CN107828801A - Application of the Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled - Google Patents
Application of the Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled Download PDFInfo
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- CN107828801A CN107828801A CN201711231270.3A CN201711231270A CN107828801A CN 107828801 A CN107828801 A CN 107828801A CN 201711231270 A CN201711231270 A CN 201711231270A CN 107828801 A CN107828801 A CN 107828801A
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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
- 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
Abstract
The invention discloses Chlamydomonas reinhardtiiRFC1Application 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 the fragments of aph VIII of the resistant gene containing paromomycin（SEQ ID NO:3）The Chlamydomonas reinhardtii mutation algae strain of obvious anti-chlorine cadmium is obtained in radom insertion chlamydomonas, further identification finds that the Cadmium resistance of mutation algae strain is due to that the fragments of aph VIII are inserted intoRFC1Gene（CDS sequences such as SEQ ID NO:Shown in 1）In caused by.By reducing Chlamydomonas reinhardtiiRFC1The expression of gene, makeRFC1Gene inactivates or missingRFC1Gene can improve the cadmium tolerance of Chlamydomonas reinhardtii；The Chlamydomonas reinhardtii algae strain that the cadmium tolerance obtained by genetic engineering means strengthens be can be applied in the monitoring and improvement in cadmium pollution waters, and new direction is provided for the improvement of sewage.
Description
Technical field
The present invention relates to biological technical field and field of environment pollution control, and in particular to Chlamydomonas reinhardtii RFC1 genes are being adjusted
Control the application in Chlamydomonas reinhardtii cadmium tolerance.
Background technology
With the continuous improvement of modern industrialization, the problem of environmental pollution has become globalization, due to some factories
The unreasonable development of unreasonable exploitation and industry result in a series of problem of environmental pollution, wherein polluting, the most serious is water body
And air.The pollution of heavy metal cadmium is exactly a kind of key industry pollution therein, no matter industrial waste gas and waste water, or car tail
All contain substantial amounts of cadmium inside gas etc., with the continuous discharge of industrial wastewater waste gas and vehicle exhaust, and because water body and air
All there is mobility, heavy metal pollution is constantly accumulated in water body, soil, air, last ecological environment is destroyed, people
The survival and development of class are on the hazard.The pollution of heavy metal cadmium is increasingly severe at present, how scientifically to carry out heavy metal
The pollution control and recovery of cadmium have become emphasis and hot issue in scientific research.
At present, the improvement of heavy metal pollution mainly has physical-chemical process and bioanalysis.Comparatively speaking, traditional physics
Chemical method the characteristics of often cost is higher, complex operation, it is more suitable for the heavy metal pollution region for focusing on high concentration.It is and relative
For large watershed, low concentration Heavy Metal Pollution Control for biological rule there are more advantages.It can adsorb and be metabolized weight
The microbe species of metal mainly include some engineered bacterials, fungi and algae etc..Easily made relative to engineered bacterial and fungi
Into for secondary pollution, algae is a kind of ideal material for carrying out heavy metal-polluted water harnessing and recovery.
Chlamydomonas reinhardtii is a kind of single celled eucaryote, can carry out photosynthesis, is otherwise known as " green yeast ".Lay
The work of mattress chlamydomonas gene order-checking has been completed, and is advantageous to the metabolic regulation network of further research heavy metal cadmium.Rhein simultaneously
Chlamydomonas genetic conversion system is more ripe, easily obtains mutant, and reinhardtii cell is easily cultivated, and growth and breeding is fast, is a kind of weight
The research mode biology wanted.Algae strain is mutated by the anti-type of caddy of acquisition in the present invention, further identification has obtained and a huge sum of money
Belong to cadmium regulation and control related gene RFC1 and its encoding proteins, can be applied in the improvement in cadmium pollution waters, while further research
Effect of the RFC1 genes in chlamydomonas heavy metal cadmium metabolic regulation, can be that theoretical base is established in the improvement of bioanalysis heavy metal cadmium
Plinth.
The content of the invention
The primary and foremost purpose of the present invention is in the answering in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled in offer Chlamydomonas reinhardtii RFC1 genes
With.Based on the function of RFC1 gene regulation Chlamydomonas reinhardtii cadmium tolerances, the present invention also aims to provide Chlamydomonas reinhardtii RFC1
Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is improved, there is provided Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain preparation method and
Application of cadmium tolerance enhancing algae strain etc..
