生物信息学软件使用

生物信息学软件的使用（以MC4R基因为例）

第一章从NCBI上查找DNA、mRNA、蛋白质序列

一、以猪的黑素皮质素受体4(MC4R, melanocortin-4 re-ceptor)基因为例，介绍如何从NCBI 上查找DNA、mRNA、氨基酸序列。

1.首先查找MC4R的DNA序列。

在百度里输入NCBI，打开后得到的结果如下网页：

在Search 栏输入“MC4R pig”，在下拉菜单里选择Gene，然后点击Search，得到如下结果：

点击第一个ID为397359的链接，得到如下的结果：

可以看到该基因位于猪的1号染色体上，在右下方有个“Go to nucleotide”即进入核酸序列，有三种格式（用红圈标记的），经常用的是“FASTA”和“GenBank”，“FASTA”格式的比较简洁，不包含任何的数字，就全部是碱基，序列的对比和分析是就要用到这种格式；而“GenBank”格式就比较详细，可以查看到很多信息，比如碱基数、mRNA序列、内含子、外显子、CDS，以及氨基酸序列等等之类的。点击GenBank后得到如下结果：

Sus scrofa breed mixed chromosome 1,

Sscrofa10.2 DNA

LOCUS NC_010443 2265 bp DNA linear CON 29-SEP-2013 DEFINITION Sus scrofa breed mixed chromosome 1, Sscrofa10.2.

ACCESSION NC_010443 REGION: complement(178553488..178555752) GPC_000000583 VERSION NC_010443.4 GI:347618793

DBLINK BioProject: PRJNA28993

Assembly: GCF_000003025.5

KEYWORDS RefSeq.

SOURCE Sus scrofa (pig)

ORGANISM Sus scrofa

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 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 is from the genome

assembly released by the Swine Genome Sequencing Consortium as

Sscrofa10.2 in August 2011 (see

https://www.360docs.net/doc/549958585.html,/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##

Annotation 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; ncRNA

##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; Derived 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"

/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: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 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="MNSTHHHGMHTSLHFWNRSTYGLHSNASEPLGKGYSEGGCYEQL FVSPEVFVTLGVISLLENILVIVAIAKNKNLHSPMYFFICSLAVADMLVSVSNGSETI VITLLNSTDTDAQSFTVNIDNVIDSVICSSLLASICSLLSIAVDRYFTIFYALQYHNI MTVKRVGIIISCIWAVCTVSGVLFIIYSDSSAVIICLITVFFTMLALMASLYVHMFLM ARLHIKRIAVLPGTGTIRQGANMKGAITLTILIGVFVVCWAPFFLHLIFYISCPQNPY CVCFMSHFNLYLILIMCNSIIDPLIYALRSQELRKTFKEIICCYPLGGLCDLSSRY" 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 ctaggaaaat 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 tgaggctacc

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

813 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 tgactcagtg 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

1321 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 catcatcgat 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 attctgctca 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 ttctagcatt 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序列的一样，先打开NCBI，得到如下主页。

2.1 点击主页面的“Nucleotide”，得到下面的网页：

2.2 在“Search”栏里，输入“MC4R pig”，然后点击“Search”，得到如下结果：

出现了很多的搜索结果，可以按照自己的需要点击不同的链接，比如我想要查找mRAN 的完全编码序列，我就点击第一个“Sus scrofa MC4R mRNA, complete cds”，得到如下结果：

Sus scrofa MC4R mRNA, complete cds

GenBank: DQ388767.1

FASTA Graphics

Go to:

LOCUS DQ388767 999 bp mRNA linear MAM 15-FEB-2006 DEFINITION Sus scrofa MC4R mRNA, complete cds.

ACCESSION DQ388767

VERSION DQ388767.1 GI:87137928

KEYWORDS .

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,H. and Liu,D.

TITLE The comparative analysis on MC4R gene of wild boar, domestic pig

and their crossbred

JOURNAL Unpublished

REFERENCE 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 & Technology, Xiangfang Block Gongbin Road Mucai Street,

Harbin, Heilongjiang 150030, China

FEATURES 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="ABD28176.1"

/db_xref="GI:87137929"

/translation="MNSTHHHGMHTSLHFWNRSTYGLHSNASEPLGKGYSEGGCYEQL FVSPEVFVTLGVISLLENILVIVAIAKNKNLHSPMYFFICSLAVADMLVSVSNGSETI VITLLNSTDTDAQSFTVNIDNVIDSVICSSLLASICSLLSIAVDRYFTIFYALQYHNI MTVKRVGIIISCIWAVCTVSGVLFIIYSDSSAVIICLITVFFTMLALMASLYVHMFLM ARLHIKRIAVLPGTGTIRQGANMKGAITLTILIGVFVVCWAPFFLHLIFYISCPQNPY CVCFMSHFNLYLILIMCNSIIDPLIYALRSQELRKTFKEIICCYPLGGLCDLSSRY"

variation 171

/replace="g"

variation 175

/replace="c"

variation 551

/replace="c"

variation 758

/replace="c"

variation 892

/replace="a"

ORIGIN

1 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 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 agttgtatct 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 ctgggccccc 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，就可以得到我们想要的mRNA序列和氨基酸序列。

第二章 PCR引物的设计

1.用NCBI设计PCR引物

打开NCBI的首页，然后点击BLAST，得到如下结果：

点击“Specialized BLAST”中的“Primer-BLAST”，得到如下界面：

如果我要设计扩增mRNA的引物，那么就把mRNA序列输入进去，其他参数可以根据自己

需要进行设定，一般默认就好，然后点击“Get Primers”，得到如下界面：

第三章利用生物软件对RNA的二级结构进行预测

1. 利用生物软件（http://www.genebee.msu.su/services/rna2_reduced.html）对mRNA的二级结构进行预测，打开网址后得到如下网页：

