CN105483094A - Pinctada martensii superoxide dismutase (PmECSOD) and application thereof - Google Patents
Pinctada martensii superoxide dismutase (PmECSOD) and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly discloses pinctada martensii superoxide dismutase (PmECSOD) and an application thereof. The amino acid sequence of the PmECSOD is shown as SEQ ID NO:1; the nucleotide sequence of a PmECSOD gene is shown as SEQ ID NO:2 or SEQ ID NO:3. Compared with traditional SOD, the PmECSOD has many differences and is new SOD through preliminary identification, and the PmECSOD has the effects of inhibiting growth of escherichia coli and growth of micrococcus luteus and has good application prospects.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to pteria martensii superoxide-dismutase PmECSOD and application thereof.
Background technology
Pteria martensii (Pinctadamartensii) is one of China's artificial seawater main Margarita of growing cultured pearls, in recent years because of the deterioration increasingly of coastal water quality, various disease also frequently occurs, cause pteria martensii because of disease generation mortality, huge financial loss is caused to China's pearl sector.Therefore, understand pteria martensii immunologic mechanism and improve pteria martensii on this basis and resist the ability of coercing to be need the problem of solution at present badly.
Invertebrates, does not possess the specific immune system of higher animal, so it is very important to play immunologic function in vivo for innate immune molecule superoxide-dismutase (SOD).Superoxide-dismutase can remove the free radical (O in body
2-), maintain free radical generate with remove between unbalance.Superoxide-dismutase is generally dimer or the tetramer, and molecular weight is between 20kDa ~ 80kDa.Large quantifier elimination finds superoxide-dismutase at some diseases as inflammation, autoimmune disorder, radiation and anti-ageing therapeutic action of having waited for a long time.In recent years, both at home and abroad the research of superoxide-dismutase is increased gradually, relatively play vertebrates, only have the superoxide-dismutase of minority species as bay scallop, the long oyster in the Pacific Ocean and razor clam etc. of hanging studied shellfish.For pteria martensii, yet there are no the report research of superoxide-dismutase, the superoxide-dismutase of research pteria martensii not only can understand the immunologic mechanism of pteria martensii, and accomplish diseases prevention and treatment, also the indirect life to the mankind is significant.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of pteria martensii superoxide-dismutase PmECSOD is provided.
Another object of the present invention is to provide a kind of pteria martensii superoxide-dismutase PmECSOD gene.
Another object of the present invention is to provide the application of pteria martensii superoxide-dismutase PmECSOD.
To achieve these goals, the present invention is achieved by the following technical programs:
A kind of pteria martensii superoxide-dismutase PmECSOD, its aminoacid sequence is as shown in SEQIDNO:1.
A kind of pteria martensii superoxide-dismutase PmECSOD gene, its nucleotide sequence is as shown in SEQIDNO:2 or SEQIDNO:3.Sequence shown in SEQIDNO:2 is the DNA full length sequence of PmECSOD gene, and total length 1846bp, comprises the 3'UTR of 5'UTR and 382bp of 42bp.Sequence shown in SEQIDNO:3 is the open reading frame sequence of PmECSOD gene, open reading frame 1422bp, 473 amino acid of encoding.
At present to SOD research many, mainly it can remove the free radical in body, treat the some diseases caused by free radical, as anti-ageing, antitumor and some inflammation etc., and have in a small amount of report and be found the effect that CuZnSOD has participating in activation antibacterial peptide and lyase bacterium.The present invention finds that pteria martensii superoxide-dismutase PmECSOD has the effect suppressing intestinal bacteria and M. luteus bacteria growing.Therefore application claims protection pteria martensii superoxide-dismutase PmECSOD is suppressing the purposes in intestinal bacteria and M. luteus bacteria growing.
In addition; the present invention devises the fluorescent quantitation primer of a pair detection pteria martensii superoxide-dismutase described above PmECSOD gene; for analyzing the expression of pteria martensii superoxide-dismutase PmECSOD gene in each tissue; therefore application claims protects the fluorescent quantitation primer of a pair detection pteria martensii superoxide-dismutase described above PmECSOD gene, and the nucleotide sequence of primer is as shown in SEQIDNO:4 ~ 5.
The present invention also protection package contains the recombinant vectors of pteria martensii superoxide-dismutase PmECSOD gene described in SEQIDNO:2 or 3.The present invention also protection package contains the recombinant bacterium of recombinant vectors described above.