The purpose of the present invention is achieved through the following technical solutions：
The present invention in the fragment radom insertion chlamydomonas of aph VIII by the resistant gene containing paromomycin by having obtained obvious anti-chlorine
The Chlamydomonas reinhardtii mutation algae strain of cadmium, further identification find that the Cadmium resistance of mutation algae strain is due to that the fragments of aph VIII are inserted into
Caused by 6th extron of RFC1 genes.Gene of the RFC1 genes in JGI chlamydomonas databases is numbered
Cre12.g521200, its CDS sequence such as SEQ ID NO:Shown in 1.
Based on above-mentioned discovery, it is resistance in regulation and control Chlamydomonas reinhardtii cadmium that an object of the present invention is to provide Chlamydomonas reinhardtii RFC1 genes
Application in by property.
The nucleotides sequence of the RFC1 genes is classified as one kind in following sequences：
(1) such as SEQ ID NO are included:The nucleotide sequence of 3552bp 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 RFC1 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 is to provide above-mentioned Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is improved
Using.Chlamydomonas reinhardtii is improved by reducing the expression of Chlamydomonas reinhardtii RFC1 genes, RFC1 genes is inactivated or lack RFC1 genes
Cadmium tolerance.
The third object of the present invention is to provide improves Chlamydomonas reinhardtii cadmium tolerance using above-mentioned Chlamydomonas reinhardtii RFC1 genes
Method.Methods described is to be carried by reducing the expression of Chlamydomonas reinhardtii RFC1 genes, RFC1 genes is inactivated or lack RFC1 genes
High Chlamydomonas reinhardtii cadmium tolerance.
The fourth object of the present invention is to provide a kind of Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain.The cadmium tolerance strengthens algae
Strain for without RFC1 gene activities RFC1 gene mutations strain or RFC1 gene-deleted strains, or RFC1 gene expressions decline RFC1
Gene expression defect strain.
The fifth object of the present invention is to provide a kind of preparation method of Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain.The acquisition
Method includes：Chlamydomonas reinhardtii RFC1 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 RFC1 gene-deleted strains, Chlamydomonas reinhardtii is obtained by building RNAi interference vectors
RFC1 gene expression defect strains.
The sixth object of the present invention is to provide prison of the above-mentioned Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain in heavy metal cadmium
Survey, the application in detection and improvement.According to the strain of RFC1 gene expression defects, RFC1 gene mutations strain or the RFC1 bases of Chlamydomonas reinhardtii
Because of the characteristic that gene-deleted strain can grow in the environment containing heavy metal cadmium of higher concentration, its can be applied to the monitoring of cadmium pollution,
In detection and improvement, it is particularly applicable in the monitoring, detection and improvement of cadmium in sewage, one kind is provided for bioanalysis sewage disposal
New thinking and new material.Meanwhile mechanism of action of the RFC1 genes in chlamydomonas heavy metal cadmium metabolic regulation network is studied,
Bioanalysis waste water control is may further be to provide fundamental basis.
The beneficial effects of the invention are as follows：
(1) present invention discover that RFC1 genes and its encoding proteins, be a kind of new base related to heavy metal cadmium regulation and control
Cause and albumen, it yet there are no correlative study report.
(2) method of the acquisition heavy metal cadmium anti-type mutation algae strain of the invention used is simple, technical system is ripe, can enter one
Step is improved and is applied in other algae or model plant.
(3) in the present invention RFC1 genes because of the anti-type mutant of heavy metal cadmium and wild type chlamydomonas phase that insertion mutation is formed
Than there is the characteristic of significant preventing from heavy metal cadmium, can be further used for studying RFC1 genes in cadmium absorption and metabolic regulation
Mechanism of action.
(4) algae is the important model biology of research biology ruling by law reason heavy metal water body pollution, is obtained in the present invention
RFC1 gene mutation bodies (TC8) show the characteristic of significant preventing from heavy metal cadmium, for wild type chlamydomonas, Neng Gou
Survived in the environment of cadmium containing higher concentration, future is expected to be applied in the improvement in cadmium pollution waters and the recovery of heavy metal cadmium, is
Waste water control provides new direction and neoformation material.
Brief description of the drawings
Fig. 1 is agarose gel electrophoresis figure after the plasmid enzyme restrictions of pJMG-aph VIII.
Fig. 2 is wild type Chlamydomonas reinhardtii 21gr and caddy resistance chlamydomonas mutant (TC8) in the caddy containing 0.6mM
Growth comparison diagram on solid plate.
Fig. 3 is caddy resistance chlamydomonas mutant (TC8) agarose gel electrophoresis figure after RESDA-PCR.
Fig. 4 is the insertion mutation site schematic diagram of the RFC1 genes of caddy resistance chlamydomonas mutant (TC8).
