CN107828799A - Application of the Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled - Google Patents

Abstract

The invention discloses Chlamydomonas reinhardtiiCre01.g046237.t1.1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled.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 intoCre01.g046237.t1.1Caused by gene.By reducing Chlamydomonas reinhardtiiCre01.g046237.t1.1Gene（CDS sequences such as SEQ ID NO:Shown in 1）Expression, the gene is inactivated or is lacked the cadmium tolerance that the gene improves 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 waste water control.

Description

Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes are in regulation and control Chlamydomonas reinhardtii cadmium tolerance In application

Technical field

The present invention relates to biological technical field and field of environment pollution control, and in particular to Chlamydomonas reinhardtii Application of the Cre01.g046237.t1.1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled.

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 regulating and controlling phase with heavy metal cadmium The gene C re01.g046237.t1.1 and its encoding proteins of pass, can be applied in the improvement in cadmium pollution waters, while further Effect of the Cre01.g046237.t1.1 genes in chlamydomonas heavy metal cadmium metabolic regulation is studied, can be bioanalysis heavy metal cadmium Improvement based theoretical.

The content of the invention

The primary and foremost purpose of the present invention is to provide Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes in regulation and control Chlamydomonas reinhardtii Application in cadmium tolerance.Based on the function of Cre01.g046237.t1.1 gene regulation Chlamydomonas reinhardtii cadmium tolerances, the present invention Purpose also reside in application of the Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes in Chlamydomonas reinhardtii cadmium tolerance is improved be provided, The preparation method of Chlamydomonas reinhardtii cadmium tolerance enhancing algae strain and the application of cadmium tolerance enhancing algae strain etc. are provided.

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 (HM24) of cadmium, further identification find that the Cadmium resistance of mutation algae strain is due to that the fragments of aph VIII are inserted Enter caused by into the 9th extron of Cre01.g046237.t1.1 genes.Cre01.g046237.t1.1 genes are located at clothing The 6484612-6500252 positions of No. 1 chromosome of algae, 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 Cre01.g046237.t1.1 genes and adjusted Control the application in Chlamydomonas reinhardtii cadmium tolerance.

The nucleotides sequence of the Cre01.g046237.t1.1 genes is classified as one kind in following sequences：

(1) such as SEQ ID NO are included:The nucleotide sequence of 6795bp 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 Cre01.g046237.t1.1 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 Cre01.g046237.t1.1 genes and is improving Rhein clothing Application in algae cadmium tolerance.By reducing the expression of Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes, making Cre01.g046237.t1.1 genes inactivate or missing Cre01.g046237.t1.1 genes improve Chlamydomonas reinhardtii cadmium tolerance.

The third object of the present invention is to provide improves Rhein using above-mentioned Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes The method of chlamydomonas cadmium tolerance.Methods described is by reducing the expression of Chlamydomonas reinhardtii Cre01.g046237.t1.1 genes, making Cre01.g046237.t1.1 genes inactivate or missing Cre01.g046237.t1.1 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 Cre01.g046237.t1.1 gene activities Cre01.g046237.t1.1 gene mutations strain or Cre01.g046237.t1.1 gene-deleted strains, or Cre01.g046237.t1.1 gene expressions decline Cre01.g046237.t1.1 is because expressing 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 Cre01.g046237.t1.1 gene mutations are obtained by the method for insertion mutation or rite-directed mutagenesis Strain, Chlamydomonas reinhardtii Cre01.g046237.t1.1 gene-deleted strains are obtained by building Crispr carriers, pass through structure RNAi interference vectors and then acquisition Chlamydomonas reinhardtii Cre01.g046237.t1.1 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 Cre01.g046237.t1.1 gene expression defects strain of Chlamydomonas reinhardtii, Cre01.g046237.t1.1 gene mutations strain or Cre01.g046237.t1.1 gene-deleted strains can containing in higher concentration The characteristic grown in the environment of heavy metal cadmium, it can be applied in the monitoring of cadmium pollution, detection and improvement, be particularly applicable to dirt In water in monitoring, detection and the improvement of cadmium, a kind of new biomaterial is provided for bioanalysis sewage disposal.Meanwhile study Mechanism of action of the Cre01.g046237.t1.1 genes in chlamydomonas heavy metal cadmium metabolic regulation network, may further be bioanalysis Waste water control is provided fundamental basis.

The beneficial effects of the invention are as follows：

(1) present invention discover that Cre01.g046237.t1.1 genes and its encoding proteins, be it is a kind of it is newfound with again Cadmium metal regulation and control related gene and albumen, 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 Cre01.g046237.t1.1 genes because of the anti-type mutant of heavy metal cadmium that insertion mutation is formed and Wild type chlamydomonas is compared, and has the characteristic of significant preventing from heavy metal cadmium, can be further used for studying Mechanism of action of the Cre01.g046237.t1.1 genes in cadmium absorption and metabolic regulation.

(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 Cre01.g046237.t1.1 gene mutation bodies (HM24) show the characteristic of significant preventing from heavy metal cadmium, relative to wild type It for chlamydomonas, can be survived in the environment of cadmium containing higher concentration, may be used on improvement and the heavy metal cadmium in cadmium pollution waters In recovery, neoformation material is provided for waste water control.

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 HM24 consolidating in the caddy containing 0.6mM Growth comparison diagram on body flat board.

Fig. 3 is caddy resistance chlamydomonas mutant (HM24) agarose gel electrophoresis figure after RESDA-PCR.

