Disassembly with Two Distinct Phases of Regulation,Cell
Reports,2015,10(11):1803-1810), VIII fragments of aph (sequence SEQ ID NO are obtained:Shown in 3), which contains
The mould resistant genes of Ba Long.By agarose gel electrophoresis, VIII fragments of aph are then recycled.As shown in Figure 1, M is DL5000DNA
Marker, 1 is samples of the pJMG-aph VIII after I-HF enzyme digestions of EcoR, and the band between 2k-3k is to need to carry out glue reclaim
VIII fragments of aph.
(3) it is 4 × 10 that above-mentioned concentration is taken in super-clean bench6The 21gr cells 20mL of/mL is in 50mL centrifuge tubes, 2500rpm/
Min centrifuges 3min, outwells supernatant in super-clean bench, and with shift to an earlier date -20 DEG C of precoolings TAP+60mM sorbierites (sorbierite containing 60mM
TAP fluid nutrient mediums) it is resuspended, the resuspension of 500 μ L TAP+60mM sorbierites is added after centrifuging again.Take two electric shock cup (super-clean benches
It is interior to be washed once with absolute ethyl alcohol, -20 DEG C of precoolings after TAP+60mM sorbierites are washed twice), it is separately added into 250 μ L re-suspension liquids and 10ng
VIII fragments of aph, place 10min on ice.The parameter of adjustment electric shock instrument (model BTX ECM630) is voltage 800V, resistance 1575
Ω, 50 μ F of capacitance, shock by electricity 10-14s, is transferred in 50mL centrifuge tubes after being put in 10min on ice again after the completion of electric shock, finally uses
TAP+60mM sorbierites are settled to 10mL, and tinfoil wraps on shaking table that (100r/min) shakes overnight reparation at a slow speed.
(4) 20% starch solution is matched somebody with somebody：According to done turn over number calculate needed for starch solution amount, weigh corresponding starch in
50mL centrifuge tubes, are first washed 1 time with absolute ethyl alcohol, then are washed 2 times with aseptic double-distilled water, 1000rpm/min centrifugation 2min, then with 70%
Ethanol is resuspended to final concentration of 20%.1000rpm/min centrifuges 1min before coated plate, is added after washing 4 times with TAP+60mM sorbierites
Suitable TAP+60mM sorbitol solutions are resuspended.
(5) 2500rpm/min centrifuges 3min and collects reinhardtii cell after the electricity repaired overnight turns, and adds the starch of 1mL 20%
Solution is resuspended, and is uniformly coated on the TAP solid mediums containing the mould antibiotic of Ba Long (10 μ g/mL), is dried up in super-clean bench
Liquid, sealed membrane sealing, is inverted under the photoperiod and is cultivated.Son to be transformed grows rear picking transformant in TAgP solid cultures
Photoperiod (14h illumination/10h is dark) culture 3-5 days on base.
The acquisition of 2 caddy sensitive mutant algae of embodiment strain
(1) it is optimal dense for the algae strain of screening sensitive mutants according to the result of study of applicant's early period, 0.4mM caddies
Spend (being specifically shown in patent 201611022254.9).By the transformant on TAgP solid mediums when cultivating 3 days or so the photoperiod,
Correspond numbering to be coated on the TAgP solid mediums of 0.4mM caddies, cultivated under the photoperiod, observe the state of transformant,
If transformant on 0.4mM caddy culture mediums can not normal growth and wild type can normal growth, it was initially believed that
It is that the mutant is the strain of caddy sensitive mutant algae.
(2) repeat screening several times, the wild type algae strain of intimate equivalent is inoculated with the TAgP culture mediums of 0.4mM caddies
The strain of the mutation algae of 21gr and the caddy responsive type tentatively assert, the growth conditions of two kinds of algae strains of paired observation, if in wild type
Can normal growth all the time, and mutant is unable to normal growth and either grows be suppressed or directly dead, then can determine
The mutant is the strain of caddy sensitive mutant algae.As shown in Fig. 2, the mutant (KY1) screened is caddy responsive type
Mutant, compared with wild type chlamydomonas 21gr, 21gr can on the TAgP culture mediums of 0.4mM caddies normal growth, and KY1
Mutant is not grown even progressively in death substantially, further illustrates that KY1 mutant has significant caddy sensitiveness.
3 sensitive mutant algae of the embodiment strain insertion mutation gene of KY1 and the acquisition in mutational site
(1) by mutation algae strain KY1 pickings in good condition on TAP solid mediums to blowing equipped with TAP fluid nutrient mediums
In gas cylinder, ventilation culture to cell concentration is 4 × 106During/mL, cell is collected, liquid nitrogen flash freezer, -80 DEG C of placements are spare.
(2) genomic DNA for the KY1 algaes strain collected with the extraction of CTAB methods.
