CN107760693A - Application of the Chlamydomonas reinhardtii LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled - Google Patents
Application of the Chlamydomonas reinhardtii LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled Download PDFInfo
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Abstract
The invention discloses Chlamydomonas reinhardtiiLRRApplication 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 mutated algae strain by having obtained the Chlamydomonas reinhardtii of obvious anti-chlorine cadmium in the fragment radom insertion chlamydomonas of aph VIII by the resistant gene containing paromomycin, and further identification finds that the Cadmium resistance of mutation algae strain is due to the fragments of aph VIII（SEQ ID NO:3）It is inserted intoLRRGene（CDS sequences such as SEQ ID NO:Shown in 1）In caused by.By reducing Chlamydomonas reinhardtiiLRRThe expression of gene, makeLRRGene inactivates or missingLRRGene 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 cadmium pollution water environment is administered in the Chlamydomonas reinhardtii algae strain that is strengthened using cadmium tolerance, and operation is simple, and cost is low.
Description
Technical field
The present invention relates to biological technical field and field of environment pollution control, and in particular to Chlamydomonas reinhardtii LRR genes are being adjusted
Control the application in Chlamydomonas reinhardtii cadmium tolerance.
Background technology
With the development of heavy industry, the pollution of environment is on the rise, and wherein heavy metal pollution is particularly acute.Heavy metal is phase
5g/cm is more than to density3Metal, heavy metal is in soil, air, is difficult to be degraded in water environment.Improvement side main at present
Method is administered for physics, and chemical harnessing and biological treating, wherein physics, chemical harnessing consumptive material is more expensive, cumbersome.And biological treating
It is that enriching heavy metal is carried out by some microorganisms and algae, then reaches the mesh of recovery heavy metal by reclaiming microorganism and algae
, biological treating consumptive material is less for arrangement, easy to operate, is following sewage heavy metal accumulation and a kind of important means of recovery
One of.
For Heavy Metal Pollution in Water Environment of China administer using algae and numerous advantages be present.Algae adaptability is very first
By force, it is easy to cultivate, the cell membrane of algae inherently plays the role of certain absorption heavy metal, can contain certain heavy metal
Survived in water environment, using the water environment rich in heavy metal as living area, by the metabolism of itself by heavy metal accumulation, by returning
The purpose of recovery heavy metal can be reached by receiving algae, so as to effectively solve the pollution of heavy metal water environment.Secondly, Chlamydomonas reinhardtii
Gene sequencing completed, the method that mutant is obtained using insertion mutation is ripe (see patent 201611022254.9), compares
It is readily obtained the mutation algae strain for having enrichment to each heavy metal species.
The present invention obtains heavy metal cadmium using the method for insertion mutation has the mutation algae strain of obvious resistance, identified
The gene LRR and its encoding proteins related to chlamydomonas cadmium metabolic regulation is obtained.The encoding proteins of LRR gene mutation bodies have one section
Domain rich in leucine (Leucine Rich repeat), rich leucine sequence basic framework are
LxxLxxLxxLxLxGxxLxxxIPxx (L is leucine, and x is uncertain amino acid), its basic structure is by β lamellas and α spirals
Form.Studies have reported that leucine-rich repeat is widely present in the disease-resistant gene of plant, and in relevant heavy metal cadmium generation
The research report of correlation is there is no in terms of thanking to regulation and control.The algae strain for the LRR gene mutations that the present invention obtains, heavy metal cadmium have very
It obvious resistance, can be survived in the environment of higher concentration cadmium, can further study chlamydomonas heavy metal cadmium metabolic regulation mechanism,
Laid the foundation to carry out sewage disposal using algae.
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 LRR genes
With.Based on the function of LRR gene regulation Chlamydomonas reinhardtii cadmium tolerances, the present invention also aims to provide Chlamydomonas reinhardtii LRR bases
Because of the application in Chlamydomonas reinhardtii cadmium tolerance is improved, there is provided Chlamydomonas reinhardtii cadmium tolerance strengthens the preparation method and cadmium of algae strain
Application of 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 LRR
Caused by first extron of gene.Compared by Chlamydomonas reinhardtii database, the numbering of LRR genes is
Cre06.g302700, the 7815997-7822578 regions of No. 6 chromosomes of chlamydomonas are positioned at, its CDS sequence such as SEQ ID NO:1
It is shown.
