CN1563379A - Gene order of b subtype of 4 receptor of hydroxytryptarmine in pig - Google Patents
Gene order of b subtype of 4 receptor of hydroxytryptarmine in pig Download PDFInfo
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Abstract
This invention relates to a serotonin 4 receptor b subtype gene sequence of pigs amplifying a serotonin 4 receptor b subtype gene nucleotide sequence form a master RNA of a pig's connective tissue, the tolal length of which is 1209bp, the open reading frame 1bp-1209bp segments coded with 402 amino acid protein, the molecular weight is 44960 dalton, pl is 8.08, which is important to the regulation of stomach intestine movement of pigs and an important standby gene for studying pig stress diarrhea and pig molecular marked assistant breeding.
Description
Technical field
What the present invention relates to is the gene order of the five hydroxytryptamine 4 acceptor b hypotypes of a kind of albumen coded sequence that is used for gene engineering technology field, particularly a boar.
Background technology
No matter on physianthropy still was animal medicine, irritability diarrhoea all was common clinical disease, and diarrhea of weaned piglets promptly is a representative instance of animal irritability diarrhoea.Although century more than one has been experienced in understanding and research to irritability diarrhoea, and is still very unclear to its cause of disease, pathogeny at present.In recent years, some scholars explore neurotransmitter five hydroxytryptamine and the five hydroxytryptamine 4 acceptors biological effect in mediation irritability diarrhoea one after another both at home and abroad.
(5-hydroxytryptamine receptor 4 5-HT4R) is a class bind receptor of neurotransmitter five hydroxytryptamine to five hydroxytryptamine 4 acceptors, and it belongs to G protein coupling receptor superfamily, extensively is distributed in mammiferous central nervous system and unifies in the peripheral tissues.It mainly is present among Auerbach's plexus neurone, smooth muscle cell and the enterochromaffin cell in Digestive tract, can combine with the five hydroxytryptamine in enterochromaffin cell, the enteric nervous unit, mediate the diastole and the contraction of the level and smooth muscle bundle of ring-type, cause the liquid secretion of small intestine and colon.Five hydroxytryptamine 4 acceptors also can mediate the release of some neurotransmitter in the enteron aisle, comprise vagusstoff, P material, vasoactive intestinal peptide and the calcitonin-gene-related peptide that stimulates the wriggling reflection, and these materials are playing an important role aspect the adjusting gastrointestinal tract dynamia.Thereby five hydroxytryptamine 4 acceptors and gastrointestinal tract dynamia are regulated substantial connection are arranged.Five hydroxytryptamine 4 acceptors are found (1988) at first in cavy, in research subsequently, successively proved that there are several hypotypes in five hydroxytryptamine 4 acceptor genes in different plant species, comprise comparatively clearly 9 kinds of five hydroxytryptamine 4 acceptor a hypotypes, b hypotype, c hypotype, d hypotype, e hypotype, f hypotype, g hypotype, n hypotype, h hypotypes etc., the expression amount of several hypotypes in different tissues has than big-difference.
In retrieval and analysis to the prior art document, " though FEBS Letters; 1995; 370:215-221 " (Expression of serotonin receptor mRNAs in blood vessels, the expression of seretonine receptor mRNA in blood vessel) clone of the method by RT-PCR has obtained the part coding protein sequence of five hydroxytryptamine 4 acceptors of pig, and five hydroxytryptamine 4 acceptor genes have been analyzed the people, mouse, expression rule on the different plant species such as pig, but find as yet so far that the clone obtains the complete coding protein sequence of pig five hydroxytryptamine 4 acceptor genes and to the report of Mammals five hydroxytryptamine 4 acceptor gene evolution rule researchs.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, the gene order of a boar five hydroxytryptamine 4 acceptor b hypotypes is provided.Make its albumen that can pass through this gene of preparation on the one hand, pig gastrointestinal motility function is regulated the important regulating and controlling effect; Can provide fundamental basis for the animal model of setting up irritability diarrhoea on the other hand, and can play the directiveness effect, have very big using value for the pig molecule mark assistant breeding.
The present invention is achieved by the following technical solutions, the cDNA sequence of the pig five hydroxytryptamine 4 acceptor b subtype gene among the present invention is by being template with total RNA in the colon of pig, three pairs of primers of homologous sequence design of the five hydroxytryptamine 4 acceptor b subtype gene of reference men and women, mouse, carry out reverse transcription PCR, the clone obtains the partial sequence of three new genes.Utilization biological software DNASTAR splices the overlap of three partial sequences, thereby obtains complete new gene order.The pig five hydroxytryptamine 4 acceptor b subtype gene cDNA sequences that obtain are seen SEQ ID NO.7.The protein coding sequence that encoding sequence (CDS) in the pig five hydroxytryptamine 4 acceptor b subtype gene cDNA sequences is translated into by universal code is seen SEQ ID NO.7.
