CN102477429A - Scylla paramamosain ETFB gene and clone, detection method and application thereof - Google Patents
Scylla paramamosain ETFB gene and clone, detection method and application thereof Download PDFInfo
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- CN102477429A CN102477429A CN201110382739XA CN201110382739A CN102477429A CN 102477429 A CN102477429 A CN 102477429A CN 201110382739X A CN201110382739X A CN 201110382739XA CN 201110382739 A CN201110382739 A CN 201110382739A CN 102477429 A CN102477429 A CN 102477429A
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Abstract
The invention relates to a gene sequence expressed in crab main metabolic organs, and particularly relates to a scylla paramamosain ETFB gene and a clone, a detection method and an application thereof. According to the invention, a cDNA full length sequence of a scylla paramamosain ETFB gene is obtained by cloning through 5'-RACE and 3'-RACE technology; the obtaining of the gene of the invention provides preconditions for subsequent gene function research through RNAi gene expression experiment, and research of protein products from transcription expression of the gene from the point of view of structural biology through obtaining protein with biological activity by eukaryotic expression purification, and finally lays a foundation for the illustration of the functions and the generation mechanism of the crab ETFB gene and its expressed protein products in mitochondrial electron transport chains and energy metabolism.
Description
Technical field
The present invention relates to the gene order expressed in the crab class main metabolic organ, more specifically relate to nucleotide sequence, aminoacid sequence and cloning process thereof, detection method and the application of a kind of Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB.
Background technology
Flavoprotein be one type with the composition of flavin nucleotide as coenzyme or prothetic group, participation mitochondrial respiratory chain, can accept and provide two protons and two electronics through coenzyme or prothetic group, play the effect of oxydo-reductase or electron transit mediator.Electron transfer flavoprotein (Electron Transfer Flavoprotein, ETF) be one type with flavin adenine dinucleotide (flavin adenine dinucleotide FAD) is the protein of coenzyme.At present ETF is successfully from Mammals such as people, mouse and pig, and in some bacteriums as Paracoccus denitrificans, Megasphaera elsdenii and food methyl have a liking in the methyl bacterium evaluation and come out.In these biologies; ETF is that the β subunit by the α subunit of 30KDa and 28KDa constitutes heterodimer; Each subunit comprises a flavin adenine dinucleotide (flavin adenine dinucleotide; FAD) and amp (Adenosine Monophosphate, AMP).ETF is the important electron transit mediator of at least 11 kinds of desaturases in plastosome and bacterium alienation approach and the respiratory chain; Typical case's approach produces proton potential energy for through ETF-ubiquinone oxide-reductase enzyme electronics is transferred to ubiquinone from initial desaturase to guarantee the electron transport chain continued running.
The required energy metabolism of crab class growth receives the adjusting of multiple internal and external factor, and after all, it is the regulating and controlling that receives genes involved.In relevant mammals research, show: ETF is the nuclear gene group coding; Transferring to the plastosome processing treatment after in tenuigenin, synthesizing is mature peptide; Through ETF-ubiquinone oxide-reductase enzyme with transfer transport to ubiquinone; Make multiple one-level desaturase and ubiquinone-Lrax get in touch, thereby guarantee that the running of electron transport chain generates institute's energy requirement of vital movement.Similar mechanism is also found in research in bacterium.In the crab class, also do not identify the electron transfer flavoprotein gene at present, if can find the gene of getting in touch in the respiratory chain in transfer transport and the desaturase; Then multiple research means be can pass through, (RNAi), gene overexpression disturbed like RNA; Promoter Analysis; Eukaryotic expression and the separation and purification of tool biological activity protein, external biochemical function analysis, thereby for illustrating function and the mechanism based theoretical thereof of crab class ETFB gene in electron transport mechanism and energy metabolism.
Summary of the invention
The present invention aims to provide a kind of 5 '-RACE and 3 '-RACE technology of passing through and clones and obtain Scylla paramamosain ETFB gene, and cloning process, detection method and the application of said ETFB gene also are provided.