Compared with prior art, the present invention has following beneficial effect:
The present invention first from pteria martensii serum separation and purification obtain a kind of new superoxide-dismutase, this enzyme has 3 structural domains, FRI(Frizzled respectively) structural domain, IGc2(immunoglobulin (Ig)) structural domain and SOD_Cu structural domain, but comparison can only have SOD_Cu structural domain in Blastp, tentatively be judged as a kind of new SOD.By research, contriver finds that pteria martensii superoxide-dismutase PmECSOD has the effect suppressing intestinal bacteria and M. luteus bacteria growing in addition.
The ECSOD of PmECSOD expression contents and other species is in the tissue completely different, and it all has expression in closed shell flesh, mantle, hepatopancreas, the gill, sexual gland and blood, and the gill is the main expression place of PmECSOD; Then be mantle, blood and sexual gland, this three's content difference is few; What expression amount was minimum is hepatopancreas and closed shell flesh.After stimulating with the intestinal bacteria after sterilizing and micrococcus luteus, PmECSOD is not identical at the content in hemocyte each period.After stimulation, the content of 4h, PmECSOD reduces, and 8h raises a little afterwards, from after 8h again reduce, until the bottom out of 36h content, until the content of 48h, PmECSOD reaches maximum value, have obvious difference with section At All Other Times.According to some research, can infer, when certain species are subject to foreign objects (as bacterium, fungi and parasite etc.) stimulation, active oxygen ROS starts a large amount of gathering, engulf foreign matter, a large amount of ROS can by the expression of some regulatory factors regulation and control ECSOD, and the ECSOD of generation and excessive ROS effect, prevent excessive ROS from damaging body.
Accompanying drawing explanation
Fig. 1 is pteria martensii serum Sunfire
tMthe separating obtained color atlas of semi-preparative C18 post, collects and has the object peak of bacteriostatic activity " * * " mark.
The further separation and purification of peak analysis mode VydacC18 post for the purpose of Fig. 2, the elutriant that collecting has the peak C1 of bacteriostatic activity to indicate correspondence does structural analysis.
Fig. 3 is that pteria martensii serum separated portion is to micrococcus luteus and Escherichia coli Growth suppression figure.
Fig. 4 is the mass spectrum of PmECSOD peptide section.
Fig. 5 is the intermediate segment electrophorogram of PmECSOD gene; M:Marker; 1:PmECSOD intermediate segment product.
Fig. 6 is PmECSOD gene RACE-PCR product agarose gel electrophoresis; M:Marker; A-1:3'RACE product; B-2:5'RACE product.
Fig. 7 is cDNA sequence and the prediction of aminoacid sequence functional site of pteria martensii PmECSOD; ORF region capitalization in figure represents, 5 ' UTR and 3 ' UTR lowercase represents, the conserved domain of PmECSOD represents with black surround, and two horizontal line represents poly A tailing signal, the amino acid that thick line representative order-checking obtains.
Fig. 8 is SMART on-line analysis PmECSOD sequential structure field result figure.
Fig. 9 is the compare of analysis of the aminoacid sequence of the aminoacid sequence of PmECSOD and the ECSOD of other species.
Figure 10 is the PmECSOD family member aminoacid sequence systematic evolution tree built with NJ method.
Figure 11 is PmECSOD Quantitative analysis of gene expression; X-coordinate is tissue, and ordinate zou is repeat averaging of income value ± standard deviation five times.
Figure 12 is that bacterium stimulates rear PmECSOD gene at the temporal expression of pteria martensii hemolymph; Significant difference 0.01<P<0.05 asterisk represents, P<0.01 two asterisks show; Ordinate zou is mean value ± standard deviation (n=3).
Embodiment
To make the present invention below in conjunction with Figure of description and specific embodiment and elaborating further, described embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.The test method used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc. if no special instructions, are the reagent that can obtain from commercial channels and material.
Embodiment 1
1, the induction of pteria martensii and the collection of serum: after pteria martensii raises three days, the closed shell intramuscular that colibacillus deactivating and micrococcus luteus inject pteria martensii is induced, every only injection 10 μ L colibacillus deactivatings and 20 μ L deactivation micrococcus luteuses, be reentered in seawater after raising 12h and collect hemolymph with aseptic syringe from the closed shell flesh of pteria martensii, 4 DEG C, the centrifugal 15min of 3000rpm, gets supernatant, and lyophilize obtains the serum dry powder of pteria martensii.