交”，得到如下结果：

第四章 DNA序列、蛋白质序列的BLAST对比分析

1.我就以MC4R的蛋白序列为例，首先打开NCBI，得到如下界面：

点击右边的“BLAST”，得到如下界面：

一般使用“nucleotide blast”和“protein blast”，前者是核酸的对比，后者是蛋白质的对比，我查找的是蛋白质对比，所以点击“protein blast”，得到如下结果：

输入猪MC4R基因编码的蛋白质序列，然后点击BLAST，得到如下界面：

第五章预测分析蛋白质的一级结构、二级结构以及三级结构

一，以猪的以猪的黑素皮质素受体4(MC4R,melanocortin-4 re-ceptor)基因的氨基酸序列为例，通过不同的在线生物分析软件对MC4R编码的蛋白质的一级结构进行分析。

1. 通过ExPASy中的protparam(https://www.360docs.net/doc/549958585.html,/tools/protparam.html)对蛋白质的分子量、等电点进行预测，输入网址后结果如下：

输入氨基酸序列后，点击“Compare parameters”，得到如下结果：

ProtParam

User-provided sequence:

10 20 30 40 50 60 MNSTHHHGMH TSLHFWNRST YGLHSNASEP LGKGYSEGGC YEQLFVSPEV FVTLGVISLL

70 80 90 100 110 120

ENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIVITLLNST DTDAQSFTVN

130 140 150 160 170 180

IDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAVCTVS

190 200 210 220 230 240

GVLFIIYSDS SAVIICLITV FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGTIRQGAN

250 260 270 280 290 300

MKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPL

310 320 330

IYALRSQELR KTFKEIICCY PLGGLCDLSS RY

Number of amino acids: 332 （氨基酸数目）

Molecular weight: 36946.7 （分子量）

Theoretical pI: 7.13 （等电点）

Amino acid composition:（氨基酸组成）

Ala (A) 19 5.7% Arg (R) 9 2.7% Asn (N) 15 4.5% Asp (D) 9 2.7% 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) 0 0.0% (X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 17

Total number of positively charged residues (Arg + Lys): 17

Atomic composition: （原子组成）

Carbon C 1692

Hydrogen H 2645

Nitrogen N 415

Oxygen O 455

Sulfur S 27

Formula: C1692H2645N415O455S27

Total number of atoms: 5234 （总原子数）

Extinction coefficients: （消光系数）

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 39725

Abs 0.1% (=1 g/l) 1.075, assuming all pairs of Cys residues form cystines Ext. coefficient 38850

Abs 0.1% (=1 g/l) 1.052, assuming all Cys residues are reduced

Estimated half-life: （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: （不稳定系数）

The instability index (II) is computed to be 46.15

This classifies the protein as unstable.

Aliphatic index: 119.76 （脂肪系数）

Grand average of hydropathicity (GRAVY): 0.765 （总平均亲水性）由以上结果可以知道猪MC4R基因所编码的蛋白质的分子量为36946.7，等电点为7.13，脂肪系数为119.76，总平均亲水性为0.765 ，以及其他的指标。

2.利用ProtScale(https://www.360docs.net/doc/549958585.html,/cgi-bin/protscale.pl)对氨基酸序列做疏水性分析;输入网址后结果如下：

输入MC4R的氨基酸序列，点击“Submit”，得到如下的结果：

ProtScale

User-provided sequence:

10 20 30 40 50 60 MNSTHHHGMH TSLHFWNRST YGLHSNASEP LGKGYSEGGC YEQLFVSPEV FVTLGVISLL

70 80 90 100 110 120

ENILVIVAIA KNKNLHSPMY FFICSLAVAD MLVSVSNGSE TIVITLLNST DTDAQSFTVN

130 140 150 160 170 180

IDNVIDSVIC SSLLASICSL LSIAVDRYFT IFYALQYHNI MTVKRVGIII SCIWAVCTVS

190 200 210 220 230 240 GVLFIIYSDS SAVIICLITV FFTMLALMAS LYVHMFLMAR LHIKRIAVLP GTGTIRQGAN

250 260 270 280 290 300

MKGAITLTIL IGVFVVCWAP FFLHLIFYIS CPQNPYCVCF MSHFNLYLIL IMCNSIIDPL

310 320 330

IYALRSQELR KTFKEIICCY PLGGLCDLSS RY

SEQUENCE LENGTH: 332

Using the scale Hphob. / Kyte & Doolittle, the individual values for the 20 amino acids are: （所用氨基酸标度信息）

Ala: 1.800 Arg: -4.500 Asn: -3.500 Asp: -3.500 Cys: 2.500 Gln: -3.500 Glu: -3.500 Gly: -0.400 His: -3.200 Ile: 4.500 Leu: 3.800 Lys: -3.900 Met: 1.900 Phe: 2.800 Pro: -1.600 Ser: -0.800 Thr: -0.700 Trp: -0.900 Tyr: -1.300 Val: 4.200 : -3.500 : -3.500 : -0.490

Weights for window positions 1,..,9, using linear weight variation model: （分析所用参数信息）

1 2 3 4 5 6 7 8 9

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

edge center edge

经蛋白质疏水性预测分析表明，其中间部位则表现出较强的亲水性，按分值大小划分其疏水最大值为3.367，分别位于第197和第253 AA处；最小值为-1.878，位于第311AA 处。