【文章內(nèi)容簡介】 來研究玉米的進(jìn)化，他們對17個玉米基因型，12個玉蜀黍基因型和幾個墨西哥玉蜀黍變種的tb1基因的編碼區(qū)和非編碼區(qū)進(jìn)行了分析，表明在玉米的馴化過程中，選擇壓力對啟動子區(qū)域的影響比編碼區(qū)大，進(jìn)而造成表型的改變。此外，植物病原微生物和昆蟲在外來?xiàng)l件的選擇壓力下，基因常常會發(fā)生變異，使作物品種喪失抗性，藥劑防治效果下降。采用基因突變檢測技術(shù)，及時地監(jiān)測病原微生物和昆蟲基因的變異情況，對玉米的抗病蟲育種以及病蟲害防治均具有一定指導(dǎo)意義?！NP位點(diǎn)的生物信息學(xué)發(fā)掘隨著人類基因組計(jì)劃和各種模式生物的基因組計(jì)劃陸續(xù)開展，NCBI、EBI、DDBJ三大數(shù)據(jù)庫中存儲的DNA序列數(shù)據(jù)量飛速增長，這一不斷增大的數(shù)據(jù)庫資源成為尋找SNPs的重要寶藏。采用先進(jìn)的生物信息學(xué)軟件，利用計(jì)算機(jī)自動識別，從已有的DNA Database中搜尋SNP位點(diǎn)已經(jīng)成為一種簡單、有效、廉價(jià)的新策略。對于已經(jīng)完成全基因組測序的物種，可以基于不同生態(tài)型或種質(zhì)的全基因組序列對比來發(fā)現(xiàn)它們間的序列多態(tài)性位點(diǎn)[[] 陳偉，張戈，張思仲．基于生物信息學(xué)的SNP候選位點(diǎn)搜尋方法．遺傳，2001，23(2)：153156]。在2002，水稻的兩個品種（粳稻品種日本晴和秈稻品種9311）和擬南芥的兩種生態(tài)型（Col和Ler）已經(jīng)完成全基因組序列測定，在當(dāng)年建立了擬南芥全基因組序列SNP多態(tài)性數(shù)據(jù)庫[[] Jander G, Norris SR, Rounsley SD, et al. Arabidopsis mapbased cloning in the postgenome era. Plant Physiology, 2002, 129: 440450]，2004年建立了水稻的基因組序列SNP多態(tài)性數(shù)據(jù)庫[[] Shen YJ, Jiang H, Jin JP, et al. Development of GenomeWide DNA Polymorphism Database for MapBased Cloning of Rice Genes. Plant Physiology, 2004, 135: 11981205]；而對于大多數(shù)物種而言，雖沒有全基因組序列數(shù)據(jù)，但有豐富的表達(dá)序列標(biāo)簽（Expressed Sequence Tag，EST）[[] Zheng Y, James MP. The discovery and confirmation of single nucleotide polymorphisms in the human p53R2 gene by EST database analysis. Mutagenesis, 2004, 17(5): 361364,[] Mariana S, Diana B, Adhemar Z, et al. Single nucleotide polymorphisms identification in expressed genes of Schistosoma mansoni. Molecular Biochemical Parasitology, 2007, 154: 134140,[] Schultz J, Doerks T, Ponting CP. More than 1000 putative new human signaling proteins revealed by EST data mining. Nature Genetics, 2000, 25: 201204,[] Kim H, Schmid CJ, Deckerks, et al. A double screening method to identify reliable candidate nonsynonymous SNP from chicken EST data. Animal Genetics, 2003, 34: 249254,[] Schmid KJ, Sorensen TR, Strack R. Large scale identification and analysis of genome wide singlenucleotide polymorphisms for mapping in Arabidopsis thaliana. Genome Res, 2003, 13: 12501257,[] David C, Ksenija G, Ross NC, et al. Development of a set of SNP markers present in expressed genes of the apple. Genomics, 2008, 92: 353358]和序列標(biāo)記位點(diǎn)（SequenceTagged Site，STS）資源[[] Marth GT, Korf I, Mark D, et al. A general approach to singlenucleotide polymorphism discovery. Nature Genetics, 1999, 23: 452455,[] Taillon MP, Gu ZJ, Li Q, et al. Overlapping Genomic Sequence: A treasure Trove of Single nucleotide Polymorphisms. Genome Res, 1998, 8: 748754]。在GenBank中，僅玉米的EST序列已經(jīng)達(dá)到200多萬條，它是SNP位點(diǎn)發(fā)掘的有一個重要資源；此外，一些物種的基因組序列在公共數(shù)據(jù)庫中公布的序列的豐富度不高，很多實(shí)驗(yàn)室就自行構(gòu)建cDNA文庫進(jìn)行SNP位點(diǎn)的發(fā)掘[[] Morales M, Roig E, Monforte AJ. Singlenucleotide polymorphisms detected in expressed sequence tags of melon (cucumis melo L.). Genome, 2004, 47(2): 352360,[] Coles ND, Coleman CE, Christensen SA, et al. Development and use of an expressed sequenced tag library in quinoa (Chenopodium quinoa Willd.) for the discovery of single nucleotide polymorphisms. Plant Science, 2005, 168: 439447,[] Fahrenkrug SC, Freking BA, Smith TP, et al. Single nucleotide polymorphism (SNP) discovery in porcine expressed genes. Animal Genetics, 2002, 33: 186195,[] Fizsimmons CJ, Savolainen P, Amini B, et al. Detection of sequence polymorphisms in red junglefowl and white leghorn ESTs. Animal Genetics, 2004, 35: 391396]。 