CN107904248A - Application of the Chlamydomonas reinhardtii VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled - Google Patents
Application of the Chlamydomonas reinhardtii VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled Download PDFInfo
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- CN107904248A CN107904248A CN201711228405.0A CN201711228405A CN107904248A CN 107904248 A CN107904248 A CN 107904248A CN 201711228405 A CN201711228405 A CN 201711228405A CN 107904248 A CN107904248 A CN 107904248A
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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
-
- 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 reinhardtiiVMPL1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, belongs to biological technical field and field of sewage treatment.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 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 VIII fragments of aph are inserted intoVMPL1Gene（CDS sequences such as SEQ ID NO:Shown in 1）In caused by.By reducing Chlamydomonas reinhardtiiVMPL1The expression of gene, makeVMPL1Gene inactivates or missingVMPL1Gene can improve the cadmium tolerance of Chlamydomonas reinhardtii；The Chlamydomonas reinhardtii algae strain that the cadmium tolerance obtained by genetic engineering means strengthens can be applied in the monitoring and improvement in cadmium pollution waters.The present invention provides new thinking and biomaterial for waste water control.
Description
Technical field
The present invention relates to biological technical field and field of sewage treatment, and in particular to Chlamydomonas reinhardtii VMPL1 genes are regulating and controlling
Application in Chlamydomonas reinhardtii cadmium tolerance.
Background technology
With the rapid development of social economy and industrialized production, problem of environmental pollution is on the rise, particularly heavy metal
Pollution problem.The source of heavy metal pollution has very much, " three wastes " (waste water, waste residue and the exhaust gas) of factory's discharge, vehicle exhaust, agriculture
The various house refuses produced in medicine residual and people's daily routines and Waste ammunition etc. contain heavy metal, " three wastes "
Exceeded violation discharge and house refuse, random abandon etc. of Waste ammunition extremely easy cause poisonous and hazardous heavy metal
Largely accumulated in soil, air and water body.And heavy metal has chronicity and non-biodegradable, it is difficult to dropped by microorganism
Solution, as drinking water and food chain eventually progressively accumulate in human body, can directly endanger for a long time there are in soil and water body
Evil human health.Cadmium pollution is a kind of common heavy metal pollution, mainly with plating, metallurgical, mining, battery and chemical work
The discharge of the waste water and waste residue of industry etc. is run up in water body and soil.Cadmium is not human essential elements, in the blood of normal person
Cadmium concentration is less than 5 μ g/L, is less than 1 μ g/L in urine.Cadmium can constantly accumulate in human body, by forming cadmium with metallothionein
Sulfoprotein is run up in kidney and liver, influences the normal function of urinary system, and cadmium can also influence the calcium in bone cadmium, cause bone
Bone seriously softens, and causes Itai-itai diseases, and in addition cadmium may also interfere with absorption of the human body to divalent metal element, cause iron, copper, zinc etc.
The shortage of element.Early in 1984, cadmium was just included in 12 kinds of hazardous materials with global significance by United Nations Environment Programme
One of.
At present, the improvement to heavy metal pollution mainly has physical-chemical process (chemical precipitation method, active carbon adsorption, high score
Sub- trapping agent method, solvent extraction, ion-exchange, UF membrane etc.) and bioanalysis (microbiological coagulation method, biosorption process, plant
Thing repairing method etc.).Comparatively speaking, often cost is higher, complicated for traditional physical-chemical process, is adapted to centralized processing highly concentrated
The heavy metal pollution region of degree, be easy to cause secondary pollution if the use of large area.And for the weight of large watershed, low concentration
Metallic pollution bioanalysis, which is administered, has more advantages, and not only input cost is low, easy to operate, but also easily avoids and reduce two
The problem of secondary pollution.At present, the focus of attention for the researcher that heavy metal-polluted water harnessing becomes is carried out using microorganism, with dynamic plant
Thing is compared, microorganism is small, surface area is big, breeding is fast, it is adaptable, be easy to culture the features such as, be widely studied and be used for
In heavy metal-polluted water process.It is many can to adsorb and be metabolized the microbe species of heavy metal, mainly including some engineered bacterials, true
Bacterium and algae etc..Algae is one of ideal biomaterial for carrying out heavy metal-polluted water reparation, and what is be reported at present can be used for
The algal kind of heavy metal-polluted water harnessing is very much, such as：Chlamydomonas reinhardtii, chlorella, sargassum, Scenedesmus quadricauda, circular fan algae and spiral shell
Revolve algae etc..Different algae is often also different to the adsorption effect of different heavy metals, many studies have shown that algae is to an a variety of huge sum of moneys
Category has fine adsorption capacity, can be widely applied to the sides such as heavy metal accumulation recycling and the water body transformation in heavy metal pollution waters
Face.