Chlamydomonas reinhardtii LRR genes are to provide in regulation and control Chlamydomonas reinhardtii based on one of above-mentioned discovery, main object of the present invention
Application in cadmium tolerance.
The nucleotides sequence of the LRR genes is classified as one kind in following sequences：
(1) such as SEQ ID NO are included:The nucleotide sequence of 1674bp 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 LRR 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.
Domain containing one section of rich leucine in the encoding proteins of the LRR genes.
The second object of the present invention is to provide above-mentioned Chlamydomonas reinhardtii LRR genes answering in Chlamydomonas reinhardtii cadmium tolerance is improved
With.The tolerance of Chlamydomonas reinhardtii cadmium is improved by reducing the expression of Chlamydomonas reinhardtii LRR genes, LRR genes is inactivated or lack LRR genes
Property.
The third object of the present invention is to provide the side that Chlamydomonas reinhardtii cadmium tolerance is improved using above-mentioned Chlamydomonas reinhardtii LRR genes
Method.Methods described is to improve Rhein by reducing the expression of Chlamydomonas reinhardtii LRR genes, LRR genes is inactivated or lack LRR genes
Chlamydomonas 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 LRR gene activities LRR gene mutations strain or LRR gene-deleted strains, or LRR gene expressions decline LRR genes
Express 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 LRR 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 LRR gene-deleted strains, Chlamydomonas reinhardtii is obtained by building RNAi interference vectors
LRR 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 LRR gene expression defects, LRR gene mutations strain or the LRR genes of Chlamydomonas reinhardtii
The characteristic that gene-deleted strain can grow in the environment containing heavy metal cadmium of higher concentration, it can be applied to the monitoring of cadmium pollution, inspection
In surveying and administering, it is particularly applicable in the monitoring, detection and improvement of cadmium in sewage, is provided for bioanalysis sewage disposal a kind of
New thinking and new material.Meanwhile the mechanism of the tolerance of LRR gene regulation chlamydomonas heavy metal cadmiums is studied, one can be entered
Walk as bioanalysis waste water control based theoretical.
The beneficial effects of the invention are as follows：
(1) present invention is imported the fragments of aph VIII in chlamydomonas by electroporated method, has one to cadmium by resistance screening
It is simple to determine the strain of mutant algae (lrr) method of resistance, efficiency high.
(2) present invention identifies mutant mutator and insertion mutation site by RESDA-PCR technologies, for studying
Chlamydomonas heavy metal cadmium metabolic mechanism.
(3) present invention is screened by 0.6mM caddies, and is obtained pair by the solid medium secondary screening of caddy containing 0.6mM
The method used time of the resistant mutant algae strain of cadmium is short, and method is feasible, and obtained mutant character is stable.
(4) mutant that the present invention obtains LRR gene defects has certain resistance to 0.6mM caddies, available for water ring
In detection, monitoring and the improvement of cadmium pollution in border.
Brief description of the drawings
Fig. 1 is the plasmids of pJMG-aph VIII agarose gel electrophoresis figure after the digestions of EcoR I.
Fig. 2 is wild type Chlamydomonas reinhardtii 21gr and the anti-type mutant (lrr) of heavy metal cadmium under the conditions of 0.6mM caddies
Grow comparison diagram.
Fig. 3 is agarose gel electrophoresis figure of the anti-type mutant of heavy metal cadmium after RESDA-PCR.
Fig. 4 is the insertion mutation site schematic diagram of LRR genes in the anti-type mutant of heavy metal cadmium.
Embodiment
Following examples are used to further illustrate the present invention, but should not be construed as limiting the invention.If do not refer in particular to
Conventional meanses bright, that technological means used is well known to those skilled in the art in embodiment.