Fig. 4 is the insertion mutation site of the Cre01.g046237.t1.1 genes of caddy resistance chlamydomonas mutant (HM24) Schematic diagram.

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, is not restricted by the use range 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^*, Cilia Disassembly 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, M is BM5000DNA Marker, 1 is digestion products, and the signified band of arrow is the purpose fragment for needing to reclaim.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 are added, With 20 μ L Elution Buffer eluted dna fragments, make final concentration in 10ng/ μ L or so.

(2) preparation of chlamydomonas competent cell

Wild Rhein type chlamydomonas 21gr is transferred on fresh TAP solid mediums and activated, about 3 days or so, switching Blow and cultivate into TAP liquid, continuous illumination makes cell concentration grow to 1 × 10⁷Cell/mL.It is forwarded to the liquid of TAP containing 100mL It is 1 × 10 to make initial concentration in the triangular flask of culture medium⁶Cell/mL, 20h or so is being cultivated under the conditions of continuous illumination on shaking table, Reinhardtii cell concentration is set to reach 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 250 μ L concentration 1 × 10 are 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 10mL TAP+ well into competent cell (masking foil encases, and is placed in for reparation overnight in the 50mL centrifuge tubes of 60mM sorbierites (the TAP fluid nutrient mediums of the sorbierite containing 60mM) 100rpm shakes overnight slowly on shaking table), next day is coated with the flat board of the μ g/mL containing paromomycin 10, and a usual transformant can apply 2-3 Individual flat board, sealed membrane sealing, (14h illumination/10h is dark) is inverted culture under the photoperiod.

(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 mutant of heavy metal cadmium that applicant's previous experiments obtain to the upgrowth situation of variant Optimal screening concentration, be specifically shown in patent 201611022254.9).If wild type chlamydomonas be unable to normal growth or death 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 screen It is 3-4 times, 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 HM24, which then grows, there is no and be affected, and illustrate that HM24 mutation algae strains have the spy of significant preventing from heavy metal cadmium Property, 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 HM24 of the above-mentioned heavy metal cadmium screened, it is connected to pipette tips in fluid nutrient medium, treats cell Logarithmic phase is grown to, collects cell 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 (HM24) 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 DL2000DNAMarker, 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：

Primer	5′—3′
		F-Z2	TTTTACCGGCTGTTGGAC
F-Z8	AGTTCTTCTGAGGGACCTG
		Q0	CCAGTGAGCAGAGTGACG
DegAluI	CCAGTGAGCAGAGTGACGIIIIINNSWCAGCTT
		DegPstI	CCAGTGAGCAGAGTGACGIIIIINNSCTGCAGW
DegSacII	CCAGTGAGCAGAGTGACGIIIIINNSCCGCGGW
		DegTaqI	CCAGTGAGCAGAGTGACGIIIIINNSWGTCGAA

(3) the anti-type mutation algae strain HM24 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 HM24 of caddy obtained is due to that the fragments of aph VIII are reversely inserted into the 9th of Cre01.g046237.t1.1 genes The mutant formed in individual extron.

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 Cre01.g046237.t1.1 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6795