Chlamydomonas reinhardtii is a kind of single celled eucaryote, about 10 μm of diameter, has the one large-scale cup-shaped leaf green in cytoplasm
Body, can carry out photosynthesis, therefore be referred to as " green yeast ".There is two flagellums, Ke Yiyou on the top of reinhardtii cell
It is dynamic, there is eyespot that can be photosensitive in pne cell.Chlamydomonas reinhardtii has the feature of zooblast and plant cell, karyogene at the same time
Also completed for monoploid and gene order-checking work, it is often more important that Chlamydomonas reinhardtii has the genetic conversion system of maturation, dashes forward
Variation is readily available, and cell culture is easy, and the speed of growth is fast, and breeding is fast, it has also become the important model biology of scientific research.
The present invention in VIII fragment radom insertion chlamydomonas of aph, will obtain having caddy obvious anti-using electrotransformation
Property the strain of mutation algae, further identification having obtained regulating and controlling relevant gene VMPL1 and its encoding proteins with heavy metal cadmium tolerance,
Can be applied in the monitoring and improvement in cadmium pollution waters, new thinking and biomaterial provided for waste water control, at the same be also into
One step research chlamydomonas lays the foundation the tolerance mechanism of heavy metal cadmium.
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 VMPL1 genes
With.Based on the function of VMPL1 gene regulation Chlamydomonas reinhardtii cadmium tolerances, the present invention also aims to provide Chlamydomonas reinhardtii
Application of the VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is improved, there is provided the acquisition side of Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain
Application of method and the enhancing algae strain of cadmium tolerance etc..
The purpose of the present invention is achieved through the following technical solutions：
The present invention in the VIII fragment radom insertion chlamydomonas of aph 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 VIII fragments of aph are inserted into
Caused by the 3 ' UTR of VMPL1 (Endosomal R-SNARE protein, VAMP-like family (R.III)) gene,
Gene numbering of the gene in JGI Chlamydomonas reinhardtii gene databases is Cre04.g214800, positioned at No. 4 dye of Chlamydomonas reinhardtii
The 1286309-1289084 regions of colour solid, 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 VMPL1 genes in regulation and control Chlamydomonas reinhardtii cadmium
Application in tolerance.
The nucleotides sequence of the VMPL1 genes is classified as one kind in following sequences：
(1) such as SEQ ID NO are included:The nucleotide sequence of 603bp 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 VMPL1 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 VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is improved
Using.Rhein clothing is improved by reducing the expression of Chlamydomonas reinhardtii VMPL1 genes, VMPL1 genes is inactivated or lack VMPL1 genes
Algae cadmium tolerance.
The third object of the present invention is to provide improves Chlamydomonas reinhardtii cadmium tolerance using above-mentioned Chlamydomonas reinhardtii VMPL1 genes
Method.The method is by reducing the expression of Chlamydomonas reinhardtii VMPL1 genes, VMPL1 genes is inactivated or lack VMPL1 genes
Improve 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 VMPL1 gene activities VMPL1 gene mutations strain or VMPL1 gene-deleted strains, or VMPL1 gene expressions decline
VMPL1 gene expression defect strains.