The nucleotide sequence of the five hydroxytryptamine 4 acceptor b subtype gene that the present invention amplifies from the total RNA of the colon of pig, this full length gene 1209bp, open reading frame lbp-1209bp section, coding has 402 amino acid whose protein, molecular weight is 44960 dalton, and iso-electric point (pI) is 8.08.Be spliced by three RT-PCR products, length is respectively 445bp, 407bp, 952bp, and electrophoresis result is seen accompanying drawing 1,2,3, and the cDNA splicing the results are shown in accompanying drawing 4.In SEQ ID NO.7, wherein the 1-3 position is initiator codon ATG, and the 1207-1209 position is terminator codon TAG, and the 1-1209 position is coded protein zone (CDS).
The nucleotide sequence of pig five hydroxytryptamine 4 acceptor b subtype gene is as follows:
1 ATGGACAAAC?TTGATGCTAA?TGGGAGTTCC?AAGGAGGGTT?TCGGGTCCGT?GGAGAAGGTC
61 GTGTTGCTCA?CGTTTGTCTC?GGCGGTTATC?CTCATGGCTG?TCTTGGGGAA?CCTGCTGGTG
121 ATGGTGGCTG?TGTGCAGGGA?CCGGCAGCTC?AGGAAAATCA?AAACCAATTA?CTTCATTGTG
181 TCTCTTGCCT?TTGCGGATCT?GCTGGTGTCC?GTGCTGGTGA?TGCCCTTTGG?AGCCATTGAG
241 CTGGTCCAGG?ACGTCTGGAT?TTATGGGGAG?ATGTTCTGCC?TCGTCCGGAC?GTCTCTGGAT
301 GTCCTGCTCA?CGACGGCATC?GATTTTTCAC?CTGTGCTGCA?TCTCTCTGGA?CAGGTACTAC
361 GCCATCTGCT?GCCAGCCTCT?GGTCTACAGG?AACAAGATGA?CCCCTCTGCG?CGTGGCGGTG
421 CTGCTCGCAG?GCTGCTGGGC?CATCCCCGTG?CTGATCTCCT?TTCTCCCCAT?AATGCAAGGC
481 TGGAATAACA?TCGGCATAAC?TGACCTGGAA?AGGACATCCA?AACCAAGACT?GGGCCAGGAT
541 TTGCATGTGA?TAGAAAAAAG?GAAGTTCCAC?CAGAATTCGA?ACTCTACGTA?CTGTATCTTC
601 ATGGTCAACA?AGCCCTACGC?CATCACCTGC?TCTGTGGTGG?CCTTCTACAT?CCCGTTCCTC
661 CTCATGGTGC?TGGCCTATTG?GCGCATCTAC?GTGACAGCTA?AGGAGCACGC?CCACCAGATC
721 CAGATGTTGC?AACGGGCAGG?GGCCCCCGCA?GAGGGCAGGC?CTCCGTCCGC?AGACCAGCAC
781 AGCACCCATC?GCATGAGGAC?AGAGACCAAA?GCAGCCAAGA?CCCTGTGCGT?CATCATGGGT
841 TGCTTCTGCC?TCTGCTGGGC?TCCGTTCTTC?GTCACCAATG?TCGTGGACCC?TTTCGCAGAC
901 TACTCTGTCC?CCGGGCAGGT?GTGGACCGCT?TTCCTCTGGC?TCGGCTACAT?CAATTCCGGG
961 TTGAACCCCT?TTCTCTACGC?CTTCCTGAAT?AAGTCTTTCA?GACGTGCCTT?CCTCATCATC
1021 CTCTGCTGCG?ATGATGAGCG?CTACCGAAGA?CCTTGCGTGG?CGGGCCAGAC?GGTCCCCTGT
1081 TCAACCACAA?CCGTCAACGG?ATCCACTCAT?GTGCTAAGGG?ATGCAGTGGA?GTGTGGTGGC
1141 CAGTGGGAGA?GTCAGTGCCA?CCCGCCAGCA?ACTTCTCCTT?TGGTGGCTGC?TCAGCCCAGT
1201 GACACTTAG
402 amino acid of pig five hydroxytryptamine 4 acceptor b subtype gene coding among the present invention, molecular weight is 44960 dalton, iso-electric point (pI) is 8.08.