Embodiment
In order to allow the related personnel for being related to this field to be better understood on, the present invention mainly passes through following implementation case
Example illustrates, and these methods are intended merely to better illustrate the present invention, be not restricted by protection scope of the present invention.Implement below
It is conventional method if without specified otherwise in example.
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, add each component in order, be settled to
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, add each component in order, be settled to
500mL.7.5g Agar (agar) are then added to prepare solid medium.
Embodiment 1：The screening of the anti-type mutation algae pearl of Chlamydomonas reinhardtii heavy metal cadmium
(1) preparation of purpose fragment is inserted
The plasmids of PJMG-aph VIII (Zhangfeng Hu, Yinwen Liang, Wei He, Junmin Pan is extracted first*,
CiliaDisassembly with Two Distinct Phases of Regulation,Cell Reports,2015,10
(11):1803-1810；Contain the fragments of aph VIII, sequence such as SEQ ID NO:Shown in 3), then with EcoR I and cutsmart
Buffer digestions, by digestion products agarose gel electrophoresis, by purpose fragment gel extraction, as shown in figure 1, the bar that arrow is signified
The purpose fragment that band reclaims for needs.Glue reclaim mainly uses Sheng Gong companies glue reclaim kit (article No. B518191-
0100), according to the weight of adhesive tape, the Buffer B2 of 3-6 times of adhesive tape weight is added, are eluted with 20 μ L Elution Buffer
DNA fragmentation, make final concentration in 10ng/ μ L or so.
(2) preparation of chlamydomonas competent cell
Chlamydomonas reinhardtii 21gr is transferred on fresh TAP solid mediums and activated, about 3 days or so, be transferred to TAP liquid
Blow and cultivate in body, continuous illumination makes cell concentration grow to 1 × 107Cell/mL.It is forwarded to the fluid nutrient mediums of TAP containing 100mL
Triangular flask in make initial concentration be 1 × 106Cell/mL, 20h or so is being cultivated under the conditions of continuous illumination on shaking table, is making chlamydomonas
Cell concentration reaches 5 × 106The preparation of competent cell is carried out during cell/mL immediately.
(3) electroporated method obtains chlamydomonas transformant
Preprepared Insert Fragment (aph VIII) is added 250ul concentration 1 × 10 is housed8Cell/mL competence is thin
In the electric shock cup of born of the same parents, 10min, electric shock instrument electric shock (model BTX ECM630, shock parameters are placed on ice：Voltage 800V, resistance
1575 Ω, the μ F of electric capacity 50), put 10min again on ice and allow DNA to be finally transferred to equipped with 10mLTAP+ well into competent cell
In the 50mL centrifuge tubes of 60mM sorbierites (the TAP fluid nutrient mediums of the sorbierite containing 60mM), masking foil encases, 100rpm on shaking table
Slowly overnight reparation is shaken, next day is coated with the flat board of the μ g/mL containing paromomycin 10, and a usual transformant can apply 2-3 flat board, seals
Membrana oralis is sealed, and culture is inverted under the photoperiod (14h illumination/10h is dark).
(4) screening of the anti-type mutation algae strain of heavy metal cadmium
After son length to be transformed is good, the algae strain of monoclonal is chosen on TAP solid mediums, and is numbered.Growth 3-4 days
It is optimal to mutant algae strain vigor, then be transferred on the solid plate of 0.6mM caddies, observe daily and record wild type and dash forward
(screening concentration of 0.6mM caddies is the anti-type mutation of heavy metal cadmium that research team's previous experiments obtain to the upgrowth situation of variant
The optimal screening concentration of body, is specifically shown in patent 201611022254.9).If it is unable to normal growth or death in wild type chlamydomonas
In the case of, the strain of mutant algae can also be normally carried out growing, then tentatively regard as the anti-type mutation algae strain of heavy metal cadmium.Repeat to sieve
Select 3-4 times, it is as a result consistent then to can determine that the mutation algae strain is mutated algae strain for the anti-type of heavy metal cadmium.As shown in Fig. 2 wild type chlamydomonas
21gr grows on the solid plate of 0.6mM caddies to be significantly suppressed, and can not substantially be grown, and what the present invention obtained
Mutant TC8, which then grows, there is no and be affected, and illustrate that TC8 mutation algae strains have the characteristic of significant preventing from heavy metal cadmium,
Belong to the anti-type mutant of heavy metal cadmium.
