生物信息學軟件使用

1、生物信息學軟件的使用（以MC4R基因為例）第1章 從NCBI上查找DNA、mRNA、蛋白質序列1、 以豬的黑素皮質素受體4(MC4R, melanocortin-4 re-ceptor)基因為例，介紹如何從NCBI上查找DNA、mRNA、氨基酸序列。1. 首先查找MC4R的DNA序列。 在百度里輸入NCBI，打開后得到的結果如下網(wǎng)頁：在Search 欄輸入 “MC4R pig”，在下拉菜單里選擇Gene，然后點擊Search，得到如下結果：點擊第一個ID為397359的鏈接，得到如下的結果： 可以看到該基因位于豬的1號染色體上，在右下方有個“Go to nucleotide”即進入核酸序列，有

2、三種格式（用紅圈標記的），經(jīng)常用的是“FASTA”和“GenBank”，“FASTA”格式的比較簡潔，不包含任何的數(shù)字，就全部是堿基，序列的對比和分析是就要用到這種格式；而“GenBank”格式就比較詳細，可以查看到很多信息，比如堿基數(shù)、mRNA序列、內含子、外顯子、CDS，以及氨基酸序列等等之類的。點擊GenBank后得到如下結果：Sus scrofa breed mixed chromosome 1, Sscrofa10.2 DNALOCUS NC_010443 2265 bp DNA linear CON 29-SEP-2013DEFINITION Sus scrofa breed mi

3、xed chromosome 1, Sscrofa10.2.ACCESSION NC_010443 REGION: complement(178553488.178555752) GPC_000000583VERSION NC_010443.4 GI:347618793DBLINK BioProject: PRJNA28993 Assembly: GCF_000003025.5KEYWORDS RefSeq.SOURCE Sus scrofa (pig) ORGANISM Sus scrofa Eukaryota; Metazoa; Chordata; Craniata; Vertebrata

4、; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Suina; Suidae; Sus.COMMENT REFSEQ INFORMATION: The reference sequence is identical to CM000812.4. On Oct 11, 2011 this sequence version replaced gi:333795951. Assembly Name: Sscrofa10.2 The genomic sequence for this RefSeq record i

5、s from the genome assembly released by the Swine Genome Sequencing Consortium as Sscrofa10.2 in August 2011 (see http:/www.sanger.ac.uk/Projects/S_scrofa). Sscrofa10.2 is a mixed assembly of clones and contigs from the whole-genome shotgun project AEMK00000000.1. #Genome-Annotation-Data-START# Annot

6、ation Provider : NCBI Annotation Status : Full annotation Annotation Version : Sus scrofa Annotation Release 104 Annotation Pipeline : NCBI eukaryotic genome annotation pipeline Annotation Software Version : 5.1 Annotation Method : Best-placed RefSeq; Gnomon Features Annotated : Gene; mRNA; CDS; ncR

7、NA #Genome-Annotation-Data-END#FEATURES Location/Qualifiers source 1.2265 /organism="Sus scrofa" /mol_type="genomic DNA" /db_xref="taxon:9823" /chromosome="1" /breed="mixed" gene 1.2265 /gene="MC4R" /note="melanocortin 4 receptor; Deri

8、ved by automated computational analysis using gene prediction method: BestRefSeq." /db_xref="GeneID:397359" mRNA join(1.681,834.2265) /gene="MC4R" /product="melanocortin 4 receptor" /inference="similar to RNA sequence, mRNA (same species):RefSeq:NM_214173.1&qu

9、ot; /exception="annotated by transcript or proteomic data" /note="The RefSeq transcript has 2 indels compared to this genomic sequence; Derived by automated computational analysis using gene prediction method: BestRefSeq." /transcript_id="NM_214173.1" /db_xref="GI:

10、55741558" /db_xref="GeneID:397359" CDS join(534.681,834.1685) /gene="MC4R" /inference="similar to AA sequence (same species):RefSeq:NP_999338.1" /exception="annotated by transcript or proteomic data" /note="The RefSeq protein has 1 indel compared to