For realizing goal of the invention of the present invention, the contriver provides following technical scheme:
A kind of Scylla paramamosain ETFB gene has the nucleotide sequence shown in SEQ ID No. 1.
A kind of Scylla paramamosain ETFB gene, its encoded protein matter has the aminoacid sequence shown in SEQ ID No.2.
Scylla paramamosain electron transfer flavoprotein beta subunit gene of the present invention (being Scylla paramamosain ETFB gene) is a kind of electron transfer flavoprotein beta subunit gene that from the liver pancreas of Scylla paramamosain, identifies.Described electron transfer flavoprotein beta subunit gene is that its cDNA nucleotide sequence and encoded protein matter sequence are following through the gene that 5 '-RACE and 3 '-RACE technology clone obtains.This gene cDNA total length 1030bp; Is its ORFs from 18bp to the 782bp section; 254 amino acid of encoding, the long 17bp of 5 ' end non-coding region, 3 ' holds the long 248bp of non-coding region; Comprising 1 mRNA partly declines and regulates motif (ATTTA), polyadenylic acid tailing signal AATAAA and polyadenylic acid tail.In GenBank, carry out the Blast homology and measure, confirm that this cDNA coded electronic shifts the flavoprotein beta subunit gene.
Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB nucleotide sequence is (SEQ ID NO.1) as follows:
ggccattcaccgatacaATGTCTCTCCGGGTACTTGTTGGCGTCAAGAGAGTGATTGATTATGCCGTGAAGATTCGGGTCCGGCCAGACAAGCTTGGGGTGGTGACAGATGGCGTGAAACACTCCATGAACCCTTTCGATGAGATAGCCATTGAGGAGGCCGTGCGGCTCAAGGAGAAGAAGATTGCAAAGGAGGTGGTGGCTGTGTCATGTGGCCCAACCCAGGCACAGGAGACCATCCGCACTGCCCTGGCCATGGGGGCTGACAGAGGTATCCACGTAGAGATTCCAGCCGAGGAGGTTGAGAAACTAGAACCATTGCATGTAGCCAAGATTATGGCCAAGCTGGTGGAGCAGGAGAAGGCTGACTTAGTTGTGCTTGGCAAACAGGCTATTGATGATGACTCCAATGCCACTGCCCAGATGACAGCTTCCATCCTTGACTGGCCTCAGGCAACCTTTGCTTCCAAGATTGAGAAGACTGATGGTGAGCTGCAGGTGACAAGAGAGGTGGATGGAGGTCTGGAGACAATCAAAGTGAAGTTGCCAGCTGTGGTCTCAGCTGATCTCCGCCTCAATGAGCCCCGATATGCCACCCTGCCCAACATCATGAAAGCTAAGAAGAAGAAAATTGCTAAGATGAAGGCTGCTGACCTTGGAGTAGATACAACTTCTCACTTTGAAGTTTTGGAGGTGGCTGATCCACCTGTGAGGGAGGCTGGCATCAAAGTGGAGGATGTGGACACTCTTATTACAAAGTTGAAGGAGACTGGGCGCATTTAGcacagttaatcggggacgtacagtgtgactgtggttggaggaattgcaccatacctgtacacctgccaccaatcaatgcaatatacctattttctttgtgggcaagctatccttaatgaatgtgctacttttcagagatttctttaatacacaatctgtatttatattttcttgtacaaaacattctatagaaacaggtttaataaaatatataaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。
Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB amino acid sequence coded is (SEQ ID NO.2) as follows:
MSLRVLVGVKRVIDYAVKIRVRPDKLGVVTDGVKHSMNPFDEIAIEEAVRLKEKKIAKEVVAVSCGPTQAQETIRTALAMGADRGIHVEIPAEEVEKLEPLHVAKIMAKLVEQEKADLVVLGKQAIDDDSNATAQMTASILDWPQATFASKIEKTDGELQVTREVDGGLETIKVKLPAVVSADLRLNEPRYATLPNIMKAKKKKIAKMKAADLGVDTTSHFEVLEVADPPVREAGIKVEDVDTLITKLKETGRI。
The present invention also provides a kind of cloning process of Scylla paramamosain ETFB gene; Described cloning process is for utilizing 5 '-RACE and 3 '-RACE technology clone Scylla paramamosain electron transfer flavoprotein beta subunit gene cDNA total length; Comprise that with Scylla paramamosain liver pancreas reverse transcription product be template; Utilize 3-GP1 and 3-GP2 primer to carry out 3 '-RACE reaction, amplification obtains the 3 ' terminal sequence of Scylla paramamosain ETFB; Carry out 5 '-RACE reaction with 5-GP1 and 5-GP2 primer, amplification obtains the 5 ' sequence of ETFB, and wherein: described 3-GP1 primer has the nucleotide sequence shown in SEQ ID No.3, and the 3-GP2 primer has the nucleotide sequence shown in SEQ ID No.4; The 5-GP1 primer has the nucleotide sequence shown in SEQ ID No.5, and the 5-GP2 primer has the nucleotide sequence shown in SEQ ID No.6.
