如何在线粒体基因组中搜索tRNA

熊荣川

六盘水师范学院

线粒体基因组数据分析的一个重要组成部分就是在数据中搜索转运RNA（tRNA）,常常使用的工具软件是tRNAscan-SE，由于该软件官网上只有Unix系统的源代码，所以无法方便的下载程序进行本地运算搜索，所以通常需要到官网上使用作者提供的服务器进行，网址如下

下面我们就以该服务器的使用来说明怎样进行线粒体基因组中tRNA的搜索。我们以最近发表的线粒体基因组HQ324232为例。

在genbank中使用索取号HQ324232搜索并下载到该物种Nanorana pleskei的线粒体基因组保存为文件Nanorana pleskei.fasta 登录tRNAscan-SE服务器

在搜索模型（Search Mode）中选择“tRNAscan-> Cove”，数据来源（Source）中选择“Mito/Chloroplast”

在提交序列文件选项（submit a file）中选择序列文件，并选择通过E-mail接收结果（Receive results by e-mail instead）

另外还有在扩展选项（Extended Options）中的密码子类型选择线粒体密码子

提交请求，

通常提交之后，可能因为网络原因，结果网页无法显示，但是结果会发到邮箱里面来，这也是我们主张选择把结果发到邮箱的原因。当然如果能在网页上显示结果更好，因为有查看结果二级结构的选项。结果如下：

tRNA Search Results:

tRNAscan-SE v.1.23 - scan sequences for transfer RNAs

Please cite:

Lowe, T.M. & Eddy, S.R. (1997) "tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence" Nucl. Acids Res. 25: 955-964.

This program uses a modified, optimized version of tRNAscan v1.3 (Fichant & Burks, J. Mol. Biol. 1991, 220: 659-671),

a new implementation of a multistep weight matrix algorithm for identification of eukaryotic tRNA promoter regions (Pavesi et al., Nucl. Acids Res. 1994, 22: 1247-1256),

as well as the RNA covariance analysis package Cove v.2.4.2 (Eddy & Durbin, Nucl. Acids Res. 1994, 22: 2079-2088).

------------------------------------------------------------------------------

Sequence tRNA Bounds tRNA Anti Intron Bounds

Cove

Name Score -------- ------ Your-seq 30.20 Your-seq 30.63 Your-seq tRNA # Begin End ------ ---- 1 2 3 209 1213 2874 Type Codon Begin End----- ----- ----GAA TAC TAA 0 0 0 000------ ---- 278 1282 2946 Phe Val Leu 28.25 Your-seq 34.52 Your-seq 34.14 Your-seq 32.39 Your-seq 37.73 Your-seq 42.26 Your-seq 50.62 Your-seq 34.55 Your-seq 40.93 Your-seq 39.65 Your-seq 49.12 Your-seq 46.04 Your-seq 16.33 Your-seq 39.82 Your-seq 29.28 Your-seq 58.91 Your-seq 41.56

4 3905 5 4046 6 4124 7 5226 8 7221 9 9670

10 10097 11 11808 12 14364 13 7220 14 5603 15 5536 16 5440 17 5365 18 4045 19

209

3975 Ile 4114 Met 4192 Met 5295 Trp 7289 Asp 9738

Gly

10165 Arg 11876 His 14296 Glu 7150 Ser 5537 Tyr 5471 Cys 5368 Asn 5296 Ala 3975 Gln 141

Pro

GAT 0 CAT 0 CAT 0 TCA 0 GTC 0 TCC 0 TCG 0 GTG 0 TTC 0 TGA 0 GTA 0 GCA 0 GTT 0 TGC 0 TTG 0 TGG

[**************]0

=============================================================================

Run Statistics:

Started: Tue Jan 3 22:24:46 PST 2012

------------------------------------------------------------ Search Mode: Organellar Searching with: Cove only Covariance model: TRNA2.cm tRNA Cove cutoff score: 15

Alternate transl code used: from file /www/lib/tRNAscan-SE/gcode.vertmito

Max intron + var. length: 40 Pseudogene checking disabled

------------------------------------------------------------

Cove Stats: -----------

Sequences read: 1 Cove-confirmed tRNAs: 19 Bases scanned by covels: 35320 % seq scanned by covels: 100.0 % Script CPU time: 0.02 s Cove CPU time: 71.12 s

Scan speed: 496.6 bp/sec

Cove analysis of tRNAs ended: Tue Jan 3 22:25:57 PST 2012

Overall scan speed: 496.5 bp/sec

tRNAs decoding Standard 20 AA: 19 Selenocysteine tRNAs (TCA): 0 Possible suppressor tRNAs (CTA,TTA): 0 tRNAs with undetermined/unknown isotypes: 0 Predicted pseudogenes: 0