<212> DNA

<213> Chlamydomonas reinhardti

<400> 1

atgcagctgg cccgagccag gtcccgccaa gccgctgcgt cggctgccag caacgccacg 60

caacagcagt cgcgtgctgc tgcaacgggc ccggcggcgg ccgcggaggg gcacggcaag 120

cgcccggccg gtgccggcgc gggcgtggca gctgcgcggc cagtcgccgc cccagctccg 180

tctgcagccg caggcacggg acccaagcct gtaaccgtaa cagcagacgt cgtacagtca 240

accgccgcca gctcccagcc ggaagcggcg tcgtctgcag tggcgggcgc aaaagcagcg 300

gcagcgggga cggcggcggc ggcggcaggg tcggacgggg gtaagggcgg gggtggcggc 360

gcggtggcag cgacggcgac ggaggttaca ccgcaagcct caacgccggc tcagggctcg 420

gcgccgctgc tgccggagcc tgcgccgccc accggggcgc cggctaccgg gcccgcagcg 480

gcagccgcaa cagctgcgaa cgacgatggc ggcggggcac ggggcgatgt ggtgaatgcg 540

gcggcgccag cggcggatgt gatgccgccg ccgcatcagc cccagcagga gccgcagcag 600

gagcccaagc cggacgcgaa gcaggagccg aagcaggagc cgaagcagca gccggcgagg 660

aaagcgaagg gctccgccgc gtcacccgcg ggcaagggtg cgggcaaggg cgcgggcaag 720

ggcgcaaggg tgggggcggc agccaaggct aagtcagagc ccgaggccga tgcgggtgcg 780

ccagcggcag tagcgcctgc acccgcgccc gcgcccgcgc ccgcacctgc acctgcgcct 840

gcacccgcac ctgagcctgc acccgcgccg gcagccgtgc cgtgtgctgc ggtgctgaag 900

ccgactgccc tcgcggccgc gcccgacggg ccggtggcgg cacccggcag cctggagcag 960

caggagggcg gtgcggcggc ggcagaggcg gcggcggcgg cggtggctac ggcgacgggt 1020

acggcgacgg cgcctgttgc ggcggcaagt ggcggcggcg gctcagacac tgttgcagtt 1080

ggcggcggcg gtggcggcgg tgacggggtg gtggatgtgg ccgcgggctt gcccgagccg 1140

cctccgacgg cggcggtggc acaggcggcg gcggcggcgg cgacacaggc ggcggcaggt 1200

gcggcggtgg atgcaggcgg cggcatcgct gcgcagccgc aggagcagcc gcaggaggcg 1260

ccgccgccgc cgcagcagct gcggcaaccg cagcagccgc agcagccgca gcagccgcag 1320

cagccgcagc agctgcagca gctacaacag ccgcagcagc cgcagcagcc gcagggggcg 1380

cagggggcag ggcggaagcg gcctgtgcct gtgcgggggg agcagcaggc ggggctgggc 1440

gagatggagg tggaggtgga ggtggaggtg gaggtggcgg cagctgaggc cgcgggtgag 1500

gccgcgggcc cacccgccaa gcgccaggcc gtggccgcag ccgccacaca cgcgccgccg 1560

cgtcacccgc agcagcaaca acatcagcag ccgccgccac cggcgccgcc ccatccggag 1620

cagcagcagc agcagcagca gcagccgcag gcgacagcgt cagccgccgc gccagatccc 1680

gcacctgcgc ctgcgcctac ggtttccccg gccacagccg cctctcccgc ctcccctgca 1740

gcagcagccc ccaccgccat cgccgccacc tctcaggcag cggccgccac cacctcccca 1800

cccgctgccg ccgccactgc cgccacctcc ccggccgcct ccccagccgc cgccactgcc 1860

tccccggccg ccactgctcc agccgccact gctccagccg ccactgctcc agccgccact 1920

gctccagccg ccactgctcc agccgccact gctccagccg ccactgctcc agccgccgcc 1980

actgcctccc cggccgccac tgctccagcc gccactgctc cagccgccgc tgctccagcc 2040

gccactgctc cagccgccac tgctccagcc gccactgctc cagccgccac tgctccagcc 2100

gccgctgctc cagccgctgc cgccgccggc tcgctggcag cccaggaggc ggcgctgcgg 2160

cggcacctgg gcgtggtggc ggggcggctg agggagggga ccatgcccgt cagcaggcag 2220

agcaatgagc tgcgcccgct gacggatgct gagcggtcca agttccagga agccgccgcg 2280

aagctggagc gccagatcca cgccctgcag cagcagcagc aggcacaaca gcaggcacag 2340

caacctaagc aggcgcagca gacgcagcag gcgcagcagg cggcggtggc gcagcctcag 2400

gcagggccgc gcactgtcag cccggcggcc gcggcgggcg gtggggcggc gggcgcagct 2460

acagcgcccg ccggtgctgc tgctactgcc gctgctgcta ctgctgccgc tgcaggcgcc 2520

accggcctgg ctgcagtgat tgctgcccgc ggggcggtgg tatggcaggg tccgggtagg 2580

cccgccatga ctacggcccc agccgccaat ggcggtggcg gcgccgccgc acgggcagtc 2640

tccggggcgg cggcggcggc agcgacggcg gcggcggccc cgagcggcgc cggaggtggc 2700

ggctctgcgg cgccggcatt gcccgtggcg ataccggcca gcggcctgct gcgcgcggcg 2760

gcacaggcag cggtgataca tgcagcggcg caggcggcgg cgcggcaggc ggcggcggcg 2820

gcggcggcag gggcgggcgg tgcagtgggc gtgggcgcgc ccggtcgtgg cgcgggcgcc 2880

gtggccgacg cggcgggtgg cggcaaaggc ccggagcacc tggcggcgct gcgggcggtg 2940

atcgagagcc tgtccgccac aggcatggag gagctggagc cgccgccgaa gacgctgcag 3000