11、this genomic sequence; Derived by automated computational analysis using gene prediction method: BestRefSeq." /codon_start=1 /product="melanocortin receptor 4" /protein_id="NP_999338.1" /db_xref="GI:55741559" /db_xref="GeneID:397359" /translation="MN

12、STHHHGMHTSLHFWNRSTYGLHSNASEPLGKGYSEGGCYEQL FVSPEVFVTLGVISLLENILVIVAIAKNKNLHSPMYFFICSLAVADMLVSVSNGSETI VITLLNSTDTDAQSFTVNIDNVIDSVICSSLLASICSLLSIAVDRYFTIFYALQYHNI MTVKRVGIIISCIWAVCTVSGVLFIIYSDSSAVIICLITVFFTMLALMASLYVHMFLM ARLHIKRIAVLPGTGTIRQGANMKGAITLTILIGVFVVCWAPFFLHLIFYISCPQNPY CVCFMSHFNLYLILIMCNSII

13、DPLIYALRSQELRKTFKEIICCYPLGGLCDLSSRY"ORIGIN 1 tcacagactc cccaggactt ggattggtca gaaagaagca gaggaggagc cactgtgcac 61 attttttttt ccccttcaca caccataaaa atcacagagg caactaacac tcacagcaaa 121 gcttcaggtt gggaactgat tctctctgcg aggcagctga tctgagcatg cgcacacaga 181 ttcattcttc tcccaatagc acagcagccg ctaggaaa

14、at tattttgaaa agacctgaat 241 gcattaagac taaagttaaa gtggaagtga gaacaaaata tcaaacagca gactcgacag 301 agaatgagcg tcttgaagcc taagatttca aagtgatgct aatcagagcc ctacctgaaa 361 gagactaaaa actccatttc aagcttcgga gcatgtgata tttattcaca acaggcattc 421 caatttcagc ctcataactt tcagacagat aaagacttgg agaaaatcgc tgaggc

15、tacc 481 tgacccagga gcttaaatca ggtcagaggg gatctcaacc cacctggcgc aggatgaact 541 caacccatca ccatggaatg catacttctc tccacttctg gaaccgcagc acctacggac 601 tgcacagcaa tgccagtgag ccccttggaa aagagctact ctgaaggagg atgctacgag 661 caactttttg tctctcctga ggtgtttgtg actctgggtg tcataagcct gt gap 100 bp Expand Ns 81

16、3 aaacgacg gcgtctctct gaggtgtttg 841 tgactctggg tgtcataagc ctgttggaga acattctggt gattgtggcc atagccaaga 901 acaagaatct gcattcaccc atgtactttt tcatctgtag cctggctgtg gctgatatgc 961 tggtgagcgt ttccaatggg tcagaaacca ttgtcatcac cctattaaac agcacggaca 1021 cggacgcaca gagtttcaca gtgaatattg ataatgtcat tgactcag

17、tg atctgtagct 1081 ccttactcgc ctcaatttgc agcctgcttt cgattgcagt ggacaggtat tttactatct 1141 tttatgctct ccagtaccat aacattatga cagttaagcg ggttggaatc atcatcagtt 1201 gtatctgggc agtctgcacg gtgtcgggtg ttttgttcat catttactca gatagcagtg 1261 ctgttattat ctgcctcata accgtgttct tcaccatgct ggctctcatg gcttctctct 13

18、21 atgtccacat gttcctcatg gccagactcc acattaagag gatcgccgtc ctcccaggca 1381 ctggcaccat ccgccaaggt gccaacatga agggggcaat taccctgacc atcttgattg 1441 gggtctttgt ggtctgctgg gcccccttct tcctccactt aatattctat atctcctgcc 1501 cccagaatcc atactgtgtg tgcttcatgt ctcactttaa tttgtatctc atcctgatca 1561 tgtgtaattc ca