2, the separation and purification of pteria martensii serum: use SunFire
tMsemi-preparative C18 column separating purification pteria martensii serum, obtain result as shown in Figure 1, epicycle wash-out is totally four concentration gradients (5% acetonitrile, 18% acetonitrile, 40% acetonitrile and 60% acetonitrile), material through antibacterial detection discovery the IIIth gradient (i.e. 40% acetonitrile) wash-out has anti-microbial activity (in Fig. 1, asterisk * * represents), is got up by this Fraction collection.This component by analysis type reverse-phase chromatographic column C18 is separated, and the results are shown in Figure 2.As seen from Figure 2, have complicated component in the component of anti-microbial activity, collection is more than 20 kinds of elution peaks altogether.Bacteriostatic activity detection is carried out respectively after lyophilize.Wherein have the component of anti-microbial activity to be labeled as C1(and see Fig. 2), collect and make further research after lyophilize.
Adopt microplate reader method, control group deionized water, experimental group is above-mentioned component after HPLC is separated, and sample concentration is 100 μ g/mL.For pteria martensii serum component to micrococcus luteus and colibacillary restraining effect, result as shown in Figure 3.A figure is the OD value of micrococcus luteus after the addition of the sample measured by 0 and 12 hour, and from Fig. 3 A, after adding pteria martensii serum component, the growth of micrococcus luteus is subject to obvious suppression (p<0.05); Meanwhile, pteria martensii serum component produces obvious restraining effect (P<0.05) (Fig. 3 B) to colibacillary growth media.Bacteriostatic test prove component C1 to micrococcus luteus and intestinal bacteria inhibited.
3, the mass spectroscopy of pteria martensii Serum bactericidal activity albumen: bacteriostatic active ingredients C1 step 2 being separated acquisition uses MALDI-TOF/TOF5800 mass spectrograph to carry out the molecular weight identification of activated protein; Adopt second order ms in conjunction with the aminoacid sequence of the fragment of Denovo sequencing technologies analyzing proteins after trypsin digestion.Fig. 4 is seen through the check order mass spectrum of the C1 component obtained of mass spectrum.The aminoacid sequence recorded is carried out homology comparison in NCBI, find that the similarity of component C1 and superoxide-dismutase is high, respectively at the Protein Data Bank of NCBI and pteria martensii genome screening superoxide-dismutase homologous sequence, with the highest homologous sequence of score for stencil design amplification C1 component intermediate segment, 5 ' RACE and 3 ' RACE amplimer.Amplimer sequence is as shown in table 1.
4, pteria martensii hemocyte total serum IgE is extracted, RT-PCR method is utilized to obtain the intermediate segment of C1 component gene for 1342bp(Fig. 5), again according to the gene design 3'RACE of intermediate segment and the Auele Specific Primer (table 1) of 5'RACE, obtain the 3' terminal nucleotide sequence (A in Fig. 6) of 1089bp and the 5' terminal nucleotide sequence (B in Fig. 6) of 137bp by RACE technology.The cDNA sequence total length of final acquisition C1 component gene, overall length is 1846bp.Protein amino acid sequence coded by this cDNA and mass spectrum survey peptide section compare, finally determine whole aminoacid sequences of this active ingredient.By sequential analysis, find that C1 component is a kind of new superoxide-dismutase, by C1 component called after pteria martensii superoxide-dismutase (PmECSOD).The DNA total length of PmECSOD gene is 1846bp (sequence is as shown in SEQIDNO:2), comprise the 3'UTR of 5'UTR and 382bp of 42bp, open reading frame is 1422bp (sequence is as shown in SEQIDNO:3), 473 amino acid (sequence is as shown in SEQIDNO:1) of encoding.3'UTR end has tailing signal sequence A ATAAA and poly(A) tail.