(3) special primer F-Z2 and F-Z8 are designed according to the nucleotide sequence of Insert Fragment aph VIII, it is special according to chlamydomonas genome
Point designs primer Q0 and is drawn by the Q0 degeneracys formed with AluI, PstI, SacII, TaqI restriction enzyme specific recognition sequence
Thing DegAluI, DegPstI, DegSacII, DegTaqI.Template is done with the genomic DNA of the KY1 algaes strain of extraction, is carried out
RESDA-PCR, obtains four kinds of PCR products, as shown in figure 3, M is DL2000Marker, A, P, S, T represent respectively comprising AluI,
The amplified production of PstI, SacII, TaqI restriction enzyme.The blob of viscose of more than 500bp bands is cut down and send sequencing.
The primer sequence is as shown in the table：
(4) sequencing result sequence is compared with VIII fragment sequences of aph, obtains the partial order for the gene for being inserted into mutation
Row.This partial sequence is being subjected to sequence alignment on Phytozome websites in Chlamydomonas reinhardtii database, you can it is prominent to obtain insertion
Become the information such as nucleotide sequence, coding protein sequence, VIII fragment insertion sites of aph and the direction of insertion of gene.
(5) qualification result of the insertion mutation gene of cadmium sensitive mutants KY1 is shown, chlamydomonas mutant (KY1) is
Due to the insertion mutation body that VIII fragment forward directions of aph are inserted into the 5th extron position of the gene g3280.t2 and are formed, figure
4 structure diagrams for g3280.t2 genes and specific insertion mutation site.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>Jianghan University
<120>Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 894
<212> DNA
<213> Chlamydomonas reinhardti
<400> 1
atgctgcttt taggactcgt tctggccctc gctggccatg tagcggctgc gccttcaagc 60
gccatgatgg gcactggcca caccgtgggc tttggcgagc tcaaggaaga atggcgtggt 120
gaggttgtcc acctgtcgtg gtcgccgcga gccttcctgc tgaagaattt cctttcggat 180
gaggagtgcg actacatcgt cgagaaggct cgcccgaaga tggtcaagtc ttctgtggtt 240
gataacgagt cgggcaagag tgtggacagc gagatccgca ccagcaccgg cacctggttt 300
gctaagggcg aggactctgt catctccaaa atagagaagc gcgtggcgca ggtgacgatg 360
attcccctgg agaaccatga ggggctgcag gttctgcact accatgacgg tcagaagtat 420
gagccacact atgactactt ccacgacccc gtcaacgccg gccccgagca cggcggccag 480
cgcgtggtca cgatgctcat gtacctgacc acggtggagg agggcggcga gacggtgctg 540
cccaacgcgg agcagaaggt gaccggcgac ggctggtcgg agtgcgccaa gcgcggactg 600
gccgtgaagc ccatcaaggg cgatgcgctc atgttctact cgctgaagcc ggacggcagc 660
aatgaccccg ccagcctgca cggcagctgc cccacgctta agggtgacaa gtggagcgcc 720
accaagtgga tccacgtggc ccccatcggt ggaaagaaga agctaaacct gggtacaccc 780
gagtgccacg acgaggatga gcggtgccag gagtgggcat tctttggtga atgcgagaag 840
aaccctggct tcatggatgc ccagtgcaag cgctcgtgca agaagtgcac ctaa 894
<210> 2
<211> 297
<212> PRT
<213> Chlamydomonas reinhardti
<400> 2
Met Leu Leu Leu Gly Leu Val Leu Ala Leu Ala Gly His Val Ala Ala
1 5 10 15
Ala Pro Ser Ser Ala Met Met Gly Thr Gly His Thr Val Gly Phe Gly
20 25 30
Glu Leu Lys Glu Glu Trp Arg Gly Glu Val Val His Leu Ser Trp Ser
35 40 45
Pro Arg Ala Phe Leu Leu Lys Asn Phe Leu Ser Asp Glu Glu Cys Asp
50 55 60
Tyr Ile Val Glu Lys Ala Arg Pro Lys Met Val Lys Ser Ser Val Val
65 70 75 80
Asp Asn Glu Ser Gly Lys Ser Val Asp Ser Glu Ile Arg Thr Ser Thr
85 90 95
Gly Thr Trp Phe Ala Lys Gly Glu Asp Ser Val Ile Ser Lys Ile Glu
100 105 110
Lys Arg Val Ala Gln Val Thr Met Ile Pro Leu Glu Asn His Glu Gly
115 120 125
Leu Gln Val Leu His Tyr His Asp Gly Gln Lys Tyr Glu Pro His Tyr
130 135 140
Asp Tyr Phe His Asp Pro Val Asn Ala Gly Pro Glu His Gly Gly Gln
145 150 155 160