The present invention also provides a kind of detection method of Scylla paramamosain ETFB gene; Described detection method comprises at least uses a pair of sxemiquantitative PCR primer; Described a pair of sxemiquantitative PCR primer is made up of upstream primer and downstream primer; Wherein, described sxemiquantitative PCR upstream primer (ETFB-RT-F) has the nucleotide sequence shown in SEQ ID NO.7; Sxemiquantitative PCR downstream primer (ETFB-RT-R) has the nucleotide sequence shown in SEQ ID NO.8.
The present invention also provides the application of described a kind of Scylla paramamosain ETFB gene in the running of Scylla paramamosain plastosome electron transport chain.
The present invention is through the homologous clone strategy based on 5 '-RACE and 3 '-RACE technology, and pcr amplification order-checking and Blast compare, and are cloned into the full length sequence of Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB; Through to Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB design effectively expressing primer, thereby utilize the sxemiquantitative round pcr to analyze the expression map of said gene in healthy tissues.The contriver has used sxemiquantitative RT-PCR technology for detection ETFB gene express spectra in four major organs (liver pancreas, ovary, heart and muscle) in Scylla paramamosain, the result is presented at the ETFB genetic expression that all has higher level in four organs that detected.
Compared with prior art, the present invention has following advantage:
The acquisition of gene of the present invention can be the follow-up RNAi that passes through; Genetic expression experimental study gene function and the protein that passes through eukaryotic expression purifying acquisition biologically active; The protein product of studying said gene transcript expression from the structure biology angle provides precondition, and final for illustrating crab class ETFB gene and the expressed proteins product lays the foundation in the function and the mechanism thereof of plastosome electron transport chain and energy metabolism.
Description of drawings
Fig. 1 detects the distribution and expression of Scylla paramamosain electron transfer flavoprotein beta subunit gene ETFB in four tissues for sxemiquantitative RT-PCR.
Embodiment
Below in conjunction with embodiment and Figure of description, content of the present invention is described more specifically.Should be appreciated that enforcement of the present invention is not limited to following embodiment, all will fall into protection domain of the present invention any pro forma accommodation and/or the change that the present invention made.
In the present invention, if not refer in particular to, all part, per-cents are weight unit, and all equipment and raw material etc. all can be buied from market or the industry is commonly used.Do not specialize if having, the method that embodiment adopts is this area current techique.