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 VMPL1 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 VMPL1 gene-deleted strains, Rhein clothing is obtained by building RNAi interference vectors
Algae VMPL1 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 VMPL1 gene expression defects strain of Chlamydomonas reinhardtii, VMPL1 gene mutations strain or
The characteristic that VMPL1 gene-deleted strains can be grown in the environment containing heavy metal cadmium of higher concentration, it can be applied to cadmium pollution
Monitoring, detection and improvement in, be particularly applicable in the monitoring, detection and improvement of cadmium in sewage, be bioanalysis sewage disposal
A kind of new thinking and new material are provided.Meanwhile study machine of the VMPL1 gene regulations chlamydomonas to the tolerance of heavy metal cadmium
System, can be further bioanalysis waste water control based theoretical.
The beneficial effects of the invention are as follows：
(1) present invention discover that VMPL1 genes and its encoding proteins, be a kind of new with heavy metal cadmium resistant metabolic tune
Relevant gene and albumen are controlled, has not yet to see relevant research report.
(2) heavy metal cadmium regulates and controls the method for relevant gene and mutant in the acquisition used in the present invention and Chlamydomonas reinhardtii
Simply, technical system is ripe, further can improve and be applied in other algae or model plant.
(3) algae is the important model biology of research biology ruling by law reason heavy metal water body pollution, is obtained in the present invention
VMPL1 genes have the insertion mutation body (HM25) of insertion about 2k or so fragments (aph VIII) can be in caddy containing higher concentration
Under conditions of normal growth, it can be applied in the monitoring and improvement in cadmium pollution waters, for waste water control provide new thinking with
Biomaterial.
(4) Chlamydomonas reinhardtii VMPL1 gene mutation bodies show the characteristic of obvious anti-chlorine cadmium, are conducive to further grind
Study carefully mechanism of action of the VMPL1 genes in the absorption of chlamydomonas heavy metal cadmium and metabolic regulation, can further be controlled for bioanalysis sewage
Manage based theoretical.
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 the anti-type chlamydomonas mutant HM25 of caddy consolidating in the caddy containing 0.6mM
Growth comparison diagram on body tablet.
Fig. 3 is anti-type chlamydomonas mutant (HM25) agarose gel electrophoresis figure after RESDA-PCR of caddy.
Fig. 4 is the VMPL1 gene insertion mutations site schematic diagram of the anti-type chlamydomonas mutant (HM25) of caddy.
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 explanation.The method not clearly stated in following embodiments 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.
Embodiment 1
First, Chlamydomonas reinhardtii transformant is obtained by electrotransformation
(1) VIII fragments of aph are prepared
By containing VIII fragments of aph VIII plasmids of PJMG-aph (Zhangfeng Hu, Yinwen Liang, Wei He,
Junmin Pan*,Cilia Disassembly with Two Distinct Phases of Regulation,Cell
Reports,2015,10(11):1803-1810) stayed overnight with EcoR I enzyme, 37 DEG C of digestions, 1% agarose gel electrophoresis separation aph
VIII fragment (2118bp, sequence such as SEQ ID NO:3), the results are shown in Figure 1 for digestion rear electrophoresis, and wherein M is DL2000DNA
Marker, 1 for the bands of VIII plasmid EcoR of PJMG-aph, I enzyme digestion products, 2k or so is VIII fragments of aph.With Sheng Gong companies
Gel reclaims kit (article No. B518191-0100) carries out VIII fragments of glue reclaim aph, and Q5000 ultramicron detection of nucleic acids instrument is surveyed
Surely the concentration of DNA fragmentation is recycled, -20 DEG C save backup.
(2) reinhardtii cell for being used for electricity conversion is prepared
Chlamydomonas reinhardtii 21gr is activated on TAP solid plates, is then forwarded in TAP fluid nutrient mediums and is blown
Culture, is placed under the conditions of continuous illumination culture of blowing and can reach 1 × 10 in 3-4 days7Cell/mL.The above-mentioned reinhardtii cells of 10mL are taken to turn
Being connected in the triangular flask of the fluid nutrient mediums of TAP containing 100mL makes initial concentration be 1 × 106Cell/mL, is placed on shaking table
(200rpm), 20h or so is cultivated under the conditions of continuous illumination, reinhardtii cell concentration is reached 5 × 106Cell/mL.