The aminoacid sequence of pig five hydroxytryptamine 4 acceptor b hypotypes is as follows:
1 MDKLDANGSS?KEGFGSVEKV?VLLTFVSAVI?LMAVLGNLLV?MVAVCRDRQL?RKIKTNYFIV
61 SLAFADLLVS?VLVMPFGAIE?LVQDVWIYGE?MFCLVRTSLD?VLLTTASIFH?LCCISLDRYY
121 AICCQPLVYR?NKNTPLRVAV?LLAGCWAIPV?LISFLPIMQG?WNNIGITDLE?RTSKPRLGQD
181 LHVIEKRKFH?QNSNSTYCIF?MVNKPYAITC?SVVAFYIPFL?LMVLAYWRIY?VTAKEHAHQI
241 QMLQRAGAPA?EGRPPSADQH?STHRMRTETK?AAKTLCVIMG?CFCLCWAPFF?VTNVVDPFAD
301 YSVPGQVWTA?FLWLGYINSG?LNPFLYAFLN?KSFRRAFLII?LCCDDERYRR?PCVAGQTVPC
361 STTTVNGSTH?VLRDAVECGG?QWESQCHPPA?TSPLVAAQPS?DT
Pig five hydroxytryptamine 4 acceptor b subtype gene among the present invention are searched homologous sequence in GenBank Nucleotide database, find that the nucleotide sequence (CDS) of this gene coded protein has the homology of height with other mammiferous homologous gene.Portion C DS sequence homology with known two pig five hydroxytryptamine 4 acceptor genes among the GenBank is 100% (Z48176, Z48175); Five hydroxytryptamine 4 acceptor b subtype C DS sequence homologies with the people are 91% (AJ278980); Homology with rat is 79.49% (U20907); Homology with mouse is 84.53% (Y09585); Homology with dog is 89.99% (AX068008); Homology with cavy is 84.86% (Y13585).
Pig five hydroxytryptamine 4 acceptor b subtype gene among the present invention are searched homologous sequence in the GenBank Protein Data Bank, find that the aminoacid sequence of this genes encoding has the homology of height equally with other mammiferous homogenic aminoacid sequence.Five hydroxytryptamine 4 acceptor b hypotype amino acid sequence homologies with the people are 88.81% (CAC22249); Homology with rat is 80.95% (AAC52233); Homology with mouse is 86.07% (CAA70773); Homology with cavy is 88.31% (CAA73912); Homology with dog is 90.55%.
Pig five hydroxytryptamine 4 acceptor b subtype gene among the present invention are carried out Nucleotide and protein homology retrieval in ncbi database, found that it and people's five hydroxytryptamine 4 acceptor b subtype gene (AJ278980) have 91% homogeny on nucleotide level, for details see attached table 1; On amino acid levels, it and people's five hydroxytryptamine 4 acceptor b subtype gene (CAC22249) also have 88% homogeny, and for details see attached table 2.This shows that all there are higher homology in pig five hydroxytryptamine 4 acceptor b subtype gene and people's five hydroxytryptamine 4 acceptor b subtype gene on nucleic acid still is protein level.People's five hydroxytryptamine 4 acceptor b subtype gene (AJ278980) have been proved to be the effect with tangible adjusting gastrointestinal motility, can think that pig five hydroxytryptamine 4 acceptor b subtype gene also have similar effect on the gastrointestinal motility function of regulating pig.
Pig five hydroxytryptamine 4 acceptor b subtype gene among the present invention are found in the comparison of the five hydroxytryptamine 4 acceptor b subtype gene nucleotide sequences of species such as same people, mouse, cavy, an exon that contains 42 bases is inserted in nucleotide sequence the 507th site in pig five hydroxytryptamine 4 acceptor b hypotypes, and the 169th of its aminoacid sequence inserts 14 amino acid.This structure of pig is identical with dog, and the variation of this structure may cause the variation of protein function.
In the present invention, carry out cluster analysis by coding protein sequence to 18 five hydroxytryptamine 4 acceptor genes of 6 species of Mammals, the result shows that SEQ ID NO.7 that the present invention obtains meets the evolution rule of five hydroxytryptamine 4 acceptor genes, indirect proof resulting sequence really be five hydroxytryptamine 4 acceptor gene family members.