Main raw, reagent and plant and instrument: scalpel, eye scissors, tweezers, Dissecting tray, oxygen charging pump, water tank, 1.5ml centrifuge tube, micropipet, micropipet rifle head, disposable petridish, ceramic mortar.Deoxyribonucleotide dNTP, thermopolymerization Taq enzyme, Trnzol-A
+The total RNA extraction reagent box, 5 '-Full RACE Kit (TaKaRa), 3 '-Full RACE Core Set Ver.2.0 (TaKaRa), Quant cDNA first chain synthetic agent box and DH5 α competent cell, compact centrifuge (Qspin
TM, BAYGENE), and the vortex vibrator (the QL-866 type, QILINEBEIER), nucleic acid-protein appearance (SmartSpace
TMPlus; Bio-Rad); Bechtop (SW-CJ-1G type, the star of famous brand), constant incubator (PH070A type; Shanghai Yiheng Scientific Instruments Co., Ltd);-80 ℃ of Ultralow Temperature Freezers (Thermo scientific), quantitative fluorescent PCR system (ABI 7300 types), pH meter (Mettler Toledo 320PH Meter), autoclaving pot (SANYO), high speed freezing centrifuge (Centrifuge 5417R; Eppendorf), electrophoresis apparatus (DYY-6C type, Beijing 6 1), water-bath (it is grand to go up the Nereid), gel imaging system (Bio-Rad GD2000), PCR appearance (ABI Veriti 96well Thermal Cycler), automatic DNA sequencer DNA (ABI 3730 types).
The conventional medicine chloroform (analytical pure) that the embodiment of the invention is used, Virahol (analytical pure), paraxin; Sodium-chlor (analytical pure) is available from traditional Chinese medicines group, isopropylthio-(IPTG), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal); Tryptones, yeast extract, agarose; Ethidium bromide, diethylpyrocarbonates (DEPC) etc. are available from Takara company, and liquid nitrogen is Zhoushan, Zhejiang Province hundred million foreign flavour body ltd products; Deoxyribonucleotide dNTP, thermopolymerization Taq enzyme, Trnzol-A
+The total RNA extraction reagent box, RealMasterMix (SYBRGreen) reagent, Quant cDNA first chain synthetic agent box and DH5 α competent cell are available from TIANGEN company, SMART
TMCDNA Library Construction Kit is available from Clontech company, and the plasmid library order-checking is accomplished by the big genome company of China, and primer is synthetic by Nanjing Genscript Biotechnology Co., Ltd..
The experiment sample Scylla paramamosain is available from Dinghai District Nan Zhen food market, Zhoushan, Zhejiang Province city.
The experimental technique of unreceipted actual conditions among the embodiment; Be according to normal condition; Author's such as Sambrook molecular cloning laboratory manual (New York:Cold Spring Harbor Laboratory Press for example; 1989) condition described in, or according to the condition of manufacturer's specification proposes.
Embodiment 1
1. utilize Trnzol-A
+Total RNA extraction reagent box (TIANGEN) extracts the total RNA of Scylla paramamosain liver pancreas.
Get healthy Scylla paramamosain (body weight is about 500g) after the oxygenation water tank is cultured 7, dissect the crab body and take out liver pancreas, ovary, heart and muscle, it is subsequent use that liquid nitrogen flash freezer is put into-80 ℃ of refrigerators.Get the Trnzol-A that the subsequent use about 100mg of liver pancreas tissue adds 1ml after with liquid nitrogen grinding respectively
+Reagent homogenate (TIANGEN) is extracted total RNA according to experimental technique shown in this test kit operational manual.Get total RNA that 2 μ l extract, use spectrophotometer and electrophoresis detection total rna concentration and quality respectively.
2. utilize 5 '-RACE and 3 '-RACE technology clone Scylla paramamosain electron transfer flavoprotein beta subunit gene cDNA full length sequence.
Through comparing the nucleotide sequence of other species electron transfer flavoprotein beta subunit genes ETFB, find conservative section, as stencil design 5-GP1/5-GP2 primer and 3-GP1/3-GP2 primer.5 '-Full RACE Kit (TaKaRa) and 3 '-Full RACE Core Set Ver.2.0 (TaKaRa) test kit are used in 5 '-RACE and 3 '-RACE experiment.Concrete operation method is seen the test kit specification sheets, briefly as follows:
With Scylla paramamosain liver pancreas reverse transcription product is template; Utilize 3-GP1:ATGGGBGCWGACMGAGGYATCCACGT (SEQ ID No.3) and 3-GP2:TGCCCAACATCATGAAAGCYAAGAAGA (SEQ ID No.4) to carry out 3 '-RACE reaction, amplification obtains the 3 ' terminal sequence of ETFB; Carry out 5 '-RACE reaction with 5-GP1:CCTTTGATGCCAGCCTCCCTCACAGGT (SEQ ID No.5) and 5-GP2: GGAGATCAGCTGAGACCACAGCTGGC (SEQ ID No.6), amplification obtains the 5 ' sequence of ETFB.