Embodiment 2：The identification of the anti-type mutation algae plant mutant gene of Chlamydomonas reinhardtii heavy metal cadmium
(1) mutant gene group DNA extraction
a：By the anti-type mutant TC8 of the above-mentioned heavy metal cadmium screened, it is connected to pipette tips in fluid nutrient medium, treats that cell is grown
To logarithmic phase, cell is collected into 1.5mL EP pipes, room temperature 14000rpm centrifugation 1min, abandons supernatant, precipitation liquid nitrogen is cold
Freeze, -80 DEG C save backup.
b：The cell of above-mentioned collection is taken out with liquid nitrogen, adds the CTAB lysates of 700 μ L preheatings, is placed on 65 DEG C of water-bath
It is or so one hour of water-bath in pot, repeatedly reverse therebetween to mix, cell is fully cracked.
c：After cell fully cracks, 700 μ L phenol are added into the EP pipes：Chloroform：Isoamyl alcohol=25:24:1 mixing
Solution, EP pipes are rocked, be sufficiently mixed uniformly, 14000rpm centrifugations 10min.
d：After having centrifuged, supernatant is drawn onto in new EP pipes, adds isometric chloroform：Isoamyl alcohol=24:1 mixing is molten
Liquid, the EP that turns upside down pipes, 14000rpm centrifugations 10min.
e：The supernatant after centrifugation is taken then to add the isopropanol of 3/4ths volumes, gently into another new EP pipe
Ground is overturned several times, and 15-20min is placed in -20 DEG C, until there is floccule appearance.
f：Above-mentioned EP pipes are taken out, 14000rpm centrifugation 10min, supernatant is abandoned, washs precipitation with 70% ethanol, about
3-5 times, each 14000rpm centrifuges 5min.
g：Finally with 50 μ L ddH2O dissolving DNAs.
(2)RESDA-PCR
Special primer F-Z2 and F-Z8 are designed according to Insert Fragment aph VIII nucleotide sequence, according to chlamydomonas genome characteristics
Design primer Q0 and the degenerate primer being made up of Q0 and AluI, PstI, SacII, TaqI restriction enzyme specific recognition sequence
DegAluI、DegPstI、DegSacII、DegTaqI.With the genome of the anti-type mutant algae strain (TC8) of the heavy metal cadmium of extraction
DNA does template, carries out RESDA-PCR, four kinds of PCR primers is obtained, as shown in figure 3, M is DL2000DNA Marker, A, P, S, T
The amplified production for including AluI, PstI, SacII, TaqI restriction enzyme is represented respectively.By the blob of viscose of more than 500bp bands
Cut down and send sequencing.
The primer sequence see the table below shown：
(3) the anti-type mutation algae strain TC8 of analysis heavy metal cadmium insertion mutation gene and insertion mutation site
Sequencing result is compared with the fragment sequences of aph VIII, obtains Insert Fragment aph VIII partial sequence and its flank
Sequence, sequence alignment is carried out in the chlamydomonas database on Phytozome websites, can obtain the nucleotide sequence of insertion mutation gene
And the amino acid sequence of encoding proteins, further analysis can obtain specific insertion mutation site.As shown in figure 4, the present invention is obtained
The anti-type mutant TC8 of caddy obtained is due to that the fragments of aph VIII are inserted into the 6th extron of RFC1 genes and formed prominent
Variant.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by 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 RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3552