5, the cDNA sequence of pteria martensii PmECSOD and aminoacid sequence functional site predict the outcome and see Fig. 7.PmECSOD albumen theoretical molecular (MW) is 49.6KDa, iso-electric point pI is 8.62; Contain 20 amino acid whose signal peptides at N-end, and general CuZnSOD does not have signal peptide, this illustrates that PmECSOD belongs to born of the same parents external form CuZnSOD, therefore direct called after PmECSOD.For the CuZnSOD of shellfish, that major part is studied is plasmotype CuZnSOD, only has in the long oyster in the Pacific Ocean and bay scallop at present and be found born of the same parents external form CuZnSOD(ECSOD in shellfish).3 N-glycosylation sites are comprised, 2 amidation sites, 4 CAXX boxes, 6 microbody C-end localization signal sequence, 4 casein II phosphoric acid sites, 6 PKC phosphoric acid sites and 15 myristoylation site in PmECSOD aminoacid sequence.Wherein aminoacid sequence contains 2 typical Cu/Zn superoxide dismutase signal sequences, lay respectively at this section of amino acid 359 ~ 369, amino acid characteristics is: [GA]-[IMFAT]-H-[LIVF]-H-[S]-x-[GP]-[SDG]-x-[STAGDE]; Characteristic sequence contained by pteria martensii PmECSOD amino acid is: GFHIHEYGGME; Also have 453 ~ 464 these sections, amino acid characteristics is: G-[GNHD]-[SGA]-[GR]-x-R-x-[SGAWRV]-C-x (2)-[IV] and characteristic sequence contained by pteria martensii CuZnSOD amino acid is: GNAGTRIACCTI.
6, the albumen that the open reading frame coding of PmECSOD gene generates has 473 amino acid, and wherein signal peptide has 20 amino acid, and mature peptide has 453 amino acid.From SMARTmainpage, obtain structural domain, as shown in Figure 8, showing it in figure has 3 structural domains, is FRI(Frizzled respectively) structural domain, IGc2(immunoglobulin (Ig)) structural domain and SOD_Cu structural domain.But comparison can only have SOD_Cu structural domain in blastp, tentatively be judged as a kind of new SOD.
PmECSOD and other species are carried out sequence alignment, and result as shown in Figure 9.The similarity of PmECSOD and other species is very low, 37.01% with the similarity of zebra fish (D.rerio), 40.52% with the similarity of fruit bat (D.nasuta), 19.9% with the similarity of homo sapiens (H.sapiens), compare with bay scallop (A.irradians) with the long oyster in the Pacific Ocean (C.gigas), similarity is still very low, is 28.32% and 36.4% respectively.
MEGA6.0 software (Neighbor-Joining method) is used to set up evolutionary tree.Because the structural domain of PmECSOD and the ECSOD of other species have some difference, evolutionary tree set up result as Figure 10, show evolutionary tree in figure and be mainly divided into Liang great branch, the long oyster of PmECSOD and the Pacific Ocean is on same, and the bay scallop ECSOD of Bao Yongbo research is in an other branch.It can be said that this superoxide-dismutase of bright ECSOD is except small part sequence preservative, different plant species, their amino acid has obvious difference.
Embodiment 2
1, the histological difference expression analysis of pteria martensii PmECSOD: get 10 mature horses martensiis and do histological difference expression analysis, the tissue got comprises closed shell flesh, mantle, hepatopancreas, the gill, sexual gland and hemocyte, extract their total serum IgE respectively, reverse transcription becomes cDNA, for tissue specific expression analysis.According to the conserved sequence of pteria martensii PmECSOD gene, design fluorescent quantitation primer, internal reference used is GAPDH.The primer of quantitative fluorescence analysis is in table 2.
As shown in figure 11, the highest expression tissue is the gill to fluorescent quantitation result, is about 2.9 times of blood, has significant difference (P<0.05); Next is 0.29 times that hepatopancreas is about hemolymph, also has significant difference (P<0.05); And mantle and sexual gland are about 1.15 times and 0.67 times of blood.Minimum is closed shell flesh, is about 0.11 times of blood, has significant difference (P<0.05);
The ECSOD of the PmECSOD of pteria martensii expression contents and other species is in the tissue completely different, and it all has expression in closed shell flesh, mantle, hepatopancreas, the gill, sexual gland and blood, and the gill is the main expression place of PmECSOD; Then be mantle, blood and sexual gland, this three's content difference is few; What expression amount was minimum is hepatopancreas and closed shell flesh.This has obvious difference with the bay scallop being all shellfish, and bay scallop superoxide-dismutase (AiECSOD) expression amount in hemocyte is the highest, lower in the gill, does not almost have in sexual gland and mantle.The ECSOD that Gonzalez etc. study the long oyster in the Pacific Ocean finds in circulatory system and intercellular substance.Compared with vertebrates mouse, also different, contain a lot of ECSOD in the lung of the discovery such as Folz mouse and kidney, and do not detect in skeletal muscle.Visible different species, ECSOD content is in the tissue different, just same species at last, and different tissue expression content is also differentiated.