3. amplification is obtained order-checking of RACE-PCR product cloning and splicing acquisition ETFB gene cDNA full length sequence.
Agarose gel electrophoresis with 1.% detects amplified production and utilizes sepharose DNA to reclaim test kit (TIANGEN) purifying and recovering PCR product.The PCR product of purifying and recovering connected under 4 ℃ of conditions with pGEM-T carrier (Promega) spend the night the preparation of linked system by specification.The connection product that will spend the night is transformed in the DH5 α competent cell.Be coated with flat board and (include paraxin, isopropylthio-(IPTG) and 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal), overnight cultures in 37 ℃ of constant incubators.Contain cDNA through the screening of blue hickie and insert segmental positive recombinant, each mono-clonal of picking is inoculated into respectively in the LB nutrient solution of 1ml chlorampenicol resistant, 37 ℃ were shaken bacterium 2 hours, and bacterium liquid PCR confirms positive colony, rejects false positive clone.To pass through positive colony of bacterium liquid PCR evaluation and send the order-checking of the big genome company of China, order-checking is carried out on ABI 3730 sequenators.5 ' terminal sequence of gained and 3 ' terminal sequence spliced in MEGA4.1 software (biosoftware net, http://www.bio-soft.net/) obtain ETFB gene cDNA full length sequence.The result is shown in SEQ ID NO.1 and SEQ ID NO.2.
The institute's calling sequence that will check order carries out the Blast homology in GenBank measures, and identifies Scylla paramamosain electron transfer flavoprotein beta subunit gene, this gene cDNA total length 1030bp; Is its ORFs from 18bp to the 782bp section; 254 amino acid of encoding, the long 17bp of 5 ' end non-coding region, 3 ' holds the long 248bp of non-coding region; Comprising 1 mRNA partly declines and regulates motif (ATTTA), polyadenylic acid tailing signal AATAAA and polyadenylic acid tail.
4. the semi-quantitative expressed collection of illustrative plates of ETFB gene in the healthy tissues.
For detecting the distribution situation of ETFB gene in normal Scylla paramamosain main metabolic organ, take out each the about 100mg of four tissues (liver pancreas, ovary, heart and muscle) that preserves respectively, use Trnzol-A
+Total RNA extraction reagent box (TIANGEN) extracted total RNA.Get the total RNA of 1 μ g with synthetic cDNA first chain of the Quant cDNA first chain synthetic agent box, as the template of sxemiquantitative RT-PCR.ETFB designs a pair of special primer according to Scylla paramamosain electron transfer flavoprotein beta subunit gene, sxemiquantitative PCR upstream primer (ETFB-RT-F): TTCCAGCCGAGGAGGTTGAG (SEQ ID No.7); Sxemiquantitative PCR downstream primer (ETFB-RT-R): TGTTGGGCAGGGTGGCATAT (SEQ ID No.8) increases to the cDNA of ETFB gene; (this sequence is included the data in GenBank as internal control gene according to Scylla paramamosain β-actin gene of having reported; Accession number is HM217821), design a pair of special primer (upstream primer (β-actin-RT-F): TCACCAACTGGGACGACATG (SEQ ID No.9); Downstream primer (β-actin-RT-R): ATAGCGTGAGGAAGGGCATA (SEQ ID No.10).Sxemiquantitative RT-PCR reaction system is: ddH
2O 17.3 μ l, 10 * PCR Buffer (TIAGEN), 2.5 μ l, dNTP (2.5mM, TIANGEN) 2 μ l, each 1 μ l of upstream and downstream primer, cDNA template 1 μ l, (the reaction TV is 25 μ l to the Taq enzyme for 5U/ μ l, TIANGEN) 0.2 μ l.Reaction conditions is set to: 95 ℃, and 3 minutes; 95 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, 30 seconds, totally 30 circulations; 72 ℃, 5 minutes.After reaction finishes, get 5 μ l PCR products with 1.5% the agarose gel electrophoresis detection and the preservation of taking pictures.