gtgactgtgc tgcgccacca gcgcatggcc ctggcctgga tgctgcggcg ggagacgggg 3060

ccggagccgc ggggcggcat actggcagac gaccagggcc tgggcaagac cgtcaccacc 3120

atctccctca tcgtcacgca caccaacccc gaggacagcg caccgggcgc agccgaagcg 3180

gcagcagcag cagcagcagc agccgcggcc gagggaggcg gtgccgaagg cgctgccgcc 3240

aatggcgccg gcgcaacggc agcagctgca gctgcagcgg caggggcccc aggggttgcg 3300

gcgaccagcg gcggtggggc cgggcacggg cacttgggcg ggccgggcag ctcccaatct 3360

ccgccgcggc agcagcagca gcggcgccgc ggggggcgcg gcagcagcgg cagcgctgac 3420

ggggatcaag gggaggacca ggatcgagat caggatcagg atcgggatga gggtgaggat 3480

caggaggatc agcaggagga ggaggatgag atggtggcgg aggatgagag cgaggagcgg 3540

gtggacgagc agggggtgcg gctgggggcg ctgagggctg agcgcgaggc ggcggcggct 3600

gaagcggcgg cggaggcggg cggcgcggcg gcggatatgc tggcggagga ggctgcggcg 3660

gcaggggcgg cggaggaggt ggcggcggag gtggcggcgg aggcggtgga ggcggcggag 3720

gcggcggctg cagaagaggt gccgctcgct gtgaaggcag ctgcggcgaa ggcgaaggcg 3780

aaggcaaagg gcggctggca gcggctgcag ccgggggcgt cgccaaggct ggagccgggg 3840

gcggtggcgg gcgctgctgg cgctgctgct gctgctgcgg ctatgggtgt gaaggctgag 3900

ggcggtgcgg aggcggcgcc gggggatgtg gggctggtcg gggctgcggt gaaggtggaa 3960

ggggcggcgg aggcagaagg ggcagaggag gcggaggcag cgggcgtcaa ggccgagcct 4020

gaggccagtc ctgatgctga cgtccacatg gcggatgtca tcaagcccga ggtcatcaag 4080

cctgaggtcc ggatcaagga tgacatcggc aacatcaaag acgaggtcgg ggtcaaggcc 4140

gaggtcaaag atgagatcaa gcctgacccc atggatgtcg atgacgccgc gccctcgccc 4200

tcgccccagc tacagccgcc ccagctacag cagcagcagc agcagcagcg gtgggcgcag 4260

gtgctgcagc ccgggcctgt ggggccggtg gggcccgtgc ctgactttgt ggacctggcg 4320

ggaggcggcg atggcgagta cgacgaagag gaggaggagg aggaggatcc caccaccggc 4380

atctacacac aggagcaatc ccccgacatc ctatccatca cggacggccc ctcgccggca 4440

tcagccgccg ccagaggcgg cggcggcgcc gccgcaggcg cgcacggcag cggcgctgcg 4500

gcggcggcgg cgccgccgca gcctcggccg gcgggcgcgg gcgccgccgc ctccgccgcc 4560

ttcggcggcc gggccaaggg cgtcaagggt gccgaggtgg tgtatgcgcc ggagccgccc 4620

gggctgctgc tggggggcac actggttgtg tgtcccacca gtgtgctgca ccagtgggcg 4680

cgggagatca aggccaaggt gtcccccgcc gcggggctgg tggtgcacgt gtaccacggc 4740

aaggagcggg cgggcagcgc gagggcgctg gcgggcatgg gcgtggtgct caccacctac 4800

ggtacactgg cgcaggaggc gccggccagg gacagggcgg cggcggtgag gctgggcgcg 4860

aggcagggag gcggcgagga cagcggaggc gacgagtatg gtggcggcgg cggccgcggc 4920

cgtggtggcg gcggtggcgg tggcggcggc aggggccgcg gcaagggcgg tgcgggtggc 4980

ggcagcggcg cgggcggcat tatctaccag gtcaaatgga ggcgtgtggt gctggacgag 5040

gctcagtcca tcaagaaccc gcgcaccctg gccgcgcacg ccgcctgggc gctgcacgcg 5100

cactgccgct ggtgcctcag cggcacgccc atacagaact ccgtggacga cctgtacagc 5160

tacttcaggt tcctgcgcta cgcgccctac tgcgacccgc ggcagtggcg cgagctgatc 5220

aaggggcgca tcacgtccga cccctcccgg gggtacaagt tcctgcaggc agtgctgcag 5280

gctgtgctgc tccgccgcac caagcagtcc accctcaacg gcgagcccat catccgcctg 5340

ccggggcggc agcaggcgct ggtgcaggag cgcttcagcg cggcggaggc ggccttctac 5400

agtcaggtac agcaggattc cctcaaggcg ctacacgagg cccagagcca agccgacacc 5460

accactgccg ccggaggcgc agccgccaat ggcagcggcg gcggcggcgg ttcgcggcag 5520

tacgtcaaca tgctgcactc gctgttgaag ctgcggcagg cctgcaacca cccctggctg 5580

gtgcggggcg gccggcagtg gcacaagagt ggcgccccca gtgctgcgga ggtggaggcg 5640

gcgcgcaagc tgtcgccgga ggcgcgggcc gcgctggcgg cggcgctggc ggcgggcggc 5700

ggcggcggcg gcgcgggtgg tggcggcgac gtgccgtgtg cctgctgtgg cgacattgcg 5760

gaggcgcctg tggcggcggt gccgtgcggc cacgtgttct gtgcgcagtg cctggcgggg 5820

cagcgggagg gagccggtgt ggagggcgag ctgctgtgcc ccgcctgcag ccgcctgctg 5880

cgccccgccg acctgcacag cgccgccgca ctggccgcag ccgacccggc ggcattcggg 5940

gggctgctgg gcgggccggc gccgggcgag ggcggcgacg gcagccccgg tgctgccgcc 6000

acctctgcct ccgctgccgc caccgccgcc acctctgcca cctctgccgc ctccgcctcc 6060