Table 1
91％?identity?in?609nt?overlap
Query：548 tgatagaaaaaaggaagttccaccagaattcgaactctacgtactgtatcttcatggtca?607
||||||||||?|||||||||?|||||||?||?|||||||||||||||?||||||||||||
Sbjct：512 tgatagaaaagaggaagttcaaccagaactctaactctacgtactgtgtcttcatggtca?571
Query：608 acaagccctacgccatcacctgctctgtggtggccttctacatcccgttcctcctcatgg?667
||||||||||||||||||||||||||||||||||||||||||||||?||?||||||||||
Sbjct：572 acaagccctacgccatcacctgctctgtggtggccttctacatcccatttctcctcatgg?631
Query：668 tgctggcctattggcgcatctacgtgacagctaaggagcacgcccaccagatccagatgt?727
|||||||||||| ||||||||?||?||||||||||||||?|||||?|||||||||||||
Sbjct：632 tgctggcctattaccgcatctatgtcacagctaaggagcatgcccatcagatccagatgt?691
Query：728 tgcaacgggcagaggcccccgcagagggcaggcctccgtccgcagaccagcacagcaccc?787
|?|||||||||||?|||?||?|?|||?|||||||||?|||?|||||||||||?|||||?|
Sbjct：692 tacaacgggcaggagcctcctccgagagcaggcctcagtcggcagaccagcatagcactc?751
Query：788 atcgcatgaggacagagaccaaagcagccaagaccctgtgcgtcatcatgggttgcttct?847
|||||||||||||||||||||||||||||||||||||||||?||||||||||||||||||
Sbjct：752 atcgcatgaggacagagaccaaagcagccaagaccctgtgcatcatcatgggttgcttct?811
Query：848 gcctctgctgggctccgttcttcgtcaccaatgtcgtggaccctttcgcagactactctg?907
|||||||||||||?||?|||||?|||||||||?|?|||||?|||||| |||||||?|||
Sbjct：812 gcctctgctgggcaccattctttgtcaccaatattgtggatcctttcatagactacactg?871
Query：908 tccccgggcaggtgtggaccgctttcctctggctcggctacatcaattccgggttgaacc?967
||||?||||||||||||||?||||||||||||||||||||?|||||||||||||||||||
Sbjct：872 tccctgggcaggtgtggactgctttcctctggctcggctatatcaattccgggttgaacc?931
Query：968 cctttctctacgccttcctgaataagtctttcagacgtgccttcctcatcatcctctgct?1027
|?|||||||||||||||?|||||||||||||?||||||||||||||||||||||||||||
Sbjct：932 cttttctctacgccttcttgaataagtcttttagacgtgccttcctcatcatcctctgct?991
Query：1028?gcgatgatgagcgctaccgaagaccttgcgtggcgggccagacggtcccctgttcaacca?1087
|?|||||||||||||||||||||||||?|?| |||||||||?|||||?||||||||||
Sbjct：992 gtgatgatgagcgctaccgaagaccttccattctgggccagactgtcccttgttcaacca?1051
Query：1088?caaccgtcaacggatccactcatgtgctaagggatgcagtggagtgtggtggccagtggg?1147
|||||?|?||?||||||||?|||||?||||||||||||||||||||||||||||||||||
Sbjct：1052?caaccattaatggatccacacatgtactaagggatgcagtggagtgtggtggccagtggg?1111
Query：1148?agagtcagtgccacccgccagcaacttctcctttggtggctgctcagcccagtgacactt?1207
||||||||||?|||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1112?agagtcagtgtcacccgccagcaacttctcctttggtggctgctcagcccagtgacactt?1171
Query：1208?ag?1209
||
Sbjct：1172?ag?1173
Query: the nucleotide sequence of pig five hydroxytryptamine 4 acceptor b hypotypes
Sbjct: the nucleotide sequence (AJ278980) of people's five hydroxytryptamine 4 acceptor b hypotypes
Table 1 is the homology comparison sheet of the nucleotide sequence of pig five hydroxytryptamine 4 acceptor b hypotypes of the present invention and people's five hydroxytryptamine 4 acceptor b hypotypes.
Table 2
88％identity?in?357aa?overlap，93％similarity?in?374aa?overlap
Query：1 MDKLDANGSSKEGFGSVEKVVLLTFVSAVILMAVLGNLLVMVAVCRDRQLRKIKTNYFIV?60
MDKLDAN?SS+EGFGSVEKVVLLTF+S?VILMA+LGNLLVMVAVC?DRQLRKIKTNYFIV
Sbjct：1 MDKLDANVSSEEGFGSVEKVVLLTFLSTVILMAILGNLLVMVAVCWDRQLRKIKTNYFIV?60
Query：61 SLAFADLLVSVLVMPFGAIELVQDVWIYGEMFCLVRTSLDVLLTTASIFHLCCISLDRYY?120
SLAFADLLVSVLVMPFGAIELVQD+WIYGE+FCLVRTSLDVLLTTASIFHLCCISLDRYY
Sbjct：61 SLAFADLLVSVLVMPFGAIELVQDIWIYGEVFCLVRTSLDVLLTTASIFHLCCISLDRYY?120
Query：121?AICCQPLVYRNKMTPLRVAVLLAGCWAIPVLISFLPIMQGWNNIGITDLERTSKPRLGQD?180
AICCQPLVYRNKMTPLR+A++L?GCW?IP ISFLPIMQGWNNIGI?DL
Sbjct：121?AICCQPLVYRNKMTPLRIALMLGGCWVIPTFISFLPIMQGWNNIGIIDL-----------?169
Query：181?LHVIEKRKFHQNSNSTYCIFMVNLPYAITCSVVAFYIPFLLMVLAYWRIYVTAKEHAHQI?240
IEKRKF+QNSNSTYC+FMVNKPYAITCSVVAFYIPFLLMVLAY+RIYVTAKEHAHQI
Sbjct：170?---IEKRKFNQNSNSTYCVFMVNKPYAITCSVVAFYIPFLLMVLAYYRIYVTAKEHAHQI?226
Query：241?QMLQRAGAPAEGRPPSADQHSTHRMRTETKAAKTLCVIMGCFCLCWAPFFVTNVVDPFAD?300
QMLQRAGA?+E?RP?SADQHSTHRMRTETKAAKTLC+IMGCFCLCWAPFFVTN+VDPF?D
Sbjct：227?QMLQRAGASSESRPQSADQHSTHRMRTETKAAKTLCIIMGCFCLCWAPFFVTNIVDPFID?286
Query：301?YSVPGQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYRRPCVAGQTVPC?360
Y+VPGQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYRRP?+?GQTVPC
Sbjct：287?YTVPGQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYRRPSILGQTVPC?346
Query：361?STTTVNGSTHVLRDAVECGGQWESQCHPPATSPLVAAQPSDT?402
STTT+NGSTHVLRDAVECGGQWESQCHPPATSPLVAAQPSDT
Sbjct：347?STTTINGSTHVLRDAVECGGQWESQCHPPATSPLVAAQPSDT?388
Query: the aminoacid sequence of pig five hydroxytryptamine 4 acceptor b hypotypes
Sbjct: the aminoacid sequence (CAC22249) of people's five hydroxytryptamine 4 acceptor b hypotypes
Table 2 is that the homology of the aminoacid sequence of pig five hydroxytryptamine 4 acceptor b hypotypes of the present invention and people's five hydroxytryptamine 4 acceptor b hypotypes compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
At present, in the research of pig five hydroxytryptamine 4 acceptor genes, has only partial C DS sequence (GenBank Z48176 and Z48175).The present invention clones total length CDS sequence first.