The contriver has used sxemiquantitative RT-PCR technology for detection ETFB gene express spectra in four major organs in Scylla paramamosain; The result is as shown in Figure 1; The ETFB genetic expression that in four organs that detected, has higher level; The ETFB gene that this prompting is cloned into is really brought into play continuous action in four major organs of Scylla paramamosain, thereby possesses higher transcriptional activity and transcriptional level.
Although the contriver has done comparatively detailed elaboration to technical scheme of the present invention and has enumerated; Be to be understood that; For the those skilled in the art in this area, be obvious to the replacement scheme that the foregoing description modifies and/or flexible perhaps employing is equal to, all can not break away from the essence of spirit of the present invention; The term that occurs among the present invention is used for can not being construed as limiting the invention the elaboration of technical scheme of the present invention and understanding.
SEQUENCE LISTING
< 110>Oceanography Institute Of Zhejiang
< 120>a kind of Scylla paramamosain ETFB gene and clone, detection method and application
<130> Z111587
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<170> PatentIn version 3.3
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ggccattcac cgatacaatg tctctccggg tacttgttgg cgtcaagaga gtgattgatt 60
atgccgtgaa gattcgggtc cggccagaca agcttggggt ggtgacagat ggcgtgaaac 120
actccatgaa ccctttcgat gagatagcca ttgaggaggc cgtgcggctc aaggagaaga 180
agattgcaaa ggaggtggtg gctgtgtcat gtggcccaac ccaggcacag gagaccatcc 240
gcactgccct ggccatgggg gctgacagag gtatccacgt agagattcca gccgaggagg 300
ttgagaaact agaaccattg catgtagcca agattatggc caagctggtg gagcaggaga 360
aggctgactt agttgtgctt ggcaaacagg ctattgatga tgactccaat gccactgccc 420
agatgacagc ttccatcctt gactggcctc aggcaacctt tgcttccaag attgagaaga 480
ctgatggtga gctgcaggtg acaagagagg tggatggagg tctggagaca atcaaagtga 540
agttgccagc tgtggtctca gctgatctcc gcctcaatga gccccgatat gccaccctgc 600
ccaacatcat gaaagctaag aagaagaaaa ttgctaagat gaaggctgct gaccttggag 660
tagatacaac ttctcacttt gaagttttgg aggtggctga tccacctgtg agggaggctg 720
gcatcaaagt ggaggatgtg gacactctta ttacaaagtt gaaggagact gggcgcattt 780
agcacagtta atcggggacg tacagtgtga ctgtggttgg aggaattgca ccatacctgt 840
acacctgcca ccaatcaatg caatatacct attttctttg tgggcaagct atccttaatg 900
aatgtgctac ttttcagaga tttctttaat acacaatctg tatttatatt ttcttgtaca 960
aaacattcta tagaaacagg tttaataaaa tatataaaac aaaaaaaaaa aaaaaaaaaa 1020
aaaaaaaaaa 1030
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Met Ser Leu Arg Val Leu Val Gly Val Lys Arg Val Ile Asp Tyr Ala
1 5 10 15
Val Lys Ile Arg Val Arg Pro Asp Lys Leu Gly Val Val Thr Asp Gly
20 25 30
Val Lys His Ser Met Asn Pro Phe Asp Glu Ile Ala Ile Glu Glu Ala
35 40 45
Val Arg Leu Lys Glu Lys Lys Ile Ala Lys Glu Val Val Ala Val Ser
50 55 60
Cys Gly Pro Thr Gln Ala Gln Glu Thr Ile Arg Thr Ala Leu Ala Met
65 70 75 80
Gly Ala Asp Arg Gly Ile His Val Glu Ile Pro Ala Glu Glu Val Glu
85 90 95
Lys Leu Glu Pro Leu His Val Ala Lys Ile Met Ala Lys Leu Val Glu
100 105 110
Gln Glu Lys Ala Asp Leu Val Val Leu Gly Lys Gln Ala Ile Asp Asp
115 120 125
Asp Ser Asn Ala Thr Ala Gln Met Thr Ala Ser Ile Leu Asp Trp Pro