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：Electrotransformation obtains the chlamydomonas transformant of the fragments of radom insertion aph VIII
(1) using build early stage plasmid pJMG-aph VIII (Zhangfeng Hu, Yinwen Liang, Wei He,
JunminPan*,Cilia Disassembly with Two Distinct Phases of Regulation,Cell
Reports,2015,10(11):1803-1810), the fragments of aph VIII containing coding paromomycin resistance in the plasmid, will
The plasmids of pJMG-aph VIII are carried out staying overnight digestion processing (digestions of EcoR I), then the plasmid of digestion is carried out into DNA agarose gel electrophoresis
The fragments of aph VIII (as shown in Figure 1) are separated, glue reclaim obtains the fragments of aph VIII (sequence such as SEQ ID NO:Shown in 3).
(2) wild type chlamydomonas 21gr is forwarded in the blowing cylinder containing TAP fluid nutrient mediums from TAP solid mediums,
Blow and cultivate 3 days under the conditions of continuous illumination, its concentration is reached 1.5 × 107Individual cell/mL.Cultured cell is transferred
Into the triangular flask containing TAP fluid nutrient mediums (100mL culture mediums add in 10mL blowing cylinders the cell cultivated), make its initial
Concentration is 1.5 × 106Individual cell/mL, is placed on shaking table, is cultivated 20 hours under the conditions of continuous illumination, and shaking speed is
200rpm/min。
Reinhardtii cell concentration reaches 6 × 10 after (3) 20 hours6Individual cell/mL, 60mL cells are collected in superclean bench
Into 50mL sterile centrifugation tubes, 2500rpm centrifugations 3min.Supernatant is removed in superclean bench, with the TAP+60mM mountains of precooling
Pears alcohol liquid (the TAP fluid nutrient mediums of the sorbierite containing 60mM) is resuspended, 2500rpm centrifugations 3min.Gone in superclean bench
Supernatant, 1mL TAP+60mM sorbierite liquid is added, places 10min on ice.Now, by it is sterile electric shock cup precooling, by aph VIII
Fragment is ready to.
(4) after 10min, the cell of 250 μ L precoolings is added in sterile electric shock cup in superclean bench, then added
The 100-200ng fragments of aph VIII, are transferred to and place on ice.Adjustment electric shock instrument parameter (voltage 800V, the Ω of resistance 1575, electric capacity 50
μ F), dry electric shock rapidly with paper and cup shell and be put into electric shock instrument, clamp electric shock cup, put down safety guard and shocked by electricity, during click
Between should control in 10-14ms.Electric shock cup is transferred to and places 10min on ice by electric shock after terminating.
(5) cell after electric shock is added to the 50mL sterile centrifugation tubes of the sorbierites of TAP+60mM containing 10mL after 10min
In, sealing, masking foil is wrapped, at room temperature, on shaking table at a slow speed and lucifuge reparation is stayed overnight.Now prepare 20% starch：Weigh 4g
Starch adds absolute ethyl alcohol and washed one time in 50mL centrifuge tubes, 1000rpm centrifugation 2min, removes supernatant, adds RO washings two
Time, 1000rpm, 2min is centrifuged, remove supernatant, added 70% ethanol, stand overnight.
(6) in the second day starch taken out in a certain amount of 70% ethanol, 1000rpm centrifugation 1min, in superclean bench
Supernatant is outwelled, is washed 4 times with TAP+60mM sorbierites, 1000rpm centrifugations 1min.After starch washes, by the overnight clothing of lucifuge reparation
Frustule is centrifuged, 2500rpm, 3min.The starch re-suspension liquid that 1mL adds TAP+60mM sorbierites is added in per solencyte,
And be coated on the TAP solid mediums containing paromomycin (10 μ g/mL), after its drying, sealed with sealed membrane, in the photoperiod
Culture is inverted under the conditions of (14h illumination/10h is dark), transformant is grown within about 7 days or so.