Kohtaro etc. study at the function of 4 acceptors of five hydroxytryptamine in the peripheral tissues in gastro-intestinal system.The result shows five hydroxytryptamine 4 acceptors main function that participates in regulating gastrointestinal motility in gastro-intestinal system, and along with the enhancing of receptor active often causes irritability diarrhoea (J Gastroenterol 2000; 35:575-582).
The present invention has substantive distinguishing features and marked improvement, and pig five hydroxytryptamine 4 acceptor b subtype gene of the present invention are regulated pig gastrointestinal motility function the important regulating and controlling effect, is to study pig irritability diarrhoea and carry out the important candidate gene of pig molecule mark assistant breeding.An aspect of of the present present invention can be used for studying this gene in the effect of regulating pig gastrointestinal motility function by the albumen of this gene of preparation; Can provide fundamental basis for the animal model of setting up irritability diarrhoea on the other hand, and can play the directiveness effect, so the present invention has very big using value for the pig molecule mark assistant breeding.
Description of drawings
First section product electrophorogram of Fig. 1 pig five hydroxytryptamine 4 acceptor b subtype gene RT-PCR
Electrophoretic band is 445bp through gel imaging system analysis size among the figure.
Second section product electrophorogram of Fig. 2 pig five hydroxytryptamine 4 acceptor b subtype gene RT-PCR
Electrophoretic band is 407bp through gel imaging system analysis size among the figure.
The 3rd section product electrophorogram of Fig. 3 pig five hydroxytryptamine 4 acceptor b subtype gene RT-PCR
Electrophoretic band is 952bp through gel imaging system analysis size among the figure.
Fig. 4 pig five hydroxytryptamine 4 acceptor b subtype gene CDS sequence assembly figure
Obtain complete CDS sequence after three Partial cDNA Sequence overlap splicings among the figure, size is 1209bp.
Fig. 5 pig is with other species five hydroxytryptamine 4 acceptor gene encoding sequence cluster analysis figure
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of pig 5 one hydroxy-tryptamines 4 acceptor b subtype gene cDNA sequences
1. separate tissue (Isolation)
Butcher 30 age in days DLY commodity piglets on the pig farm, get colon's sample, put into liquid nitrogen rapidly, be stored in ℃ refrigerator of laboratory-70 after freezing, prepare to extract total tissue RNA.
2. the separation (Total RNA isolation) of total RNA
(1) preparation work of extraction RNA
The NaOH soaked overnight of used in glass products 0.1M is washed repeatedly with tap water, uses distilled water flushing again 2 times, and 180 ℃ were toasted 4 hours.
Homogenizer, electrophoresis chamber be with 3% hydrogen peroxide dipping 20-30 minute, again with 0.1% DEPC water flushing. because the DEPC of deactivation is not influential to the PCR reaction, available 0.5% SDS handles.
The DEPC water logging bubble of Tip head, EP effective 0.1% spends the night, and autoclaving makes the DEPC inactivation.
Distilled water and solution are handled with DEPC, and promptly every 100ml water or solution add 0.1mlDEPC liquid, and room temperature is placed and spent the night 30 minutes DEPC inactivations of autoclaving.