<212> DNA
<213> Chlamydomonas reinhardti
<400> 1
atgggcaagt cagagcccgc ggacagcaag tctcccaaga aggaggagaa gcagccagcg 60
aagccagtgg gcaaggacat tcgcagcttc ttcagcaaac cccctggcgg agccagcaag 120
ccggctgccc cagccgcgaa ggctgaggcc ggtggcggtg ccgtcgcgtc ggcggccgtg 180
aaggagccag cggcgacagc agtgaaggcg gagccgaagg ctgcggcagc gccggctgcg 240
gccaagcctg cggcctcggc ctccccgccg gccgccaagc ccgcagcaaa gaagagcagg 300
gtcatcatcg acgaggatga tgacgatgac ctggtggtgg tggagaagaa gaaggcctct 360
actccgaaga agcaggcggg cgccgccgct gcgggcacgg actcgaagaa gcagccgccg 420
ggcagcggcg gcaaaggctc cgggggcaag aaggctgcta gcggcaaggc gcctgccgcc 480
aagaagcggc gtgtggtggc ggatgacggc gacgacgatg acgtggtgga ggtgtcgagt 540
gagggcacgg acggagacga ggagtaccag ggcggcgacg agagcagcga cgacgacagt 600
gacgtggtga tggaggagga ggacgacgac gatgacagcg atgtggtgat ggaggacagc 660
gaggacgagg aggcgggggg caaggccggc aaggccaagg ccaaggccac gccgaaggcc 720
gctgcggcca agcccgcggc tgcggccgcc aagcagccgg ccaagggcaa gggcaaaaag 780
gtcgcgggcc ccgccatgac catcacaccc aagggctcgg agccggagaa gaagaagccg 840
ctggcgggca ccaagcacgc gcgctcgccg gccaaggacg agcagggctc tggtggcaag 900
gctacagggg gcccaaagaa ggcgcctgcc cgagcagcga gcccgaccgc ggccgcccgg 960
cgcaagtccg ctgccaccaa ggccgtcttc tccaccaacc ccggctccgc gtctgccatt 1020
gctgcggtgg acgcagcggt ggcacagctg ccgccggaga ctgaggtgga cttcagcctg 1080
ctgcctgagg gccaggacgg cgccgcctac gccagtgcgc cgccgcccaa ccagggcaac 1140
aaggaggcgc cgcgcggcca ccctgactgc ctgaccggca agacctttgt gatcagcggc 1200
gtgctggaca gcctgggccg cgaggaagcc accgactaca tcaagcgcca cggcggccgc 1260
gtcacggggg cggtgtcggg caagacgagc ttcctggtgg tgggacacca cacgggcagg 1320
agcaagtacc ggaaggcccg cgagcacggc accaaggtca ttgacgagga cggcctgttc 1380
tcactcatcc gcgcctccga gcctttcatc ccggcagaaa acgccgcagc accgcccgca 1440
tccgcacccg ccgctgccgc gcccgcctcg cagcccgcct cacagcctcc gtctcagcaa 1500
ccggccagcc agcctgccgc cgcgcagcgg cccggcgccg gcgcagctgc cggcccctcc 1560
tccgcccggc cagccggcgt cactcggcag cctgccgccg ccggggacgc cggccagtac 1620
cagctgtggg tggacaagta caagccgcgc aacagcgcag agctggtggg caacaacaca 1680
ctggtcgaca acctcaagtc atggctgatg aattgggagc aggttcacct ccgcggcggc 1740
gccgccccca cggccaaggg cggcggcagc aagcctaagg acctgtccaa gaaggcggcg 1800
ctgctgagcg gcccgcccgg catcggcaag accagcgccg cacacatcat ggcgcgggag 1860
gcgggcttcg aggtggtgga aatgaacgcc agcgacacgc gcaacaaggc cggcaagacc 1920
agcgagggca tcgccggcaa gcagtccaac atcatcaagg agatggtcac cagcaccaca 1980
ctgccgcccg ggctgttcgg aggcggctgc ggaggcggct tgggcagcac ggcgccgcgg 2040
cggcagcttc tgatcatgga cgaggtggac ggcatgtcgg gcggcgaccg cggcggcgtc 2100
caggacctga tcgacaccat caagcgcagc aagatcccca tcatctgcat ctgcaacgac 2160
aagtacaacc agaagctcaa gagcctgcgc aaccactgcc tggagctgga gttccgcaag 2220
cccacggtgc tgcagatctc caagcgcatg agcgagatcg cggccaagga gggcctggcc 2280
atcaaccagg ccaccatgga tgcacttgtg acgggcgcgg gcggcgacct gcgcctcatc 2340
ctggggcagc tgcagatggt gcgcctgcgc agtgtggcgg tcagcttcga cgacgtgagg 2400
agcggccgcc tgggctccag caaggacatg gaccgctcgc ccttcgagtg cagccggcag 2460
ctgctggagc cgtccagcgg ccagctgtcg ctgggcgacc ggctggagct ggtgttcgcg 2520
gacagcgacc tggtgccgct gctgctgcag gagaactacg tcaaccacaa gcccgccatc 2580
acaacggacg ccgcctcccg cctgcgcgcg ctggccaagg ccgccgacgc cttctccgcc 2640
ggcgacgtgc tcaacaccag catccgccgc caccagaact ggggcctcat gcccgccatg 2700
gccgtggtgg gctgcgtgct gccctgcgcc tacatgcgcg gcggccgcga agtgtttggg 2760
ctgttcccgg gagagatgaa cttcccgcgc ttctccgcct ggttcggcaa caactccacc 2820
agcggcaagc agcggcggct gctgggcgag ctgtccacca gcatgacggc ctcggggctg 2880