130 135 140
Gln Ala Thr Phe Ala Ser Lys Ile Glu Lys Thr Asp Gly Glu Leu Gln
145 150 155 160
Val Thr Arg Glu Val Asp Gly Gly Leu Glu Thr Ile Lys Val Lys Leu
165 170 175
Pro Ala Val Val Ser Ala Asp Leu Arg Leu Asn Glu Pro Arg Tyr Ala
180 185 190
Thr Leu Pro Asn Ile Met Lys Ala Lys Lys Lys Lys Ile Ala Lys Met
195 200 205
Lys Ala Ala Asp Leu Gly Val Asp Thr Thr Ser His Phe Glu Val Leu
210 215 220
Glu Val Ala Asp Pro Pro Val Arg Glu Ala Gly Ile Lys Val Glu Asp
225 230 235 240
Val Asp Thr Leu Ile Thr Lys Leu Lys Glu Thr Gly Arg Ile
245 250
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ttccagccga ggaggttgag 20
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Claims (5)
1. a Scylla paramamosain ETFB gene has the nucleotide sequence shown in SEQ ID No. 1.
2. Scylla paramamosain ETFB gene, its encoded protein matter has the aminoacid sequence shown in SEQ ID No.2.
3. the cloning process of a Scylla paramamosain ETFB gene; It is characterized in that; Described cloning process is for utilizing 5 '-RACE and 3 '-RACE technology clone Scylla paramamosain electron transfer flavoprotein beta subunit gene cDNA total length; Comprise that with Scylla paramamosain liver pancreas reverse transcription product be template, utilize 3-GP1 and 3-GP2 primer to carry out 3 '-RACE reaction, amplification obtains the 3 ' terminal sequence of Scylla paramamosain ETFB; Carry out 5 '-RACE reaction with 5-GP1 and 5-GP2 primer, amplification obtains the 5 ' sequence of ETFB, and wherein: described 3-GP1 primer has the nucleotide sequence shown in SEQ ID No.3, and the 3-GP2 primer has the nucleotide sequence shown in SEQ ID No.4; The 5-GP1 primer has the nucleotide sequence shown in SEQ ID No.5, and the 5-GP2 primer has the nucleotide sequence shown in SEQ ID No.6.
4. the detection method of a Scylla paramamosain ETFB gene; It is characterized in that; Described detection method comprises at least uses a pair of sxemiquantitative PCR primer; Described a pair of sxemiquantitative PCR primer is made up of upstream primer and downstream primer, and wherein, described sxemiquantitative PCR upstream primer has the nucleotide sequence shown in SEQ ID NO.7; Sxemiquantitative PCR downstream primer has the nucleotide sequence shown in SEQ ID NO.8.
5. according to claim 1 or claim 2 the application of a kind of Scylla paramamosain ETFB gene in Scylla paramamosain plastosome electron transport chain running.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667320A (en) * | 2013-11-04 | 2014-03-26 | 内蒙古科技大学 | Scylla paramamosain peroxy gene Sp-PX, amino acid sequence encoded by using scylla paramamosain peroxy gene Sp-PX and clone method |
| CN115058430A (en) * | 2022-06-12 | 2022-09-16 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
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|---|---|---|---|---|
| WO2002002580A2 (en) * | 2000-07-05 | 2002-01-10 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the etfb gene |
Non-Patent Citations (2)
| Title |
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| CN115058430A (en) * | 2022-06-12 | 2022-09-16 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
| CN115058430B (en) * | 2022-06-12 | 2023-06-09 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
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