(2) total tissue RNA is extracted
Get 100mg frozen sample of organizing in liquid nitrogen and put into 1ml Trizol (trizal reagent, invitrogenBRL, homogenate in homogenate organ pipe USA).Centrifugal 10 minutes of 4 ℃ of 12000g.Draw supernatant liquor, 15-30 ℃ of incubation 5 minutes.Add the 0.2ml chloroform, cover lid is used hand concuss 15 seconds.15-30 ℃ incubation 2-3 minute.Centrifugal 15 minutes of 4 ℃ of 12000g.Draw supernatant liquor, add the 0.8ml Virahol, mix.15-30 ℃ of incubation 10 minutes, centrifugal 10 minutes of 4 ℃ of 12000g, centrifuge tube bottom white precipitate is RNA.Outwell supernatant, add 1ml 75% washing with alcohol RNA, centrifugal 10 minutes of 4 ℃ of 7500g.Packing among seasoning or the vacuum-drying RNA. water-soluble (RNasefree) ,-70 ℃ of preservations.
(3) evaluation of total tissue RNA
By measuring rna content on the spectrophotometer and with the quality of agarose electrophoretic analysis RNA, concrete grammar is as follows:
Electrophoresis chamber soaked 4-5 hour with RNA scavenging solution (100mM NaOH, 1mM EDTA), washed repeatedly with the DEPC treated water that to pour 1 * TBE into behind the several stand-by again.Sepharose is prepared with 1 * TBE.The 10ul sample-loading buffer (2 * TBE, 13% phenanthrene can, 0.1% bromjophenol blue, 7M urea) in add the above total tissue RNA of 1ul, 65 ℃ of sex change were put into after 10 minutes frozen water 2-3 minute immediately.Point sample is electrophoresis in sepharose.
3. Full Length cDNA Cloning (Cloning of Full-length cDNA)
(1) design of primers and synthetic
People (the AJ278980 that delivers according to GenBank, NM000870), rat (NM008313), mouse (NM012853), the five hydroxytryptamine 4 acceptor gene CDS homologous sequences of cavy species such as (Y13585), designing that six primers [BP001 (SEQID NO.1), BP002 (SEQ ID NO.2), BP003 (SEQ ID NO.3), BP004 (SEQ ID NO.4), BP005 (SEQ ID NO.5), BP006 (SEQ ID NO.6)] are used for the pig cDNA is the pcr amplification of masterplate.Primer is with Primer Premier 5.0 design voluntarily, and is synthetic by Shanghai Hua Nuo Bioisystech Co., Ltd.
(2) reverse transcription reaction (RT)
Carry out reverse transcription by precious biological reverse transcription test kit (the RNA PCR Kit Ver.2.1) requirement in Dalian.Inverse transcription reaction liquid is formed: 2ul 10 * RNA PCR Buffer, 4ul 25Mm MgCl, 2ul dNTP Mixture, 0.5ul RnaseInhibitor (40U/ul), 1ul AMV Reverse Transcriptase (5U/ul), 1ul Oligo dTPrimer, 1ul RNA Sample, 8.5ul Rnase Free dH
2O totally is 20ul.The reverse transcription reaction condition: 30 ℃ 5 minutes, 42 ℃ 30 minutes, 99 ℃ 5 minutes, 5 ℃ 5 minutes.
(3) PCR reaction
With the RT product is that template is carried out the PCR reaction.Its reaction parameter is: 95 ℃ of sex change 5 minutes; 95 ℃ 30 seconds → 55 ℃ 30 seconds → 72 ℃ 1 minute, so carry out 30 times the circulation; 72 ℃ were extended 4 ℃ of preservations 10 minutes.PCR[BP001 (SEQ ID NO.1)+BP002 (SEQ ID NO.2)] obtain 445bp cDNA fragment (seeing accompanying drawing 1), PCR[BP003 (SEQ ID NO.3)+BP004 (SEQ ID NO.4)] obtain 407bp cDNA fragment (seeing accompanying drawing 2), PCR[BP005 (SEQ ID NO.5)+BP006 (SEQ ID NO.6)] obtain 952bp cDNA fragment (seeing accompanying drawing 3).
(4) cloning and sequencing
The PCR product is reclaimed the back be connected, be transformed in the competent e. coli jm109 with PMD 18-T Vector (TaKaRa)., get chief of a tribe's liquid 10ml and deliver biotech company's order-checking in 37 ℃ of overnight incubation of LB substratum through the bacterial strain after bacterial strain PCR and the double digestion evaluation.
(5) splicing of full-length cDNA
Utilization biological software DNAMAN analyzes the sequencing result of three RT-PCR, obtains three pig five hydroxytryptamines, 4 acceptor b subtype gene portion C DS sequences, and length is respectively 445bp, 407bp, 952bp.By the SeqManII instrument of DNASTAR, the overlap of three portion C DS sequences is spliced, obtain the complete CDS sequence of pig five hydroxytryptamine 4 acceptor b subtype gene, splicing the results are shown in accompanying drawing 4.