基于表達(dá)序列標(biāo)簽的SNP發(fā)掘 表達(dá)序列標(biāo)簽概述表達(dá)序列標(biāo)簽是從已經(jīng)建立好的cDNA文庫中隨機(jī)挑取克隆，從539。 或者339。 末端對插入的cDNA片段進(jìn)行一輪單向測序，所獲得的一段60～500 bp大小的cDNA序列。每一條EST序列均代表文庫建立時所采樣品特定發(fā)育時期和生理狀態(tài)下的某個基因的部分表達(dá)序列[[] Adams MD, Kelley JM, Jeannine D, et al. Complementary DNA sequencing expressed sequence tags and human genome project. Science, 1991, 252: 16511656.]。EST可用于搜尋同一物種中基因家族的新成員[[] Still IH, Vince P, Cowell JK．The third member of the transforming acidic coiled coilcontaining gene family TACC3, maps in 4p16, close to translocation breakpoints in multiplemyeloma and is upregulated in various cancer cell lines. Genomics, 1999, 58(2): 165l70]；搜尋不同物種間功能相同的基因[[] 劉春宇，張春玲，夏家輝．?dāng)?shù)據(jù)綜合分析鑒定人類Auxilin基因．生物化學(xué)與生物物理進(jìn)展，1998，25(5)：434439]；搜索已知基因的不同剪切模式[[] Modrek B, Resch A, Grasso C, et al. Genomewide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res, 2001, 29 (13): 28502859]；搜索等位基因在基因表達(dá)中的變異[[] Lanfranchi G, Muraro T, Caldara F, et a1. Identification of 4370 expressed sequence tags from a 339。endspecific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization. Genome Res, 1996, 6(1): 3542]等。公共數(shù)據(jù)庫中的EST序列來自不同機(jī)構(gòu)的不同文庫，這些EST序列往往體現(xiàn)著很高的多態(tài)性，因而可用于發(fā)掘表達(dá)序列中存在的SNP位點(diǎn)，這些SNP在進(jìn)行遺傳、表型相關(guān)性分析時是非常重要的。 常用的SNP發(fā)掘軟件（1）Polybayes Polybayes是在UNIX環(huán)境下開發(fā)來專門檢測SNP的軟件，可對任意DNA序列的突變堿基進(jìn)行檢測。SNP等位基因頻率、序列準(zhǔn)確度、EST序列長度及PSNP最小值的選擇都對其靈敏度有很大影響，被分析序列的準(zhǔn)確度和SNP等位基因頻率越高，所包含EST序列越短，選擇PSNP的最小值越大，則Polybayes計(jì)算法檢測SNP靈敏度越高，可達(dá)100%[[] March GT, Korf I, Yandell MD, et al. A general approach to single nucleotide polymorphism discovery. Nat Genet, 1999, 23: 452456]。（2）SNPpipelineSNPpipeline是利用信息學(xué)工具設(shè)計(jì)的檢測任意DNA序列中SNP位點(diǎn)存在情況的一套半自動裝置，分為PHRED，PHRAP和DEMIGIACE三個部分。PHRED負(fù)責(zé)分析大量基因組序列，收集含有EST的序列，并去除不準(zhǔn)確的序列；PHRAP進(jìn)行集中排列PHRED所選擇的有多態(tài)性位點(diǎn)的序列；連接色譜儀，此時選擇某一個特定核苷酸，顯示器會給出其色譜儀洗峰、等位基因頻率、洗峰振幅等信息，然后根據(jù)峰度的不同判定SNP位點(diǎn)；最后用DEMIGIACE進(jìn)行篩選、檢驗(yàn)并確認(rèn)真正的SNP，去除錯誤的信息，敏感度在99%以上[[] Touching base: More SNPs in the pipeline. Nat Genet, 1999, 22: 221]。Buetow等[[] Buetow KH, Edmonson MN, Cassidy A B. Reliable identification of large number of candidate SNPs from public EST data. Nat Genet, 1999, 21: 323325]利用該軟件發(fā)現(xiàn)了10000個候選SNP；Cheng等[[] Cheng TC, Xia QY, Qian JF, et al. Mining single nucleotide polymorphisms from EST data of silkworm, Bombyx mori, inbred strain Dazao. Insect Biochemistry and Molecular Biology, 2004, 34: 523530]以來自12個不同cDNA 文庫的81635條家蠶EST序列，用Phred/Phrap軟件包發(fā)現(xiàn)了101個高質(zhì)量SNP候選位點(diǎn)以及27個單堿基的插入/缺失位點(diǎn)。Yamamoto等[[] Naoki Y, Taneaki T, Manabu W, et al. Expressed sequence tags from the laboratorygrown miniature tomato(Lycopersicon esculentum) cultivar MicroTom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars. Gene, 2005, 356: 127134]，用Phrap在35824條番茄得EST序列中發(fā)現(xiàn)2039個SNP位點(diǎn)，和121個插入/缺失（InDel）位點(diǎn)。（3）AutoSNPAutoSNP軟件中包含一個發(fā)掘SNP 位點(diǎn)的核心程序auto_snip_cap3，該核心程序中包含2個應(yīng)用程序d2_cluster和cap3，前者用于同源序列聚類，后者用于聚類序列間的比對。AutoSNP是根據(jù)單體型得