(3) electroporated reinhardtii cell
With 50mL centrifuge tubes collect reinhardtii cell, add precooling TAP+60mM sorbitol solutions (sorbierite containing 60mM
TAP fluid nutrient mediums) it is resuspended, 2500rpm centrifugation 3min, adding the TAP+60mM sorbitol solutions resuspension of suitable precooling makes
Final concentration of 1-2 × 10 of reinhardtii cell8Cell/mL, places 10min on ice.
In superclean bench wash-in electric shock cup, first washed once with absolute ethyl alcohol, then three are washed with TAP+60mM sorbitol solutions
It is secondary, subsequent precooling electric shock cup.Precooled 1-2 × 10 of 250 μ L are added into electric shock cup8The reinhardtii cell of cell/mL, adds
VIII segment DNA of 150ng aph, gently inhales and plays mixing, in placing 10min on ice.
The parameter for adjusting electric shock instrument (model BTX ECM630) is voltage 800V, 1575 Ω of resistance, 50 μ F of capacitance.Dry electricity
The moisture of glass shell is hit, the electric shock clamping connection of both sides and electric shock instrument of the electric shock cup with metal strip is touched and is clamped electric shock cup, puts down
Safety guard, starts electric shock (the electric shock time should be controlled in 10-14ms).After electric shock, electric shock cup is placed into 10min on ice.
The reinhardtii cell of electric shock is transferred in the 50mL centrifuge tubes containing 10mL TAP+60mM sorbitol solutions, tinfoil paper
Paper bag is lived, and is placed in 100rpm on shaking table and is shaken overnight.
2500rpm centrifugations 3min collects the reinhardtii cell repaired overnight, adds cornstarch (the starch nothing of 1mL 20%
Water-ethanol is washed 1 time, then is washed 3 times with sterile distilled water, is finally washed 4 times with TAP+60mM sorbitol solutions, and with TAP+60mM mountains
Pears alcoholic solution is resuspended.), mixing is played in suction, is laid on the TAP solid plates containing paromomycin (10 μ g/mL), in ultra-clean work
Liquid is dried up in platform, is sealed, (14h illumination/10h is dark) culture is inverted under the photoperiod, conversion can be grown within about 7 days or so
Son.
2nd, screen chlamydomonas transformant and obtain the anti-type mutant of caddy
Chlamydomonas transformant is chosen on TAgP solid plates and being numbered with the toothpick of sterilizing, grows 3 under the photoperiod
After it, then transformant chosen to being screened on the TAgP solid plates containing 0.6mM caddies, wild type 21gr as pair
According to.The screening concentration of 0.6mM caddies is obtained by testing team's early-stage study, referring to patent 201611022254.9.
The chlamydomonas transformant of acquisition is screened 3-4 days on the TAgP solid plates of the caddy containing 0.6mM, observes transformant
Growing state.When if wild type chlamydomonas 21gr is dead mutant can normal growth, illustrate that mutant has certain anti-chlorine
The ability of cadmium, corresponding transformant is chosen from TAgP solid plates to carrying out on the TAgP solid plates of the caddy containing 0.6mM
Secondary screening.So repeatedly after secondary screening 3-5 time, mutant remain unchanged can normal growth and 21gr is unable to normal growth or death, then
It is the anti-type mutant of caddy to illustrate the mutant.Fig. 2 is by wild type Chlamydomonas reinhardtii 21gr and the screening anti-type chlamydomonas of caddy
Growth comparison diagrams of the mutant HM25 on the TAgP solid plates of the caddy containing 0.6mM.
3rd, the identification of the mutator of the anti-type mutant of caddy
(1) genomic DNA of the anti-type mutant of caddy is extracted
The anti-type mutant of the caddy of above-mentioned acquisition is inoculated into TAP fluid nutrient mediums from TAgP solid plates to be blown
Air culture is supported, and is cultivated 4 days or so under the photoperiod and be can reach growth logarithmic phase.Reinhardtii cell is collected, is extracted and is mutated using CTAB methods
The genomic DNA of body chlamydomonas, 1% agarose gel electrophoresis detection DNA extraction quality, Q5000 ultramicron nucleic acid-protein analyzers
The extracted DNA concentration of measure, -20 DEG C store for future use.