Embodiment 2
4 acceptor b subtype gene sequential analyses of pig five hydroxytryptamine and homology are relatively
1, pig five hydroxytryptamine 4 acceptor b subtype gene sequential analyses
The length of the pig five hydroxytryptamine 4 acceptor b subtype gene cDNA that the present invention is new is 1029bp, and detailed sequence is seen SEQ ID NO.7, and wherein open reading frame is positioned at 1-1029 position Nucleotide (1029 Nucleotide).Derive the aminoacid sequence of pig five hydroxytryptamine 4 acceptor b hypotypes according to cDNA, totally 402 amino-acid residues, molecular weight is 44960 dalton, iso-electric point (pI) is 8.08.Detailed sequence is seen SEQ ID NO.7.
2, five hydroxytryptamine 4 acceptor b subtype gene homologys relatively
Pig five hydroxytryptamine 4 acceptor b subtype gene cDNA sequences and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+ SwissProt+Superdate+PIR database, found that it and people's five hydroxytryptamine 4 acceptor b subtype gene (AJ278980) have 91% homogeny, (subordinate list 1) on nucleotide level; On amino acid levels, it and people's five hydroxytryptamine 4 acceptor b subtype gene (CAC22249) also have 88.81% homogeny (subordinate list 2).This shows that all there are higher homology in pig five hydroxytryptamine 4 acceptor b subtype gene and people's five hydroxytryptamine 4 acceptor b subtype gene on nucleic acid still is protein level.People's five hydroxytryptamine 4 acceptor b subtype gene (AJ278980) have been proved to be the effect with tangible adjusting gastrointestinal motility, can think that pig five hydroxytryptamine 4 acceptor b subtype gene also have similar effect on the gastrointestinal motility function of regulating pig.
3, five hydroxytryptamine 4 acceptor gene encoding sequence cluster analyses
The coding protein sequence of 17 five hydroxytryptamine 4 acceptor genes of 5 species such as search and acquisition people, mouse, dog on GenBank, add the SEQ ID NO.7 sequence that the present invention obtains, utilize molecular biology software PHYLIP to make up cluster analysis figure (seeing accompanying drawing 5).The result shows: five hydroxytryptamine 4 acceptor genes are evolved consistent with species evolution rule; The SEQ ID NO.7 that the present invention obtains meets the evolution rule of five hydroxytryptamine 4 acceptor genes, indirect proof resulting sequence really be five hydroxytryptamine 4 acceptor gene family members.
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 18bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
TTCCTCCTCATGGTGCTG
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 21bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.2
CGTTGACGGTTGTGGTTGAAC
(3) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 19bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.3
AGACCAAAGCAGCCAAGAC
(4) information of SEQ ID NO.4
(i) sequence signature:
(A) length: 19bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.4
TCAGCCCAGTGACACTTAG
(5) information of SEQ ID NO.5
(i) sequence signature:
(A) length: 18bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.5
ATGGACAAACTTGATCCT
(6) information of SEQ ID NO.6
(i) sequence signature:
(A) length: 19bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.6
TGATGTAGCCGAGCCAGAG
(7) information of SEQ ID NO.7
＜110〉Shanghai Communications University
＜120〉pig serotonin 4 acceptor b subtype gene albumen coded sequences
<140>
<141>2004-03-12
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1209
<212>DNA
＜213〉pig (Sus scrofa)
<400>1
1 ATGGACAAAC?TTGATGCTAA?TGGGAGTTCC?AAGGAGGGTT?TCGGGTCCGT?GGAGAAGGTC
61 GTGTTGCTCA?CGTTTGTCTC?GGCGGTTATC?CTCATGGCTG?TCTTGGGGAA?CCTGCTGGTG
121 ATGGTGGCTG?TGTGCAGGGA?CCGGCAGCTC?AGGAAAATCA?AAACCAATTA?CTTCATTGTG
181 TCTCTTGCCT?TTGCGGATCT?GCTGGTGTCC?GTGCTGGTGA?TGCCCTTTGG?AGCCATTGAG
241 CTGGTCCAGG?ACGTCTGGAT?TTATGGGGAG?ATGTTCTGCC?TCGTCCGGAC?GTCTCTGGAT
301 GTCCTGCTCA?CGACGGCATC?GATTTTTCAC?CTGTGCTGCA?TCTCTCTGGA?CAGGTACTAC