Embodiment 2：Screen transformant and obtain the anti-type mutant algae strain of caddy
(1) chlamydomonas transformant is chosen to TAgP solids with the toothpick of sterilizing from the TAP solid mediums containing paromomycin
On culture medium, the corresponding numbering of each transformant.Then cultivated 3 days under being put to the photoperiod, then chosen to containing
On the TAgP solid mediums of 0.6mM caddies, wild type 21gr conduct corresponding with the numbering on TAgP solid mediums is numbered
Control.The screening concentration of 0.6mM caddies is the optimal screening for the anti-type mutant of heavy metal cadmium that applicant's previous experiments obtain
Concentration, it is specifically shown in patent 201611022254.9.
(2) cultivate 3-4 days under the 0.6mM caddy solid mediums containing transformant were put to the photoperiod, then observe
With growing state of the record transformant on 0.6mM caddy solid mediums, if having transformant in wild type chlamydomonas death
It can be grown above, corresponding transformant is chosen to multiple on 0.6mM caddy solid mediums from TAgP solid mediums
Sieve.Repeatedly after secondary screening 3-5 times, transformant remains unchanged normal growth and 21gr is unable to normal growth, then this transformant resists for caddy
Type mutant, Fig. 2 is is screened LRR gene mutation bodies (lrr) and wild type chlamydomonas on the solid medium of caddy containing 0.6mM
Growing state.
Embodiment 3：Extract the genome of mutant
(1) mutant is chosen from TAgP solid mediums with sterilized toothpick and cultivates 4 into TAP fluid nutrient mediums
My god.
(2) cell is collected, 2500rpm centrifugation 3min, supernatant is abandoned, cell is resuspended with 4mL fluid nutrient mediums, is inhaled with liquid-transfering gun
Take 1mL cells to be transferred in 1.5mL EP pipes, 14000rpm centrifugation 1min, abandon supernatant, cell is put into liquid nitrogen and freezed, -80
DEG C store for future use.
(3) cell of freezing is taken out to the CTAB lysates for adding 650 μ L preheatings, fully piping and druming mixes cell, is placed on
Water-bath 1 hour in 65 DEG C of water-baths, overturning EP pipes every 10min makes it fully crack.
(4) 650 μ L PCI (phenol is added：Chloroform：Isoamyl alcohol=25:24:1), PCI should fully shake up before addition, add
The EP pipes that turned upside down after entering make it fully mix, 14000rpm centrifugations 10min.
(5) the μ L of supernatant about 500 are moved in new 1.5mL EP pipes, adds isometric 500 μ L CI (chloroforms：Isoamyl
Alcohol=24:1), CI should fully shake up before addition, and the EP pipes that turned upside down after addition make it fully mix, 14000rpm centrifugations
10min。
(6) the μ L of supernatant about 400 are moved in new 1.5mL EP pipes, add the isopropanol of 0.7 times of volume (280 μ L),
Turn upside down and mix EP pipes, DNA white precipitates occur after -20 DEG C of placement 15min, 14000rpm centrifugation 10min, remove supernatant.
(7) 70% ethanol is configured with absolute ethyl alcohol, washing DNA precipitations three times, use the ethanol of 1mL 70%, 14000rpm every time
5min is centrifuged, after last time is washed, 70% ethanol of residual is sucked with 20 μ L liquid-transfering gun.(60 DEG C of dry DNA
2min), 40 μ L ddH are added2O dissolving DNAs, 1 μ L RNase are added, 37 DEG C of digestion are overnight.
(8) 2 μ L DNA are drawn and enter row agarose gel electrophoresis, detection mutant gene group DNA extraction situations.
Embodiment 4：Obtain mutant gene sequence and the insertion mutation site of the anti-type Chlamydomonas reinhardtii mutant of caddy
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.Using the genomic DNA of mutant as template, obtained by RESDA-PCR
To four kinds of amplified productions, (shown in Fig. 3, wherein A, P, S, T represent comprising AluI, PstI, SacII, TaqI restriction enzyme respectively
The amplified production of enzyme), more than 500bp sequence is cut by glue by agarose gel electrophoresis and send sequencing, by sequencing result and aph VIII
Fragment is compared by NCBI blast, obtains being inserted into the partial sequence of the gene of mutation, on Phytozome websites
Sequence alignment is carried out in chlamydomonas database, the information such as the nucleotide sequence, encoding proteins and insertion point of mutator can be obtained.