(2) mutant gene group DNA carries out RESDA-PCR (Restriction enzyme site-direced
Amplification PCR) expand acquisition Insert Fragment and its flanking sequence information
Special primer F-Z2 and F-Z8 are designed according to the nucleotide sequence of Insert Fragment aph VIII, according to chlamydomonas genome characteristics
Design primer Q0 and the degenerate primer being made of Q0 and AluI, PstI, SacII, TaqI restriction enzyme specific recognition sequence
DegAluI、DegPstI、DegSacII、DegTaqI.Primer sequence see the table below：
| Primer | 5′—3′ |
| F-Z2 | TTTTACCGGCTGTTGGAC |
| F-Z8 | AGTTCTTCTGAGGGACCTG |
| Q0 | CCAGTGAGCAGAGTGACG |
| DegAluI | CCAGTGAGCAGAGTGACGIIIIINNSWCAGCTT |
| DegPstI | CCAGTGAGCAGAGTGACGIIIIINNSCTGCAGW |
| DegSacII | CCAGTGAGCAGAGTGACGIIIIINNSCCGCGGW |
| DegTaqI | CCAGTGAGCAGAGTGACGIIIIINNSWGTCGAA |
RESDA-PCR points expand for two-wheeled.The first round expands：Using mutant gene group DNA as template, with forward primer F-
Z2 and reverse primer DegAluI, DegPstI, DegSacII, DegTaqI carry out PCR amplification respectively.Second wheel amplification：By first
Wheel PCR product is used as template after diluting 25 times, and the second wheel PCR amplification is carried out with forward primer F-Z8 and reverse primer Q0.Second
After taking turns PCR, the second wheel PCR product is subjected to electrophoresis on 1% Ago-Gel, more than 500bp bands is cut and sends to sequencing.
Fig. 3 is electrophoretograms of the anti-type mutant HM25 of caddy after RESDA-PCR, wherein A, P, S, T represent respectively containing AluI,
The degenerate primer amplified production of PstI, SacII, TaqI restriction endonuclease recognition sequence, M are DL2000 DNA Marker.
(3) analysis and identification in the anti-type mutant mutant gene sequence of caddy and insertion mutation site
Sequencing result is compared with VIII fragment sequences of aph, obtains being inserted into the partial sequence of the gene of mutation,
Sequence alignment is carried out in chlamydomonas database on Phytozome websites, the nucleotide sequence and its coding egg of mutator can be obtained
Casamino acid sequence, further analysis can obtain specific insertion point.As shown in figure 4, the anti-type of caddy that the present invention is obtained
Mutant HM25 is due to that VIII fragment forward directions of aph are inserted into the mutant formed in 3 ' UTR of VMPL1 genes.
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 VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 603
<212> DNA
<213> Chlamydomonas reinhardti
<400> 1
atgccgacac gcaagccgca ggagccgcgc aaggcgatac cggcgcccca gccggtggcg 60
ccaccggaga ccccacgcgt tcacaagaag gctggcaaga gaatagtcct cgccgttctc 120
aagttgcgtc gacaccttca gcacaagttc aagaagctcg tcacagaggg caggcccaca 180
gtgcctgccc ctgccccggc cataccgccc cttcccccac ccgaggccct tgaagacagg 240
atttcccact tgacgttgaa ggtggaggag gtcacggcgg cggcgcgcga gctgaccgcc 300
caggtgctgg cgcgcggcga gaacctggag gtcctgtgcg agaaggcgga gcagctgctg 360
caggcgtccc aggactttgg aaagcgctgc aagcggctca aggggatgcc ctggtgggcc 420
cgcctggcga cggctggcgc cgtcaccgtt gcggcggtcg tgtgcgcggt gtggggcgtc 480
cgccgcggcg cgccgccgtt cggccgcgtc tcgccaccag gcggccgcgt cttcttcact 540
cgcagccggc gctacgaccg aatgcacttc tggcctgcgg gccgtctaat ggagaagcag 600
taa 603
<210> 2
<211> 200
<212> PRT
<213> Chlamydomonas reinhardti
<400> 2
Met Pro Thr Arg Lys Pro Gln Glu Pro Arg Lys Ala Ile Pro Ala Pro
1 5 10 15
Gln Pro Val Ala Pro Pro Glu Thr Pro Arg Val His Lys Lys Ala Gly
20 25 30
Lys Arg Ile Val Leu Ala Val Leu Lys Leu Arg Arg His Leu Gln His
35 40 45
Lys Phe Lys Lys Leu Val Thr Glu Gly Arg Pro Thr Val Pro Ala Pro
50 55 60
Ala Pro Ala Ile Pro Pro Leu Pro Pro Pro Glu Ala Leu Glu Asp Arg
65 70 75 80
Ile Ser His Leu Thr Leu Lys Val Glu Glu Val Thr Ala Ala Ala Arg
85 90 95
Glu Leu Thr Ala Gln Val Leu Ala Arg Gly Glu Asn Leu Glu Val Leu
100 105 110
Cys Glu Lys Ala Glu Gln Leu Leu Gln Ala Ser Gln Asp Phe Gly Lys
115 120 125
Arg Cys Lys Arg Leu Lys Gly Met Pro Trp Trp Ala Arg Leu Ala Thr
130 135 140
Ala Gly Ala Val Thr Val Ala Ala Val Val Cys Ala Val Trp Gly Val
145 150 155 160
Arg Arg Gly Ala Pro Pro Phe Gly Arg Val Ser Pro Pro Gly Gly Arg
165 170 175
Val Phe Phe Thr Arg Ser Arg Arg Tyr Asp Arg Met His Phe Trp Pro
180 185 190
Ala Gly Arg Leu Met Glu Lys Gln
195 200
<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 reinhardtiiVMPL1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, it is characterised in that：It is describedVMPL1Base
The nucleotides sequence of cause 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 describedVMPL1The encoding proteins of gene are in following albumen
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 reinhardtiiVMPL1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is improved, it is characterised in that：By reducing Rhein
ChlamydomonasVMPL1The expression of gene, makeVMPL1Gene inactivates or missingVMPL1Gene improves Chlamydomonas reinhardtii cadmium tolerance.
A kind of 4. method for improving Chlamydomonas reinhardtii cadmium tolerance, it is characterised in that：By reducing Chlamydomonas reinhardtiiVMPL1The table of gene
Reach, makeVMPL1Gene inactivates or missingVMPL1Gene improves Chlamydomonas reinhardtii cadmium tolerance.
A kind of 5. Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain, it is characterised in that：Cadmium tolerance enhancing algae strain be withoutVMPL1
Gene activityVMPL1Gene mutation strain orVMPL1Gene-deleted strain, orVMPL1What gene expression declinedVMPL1Gene expression
Defect strain.
A kind of 6. Chlamydomonas reinhardtii cadmium tolerance enhancing 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 mutagenesisVMPL1Gene mutation strain；
Chlamydomonas reinhardtii is obtained by building Crispr carriersVMPL1Gene-deleted strain；
Chlamydomonas reinhardtii is obtained by building RNAi interference vectorsVMPL1Gene 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 reinhardtiiVMPL1Application of the gene in the monitoring or detection of cadmium pollution.
10. Chlamydomonas reinhardtiiVMPL1Application of the gene in the improvement of cadmium pollution.
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|---|---|---|---|
| CN201711228405.0A CN107904248A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711228405.0A CN107904248A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| CN107904248A true CN107904248A (en) | 2018-04-13 |
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|---|---|---|---|
| CN201711228405.0A Withdrawn CN107904248A (en) | 2017-11-29 | 2017-11-29 | Application of the Chlamydomonas reinhardtii VMPL1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled |
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| CN (1) | CN107904248A (en) |
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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| US20140242676A1 (en) * | 2013-02-01 | 2014-08-28 | Los Alamos National Security, Llc | Artificial leaf-like microphotobioreactor and methods for making the same |
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2017
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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 |
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| 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: 20180413 |