361 GCCATCTGCT?GCCAGCCTCT?GGTCTACAGG?AACAAGATGA?CCCCTCTGCG?CGTGGCGGTG
421 CTGCTCGCAG?GCTGCTGGGC?CATCCCCGTG?CTGATCTCCT?TTCTCCCCAT?AATGCAAGGC
481 TGGAATAACA?TCGGCATAAC?TGACCTGGAA?AGGACATCCA?AACCAAGACT?GGGCCAGGAT
541 TTGCATGTGA?TAGAAAAAAG?GAAGTTCCAC?CAGAATTCGA?ACTCTACGTA?CTGTATCTTC
601 ATGGTCAACA?AGCCCTACGC?CATCACCTGC?TCTGTGGTGG?CCTTCTACAT?CCCGTTCCTC
661 CTCATGGTGC?TGGCCTATTG?GCGCATCTAC?GTGACAGCTA?AGGAGCACGC?CCACCAGATC
721 CAGATGTTGC?AACGGGCAGG?GGCCCCCGCA?GAGGGCAGGC?CTCCGTCCGC?AGACCAGCAC
781 AGCACCCATC?GCATGAGGAC?AGAGACCAAA?GCAGCCAAGA?CCCTGTGCGT?CATCATGGGT
841 TGCTTCTGCC?TCTGCTGGGC?TCCGTTCTTC?GTCACCAATG?TCGTGGACCC?TTTCGCAGAC
901 TACTCTGTCC?CCGGGCAGGT?GTGGACCGCT?TTCCTCTGGC?TCGGCTACAT?CAATTCCGGG
961 TTGAACCCCT?TTCTCTACGC?CTTCCTGAAT?AAGTCTTTCA?GACGTGCCTT?CCTCATCATC
1021 CTCTGCTGCG?ATGATGAGCG?CTACCGAAGA?CCTTGCGTGG?CGGGCCAGAC?GGTCCCCTGT
1081 TCAACCACAA?CCGTCAACGG?ATCCACTCAT?GTGCTAAGGG?ATGCAGTGGA?GTGTGGTGGC
1141 CAGTGGGAGA?GTCAGTGCCA?CCCGCCAGCA?ACTTCTCCTT?TGGTGGCTGC?TCAGCCCAGT
1201 GACACTTAG
<210>2
<211>402
<212>PRT
＜213〉pig (Sus scrofa)
<400>2
1 MDKLDANGSS?KEGFGSVEKV?VLLTFVSAVI?LMAVLGNLLV?MVAVCRDRQL?RKIKTNYFIV
61 SLAFADLLVS?VLVMPFGAIE?LVQDVWIYGE?MFCLVRTSLD?VLLTTASIFH?LCCISLDRYY
121 AICCQPLVYR?NKMTPLRVAV?LLAGCWAIPV?LISFLPIMQG?WNNIGITDLE?RTSKPRLGQD
181 LHVIEKRKFH?QNSNSTYCIF?MVNKPYAITC?SVVAFYIPFL?LMVLAYWRIY?VTAKEHAHQI
241 QMLQRAGAPA?EGRPPSADQH?STHRMRTETK?AAKTLCVIMG?CFCLCWAPFF?VTNVVDPFAD
301 YSVPGQVWTA?FLWLGYINSG?LNPFLYAFLN?KSFRRAFLII?LCCDDERYRR?PCVAGQTVPC
361 STTTVNGSTH?VLRDAVECGG?QWESQCHPPA?TSPLVAAQPS?DT
Claims (4)
1, the gene order of a boar five hydroxytryptamine 4 acceptor b hypotypes, it is characterized in that, the nucleotide sequence of the five hydroxytryptamine 4 acceptor b subtype gene that from the total RNA of the colon of pig, amplify, this full length gene 1209bp, open reading frame 1bp-1209bp section, coding has 402 amino acid whose protein, and molecular weight is 44960 dalton, and iso-electric point pI is 8.08.
2, the gene order of pig five hydroxytryptamine 4 acceptor b hypotypes according to claim 1, it is characterized in that, the nucleotide sequence CDS of this gene coded protein has the homology of height with other mammiferous homologous gene, is 100% (Z48176, Z48175) with the portion C DS sequence homology of known two pig five hydroxytryptamine 4 acceptor genes among the GenBank; Five hydroxytryptamine 4 acceptor b subtype C DS sequence homologies with the people are 91% (AJ278980); Homology with rat is 79.49% (U20907); Homology with mouse is 84.53% (Y09585); Homology with dog is 89.99% (AX068008); Homology with cavy is 84.86% (Y13585).
3, according to the gene order of claim 1 or 2 described pig five hydroxytryptamine 4 acceptor b hypotypes, it is characterized in that, the aminoacid sequence of this genes encoding has the homology of height equally with other mammiferous homogenic aminoacid sequence, is 88.81% (CAC22249) with people's five hydroxytryptamine 4 acceptor b hypotype amino acid sequence homologies; Homology with rat is 80.95% (AAC52233); Homology with mouse is 86.07% (CAA70773); Homology with cavy is 88.31% (CAA73912); Homology with dog is 90.55%.
4, according to the gene order of claim 1 or 2 described pig five hydroxytryptamine 4 acceptor b hypotypes, it is characterized in that, in the comparison of the five hydroxytryptamine 4 acceptor b subtype gene nucleotide sequences of species such as same people, mouse, cavy, find, an exon that contains 42 bases is inserted in nucleotide sequence the 507th site in pig five hydroxytryptamine 4 acceptor b hypotypes, and the 169th of its aminoacid sequence inserts 14 amino acid.
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|---|---|---|---|
| CN 200410017694 Pending CN1563379A (en) | 2004-04-15 | 2004-04-15 | Gene order of b subtype of 4 receptor of hydroxytryptarmine in pig |
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| Country | Link |
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2004
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