gccgcctcta cctctgcctc tgctgctgcc ggcggccccg cctggaccag cagcgccaag 6120

gtggagcggc tgatggcgct gctgggggag gtgcgcgcgc gcaacagcgc ctcggcggca 6180

ggggccggca gggccgcgcc ctccgcgctc aacccgctgg ctgtcaagtc caagtccgac 6240

aggcggctgg cgggggctct gaggaagctc acgccgctca gcgccggagc acccggcagc 6300

ggcggagcgg gacagccaga gaaggtgatt gtgttcagcc agtggacgac catgttggac 6360

ctgctggaga tcccgctcaa gaaggcgcgc atggctttcc gcaggctgga cggcaccatg 6420

tctgtggctc accgggagcg ggccatccag gactttgagt ccaagaacga ggtcatggtg 6480

cttctgacct cgctcaaggc cgccgcgctg ggtgtgaacc tggtggtggc caaccacgtg 6540

gtgctgatgg acctgtggtg gaacccgacc accgaggagc aggccataga ccgggcgcac 6600

cgcattggcc agacgcgcac ggtgtacgtg acgcgcatcg tcatcaaggg gagtgtggag 6660

gaccgcatcc tggagctgca gcagcgcaag cgcgaggtgg tggccgccgc cctgagtgag 6720

gggcgcgacg gctccgccgc cgccggccgc ctcaccctgg aggacctcaa gttcctgttc 6780

cagggactgg gctag 6795

<210> 2

<211> 2264

<212> PRT

<213> Chlamydomonas reinhardti

<400> 2

Met Gln Leu Ala Arg Ala Arg Ser Arg Gln Ala Ala Ala Ser Ala Ala

1 5 10 15

Ser Asn Ala Thr Gln Gln Gln Ser Arg Ala Ala Ala Thr Gly Pro Ala

20 25 30

Ala Ala Ala Glu Gly His Gly Lys Arg Pro Ala Gly Ala Gly Ala Gly

35 40 45

Val Ala Ala Ala Arg Pro Val Ala Ala Pro Ala Pro Ser Ala Ala Ala

50 55 60

Gly Thr Gly Pro Lys Pro Val Thr Val Thr Ala Asp Val Val Gln Ser

65 70 75 80

Thr Ala Ala Ser Ser Gln Pro Glu Ala Ala Ser Ser Ala Val Ala Gly

85 90 95

Ala Lys Ala Ala Ala Ala Gly Thr Ala Ala Ala Ala Ala Gly Ser Asp

100 105 110

Gly Gly Lys Gly Gly Gly Gly Gly Ala Val Ala Ala Thr Ala Thr Glu

115 120 125

Val Thr Pro Gln Ala Ser Thr Pro Ala Gln Gly Ser Ala Pro Leu Leu

130 135 140

Pro Glu Pro Ala Pro Pro Thr Gly Ala Pro Ala Thr Gly Pro Ala Ala

145 150 155 160

Ala Ala Ala Thr Ala Ala Asn Asp Asp Gly Gly Gly Ala Arg Gly Asp

165 170 175

Val Val Asn Ala Ala Ala Pro Ala Ala Asp Val Met Pro Pro Pro His

180 185 190

Gln Pro Gln Gln Glu Pro Gln Gln Glu Pro Lys Pro Asp Ala Lys Gln

195 200 205

Glu Pro Lys Gln Glu Pro Lys Gln Gln Pro Ala Arg Lys Ala Lys Gly

210 215 220

Ser Ala Ala Ser Pro Ala Gly Lys Gly Ala Gly Lys Gly Ala Gly Lys

225 230 235 240

Gly Ala Arg Val Gly Ala Ala Ala Lys Ala Lys Ser Glu Pro Glu Ala

245 250 255

Asp Ala Gly Ala Pro Ala Ala Val Ala Pro Ala Pro Ala Pro Ala Pro

260 265 270

Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Glu Pro Ala Pro

275 280 285

Ala Pro Ala Ala Val Pro Cys Ala Ala Val Leu Lys Pro Thr Ala Leu

290 295 300

Ala Ala Ala Pro Asp Gly Pro Val Ala Ala Pro Gly Ser Leu Glu Gln

305 310 315 320

Gln Glu Gly Gly Ala Ala Ala Ala Glu Ala Ala Ala Ala Ala Val Ala

325 330 335

Thr Ala Thr Gly Thr Ala Thr Ala Pro Val Ala Ala Ala Ser Gly Gly

340 345 350

Gly Gly Ser Asp Thr Val Ala Val Gly Gly Gly Gly Gly Gly Gly Asp

355 360 365

Gly Val Val Asp Val Ala Ala Gly Leu Pro Glu Pro Pro Pro Thr Ala

370 375 380

Ala Val Ala Gln Ala Ala Ala Ala Ala Ala Thr Gln Ala Ala Ala Gly

385 390 395 400

Ala Ala Val Asp Ala Gly Gly Gly Ile Ala Ala Gln Pro Gln Glu Gln

405 410 415

Pro Gln Glu Ala Pro Pro Pro Pro Gln Gln Leu Arg Gln Pro Gln Gln

420 425 430

Pro Gln Gln Pro Gln Gln Pro Gln Gln Pro Gln Gln Leu Gln Gln Leu

435 440 445

Gln Gln Pro Gln Gln Pro Gln Gln Pro Gln Gly Ala Gln Gly Ala Gly

450 455 460

Arg Lys Arg Pro Val Pro Val Arg Gly Glu Gln Gln Ala Gly Leu Gly

465 470 475 480

Glu Met Glu Val Glu Val Glu Val Glu Val Glu Val Ala Ala Ala Glu

485 490 495

Ala Ala Gly Glu Ala Ala Gly Pro Pro Ala Lys Arg Gln Ala Val Ala

500 505 510

Ala Ala Ala Thr His Ala Pro Pro Arg His Pro Gln Gln Gln Gln His

515 520 525

Gln Gln Pro Pro Pro Pro Ala Pro Pro His Pro Glu Gln Gln Gln Gln

530 535 540