------- Total tRNAs: 19

tRNAs with introns: 0

Isotype / Anticodon Counts:

Ala : 1 AGC: GGC: CGC: TGC: 1 Gly : 1 ACC: GCC: CCC: TCC: 1 Pro : 1 AGG: GGG: CGG: TGG: 1 Thr : 0 AGT: GGT: CGT: TGT: Val : 1 AAC: GAC: CAC: TAC: 1 Ser : 1 AGA: GGA: CGA: TGA: 1 ACT: GCT: Arg : 1 ACG: GCG: CCG: TCG: 1 CCT: TCT: Leu : 1 AAG: GAG: CAG: TAG: CAA: TAA: 1 Phe : 1 AAA: GAA: 1 Asn : 1 ATT: GTT: 1 Lys : 0 CTT: TTT: Asp : 1 ATC: GTC: 1 Glu : 1 CTC: TTC: 1 His : 1 ATG: GTG: 1 Gln : 1 CTG: TTG: 1 Ile : 1 AAT: GAT: 1 TAT: Met : 2 CAT: 2 Tyr : 1 ATA: GTA: 1 Supres: 0 CTA: TTA: Cys : 1 ACA: GCA: 1 Trp : 1 CCA: SelCys: 0 TCA: 1

=============================================================================

Predicted tRNA Secondary Structures:

Your-seq.trna1 (209-278) Length: 70 bp Type: Phe Anticodon: GAA at 33-35 (241-243) Score: 30.20 * | * | * | * | * | * | * Seq:

GCTCTTATAGCTTAAACCCAAAGTATAGTGCTGAAAACGCTAAGATGAGCCCTAAAAAGCTCTAAGGGCA

Str: >>>>>>>..>>>>.......>>>>.......>>>........

Your-seq.trna2 (1213-1282) Length: 70 bp Type: Val Anticodon: TAC at 32-34 (1244-1246) Score: 30.63 * | * | * | * | * | * | * Seq:

CAAAATGTAGCTTAACTTAAAGCCCCTCGCTTACACCGAGAAAATGTCTGTGAAACCCAAACCATTTTGA

Str: >>>>>>>..>>>>......>>>.......>.>>.......

Your-seq.trna3 (2874-2946) Length: 73 bp Type: Leu Anticodon: TAA at 34-36 (2907-2909) Score: 28.25 * | * | * | * | * | * | * | Seq:

GTTAATGAAGCAAAACCGGATATGCGAGAGATCTAAGTCCTCTTATaAGGGGGTTCAAGTCCCCTCACTAACT

Str: >>>>.>>..>>>..........>>>.........>>>>.......

Your-seq.trna4 (3905-3975) Length: 71 bp Type: Ile Anticodon: GAT at 32-34 (3936-3938) Score: 34.52 * | * | * | * | * | * | * | Seq:

GAAAACGTGCCCGAAAGATAGGGTTCTCCTTGATAGGGAGACTTaTAGAGGTTCAAACCCCCTCGTTTTCT

Str: >>>>>>>..>>>........>>>>.......>.>>.......

Your-seq.trna5 (4046-4114) Length: 69 bp Type: Met Anticodon: CAT at 31-33 (4076-4078) Score: 34.14 * | * | * | * | * | * | * Seq:

AGTAAGGTCAGCTAATTAAGCTTTTGGGCTCATGCCCCAACAATGTAGGTTTAAATCCCTCCTTTACTA Str: >>>>>>>..>>>>.....>>>>.........>>.......

Your-seq.trna6 (4124-4192) Length: 69 bp Type: Met Anticodon: CAT at 31-33 (4154-4156) Score: 32.39

* | * | * | * | * | * | * Seq:

AGTAAAGTATGCTAATTAAGCTCTTGGGCCCATACCCCAACAATGTAGGTTAAAACCCTTCCTTTACTT Str: >>>>>>>...>>>.....>>>>........>>>.......

Your-seq.trna7 (5226-5295) Length: 70 bp Type: Trp Anticodon: TCA at 32-34 (5257-5259) Score: 37.73 * | * | * | * | * | * | * Seq:

AGAAACTTAGGCTAATAAAGACCTAGAACCTTCAAAGTTCTCAGTAGGAGTTAAATTCTTCTAGTTTCTG

Str: >>>>>>>..>>.>......>>>>.......>>>>.......

Your-seq.trna8 (7221-7289) Length: 69 bp Type: Asp Anticodon: GTC at 31-33 (7251-7253) Score: 42.26 * | * | * | * | * | * | * Seq:

GAGGATTTAGTTAAAATATAACATTACTTTGTCAAAGTAAAAtTACTAGTTAAATTCTTGTAAACCTCT Str: >>>>.>>..>>>>.....>>>>.......>.>>.......