19、tcatcgat cccctgattt atgcactccg gagccaagaa ctgaggaaaa 1621 ccttcaaaga gatcatctgt tgctatcccc tgggtggcct ctgtgatttg tctagcagat 1681 attaaatggg gacagaggag acttataaat gcaagcataa gagactttct ccttacacag 1741 tctggacaat atgcttcaac aacagcattt tcttgtaagg catcagttga gacattctat 1801 tgtataaatt taagttcgtg attctgc

20、tca gtctctgtgt atttttaagg tcttgctacc 1861 ttttggctgt aaaatgttta tctatactac aggttatagg cacaatggat ttataaaaaa 1921 gaaaaaagtc cttatgaaaa gttaattaat gtatcttgtc attcgaaagg atttgacaca 1981 ttgcttgttt tagtaaaatg gaaatcacag tttcattaaa tatatcctaa taaatggttg 2041 ctaatattac actatacaac gctgaagtgt agaggtttga t

21、tctagcatt gaggggagaa 2101 atactgaaac aagtgtttaa tcattaaaaa ataagctgaa atttcaacta atttaataaa 2161 acatgctcat tctccctgtg cagaaggaga aatgaagctt ctactgggag aaaaacagtt 2221 actaaaaaaa agtgggggga tattttgagt ttgaaaacta tgttt/2. 查找mRNA和氨基酸序列第一步和查DNA序列的一樣，先打開NCBI，得到如下主頁。2.1 點擊主頁面的“Nucleotide”，得到下面的網(wǎng)頁：2.2 在“S

22、earch”欄里，輸入 “MC4R pig”，然后點擊“Search”，得到如下結果： 出現(xiàn)了很多的搜索結果，可以按照自己的需要點擊不同的鏈接，比如我想要查找mRAN的完全編碼序列，我就點擊第一個“Sus scrofa MC4R mRNA, complete cds”，得到如下結果：Sus scrofa MC4R mRNA, complete cdsGenBank: DQ388767.1FASTA GraphicsGo to:LOCUS DQ388767 999 bp mRNA linear MAM 15-FEB-2006DEFINITION Sus scrofa MC4R mRNA, com

23、plete cds.ACCESSION DQ388767VERSION DQ388767.1 GI:87137928KEYWORDS .SOURCE Sus scrofa (pig) ORGANISM Sus scrofa Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Suina; Suidae; Sus.REFERENCE 1 (bases 1 to 999) AUTHORS Yang,X.Q., Yu

24、,H. and Liu,D. TITLE The comparative analysis on MC4R gene of wild boar, domestic pig and their crossbred JOURNAL UnpublishedREFERENCE 2 (bases 1 to 999) AUTHORS Yang,X.Q., Yu,H. and Liu,D. TITLE Direct Submission JOURNAL Submitted (05-FEB-2006) Northease Agricultural University, Animal Science &

25、; Technology, Xiangfang Block Gongbin Road Mucai Street, Harbin, Heilongjiang 150030, ChinaFEATURES Location/Qualifiers source 1.999 /organism="Sus scrofa" /mol_type="mRNA" /db_xref="taxon:9823" CDS 1.999 /codon_start=1 /product="MC4R" /protein_id="ABD281

26、76.1" /db_xref="GI:87137929" /translation="MNSTHHHGMHTSLHFWNRSTYGLHSNASEPLGKGYSEGGCYEQL FVSPEVFVTLGVISLLENILVIVAIAKNKNLHSPMYFFICSLAVADMLVSVSNGSETI VITLLNSTDTDAQSFTVNIDNVIDSVICSSLLASICSLLSIAVDRYFTIFYALQYHNI MTVKRVGIIISCIWAVCTVSGVLFIIYSDSSAVIICLITVFFTMLALMASLYVHMFLM ARLHIKRIAVLPGTG