Arg Val Val Thr Met Leu Met Tyr Leu Thr Thr Val Glu Glu Gly Gly
165 170 175
Glu Thr Val Leu Pro Asn Ala Glu Gln Lys Val Thr Gly Asp Gly Trp
180 185 190
Ser Glu Cys Ala Lys Arg Gly Leu Ala Val Lys Pro Ile Lys Gly Asp
195 200 205
Ala Leu Met Phe Tyr Ser Leu Lys Pro Asp Gly Ser Asn Asp Pro Ala
210 215 220
Ser Leu His Gly Ser Cys Pro Thr Leu Lys Gly Asp Lys Trp Ser Ala
225 230 235 240
Thr Lys Trp Ile His Val Ala Pro Ile Gly Gly Lys Lys Lys Leu Asn
245 250 255
Leu Gly Thr Pro Glu Cys His Asp Glu Asp Glu Arg Cys Gln Glu Trp
260 265 270
Ala Phe Phe Gly Glu Cys Glu Lys Asn Pro Gly Phe Met Asp Ala Gln
275 280 285
Cys Lys Arg Ser Cys Lys Lys Cys Thr
290 295
<210> 3
<211> 2118
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aattcgatct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 60
aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 120
atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 180
tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt 240
ggcggccgct ctagaactag tggatccact atagggcgaa ttggagctcc accgcggtgg 300
cggccgctct agagacggcg gggagctcgc tgaggcttga catgattggt gcgtatgttt 360
gtatgaagct acaggactga tttggcgggc tatgagggcg cgggaagctc tggaagggcc 420
gcgatggggc gcgcggcgtc cagaaggcgc catacggccc gctggcggca cccatccggt 480
ataaaagccc gcgaccccga acggtgacct ccactttcag cgacaaacga gcacttatac 540
atacgcgact attctgccgc tatacataac cactcagcta gcttaagatc ccatcaagct 600
tgcatgccgg gcgcgccaga aggagcgcag ccaaaccagg atgatgtttg atggggtatt 660
tgagcacttg caacccttat ccggaagccc cctggcccac aaaggctagg cgccaatgca 720
agcagttcgc atgcagcccc tggagcggtg ccctcctgat aaaccggcca gggggcctat 780
gttctttact tttttacaag agaagtcact caacatctta aaatggccag gtgagtcgac 840
gagcaagccc ggcggatcag gcagcgtgct tgcagatttg acttgcaacg cccgcattgt 900
gtcgacgaag gcttttggct cctctgtcgc tgtctcaagc agcatctaac cctgcgtcgc 960
cgtttccatt tgcaggatgg ccactccgcc ctccccggtg ctgaagaatt tcgaagcatg 1020
gacgatgcgt tgcgtgcact gcggggtcgg tatcccggtt gtgagtgggt tgttgtggag 1080
gatggggcct cgggggctgg tgtttatcgg cttcggggtg gtgggcggga gttgtttgtc 1140
aaggtggcag ctctgggggc cggggtgggc ttgttgggtg aggctgagcg gctggtgtgg 1200
ttggcggagg tggggattcc cgtacctcgt gttgtggagg gtggtgggga cgagagggtc 1260
gcctggttgg tcaccgaagc ggttccgggg cgtccggcca gtgcgcggtg gccgcgggag 1320
cagcggctgg acgtggcggt ggcgctcgcg gggctcgctc gttcgctgca cgcgctggac 1380
tgggagcggt gtccgttcga tcgcagtctc gcggtgacgg tgccgcaggc ggcccgtgct 1440
gtcgctgaag ggagcgtcga cttggaggat ctggacgagg agcggaaggg gtggtcgggg 1500
gagcggcttc tcgccgagct ggagcggact cggcctgcgg acgaggatct ggcggtttgc 1560
cacggtcacc tgtgcccgga caacgtgctg ctcgaccctc gtacctgcga ggtgaccggg 1620
ctgatcgacg tggggcgggt cggccgtgcg gaccggcact ccgatctcgc gctggtgctg 1680
cgcgagctgg cccacgagga ggacccgtgg ttcgggccgg agtgttccgc ggcgttcctg 1740
cgggagtacg ggcgcgggtg ggatggggcg gtatcggagg aaaagctggc gttttaccgg 1800
ctgttggacg agttcttctg agggacctga tggtgttggt ggctgggtag ggttgcgtcg 1860
cgtgggtgac agcacagtgt ggacgttggg atccccgctc cgtgtaaatg gaggcgctcg 1920
ttgatctgag ccttgccccc tgacgaacgg cggtggatgg aagatactgc tctcaagtgc 1980
tgaagcggta gcttagctcc ccgtttcgtg ctgatcagtc tttttcaaca cgtaaaaagc 2040
ggaggagttt tgcaattttg ttggttgtaa cgatcctccg ttgattttgg cctctttctc 2100
catgggcggg ctggaatt 2118
Claims (9)
1. Chlamydomonas reinhardtiig3280.t2Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, it is characterised in that：It is describedg3280.t2The nucleotides sequence of gene is classified as one kind in following sequences：
（1）Include such as SEQ ID NO:Nucleotide sequence shown in 1；
（2）Include coding SEQ ID NO:The nucleotide sequence of protein shown in 2；
（3）Comprising with SEQ ID NO:Nucleotide sequence shown in 1 is with more than 90% homology and coding identical function protein
Nucleotide sequence.