It is inserted into as shown in figure 4, the anti-type mutant LRR of caddy that the present invention is obtained is due to the fragments of aph VIII positive (5 ' -3 ' end)
The mutant formed in first extron of LRR genes.
RESDA-PCR the primer sequences see the table below shown：
| Primer | 5′—3′ |
| F-Z2 | TTTTACCGGCTGTTGGAC |
| F-Z8 | AGTTCTTCTGAGGGACCTG |
| Q0 | CCAGTGAGCAGAGTGACG |
| DegAluI | CCAGTGAGCAGAGTGACGIIIIINNSWCAGCTT |
| DegPstI | CCAGTGAGCAGAGTGACGIIIIINNSCTGCAGW |
| DegSacII | CCAGTGAGCAGAGTGACGIIIIINNSCCGCGGW |
| DegTaqI | CCAGTGAGCAGAGTGACGIIIIINNSWGTCGAA |
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 LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1674
<212> DNA
<213> Chlamydomonas reinhardti
<400> 1
atgtcagttt caacccacgc cgcgggcgac tcaactgccc aaacggaagc aactcgggtg 60
acttcatcga gcagcgattc cgcgagcctg cgccaggccc gcaggcgcac ggtgacgcag 120
gctgctgagc ccgctccgca atccactggc ctcccgcctg ttgcggtcgc tgttgccgcg 180
gtcgcgggta tcggtgtgct cgcggttttg tacaacaagt tcttcgccag tgttggcagc 240
ggcgccaggt ctgcagtgga gaccctgccg caggccgtca gccagggcat gcagcaaaag 300
cggctgcaaa aggacgcgca ggcgcggctg aacgagatgt gtgatcagct gcggtcgcaa 360
cacgccgtgg atctgtcggc caagaacctg ggcgacgagg gcactgccta tgtggtggag 420
gctctcgcct tcaacaccac ctgccgcgcc ctggacctaa gcaagaacgg cattggaggc 480
gggctgggtg cggcggcgct ggtgcaggtg ctgcccagcg ccgtgatcca gacgctggtg 540
ctcaacacca acagcctggg cgacggcggc tcggagcagc tggccaaggc cattgcgggc 600
cacccgcacc tgaccactct ggacctgggc cagaacggca tcggcgacgc cggcgccacc 660
gcgctggccg aggcgctcaa gctcaacacg cggctgcagc ggctggacct gtccggcaat 720
gccatcgaca aggacggcgc ggcggcgctg gcgggcgcgg tggcggggag cagcacgctc 780
gaggtgctgg tgctgacgga caactacctg ggcgaggccg gcgcgctggc gctggcggac 840
gcactgcggg ccaacaccag cgtcaaggag ctgcacctta agggcaacga gatgggtgat 900
gcgggcgtca cggccatctg cgaggcgcta atgtcccgcg cctgcgacat caccttcctg 960
gacctgggca acaacagcat cacggaggcg ggcgccacgc acctgaaccg gctgctgttc 1020
cagaagcgct ccctcaagga gctgcacatg tacatgaacg acatcgggga cgagggcgtg 1080
ggcaagatgg ctcagtccat tgcggacaac aaggggctgt cggtgctgga cctgggcggc 1140
aacaacatcg ggcccaaggg tgtggcggtg ctggcgggcg cactgcgcaa ccacggcgcg 1200
ctgagctccc tagagctggg ctacaacccg ttggggccgg agggcactaa gaccatcacc 1260
gacctcgcaa agttccaact gcccaagctg accacgctca agctgggttg gtgcaaggtg 1320
gcgggcggcg acggcgcccg cgcgctgtct gacctgctgc tgctcaacgc cagcctcacg 1380
cacctggacc tgcgcggcaa cagcctgggc aacgacggag ccatcctgct gtcgcgcggc 1440
ctcaaggccg ccgagaacag caagctcgcg gatctggacc tgggctacaa cgagatcaag 1500
gacgacgggg cgtgcgcgct ggcccaggcg ctaaaagcca accccgaggg cgcccctcgt 1560
gagctgaagc ttaactcgaa ctacattacc cgattcggtc aggtggcgct gagcgaggcc 1620
gtggaccagg tgttcgacat gggcggcggt aagatgacca ccgtgcactt ctag 1674
<210> 2
<211> 557
<212> PRT
<213> Chlamydomonas reinhardti
<400> 2
Met Ser Val Ser Thr His Ala Ala Gly Asp Ser Thr Ala Gln Thr Glu
1 5 10 15
Ala Thr Arg Val Thr Ser Ser Ser Ser Asp Ser Ala Ser Leu Arg Gln
20 25 30
Ala Arg Arg Arg Thr Val Thr Gln Ala Ala Glu Pro Ala Pro Gln Ser
35 40 45
Thr Gly Leu Pro Pro Val Ala Val Ala Val Ala Ala Val Ala Gly Ile