gtggcggcgg ggcgcagcgg cgtgcggttg gagtacgcct cggcgctgag gcacctgctg 2940
gcgcggccgc tccagcagca gcaggaggcg gcggtgccga gggtggtggc ggagatgcac 3000
gagtactgca tagacaagga gcagttcgac tacatcctgg acgtgacctc cttcaagagc 3060
aaggcgccct gggcggcgga tctgttcaag gacgtgccgc ccaaggtcaa ggccgcattc 3120
acacggacgc tcaactccac ggcgccggcg gcgcgctgca atgcgcaggt ggaggaggtg 3180
aagctggggc gcggcaaggg caaggccaag ggcggccgca aggcggctgc ggaggaggag 3240
gacgaggagg agggcggcga gggtggcgag ggcggcgagg aggaggcgaa gcccgctgtc 3300
aagcaggagt cgggcgaggg cggggaggag gaggaagagg acgacgacga cgtggacgca 3360
cgcaccctag cgcggcggct ggccaagggc gggctggagg tgcacctgca ggacgacggc 3420
aaggctggca aagggaaaaa gggcgggacg gcgggcggtg gtggaggccg cgggaaagcg 3480
ggcgccgcgg caaagccggc gggtgggcgc ggcggaggtc ggggcggcgc caaggcggcc 3540
gggaagaagt ga 3552
<210> 2
<211> 1183
<212> PRT
<213> Chlamydomonas reinhardti
<400> 2
Met Gly Lys Ser Glu Pro Ala Asp Ser Lys Ser Pro Lys Lys Glu Glu
1 5 10 15
Lys Gln Pro Ala Lys Pro Val Gly Lys Asp Ile Arg Ser Phe Phe Ser
20 25 30
Lys Pro Pro Gly Gly Ala Ser Lys Pro Ala Ala Pro Ala Ala Lys Ala
35 40 45
Glu Ala Gly Gly Gly Ala Val Ala Ser Ala Ala Val Lys Glu Pro Ala
50 55 60
Ala Thr Ala Val Lys Ala Glu Pro Lys Ala Ala Ala Ala Pro Ala Ala
65 70 75 80
Ala Lys Pro Ala Ala Ser Ala Ser Pro Pro Ala Ala Lys Pro Ala Ala
85 90 95
Lys Lys Ser Arg Val Ile Ile Asp Glu Asp Asp Asp Asp Asp Leu Val
100 105 110
Val Val Glu Lys Lys Lys Ala Ser Thr Pro Lys Lys Gln Ala Gly Ala
115 120 125
Ala Ala Ala Gly Thr Asp Ser Lys Lys Gln Pro Pro Gly Ser Gly Gly
130 135 140
Lys Gly Ser Gly Gly Lys Lys Ala Ala Ser Gly Lys Ala Pro Ala Ala
145 150 155 160
Lys Lys Arg Arg Val Val Ala Asp Asp Gly Asp Asp Asp Asp Val Val
165 170 175
Glu Val Ser Ser Glu Gly Thr Asp Gly Asp Glu Glu Tyr Gln Gly Gly
180 185 190
Asp Glu Ser Ser Asp Asp Asp Ser Asp Val Val Met Glu Glu Glu Asp
195 200 205
Asp Asp Asp Asp Ser Asp Val Val Met Glu Asp Ser Glu Asp Glu Glu
210 215 220
Ala Gly Gly Lys Ala Gly Lys Ala Lys Ala Lys Ala Thr Pro Lys Ala
225 230 235 240
Ala Ala Ala Lys Pro Ala Ala Ala Ala Ala Lys Gln Pro Ala Lys Gly
245 250 255
Lys Gly Lys Lys Val Ala Gly Pro Ala Met Thr Ile Thr Pro Lys Gly
260 265 270
Ser Glu Pro Glu Lys Lys Lys Pro Leu Ala Gly Thr Lys His Ala Arg
275 280 285
Ser Pro Ala Lys Asp Glu Gln Gly Ser Gly Gly Lys Ala Thr Gly Gly
290 295 300
Pro Lys Lys Ala Pro Ala Arg Ala Ala Ser Pro Thr Ala Ala Ala Arg
305 310 315 320
Arg Lys Ser Ala Ala Thr Lys Ala Val Phe Ser Thr Asn Pro Gly Ser
325 330 335
Ala Ser Ala Ile Ala Ala Val Asp Ala Ala Val Ala Gln Leu Pro Pro
340 345 350
Glu Thr Glu Val Asp Phe Ser Leu Leu Pro Glu Gly Gln Asp Gly Ala
355 360 365
Ala Tyr Ala Ser Ala Pro Pro Pro Asn Gln Gly Asn Lys Glu Ala Pro
370 375 380
Arg Gly His Pro Asp Cys Leu Thr Gly Lys Thr Phe Val Ile Ser Gly
385 390 395 400
Val Leu Asp Ser Leu Gly Arg Glu Glu Ala Thr Asp Tyr Ile Lys Arg
405 410 415
His Gly Gly Arg Val Thr Gly Ala Val Ser Gly Lys Thr Ser Phe Leu
420 425 430
Val Val Gly His His Thr Gly Arg Ser Lys Tyr Arg Lys Ala Arg Glu
435 440 445
His Gly Thr Lys Val Ile Asp Glu Asp Gly Leu Phe Ser Leu Ile Arg
450 455 460
Ala Ser Glu Pro Phe Ile Pro Ala Glu Asn Ala Ala Ala Pro Pro Ala
465 470 475 480
Ser Ala Pro Ala Ala Ala Ala Pro Ala Ser Gln Pro Ala Ser Gln Pro
485 490 495
Pro Ser Gln Gln Pro Ala Ser Gln Pro Ala Ala Ala Gln Arg Pro Gly
500 505 510
Ala Gly Ala Ala Ala Gly Pro Ser Ser Ala Arg Pro Ala Gly Val Thr
515 520 525