27、TIRQGANMKGAITLTILIGVFVVCWAPFFLHLIFYISCPQNPY CVCFMSHFNLYLILIMCNSIIDPLIYALRSQELRKTFKEIICCYPLGGLCDLSSRY" variation 171 /replace="g" variation 175 /replace="c" variation 551 /replace="c" variation 758 /replace="c" variation 892 /replace="a"ORIGIN 1

28、atgaactcaa cccatcacca tggaatgcat acttctctcc acttctggaa ccgcagcacc 61 tacggactgc acagcaatgc cagtgagccc cttggaaaag gctactctga aggaggatgc 121 tacgagcaac tttttgtctc tcctgaggtg tttgtgactc tgggtgtcat aagcttgttg 181 gagaacattc tggtgattgt ggccatagcc aagaacaaga atctgcattc acccatgtac 241 tttttcatct gtagcctggc

29、 tgtggctgat atgctggtga gcgtttccaa tgggtcagaa 301 accattgtca tcaccctatt aaacagcacg gacacggacg cacagagttt cacagtgaat 361 attgataatg tcattgactc agtgatctgt agctccttac tcgcctcaat ttgcagcctg 421 ctttcgattg cagtggacag gtattttact atcttttatg ctctccagta ccataacatt 481 atgacagtta agcgggttgg aatcatcatc agttgtat

30、ct gggcagtctg cacggtgtcg 541 ggtgttttgt tcatcattta ctcagatagc agtgctgtta ttatctgcct cataaccgtg 601 ttcttcacca tgctggctct catggcttct ctctatgtcc acatgttcct catggccaga 661 ctccacatta agaggatcgc cgtcctccca ggcactggca ccatccgcca aggtgccaac 721 atgaaggggg caattaccct gaccatcttg attggggtct ttgtggtctg ctgggc

31、cccc 781 ttcttcctcc acttaatatt ctatatctcc tgcccccaga atccatactg tgtgtgcttc 841 atgtctcact ttaatttgta tctcatcctg atcatgtgta attccatcat cgatcccctg 901 atttatgcac tccggagcca agaactgagg aaaaccttca aagagatcat ctgttgctat 961 cccctgggtg gcctctgtga tttgtctagc agatattaa/由此，就可以得到我們想要的mRNA序列和氨基酸序列。第二章 PCR引物的設計

32、1. 用NCBI設計PCR引物打開NCBI的首頁，然后點擊BLAST，得到如下結果：點擊“Specialized BLAST”中的“Primer-BLAST”，得到如下界面：如果我要設計擴增mRNA的引物，那么就把mRNA序列輸入進去，其他參數(shù)可以根據(jù)自己需要進行設定，一般默認就好，然后點擊“Get Primers”，得到如下界面：第3章 利用生物軟件對RNA的二級結構進行預測1. 利用生物軟件（http:/www.genebee.msu.su/services/rna2_reduced.html）對mRNA的二級結構進行預測，打開網(wǎng)址后得到如下網(wǎng)頁： 輸入MC4R的mRNA序列后，在“Inp

33、ut Type”一欄選擇“Sequence”；然后點擊“提交”，得到如下結果：第4章 DNA序列、蛋白質序列的BLAST對比分析1.我就以MC4R的蛋白序列為例，首先打開NCBI，得到如下界面：點擊右邊的“BLAST”，得到如下界面：一般使用“nucleotide blast”和“protein blast”，前者是核酸的對比，后者是蛋白質的對比，我查找的是蛋白質對比，所以點擊“protein blast”，得到如下結果：輸入豬MC4R基因編碼的蛋白質序列，然后點擊BLAST，得到如下界面：第五章 預測分析蛋白質的一級結構、二級結構以及三級結構一，以豬的以豬的黑素皮質素受體4(MC4R,mel