Gln Gln Gln Gln Pro Gln Ala Thr Ala Ser Ala Ala Ala Pro Asp Pro

545 550 555 560

Ala Pro Ala Pro Ala Pro Thr Val Ser Pro Ala Thr Ala Ala Ser Pro

565 570 575

Ala Ser Pro Ala Ala Ala Ala Pro Thr Ala Ile Ala Ala Thr Ser Gln

580 585 590

Ala Ala Ala Ala Thr Thr Ser Pro Pro Ala Ala Ala Ala Thr Ala Ala

595 600 605

Thr Ser Pro Ala Ala Ser Pro Ala Ala Ala Thr Ala Ser Pro Ala Ala

610 615 620

Thr Ala Pro Ala Ala Thr Ala Pro Ala Ala Thr Ala Pro Ala Ala Thr

625 630 635 640

Ala Pro Ala Ala Thr Ala Pro Ala Ala Thr Ala Pro Ala Ala Thr Ala

645 650 655

Pro Ala Ala Ala Thr Ala Ser Pro Ala Ala Thr Ala Pro Ala Ala Thr

660 665 670

Ala Pro Ala Ala Ala Ala Pro Ala Ala Thr Ala Pro Ala Ala Thr Ala

675 680 685

Pro Ala Ala Thr Ala Pro Ala Ala Thr Ala Pro Ala Ala Ala Ala Pro

690 695 700

Ala Ala Ala Ala Ala Gly Ser Leu Ala Ala Gln Glu Ala Ala Leu Arg

705 710 715 720

Arg His Leu Gly Val Val Ala Gly Arg Leu Arg Glu Gly Thr Met Pro

725 730 735

Val Ser Arg Gln Ser Asn Glu Leu Arg Pro Leu Thr Asp Ala Glu Arg

740 745 750

Ser Lys Phe Gln Glu Ala Ala Ala Lys Leu Glu Arg Gln Ile His Ala

755 760 765

Leu Gln Gln Gln Gln Gln Ala Gln Gln Gln Ala Gln Gln Pro Lys Gln

770 775 780

Ala Gln Gln Thr Gln Gln Ala Gln Gln Ala Ala Val Ala Gln Pro Gln

785 790 795 800

Ala Gly Pro Arg Thr Val Ser Pro Ala Ala Ala Ala Gly Gly Gly Ala

805 810 815

Ala Gly Ala Ala Thr Ala Pro Ala Gly Ala Ala Ala Thr Ala Ala Ala

820 825 830

Ala Thr Ala Ala Ala Ala Gly Ala Thr Gly Leu Ala Ala Val Ile Ala

835 840 845

Ala Arg Gly Ala Val Val Trp Gln Gly Pro Gly Arg Pro Ala Met Thr

850 855 860

Thr Ala Pro Ala Ala Asn Gly Gly Gly Gly Ala Ala Ala Arg Ala Val

865 870 875 880

Ser Gly Ala Ala Ala Ala Ala Ala Thr Ala Ala Ala Ala Pro Ser Gly

885 890 895

Ala Gly Gly Gly Gly Ser Ala Ala Pro Ala Leu Pro Val Ala Ile Pro

900 905 910

Ala Ser Gly Leu Leu Arg Ala Ala Ala Gln Ala Ala Val Ile His Ala

915 920 925

Ala Ala Gln Ala Ala Ala Arg Gln Ala Ala Ala Ala Ala Ala Ala Gly

930 935 940

Ala Gly Gly Ala Val Gly Val Gly Ala Pro Gly Arg Gly Ala Gly Ala

945 950 955 960

Val Ala Asp Ala Ala Gly Gly Gly Lys Gly Pro Glu His Leu Ala Ala

965 970 975

Leu Arg Ala Val Ile Glu Ser Leu Ser Ala Thr Gly Met Glu Glu Leu

980 985 990

Glu Pro Pro Pro Lys Thr Leu Gln Val Thr Val Leu Arg His Gln Arg

995 1000 1005

Met Ala Leu Ala Trp Met Leu Arg Arg Glu Thr Gly Pro Glu Pro Arg

1010 1015 1020

Gly Gly Ile Leu Ala Asp Asp Gln Gly Leu Gly Lys Thr Val Thr Thr

1025 1030 1035 1040

Ile Ser Leu Ile Val Thr His Thr Asn Pro Glu Asp Ser Ala Pro Gly

1045 1050 1055

Ala Ala Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Glu Gly

1060 1065 1070

Gly Gly Ala Glu Gly Ala Ala Ala Asn Gly Ala Gly Ala Thr Ala Ala

1075 1080 1085

Ala Ala Ala Ala Ala Ala Gly Ala Pro Gly Val Ala Ala Thr Ser Gly

1090 1095 1100

Gly Gly Ala Gly His Gly His Leu Gly Gly Pro Gly Ser Ser Gln Ser

1105 1110 1115 1120

Pro Pro Arg Gln Gln Gln Gln Arg Arg Arg Gly Gly Arg Gly Ser Ser

1125 1130 1135

Gly Ser Ala Asp Gly Asp Gln Gly Glu Asp Gln Asp Arg Asp Gln Asp

1140 1145 1150

Gln Asp Arg Asp Glu Gly Glu Asp Gln Glu Asp Gln Gln Glu Glu Glu

1155 1160 1165

Asp Glu Met Val Ala Glu Asp Glu Ser Glu Glu Arg Val Asp Glu Gln

1170 1175 1180

Gly Val Arg Leu Gly Ala Leu Arg Ala Glu Arg Glu Ala Ala Ala Ala

1185 1190 1195 1200

Glu Ala Ala Ala Glu Ala Gly Gly Ala Ala Ala Asp Met Leu Ala Glu

1205 1210 1215

Glu Ala Ala Ala Ala Gly Ala Ala Glu Glu Val Ala Ala Glu Val Ala

1220 1225 1230

Ala Glu Ala Val Glu Ala Ala Glu Ala Ala Ala Ala Glu Glu Val Pro

1235 1240 1245

Leu Ala Val Lys Ala Ala Ala Ala Lys Ala Lys Ala Lys Ala Lys Gly

1250 1255 1260

Gly Trp Gln Arg Leu Gln Pro Gly Ala Ser Pro Arg Leu Glu Pro Gly