Arg Gln Pro Ala Ala Ala Gly Asp Ala Gly Gln Tyr Gln Leu Trp Val
530 535 540
Asp Lys Tyr Lys Pro Arg Asn Ser Ala Glu Leu Val Gly Asn Asn Thr
545 550 555 560
Leu Val Asp Asn Leu Lys Ser Trp Leu Met Asn Trp Glu Gln Val His
565 570 575
Leu Arg Gly Gly Ala Ala Pro Thr Ala Lys Gly Gly Gly Ser Lys Pro
580 585 590
Lys Asp Leu Ser Lys Lys Ala Ala Leu Leu Ser Gly Pro Pro Gly Ile
595 600 605
Gly Lys Thr Ser Ala Ala His Ile Met Ala Arg Glu Ala Gly Phe Glu
610 615 620
Val Val Glu Met Asn Ala Ser Asp Thr Arg Asn Lys Ala Gly Lys Thr
625 630 635 640
Ser Glu Gly Ile Ala Gly Lys Gln Ser Asn Ile Ile Lys Glu Met Val
645 650 655
Thr Ser Thr Thr Leu Pro Pro Gly Leu Phe Gly Gly Gly Cys Gly Gly
660 665 670
Gly Leu Gly Ser Thr Ala Pro Arg Arg Gln Leu Leu Ile Met Asp Glu
675 680 685
Val Asp Gly Met Ser Gly Gly Asp Arg Gly Gly Val Gln Asp Leu Ile
690 695 700
Asp Thr Ile Lys Arg Ser Lys Ile Pro Ile Ile Cys Ile Cys Asn Asp
705 710 715 720
Lys Tyr Asn Gln Lys Leu Lys Ser Leu Arg Asn His Cys Leu Glu Leu
725 730 735
Glu Phe Arg Lys Pro Thr Val Leu Gln Ile Ser Lys Arg Met Ser Glu
740 745 750
Ile Ala Ala Lys Glu Gly Leu Ala Ile Asn Gln Ala Thr Met Asp Ala
755 760 765
Leu Val Thr Gly Ala Gly Gly Asp Leu Arg Leu Ile Leu Gly Gln Leu
770 775 780
Gln Met Val Arg Leu Arg Ser Val Ala Val Ser Phe Asp Asp Val Arg
785 790 795 800
Ser Gly Arg Leu Gly Ser Ser Lys Asp Met Asp Arg Ser Pro Phe Glu
805 810 815
Cys Ser Arg Gln Leu Leu Glu Pro Ser Ser Gly Gln Leu Ser Leu Gly
820 825 830
Asp Arg Leu Glu Leu Val Phe Ala Asp Ser Asp Leu Val Pro Leu Leu
835 840 845
Leu Gln Glu Asn Tyr Val Asn His Lys Pro Ala Ile Thr Thr Asp Ala
850 855 860
Ala Ser Arg Leu Arg Ala Leu Ala Lys Ala Ala Asp Ala Phe Ser Ala
865 870 875 880
Gly Asp Val Leu Asn Thr Ser Ile Arg Arg His Gln Asn Trp Gly Leu
885 890 895
Met Pro Ala Met Ala Val Val Gly Cys Val Leu Pro Cys Ala Tyr Met
900 905 910
Arg Gly Gly Arg Glu Val Phe Gly Leu Phe Pro Gly Glu Met Asn Phe
915 920 925
Pro Arg Phe Ser Ala Trp Phe Gly Asn Asn Ser Thr Ser Gly Lys Gln
930 935 940
Arg Arg Leu Leu Gly Glu Leu Ser Thr Ser Met Thr Ala Ser Gly Leu
945 950 955 960
Val Ala Ala Gly Arg Ser Gly Val Arg Leu Glu Tyr Ala Ser Ala Leu
965 970 975
Arg His Leu Leu Ala Arg Pro Leu Gln Gln Gln Gln Glu Ala Ala Val
980 985 990
Pro Arg Val Val Ala Glu Met His Glu Tyr Cys Ile Asp Lys Glu Gln
995 1000 1005
Phe Asp Tyr Ile Leu Asp Val Thr Ser Phe Lys Ser Lys Ala Pro Trp
1010 1015 1020
Ala Ala Asp Leu Phe Lys Asp Val Pro Pro Lys Val Lys Ala Ala Phe
1025 1030 1035 1040
Thr Arg Thr Leu Asn Ser Thr Ala Pro Ala Ala Arg Cys Asn Ala Gln
1045 1050 1055
Val Glu Glu Val Lys Leu Gly Arg Gly Lys Gly Lys Ala Lys Gly Gly
1060 1065 1070
Arg Lys Ala Ala Ala Glu Glu Glu Asp Glu Glu Glu Gly Gly Glu Gly
1075 1080 1085
Gly Glu Gly Gly Glu Glu Glu Ala Lys Pro Ala Val Lys Gln Glu Ser
1090 1095 1100
Gly Glu Gly Gly Glu Glu Glu Glu Glu Asp Asp Asp Asp Val Asp Ala
1105 1110 1115 1120
Arg Thr Leu Ala Arg Arg Leu Ala Lys Gly Gly Leu Glu Val His Leu
1125 1130 1135
Gln Asp Asp Gly Lys Ala Gly Lys Gly Lys Lys Gly Gly Thr Ala Gly
1140 1145 1150
Gly Gly Gly Gly Arg Gly Lys Ala Gly Ala Ala Ala Lys Pro Ala Gly
1155 1160 1165
Gly Arg Gly Gly Gly Arg Gly Gly Ala Lys Ala Ala Gly Lys Lys
1170 1175 1180
<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 (10)
1. Chlamydomonas reinhardtiiRFC1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, it is characterised in that：It is describedRFC1Gene