50 55 60
Gly Val Leu Ala Val Leu Tyr Asn Lys Phe Phe Ala Ser Val Gly Ser
65 70 75 80
Gly Ala Arg Ser Ala Val Glu Thr Leu Pro Gln Ala Val Ser Gln Gly
85 90 95
Met Gln Gln Lys Arg Leu Gln Lys Asp Ala Gln Ala Arg Leu Asn Glu
100 105 110
Met Cys Asp Gln Leu Arg Ser Gln His Ala Val Asp Leu Ser Ala Lys
115 120 125
Asn Leu Gly Asp Glu Gly Thr Ala Tyr Val Val Glu Ala Leu Ala Phe
130 135 140
Asn Thr Thr Cys Arg Ala Leu Asp Leu Ser Lys Asn Gly Ile Gly Gly
145 150 155 160
Gly Leu Gly Ala Ala Ala Leu Val Gln Val Leu Pro Ser Ala Val Ile
165 170 175
Gln Thr Leu Val Leu Asn Thr Asn Ser Leu Gly Asp Gly Gly Ser Glu
180 185 190
Gln Leu Ala Lys Ala Ile Ala Gly His Pro His Leu Thr Thr Leu Asp
195 200 205
Leu Gly Gln Asn Gly Ile Gly Asp Ala Gly Ala Thr Ala Leu Ala Glu
210 215 220
Ala Leu Lys Leu Asn Thr Arg Leu Gln Arg Leu Asp Leu Ser Gly Asn
225 230 235 240
Ala Ile Asp Lys Asp Gly Ala Ala Ala Leu Ala Gly Ala Val Ala Gly
245 250 255
Ser Ser Thr Leu Glu Val Leu Val Leu Thr Asp Asn Tyr Leu Gly Glu
260 265 270
Ala Gly Ala Leu Ala Leu Ala Asp Ala Leu Arg Ala Asn Thr Ser Val
275 280 285
Lys Glu Leu His Leu Lys Gly Asn Glu Met Gly Asp Ala Gly Val Thr
290 295 300
Ala Ile Cys Glu Ala Leu Met Ser Arg Ala Cys Asp Ile Thr Phe Leu
305 310 315 320
Asp Leu Gly Asn Asn Ser Ile Thr Glu Ala Gly Ala Thr His Leu Asn
325 330 335
Arg Leu Leu Phe Gln Lys Arg Ser Leu Lys Glu Leu His Met Tyr Met
340 345 350
Asn Asp Ile Gly Asp Glu Gly Val Gly Lys Met Ala Gln Ser Ile Ala
355 360 365
Asp Asn Lys Gly Leu Ser Val Leu Asp Leu Gly Gly Asn Asn Ile Gly
370 375 380
Pro Lys Gly Val Ala Val Leu Ala Gly Ala Leu Arg Asn His Gly Ala
385 390 395 400
Leu Ser Ser Leu Glu Leu Gly Tyr Asn Pro Leu Gly Pro Glu Gly Thr
405 410 415
Lys Thr Ile Thr Asp Leu Ala Lys Phe Gln Leu Pro Lys Leu Thr Thr
420 425 430
Leu Lys Leu Gly Trp Cys Lys Val Ala Gly Gly Asp Gly Ala Arg Ala
435 440 445
Leu Ser Asp Leu Leu Leu Leu Asn Ala Ser Leu Thr His Leu Asp Leu
450 455 460
Arg Gly Asn Ser Leu Gly Asn Asp Gly Ala Ile Leu Leu Ser Arg Gly
465 470 475 480
Leu Lys Ala Ala Glu Asn Ser Lys Leu Ala Asp Leu Asp Leu Gly Tyr
485 490 495
Asn Glu Ile Lys Asp Asp Gly Ala Cys Ala Leu Ala Gln Ala Leu Lys
500 505 510
Ala Asn Pro Glu Gly Ala Pro Arg Glu Leu Lys Leu Asn Ser Asn Tyr
515 520 525
Ile Thr Arg Phe Gly Gln Val Ala Leu Ser Glu Ala Val Asp Gln Val
530 535 540
Phe Asp Met Gly Gly Gly Lys Met Thr Thr Val His Phe
545 550 555
<210> 3
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<212> DNA
<213> Chlamydomonas reinhardti
<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 reinhardtiiLRRApplication of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, it is characterised in that：It is describedLRRGene
Nucleotides sequence 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 describedLRRThe 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 reinhardtiiLRRApplication of the gene in Chlamydomonas reinhardtii cadmium tolerance is improved, it is characterised in that：By reducing Rhein clothing
AlgaeLRRThe expression of gene, makeLRRGene inactivates or missingLRRGene improves Chlamydomonas reinhardtii cadmium tolerance.