1265 1270 1275 1280

Ala Val Ala Gly Ala Ala Gly Ala Ala Ala Ala Ala Ala Ala Met Gly

1285 1290 1295

Val Lys Ala Glu Gly Gly Ala Glu Ala Ala Pro Gly Asp Val Gly Leu

1300 1305 1310

Val Gly Ala Ala Val Lys Val Glu Gly Ala Ala Glu Ala Glu Gly Ala

1315 1320 1325

Glu Glu Ala Glu Ala Ala Gly Val Lys Ala Glu Pro Glu Ala Ser Pro

1330 1335 1340

Asp Ala Asp Val His Met Ala Asp Val Ile Lys Pro Glu Val Ile Lys

1345 1350 1355 1360

Pro Glu Val Arg Ile Lys Asp Asp Ile Gly Asn Ile Lys Asp Glu Val

1365 1370 1375

Gly Val Lys Ala Glu Val Lys Asp Glu Ile Lys Pro Asp Pro Met Asp

1380 1385 1390

Val Asp Asp Ala Ala Pro Ser Pro Ser Pro Gln Leu Gln Pro Pro Gln

1395 1400 1405

Leu Gln Gln Gln Gln Gln Gln Gln Arg Trp Ala Gln Val Leu Gln Pro

1410 1415 1420

Gly Pro Val Gly Pro Val Gly Pro Val Pro Asp Phe Val Asp Leu Ala

1425 1430 1435 1440

Gly Gly Gly Asp Gly Glu Tyr Asp Glu Glu Glu Glu Glu Glu Glu Asp

1445 1450 1455

Pro Thr Thr Gly Ile Tyr Thr Gln Glu Gln Ser Pro Asp Ile Leu Ser

1460 1465 1470

Ile Thr Asp Gly Pro Ser Pro Ala Ser Ala Ala Ala Arg Gly Gly Gly

1475 1480 1485

Gly Ala Ala Ala Gly Ala His Gly Ser Gly Ala Ala Ala Ala Ala Ala

1490 1495 1500

Pro Pro Gln Pro Arg Pro Ala Gly Ala Gly Ala Ala Ala Ser Ala Ala

1505 1510 1515 1520

Phe Gly Gly Arg Ala Lys Gly Val Lys Gly Ala Glu Val Val Tyr Ala

1525 1530 1535

Pro Glu Pro Pro Gly Leu Leu Leu Gly Gly Thr Leu Val Val Cys Pro

1540 1545 1550

Thr Ser Val Leu His Gln Trp Ala Arg Glu Ile Lys Ala Lys Val Ser

1555 1560 1565

Pro Ala Ala Gly Leu Val Val His Val Tyr His Gly Lys Glu Arg Ala

1570 1575 1580

Gly Ser Ala Arg Ala Leu Ala Gly Met Gly Val Val Leu Thr Thr Tyr

1585 1590 1595 1600

Gly Thr Leu Ala Gln Glu Ala Pro Ala Arg Asp Arg Ala Ala Ala Val

1605 1610 1615

Arg Leu Gly Ala Arg Gln Gly Gly Gly Glu Asp Ser Gly Gly Asp Glu

1620 1625 1630

Tyr Gly Gly Gly Gly Gly Arg Gly Arg Gly Gly Gly Gly Gly Gly Gly

1635 1640 1645

Gly Gly Arg Gly Arg Gly Lys Gly Gly Ala Gly Gly Gly Ser Gly Ala

1650 1655 1660

Gly Gly Ile Ile Tyr Gln Val Lys Trp Arg Arg Val Val Leu Asp Glu

1665 1670 1675 1680

Ala Gln Ser Ile Lys Asn Pro Arg Thr Leu Ala Ala His Ala Ala Trp

1685 1690 1695

Ala Leu His Ala His Cys Arg Trp Cys Leu Ser Gly Thr Pro Ile Gln

1700 1705 1710

Asn Ser Val Asp Asp Leu Tyr Ser Tyr Phe Arg Phe Leu Arg Tyr Ala

1715 1720 1725

Pro Tyr Cys Asp Pro Arg Gln Trp Arg Glu Leu Ile Lys Gly Arg Ile

1730 1735 1740

Thr Ser Asp Pro Ser Arg Gly Tyr Lys Phe Leu Gln Ala Val Leu Gln

1745 1750 1755 1760

Ala Val Leu Leu Arg Arg Thr Lys Gln Ser Thr Leu Asn Gly Glu Pro

1765 1770 1775

Ile Ile Arg Leu Pro Gly Arg Gln Gln Ala Leu Val Gln Glu Arg Phe

1780 1785 1790

Ser Ala Ala Glu Ala Ala Phe Tyr Ser Gln Val Gln Gln Asp Ser Leu

1795 1800 1805

Lys Ala Leu His Glu Ala Gln Ser Gln Ala Asp Thr Thr Thr Ala Ala

1810 1815 1820

Gly Gly Ala Ala Ala Asn Gly Ser Gly Gly Gly Gly Gly Ser Arg Gln

1825 1830 1835 1840

Tyr Val Asn Met Leu His Ser Leu Leu Lys Leu Arg Gln Ala Cys Asn

1845 1850 1855

His Pro Trp Leu Val Arg Gly Gly Arg Gln Trp His Lys Ser Gly Ala

1860 1865 1870

Pro Ser Ala Ala Glu Val Glu Ala Ala Arg Lys Leu Ser Pro Glu Ala

1875 1880 1885

Arg Ala Ala Leu Ala Ala Ala Leu Ala Ala Gly Gly Gly Gly Gly Gly

1890 1895 1900

Ala Gly Gly Gly Gly Asp Val Pro Cys Ala Cys Cys Gly Asp Ile Ala

1905 1910 1915 1920

Glu Ala Pro Val Ala Ala Val Pro Cys Gly His Val Phe Cys Ala Gln

1925 1930 1935

Cys Leu Ala Gly Gln Arg Glu Gly Ala Gly Val Glu Gly Glu Leu Leu

1940 1945 1950

Cys Pro Ala Cys Ser Arg Leu Leu Arg Pro Ala Asp Leu His Ser Ala

1955 1960 1965

Ala Ala Leu Ala Ala Ala Asp Pro Ala Ala Phe Gly Gly Leu Leu Gly

1970 1975 1980

Gly Pro Ala Pro Gly Glu Gly Gly Asp Gly Ser Pro Gly Ala Ala Ala

1985 1990 1995 2000

Thr Ser Ala Ser Ala Ala Ala Thr Ala Ala Thr Ser Ala Thr Ser Ala

2005 2010 2015

Ala Ser Ala Ser Ala Ala Ser Thr Ser Ala Ser Ala Ala Ala Gly Gly

2020 2025 2030

Pro Ala Trp Thr Ser Ser Ala Lys Val Glu Arg Leu Met Ala Leu Leu

2035 2040 2045

Gly Glu Val Arg Ala Arg Asn Ser Ala Ser Ala Ala Gly Ala Gly Arg

2050 2055 2060

Ala Ala Pro Ser Ala Leu Asn Pro Leu Ala Val Lys Ser Lys Ser Asp

2065 2070 2075 2080

Arg Arg Leu Ala Gly Ala Leu Arg Lys Leu Thr Pro Leu Ser Ala Gly

2085 2090 2095

Ala Pro Gly Ser Gly Gly Ala Gly Gln Pro Glu Lys Val Ile Val Phe

2100 2105 2110

Ser Gln Trp Thr Thr Met Leu Asp Leu Leu Glu Ile Pro Leu Lys Lys

2115 2120 2125

Ala Arg Met Ala Phe Arg Arg Leu Asp Gly Thr Met Ser Val Ala His

2130 2135 2140

Arg Glu Arg Ala Ile Gln Asp Phe Glu Ser Lys Asn Glu Val Met Val

2145 2150 2155 2160

Leu Leu Thr Ser Leu Lys Ala Ala Ala Leu Gly Val Asn Leu Val Val

2165 2170 2175

Ala Asn His Val Val Leu Met Asp Leu Trp Trp Asn Pro Thr Thr Glu

2180 2185 2190

Glu Gln Ala Ile Asp Arg Ala His Arg Ile Gly Gln Thr Arg Thr Val

2195 2200 2205

Tyr Val Thr Arg Ile Val Ile Lys Gly Ser Val Glu Asp Arg Ile Leu

2210 2215 2220

Glu Leu Gln Gln Arg Lys Arg Glu Val Val Ala Ala Ala Leu Ser Glu

2225 2230 2235 2240

Gly Arg Asp Gly Ser Ala Ala Ala Gly Arg Leu Thr Leu Glu Asp Leu

2245 2250 2255

Lys Phe Leu Phe Gln Gly Leu Gly

2260

<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 reinhardtiiCre01.g046237.t1.1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled, its feature exist In：It is describedCre01.g046237.t1.1The nucleotides sequence of gene is classified as one kind in following sequences：

（1）Include such as SEQ ID NO:Nucleotide sequence shown in 1；

（2）Include coding SEQ ID NO:The nucleotide sequence of protein shown in 2；

（3）Comprising with SEQ ID NO:Nucleotide sequence shown in 1 is with more than 90% homology and coding identical function protein Nucleotide sequence.

2. application according to claim 1, it is characterised in that：It is describedCre01.g046237.t1.1The encoding proteins of gene For one kind in following albumen：

（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 reinhardtiiCre01.g046237.t1.1Application of the gene in Chlamydomonas reinhardtii cadmium tolerance is improved, its feature exist In：By reducing Chlamydomonas reinhardtiiCre01.g046237.t1.1The expression of gene, makeCre01.g046237.t1.1Gene inactivates Or missingCre01.g046237.t1.1Gene improves Chlamydomonas reinhardtii cadmium tolerance.

A kind of 4. method for improving Chlamydomonas reinhardtii cadmium tolerance, it is characterised in that：By reducing Chlamydomonas reinhardtiiCre01.g046237.t1.1The expression of gene, makeCre01.g046237.t1.1Gene inactivates or missingCre01.g046237.t1.1Gene 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 withoutCre01.g046237.t1.1Gene activityCre01.g046237.t1.1Gene mutation strain orCre01.g046237.t1.1 Gene-deleted strain, orCre01.g046237.t1.1What gene expression declinedCre01.g046237.t1.1Gene 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 mutagenesisCre01.g046237.t1.1Gene mutation strain；

Chlamydomonas reinhardtii is obtained by building Crispr carriersCre01.g046237.t1.1Gene-deleted strain；

Chlamydomonas reinhardtii is obtained by building RNAi interference vectorsCre01.g046237.t1.1Gene 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 reinhardtiiCre01.g046237.t1.1Application of the gene in the monitoring or detection of cadmium pollution.

10. Chlamydomonas reinhardtiiCre01.g046237.t1.1Application of the gene in the improvement of cadmium pollution.