Nucleotides sequence be 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 describedRFC1The encoding proteins of gene are in following albumen
It is a kind of：
（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 reinhardtiiRFC1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is improved, it is characterised in that：By reducing Rhein
ChlamydomonasRFC1The expression of gene, makeRFC1Gene inactivates or missingRFC1Gene improves Chlamydomonas reinhardtii cadmium tolerance.
A kind of 4. method for improving Chlamydomonas reinhardtii cadmium tolerance, it is characterised in that：By reducing Chlamydomonas reinhardtiiRFC1The table of gene
Reach, makeRFC1Gene inactivates or missingRFC1Gene improves Chlamydomonas reinhardtii cadmium tolerance.
5. a kind of Chlamydomonas reinhardtii cadmium tolerance strengthens algae strain, it is characterised in that：Cadmium tolerance enhancing algae strain be withoutRFC1
Gene activityRFC1Gene mutation strain orRFC1Gene-deleted strain, orRFC1What gene expression declinedRFC1Gene expression defect
Strain.
6. a kind of Chlamydomonas reinhardtii cadmium tolerance strengthens algae strain preparation method, it is characterised in that：For one kind in following methods：
Chlamydomonas reinhardtii is obtained by the method for insertion mutation or rite-directed mutagenesisRFC1Gene mutation strain；
Chlamydomonas reinhardtii is obtained by building Crispr carriersRFC1Gene-deleted strain；
Chlamydomonas reinhardtii is obtained by building RNAi interference vectorsRFC1Gene expression defect strain.
7. application of the Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain in the monitoring of cadmium pollution described in claim 5.
8. application of the Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain in the improvement of cadmium pollution described in claim 5.
9. Chlamydomonas reinhardtiiRFC1Application of the gene in the monitoring or detection of cadmium pollution.
10. Chlamydomonas reinhardtiiRFC1Application of the gene in the improvement of cadmium pollution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711231270.3A CN107828801A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711231270.3A CN107828801A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107828801A true CN107828801A (en) | 2018-03-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711231270.3A Withdrawn CN107828801A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii RFC1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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|---|---|
| CN (1) | CN107828801A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626092A (en) * | 2019-09-24 | 2021-04-09 | 清华大学 | Application of NAT gene as screening marker in genetic transformation of chlamydomonas |
| 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 |
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2017
- 2017-11-29 CN CN201711231270.3A patent/CN107828801A/en not_active Withdrawn
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|---|---|---|---|---|
| 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: "RFC1", 《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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626092A (en) * | 2019-09-24 | 2021-04-09 | 清华大学 | Application of NAT gene as screening marker in genetic transformation of chlamydomonas |
| 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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