2. application according to claim 1, it is characterised in that：It is describedg3280.t2The encoding proteins of gene are following albumen
In one kind：
（1）Include such as SEQ ID NO:Amino acid sequence shown in 2；
（2）Comprising by SEQ ID NO:Substitution of the amino acid sequence shown in 2 through several amino acid, missing or increase etc. change
And what is derived has and SEQ ID NO:The identical active derived protein of albumen shown in 2.
3. Chlamydomonas reinhardtiig3280.t2Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is reduced, it is characterised in that：Pass through reduction
Chlamydomonas reinhardtiig3280.t2The expression of gene, makeg3280.t2Gene inactivates or missingg3280.t2Gene is so as to reduce Rhein clothing
Tolerance of the algae to cadmium.
A kind of 4. method for reducing Chlamydomonas reinhardtii cadmium tolerance, it is characterised in that：By reducing Chlamydomonas reinhardtiig3280.t2Gene
Expression, makeg3280.t2Gene inactivates or missingg3280.t2Gene reduces tolerance of the Chlamydomonas reinhardtii to cadmium.
A kind of 5. Chlamydomonas reinhardtii cadmium responsive type algae strain, it is characterised in that：Cadmium responsive type algae strain be withoutg3280.t2Gene
Activityg3280.t2Gene mutation strain org3280.t2Gene-deleted strain, org3280.t2What gene expression declinedg3280.t2
Gene expression defect strain.
A kind of 6. preparation method of Chlamydomonas reinhardtii cadmium responsive type algae strain, it is characterised in that：For one kind in following methods：
Chlamydomonas reinhardtii is obtained by the method for insertion mutation or rite-directed mutagenesisg3280.t2Gene mutation strain；
Chlamydomonas reinhardtii is obtained by building Crispr carriersg3280.t2Gene-deleted strain；
Chlamydomonas reinhardtii is obtained by building RNAi interference vectorsg3280.t2Gene expression defect strain.
7. application of the Chlamydomonas reinhardtii cadmium responsive type algae strain in the monitoring or detection of cadmium pollution described in claim 5.
8. Chlamydomonas reinhardtiig3280.t2Application of the gene in the monitoring or detection of cadmium pollution.
9. Chlamydomonas reinhardtiig3280.t2Application of the gene in the improvement of cadmium pollution.
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| CN201711231277.5A CN108004252A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| CN201711231277.5A CN108004252A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| CN108004252A true CN108004252A (en) | 2018-05-08 |
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| CN201711231277.5A Withdrawn CN108004252A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii g3280.t2 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113549554A (en) * | 2021-02-26 | 2021-10-26 | 武汉科技大学 | Chlamydomonas reinhardtii cadmium-resistant mutant, immobilized material, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140242676A1 (en) * | 2013-02-01 | 2014-08-28 | Los Alamos National Security, Llc | Artificial leaf-like microphotobioreactor and methods for making the same |
| CN106967749A (en) * | 2016-11-16 | 2017-07-21 | 江汉大学 | A kind of Chlamydomonas reinhardtii heavy metal cadmium adsorbs the screening technique of mutant |
-
2017
- 2017-11-29 CN CN201711231277.5A patent/CN108004252A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140242676A1 (en) * | 2013-02-01 | 2014-08-28 | Los Alamos National Security, Llc | Artificial leaf-like microphotobioreactor and methods for making the same |
| CN106967749A (en) * | 2016-11-16 | 2017-07-21 | 江汉大学 | A kind of Chlamydomonas reinhardtii heavy metal cadmium adsorbs the screening technique of mutant |
Non-Patent Citations (3)
| Title |
|---|
| ANJA BRÄUTIGAM等: "Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii", 《PLANT CELL ENVIRON》 * |
| PHYTOZOME: "g3280.t2", 《PHYTOZOME》 * |
| 魏斌等: "转基因莱茵衣藻对重金属离子的抗性及对镉离子的富集", 《中国海洋湖沼学会藻类学分会第七届会员大会暨第十四次学术讨论会论文摘要集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113549554A (en) * | 2021-02-26 | 2021-10-26 | 武汉科技大学 | Chlamydomonas reinhardtii cadmium-resistant mutant, immobilized material, and preparation method and application thereof |
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Application publication date: 20180508 |