2, bacterium stimulate after the temporal expression of mRNA in hemocyte of PmECSOD: get the healthy pteria martensii 140 of supporting temporarily 3 days, points 2 groups: bacterium stimulating group, PBS control group, often organize 60 shellfishes.Adopt the method for penetrating from closed shell intramuscular injection, induce with the intestinal bacteria of deactivation and micrococcus luteus, every only the injection intestinal bacteria of 10 μ L deactivations and the micrococcus luteus of 20 μ L deactivations, experimental session, bait of not throwing something and feeding, whole experimentation does not cause death.0h, 2h, 4h, 8h, 12h, 24h, 48h after injection, get 5 shellfishes respectively as each time point sample for each group.5 shellfishes in each time point get the blood of equivalent respectively, after the centrifugal 5min of serum 800g got, remove supernatant, add 1mLTrizol, inhale beat and make its Eddy diffusion with rifle, extraction total serum IgE reverse transcription becomes cDNA, for quantitative fluorescence analysis.The primer of quantitative fluorescence analysis is with table 2.
After stimulating with the intestinal bacteria after sterilizing and micrococcus luteus, PmECSOD is at the content not identical (see Figure 12) in hemocyte each period.After induction 48h, the expression amount of PmCuZnSOD is the highest, and after comparing with blank group, discovery is 27.9 times (P<0.01) of control group, shows as pole significant difference; And when inducing 16h, PmECSOD expression amount in hemocyte is minimum, is about 0.22 times (P<0.05) of control group.
SEQUENCELISTING
<110> Guangdong Ocean University
<120> pteria martensii superoxide-dismutase PmECSOD and application thereof
<130>
<160>5
<170>PatentInversion3.3
<210>1
<211>473
<212>PRT
<213>PmECSOD aminoacid sequence
<400>1
MetTrpArgAlaThrGlyIleLeuLeuLeuAlaProIlePheValSer
151015
LeuSerValAlaGlnLysCysAsnMetValThrAspArgAsnCysLys
202530
ProTyrThrAsnTyrSerValValProAspThrSerGlySerArgThr
354045
AsnAlaMetPheSerLeuLeuAsnAsnValAsnCysGlyGluGluMet
505560
ArgSerPheLeuCysThrAlaMetTyrProSerCysAspPheProPro
65707580
LysMetProCysArgSerValCysLeuLysGlnLysThrIleCysGly
859095
ProArgLeuLysAlaLeuGlyIleGluTrpProPheAsnCysThrMet
100105110
PheGlnAspSerAsnAspAsnAsnValCysMetLeuAlaAspGlyPro
115120125
GlySerThrGlnProProLeuProThrProIleGlnThrMetAspIle
130135140
LeuGluGlyLysLysLeuArgLeuThrCysArgLysAlaAlaGlyLys
145150155160
SerValLysTrpTyrTyrAsnAspArgGlnLeuGlnLysThrLysArg
165170175
IleLysIleLysLysAsnValLeuLysIleAsnLysIleGlnThrSer
180185190
AspAlaGlyLeuTyrGluCysLysAspSerAsnGlyGlnValValAsp
195200205
IleThrTyrGlnValIleValAlaAlaLysThrAsnAlaProValLys
210215220
IleAspMetGlySerSerHisGlyGlnGlnGlySerArgAspAlaIle
225230235240
SerSerAsnLeuThrAlaLeuTrpGlyTyrValLysLysMetGluGln
245250255
AspPheGlyGluGlyGlyValHisLeuTyrMetAsnGlyAlaLysAla
260265270
ArgArgLysGlyAsnThrIleAspIleAsnLeuAsnValLysGlyAla
275280285
AspCysAspGluSerMetGlyAspAspAspGluGluArgTyrAlaThr
290295300
CysLeuValThrProAsnMetProMetAlaLysMetPheLysAlaSer
305310315320
HisArgValValGlyGlnIleArgLeuAlaGlnLysGlyArgLysAla