A kind of 4. method for improving Chlamydomonas reinhardtii cadmium tolerance, it is characterised in that：By reducing Chlamydomonas reinhardtiiLRRThe table of gene
Reach, makeLRRGene inactivates or missingLRRGene 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 withoutLRRBase
Because of activityLRRGene mutation strain orLRRGene-deleted strain, orLRRWhat gene expression declinedLRRGene expression defect strain.
A kind of 6. preparation method of Chlamydomonas reinhardtii cadmium tolerance enhancing 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 mutagenesisLRRGene mutation strain；
Chlamydomonas reinhardtii is obtained by building Crispr carriersLRRGene-deleted strain；
Chlamydomonas reinhardtii is obtained by building RNAi interference vectorsLRRGene expression defect strain.
7. application of the Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain in the monitoring or detection 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 reinhardtiiLRRApplication of the gene in the monitoring or detection of cadmium pollution.
10. Chlamydomonas reinhardtiiLRRApplication of the gene in the improvement of cadmium pollution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711228404.6A CN107760693A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711228404.6A CN107760693A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| CN107760693A true CN107760693A (en) | 2018-03-06 |
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|---|---|---|---|
| CN201711228404.6A Withdrawn CN107760693A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii LRR genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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Cited By (3)
| 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 |
| CN114671945A (en) * | 2022-01-14 | 2022-06-28 | 长沙学院 | Grass carp bacteria small peptide recognition receptor and preparation method and application thereof |
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| AU4321999A (en) * | 1998-05-28 | 1999-12-13 | Ohio State University Research Foundation, The | Organism and method for metal recovery, remediation and separation |
| US20110177546A1 (en) * | 2008-07-31 | 2011-07-21 | Labbell Inc. | Method of detecting bioactive molecules in a fluid sample |
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2017
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| AU4321999A (en) * | 1998-05-28 | 1999-12-13 | Ohio State University Research Foundation, The | Organism and method for metal recovery, remediation and separation |
| US20110177546A1 (en) * | 2008-07-31 | 2011-07-21 | Labbell Inc. | Method of detecting bioactive molecules in a fluid sample |
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Cited By (3)
| 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 |
| CN114671945A (en) * | 2022-01-14 | 2022-06-28 | 长沙学院 | Grass carp bacteria small peptide recognition receptor and preparation method and application thereof |
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