Your-seq.trna9 (9670-9738) Length: 69 bp Type: Gly Anticodon: TCC at 31-33 (9700-9702) Score: 50.62 * | * | * | * | * | * | * Seq:

ACTTTCTTAGTACAAACAGTACAAATGACTTCCAATCATTTAATCTTGGTTTAAATCCGAGAGAAAGTA Str: >>>>>>>..>>>>.....>>>>.......>>>>.......

Your-seq.trna10 (10097-10165) Length: 69 bp Type: Arg Anticodon: TCG at 31-33 (10127-10129) Score: 34.55 * | * | * | * | * | * | * Seq:

GAGCTGCTAGTCCAACAAAGACAACTGATTTCGACTCAGTTAATTATGGTTCAACCCCATAGCACCTCT Str: >>>.>>>..>>>.......>>>>.......>>>>.......

Your-seq.trna11 (11808-11876) Length: 69 bp Type: His Anticodon: GTG at 31-33 (11838-11840) Score: 40.93 * | * | * | * | * | * | * Seq:

GTGAATATAGTTTAAATAAAACCCTAGATTGTGATTCTAGGAATGAGAGATAGTATCTCTCTGTTCACC Str: >>>>>>>..>>>>.....>>>>.......>>>>.......

Your-seq.trna12 (14364-14296) Length: 69 bp Type: Glu Anticodon: TTC at 31-33 (14334-14332) Score: 39.65 * | * | * | * | * | * | * Seq:

GTTCTTATAGTGGAATTACAACAGTAGTTTTTCAGACTACCGGTCTAGGTTAAAATCCTAGTAGGAATA Str: >>>>>>>..>>.>.....>>>>.......>>>>.......

Your-seq.trna13 (7220-7150) Length: 71 bp Type: Ser Anticodon: TGA at 33-35 (7188-7186) Score: 49.12 * | * | * | * | * | * | * | Seq:

AAGAAAGTGACAGAGGGGCTATGTGTCTGACTTGAAATCAGGCTACGGGGGTTCGATTCCCTCCTTTCTCG

Str: .>>>>>>..>>>.........>>>>.......>>>>.......

Your-seq.trna14 (5603-5537) Length: 67 bp Type: Tyr Anticodon: GTA at 29-31 (5575-5573) Score: 46.04 * | * | * | * | * | * | * Seq:

GGTAAAGTGGCCGAGTAGGTGGCGGGTTGTAGCCCTGAAGACGGAGGTTCAAATCCTTCCTTTACCA Str: >>>>>>>..>>>.....>>>.......>>>>.......

Your-seq.trna15 (5536-5471) Length: 66 bp Type: Cys Anticodon: GCA at 28-30 (5509-5507) Score: 16.33 * | * | * | * | * | * | * Seq:

GGCCCTGGAGTGTcATCACGTGTGGTTGCAAACCATAAAAAGCAGAATAATTTCCTGCCGGGGCTT

Str: >>>>>>>..>>>....>>>>.......>>>.........

Your-seq.trna16 (5440-5368) Length: 73 bp Type: Asn Anticodon: GTT at 34-36 (5407-5405) Score: 39.82 * | * | * | * | * | * | * | Seq:

TAGAATGAAGCTCGCTGGAtTGAGTGTTTAGCTGTTAACTAAAATAtTACGGGATCAAGGCCCGTCTTTCTAG

Str: >>>>>.>..>>>>........>>>>.......>>>>.......

Your-seq.trna17 (5365-5296) Length: 70 bp Type: Ala Anticodon: TGC at 32-34 (5334-5332) Score: 29.28 * | * | * | * | * | * | * Seq:

AGGACTTTAGCTTAATTAAAAGTTTTTGAGTTGCATTCATAAGATGTGGGATAATTACCTGCAAGTCCTA

Str: >>>>>>>..>>>>.......>>>.......>>>>.......

Your-seq.trna18 (4045-3975) Length: 71 bp Type: Gln Anticodon: TTG at 33-35 (4013-4011) Score: 58.91 * | * | * | * | * | * | * | Seq:

TAAGGAGTAGCATAACGGAAGTGCGAAGGGCTTTGATCTCTTAGGTGTAGGTTCAATTCCTACCTTTTTAA

Str: >>>>>>>..>>>>.......>>>>.......>>>>.......

Your-seq.trna19 (209-141) Length: 69 bp Type: Pro Anticodon: TGG at 31-33 (179-177) Score: 41.56 * | * | * | * | * | * | * Seq:

CAGAACATAATTTAATAAGAATGTTGGCTTTGGGGGCCAGTAGTAGAGGTTTAACTCCTTTTGTTCTGA Str: >>>>>>>..>>>>.....>>>>.......>>>>.......