325330335
ProValGluMetAspIleHisLeuSerGlyPheAspValSerGlyPro
340345350
SerProGlnHisGluHisGlyPheHisIleHisGluTyrGlyGlyMet
355360365
GluAsnGlyCysGlnThrLeuGlyGlyHisPheAsnProGluLysVal
370375380
AsnHisGlyAlaProAspAlaLysGluArgHisHisGlyAspLeuGly
385390395400
AsnIleHisCysAspAsnPheGlyAsnSerAlaGluGluIleThrAsp
405410415
HisLysIleThrMetPheGlyArgAsnSerIleIleGlyArgSerPhe
420425430
ThrIleHisGluGlySerAspAspMetGlyLeuGlyGlyThrLysAla
435440445
SerLeuSerGlyGlyAsnAlaGlyThrArgIleAlaCysCysThrIle
450455460
AlaLeuSerAlaLysProSerGlyPro
465470
<210>2
<211>1846
<212>DNA
The cDNA full length sequence of <213> coding PmECSOD
<400>2
acatggggatccaggtttattccgtgccaggaaagcagcaagatgtggcgagcgactgga60
atacttcttttggcgccaatatttgtcagtttatctgtagcacagaagtgcaacatggtg120
acagacagaaactgcaaaccctataccaactatagtgttgtccccgacacctcaggatct180
cgcaccaacgccatgttttcacttcttaataatgtgaattgtggagaagaaatgagaagc240
tttctatgcaccgccatgtacccctcgtgcgatttcccgcccaaaatgccatgccgttcg300
gtatgtctcaaacagaaaacaatctgcggaccgcgtctgaaggcactcggtatagaatgg360
cccttcaactgtactatgttccaagattcaaacgacaacaacgtgtgtatgcttgctgat420
ggtcctggttccacccaacctcctctccccactccaatccaaacaatggatatactggag480
gggaaaaaactgaggctcacatgccgtaaagctgctggaaaatctgtcaagtggtactac540
aacgaccgacaactacaaaagacaaaaagaataaagataaagaagaacgtattgaaaata600
aacaagatacagacttcagacgctggtttatatgagtgtaaggacagcaatggtcaggta660
gtggatatcacgtatcaagttatagtggctgcaaaaacaaacgctccagttaaaatagat720
atggggtccagccatggtcaacaaggcagtcgggatgccatcagttccaacctgacagcg780
ttgtggggctatgtcaagaaaatggaacaagattttggagaaggaggagtgcacttatac840
atgaacggggctaaagccaggaggaaaggaaacactatagatataaacctcaacgtgaag900
ggcgctgattgtgatgaatctatgggagatgacgatgaagagagatatgcaacttgttta960
gttacacctaatatgccaatggcgaaaatgttcaaggccagccatcgggttgtagggcag1020
atcagactagcccaaaagggaagaaaagccccggttgagatggatatccacctgtctggg1080
tttgatgttagtggcccctcccctcagcacgaacatggattccacatccacgagtatgga1140
ggaatggaaaatggatgccagacactcggtggtcatttcaatcccgaaaaagtcaatcat1200
ggtgctccggatgccaaagaaagacaccatggcgacttagggaatattcactgtgataac1260
tttggtaattcagctgaagaaatcactgaccataaaatcacaatgttcggccgtaattcc1320
atcattggtcgatcatttacgattcacgagggaagtgatgacatgggtcttggcggcacg1380
aaagcaagtttgtcgggtggcaatgctggtaccaggatagcgtgctgtacaatagccctc1440
tctgctaaacctagtggtccttaaaggcagtggacatgcacatatgccaattggaatacg1500
cggcaattttttttctattacacgttaatatttctcgtcaccatatgatttatacatgta1560
tacgtgtgcattttttctaacaatttaaagccagtgctgaattctatgatattatttata1620
ctattttattactacatcgtagtattttgggtttgacgaatgtttgagttacgaagcatg1680
agactaaatagtcagaaattgaattctttatatgaacgaaaacactagcacaatatgtgc1740
aatacgaattgaaaatctaccatatatgtatacttctacatgtacatagtatttgttaaa1800
agcagaataaaccataaagaaaaaaaaaaaaaaaaaaaaaaaaaaa1846
<210>3
<211>1422
<212>DNA
The ORF sequence of <213> coding PmECSOD
<400>3
atgtggcgagcgactggaatacttcttttggcgccaatatttgtcagtttatctgtagca60
cagaagtgcaacatggtgacagacagaaactgcaaaccctataccaactatagtgttgtc120
cccgacacctcaggatctcgcaccaacgccatgttttcacttcttaataatgtgaattgt180
ggagaagaaatgagaagctttctatgcaccgccatgtacccctcgtgcgatttcccgccc240
aaaatgccatgccgttcggtatgtctcaaacagaaaacaatctgcggaccgcgtctgaag300
gcactcggtatagaatggcccttcaactgtactatgttccaagattcaaacgacaacaac360
gtgtgtatgcttgctgatggtcctggttccacccaacctcctctccccactccaatccaa420
acaatggatatactggaggggaaaaaactgaggctcacatgccgtaaagctgctggaaaa480
tctgtcaagtggtactacaacgaccgacaactacaaaagacaaaaagaataaagataaag540
aagaacgtattgaaaataaacaagatacagacttcagacgctggtttatatgagtgtaag600
gacagcaatggtcaggtagtggatatcacgtatcaagttatagtggctgcaaaaacaaac660
gctccagttaaaatagatatggggtccagccatggtcaacaaggcagtcgggatgccatc720
agttccaacctgacagcgttgtggggctatgtcaagaaaatggaacaagattttggagaa780
ggaggagtgcacttatacatgaacggggctaaagccaggaggaaaggaaacactatagat840
ataaacctcaacgtgaagggcgctgattgtgatgaatctatgggagatgacgatgaagag900
agatatgcaacttgtttagttacacctaatatgccaatggcgaaaatgttcaaggccagc960
catcgggttgtagggcagatcagactagcccaaaagggaagaaaagccccggttgagatg1020
gatatccacctgtctgggtttgatgttagtggcccctcccctcagcacgaacatggattc1080
cacatccacgagtatggaggaatggaaaatggatgccagacactcggtggtcatttcaat1140
cccgaaaaagtcaatcatggtgctccggatgccaaagaaagacaccatggcgacttaggg1200
aatattcactgtgataactttggtaattcagctgaagaaatcactgaccataaaatcaca1260
atgttcggccgtaattccatcattggtcgatcatttacgattcacgagggaagtgatgac1320
atgggtcttggcggcacgaaagcaagtttgtcgggtggcaatgctggtaccaggatagcg1380
tgctgtacaatagccctctctgctaaacctagtggtccttaa1422
<210>4
<211>22
<212>DNA
<213>PmECSOD-qPCR-F
<400>4
tccacctgtctgggtttgatgt22
<210>5
<211>22
<212>DNA
<213>PmECSOD-qPCR-R
<400>5
ccggagcaccatgattgacttt22
Claims (6)
1. a pteria martensii superoxide-dismutase PmECSOD, is characterized in that, its aminoacid sequence is as shown in SEQIDNO:1.
2. a pteria martensii superoxide-dismutase PmECSOD gene, is characterized in that, its nucleotide sequence is as shown in SEQIDNO:2 or 3.
3. pteria martensii superoxide-dismutase PmECSOD described in claim 1 is suppressing the purposes in intestinal bacteria and M. luteus bacteria growing.
4. the fluorescent quantitation primer of pteria martensii superoxide-dismutase PmECSOD gene described in a pair test right requirement 2, it is characterized in that, its nucleotide sequence is as shown in SEQIDNO:4 ~ 5.
5. comprise the recombinant vectors of pteria martensii superoxide-dismutase PmECSOD gene described in claim 2.
6. the recombinant bacterium containing recombinant vectors described in claim 5.
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| CN111690636A (en) * | 2020-06-24 | 2020-09-22 | 广东海洋大学 | Trypsin-like serine protease gene, encoded protein and application |
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Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "GenBank: KP689432.1", 《GENBANK》 * |
| M.W. JOHANSSON等: "A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish", 《JOURNAL OF CELL SCIENCE》 * |
| 朱家萍: "马氏珠母贝血清抗菌活性蛋白的分离纯化与鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 朱家萍等: "马氏珠母贝血清抗菌肽的初步分析", 《生命科学研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690636A (en) * | 2020-06-24 | 2020-09-22 | 广东海洋大学 | Trypsin-like serine protease gene, encoded protein and application |
| CN111690636B (en) * | 2020-06-24 | 2021-10-08 | 广东海洋大学 | Trypsin-like serine protease gene, encoded protein and application |
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