34、anocortin-4 re-ceptor)基因的氨基酸序列為例，通過不同的在線生物分析軟件對MC4R編碼的蛋白質的一級結構進行分析。1. 通過ExPASy中的protparam(/tools/protparam.html)對蛋白質的分子量、等電點進行預測，輸入網(wǎng)址后結果如下： 輸入氨基酸序列后，點擊“Compare parameters”，得到如下結果： ProtParamUser-provided sequence: 10 20 30 40 50 60MNSTHHHGMH TSLHFWNRST YGLHSNASEP LGKGYSEGGC YEQLFV

35、SPEV FVTLGVISLL 70 80 90 100 110 120ENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIVITLLNST DTDAQSFTVN 130 140 150 160 170 180IDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAVCTVS 190 200 210 220 230 240GVLFIIYSDS SAVIICLITV FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGTIRQGAN 250 260 270 280 290

36、300MKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPL 310 320 330IYALRSQELR KTFKEIICCY PLGGLCDLSS RYNumber of amino acids: 332 （氨基酸數(shù)目）Molecular weight: 36946.7 （分子量）Theoretical pI: 7.13 （等電點）窗體頂端Amino acid composition: （氨基酸組成）Ala (A) 19 5.7% Arg (R) 9 2.7% Asn (N) 15 4.5% Asp (D) 9 2.

37、7% Cys (C) 15 4.5% Gln (Q) 6 1.8% Glu (E) 8 2.4% Gly (G) 17 5.1% His (H) 12 3.6% Ile (I) 38 11.4% Leu (L) 39 11.7% Lys (K) 8 2.4% Met (M) 12 3.6% Phe (F) 19 5.7% Pro (P) 9 2.7% Ser (S) 33 9.9% Thr (T) 19 5.7% Trp (W) 3 0.9% Tyr (Y) 15 4.5% Val (V) 27 8.1% Pyl (O) 0 0.0% Sec (U) 0 0.0% (B) 0 0.0% (Z)

38、 0 0.0% (X) 0 0.0%Total number of negatively charged residues (Asp + Glu): 17Total number of positively charged residues (Arg + Lys): 17Atomic composition: （原子組成）Carbon C 1692Hydrogen H 2645Nitrogen N 415Oxygen O 455Sulfur S 27Formula: C1692H2645N415O455S27 Total number of atoms: 5234 （總原子數(shù)）Extincti

39、on coefficients: （消光系數(shù)）Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.Ext. coefficient 39725Abs 0.1% (=1 g/l) 1.075, assuming all pairs of Cys residues form cystinesExt. coefficient 38850Abs 0.1% (=1 g/l) 1.052, assuming all Cys residues are reducedEstimated half-life:

40、 （半衰期）The N-terminal of the sequence considered is M (Met).The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro). >20 hours (yeast, in vivo). >10 hours (Escherichia coli, in vivo).Instability index: （不穩(wěn)定系數(shù)）The instability index (II) is computed to be 46.15This classifies the

41、 protein as unstable.Aliphatic index: 119.76 （脂肪系數(shù)）Grand average of hydropathicity (GRAVY): 0.765 （總平均親水性） 由以上結果可以知道豬MC4R基因所編碼的蛋白質的分子量為36946.7，等電點為7.13，脂肪系數(shù)為119.76，總平均親水性為0.765 ，以及其他的指標。2.利用ProtScale(/cgi-bin/protscale.pl)對氨基酸序列做疏水性分析;輸入網(wǎng)址后結果如下： 輸入MC4R的氨基酸序列，點擊“Submit”，得到如下的結果：Pro

42、tScaleUser-provided sequence: 10 20 30 40 50 60MNSTHHHGMH TSLHFWNRST YGLHSNASEP LGKGYSEGGC YEQLFVSPEV FVTLGVISLL 70 80 90 100 110 120ENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIVITLLNST DTDAQSFTVN 130 140 150 160 170 180IDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAVCTVS 190 200 210 220 230 240GVLFIIYSDS SAVIICLITV FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGTIRQGAN 250 260 270 280 290 300MKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCV