普通小麦编码组蛋白H3基因的克隆与分析
摘要
为了获得具有组织表达特异性和与胁迫相关的小麦组蛋白H3,进一步了解组蛋白H3的功能。本文以拟南芥组蛋白H3的蛋白序列为信息探针,对小麦(Triticum aestivum)EST数据库进行同源检索筛选,克隆了小麦组蛋白H3基因。通过生物信息学的方法对得到的小麦组蛋白H3基因进行分析,分析的内容包括:内含子形式,电子基因表达谱和电子染色体定位。并采用半定量RT-PCR的方法分析了小麦组蛋白H3基因在盐处理下的表达情况。研究结果如下:
1小麦组蛋白H3基因的克隆
以拟南芥组蛋白H3的蛋白序列为信息探针,BLAST检索GenBank中的小麦EST数据库,获得了小麦组蛋白H3基因的cDNA序列。为了进一步验证电子克隆序列的正确性,在起始密码子和终止密码子区域设计特异引物,经RT-PCR进行克隆、序列分析验证,最终得到7个小麦组蛋白H3基因且与电子克隆序列相一致。经过分析,这7个基因所编码的小麦组蛋白H3可以分为两类:H3.1型和H3.2型。
2小麦组蛋白H3基因的生物信息学分析
以普通小麦基因组DNA为模板,扩增上述7个基因基因组序列,获得了3个基因的基因组序列,有一个基因含有一个长度为198bp的内含子,该内含子较其它物种类似基因内含子长一些。采用UNIGENE数据库进行的组织表达谱分析表明,克隆的基因组织特异性表达不明显。采用GrainGenes数据库将这7个基因定位于7BL,4AL和7AS染色体上。
3小麦组蛋白H3基因在盐胁迫下表达量分析
采用半定量RT-PCR的方法,对所获小麦组蛋白H3基因在盐胁迫处理下的表达情况进行了分析,获得了一个随盐处理时间的延长表达量增高的组蛋白H3基因。
In order to further undersranding the functions of histon H3,we hoped to abtain tissue specific expression or stress related histon H3 genes.In this paper,we used the EST Blast method to clone Triticum aestivum histon H3 gene,analyzed its expression changes under salt stress using semi-quatitative RT-PCR,and demonstrated its intron pattern,expression profile and in silico mapping through bioimformatic analysis.
1 Cloning and analysis of Triticum aestivum histon H3 gene
Seven Triticum aestivum histon H3 gene was cloned by Blasting the Triticum aestivum EST database with the Arabidopsis thaliana homologous protein histom H3 sequence as a querying probe.To examine the accuracy of in silico cloning cDNA sequences,these sequences were cloned by RT-PCR and sequenced.The result confirmed that the in silico cloning cDNA sequences were accurate,and they can derided into two kind:type H3.1 and type H3.2
2 in silico analysis of Triticum aestivum histon H3 gene
In order to deternine the intron pattern of wheat histon H3 gene,specific primers which were designed for cDNA cloning were also used to amplify genomic DNA, three out of seven genomic sequences of histone H3 genes were abtained,and only one of them had one 198 bp intron,which is a little longer than those homolog genes in other species.In silico expression patterns in different tissues of those seven wheat histon H3 genes were investigated by Unigene database,the results shows that they have no tissue specific expression.Also Graingene database was used to determine their In silico mapping,which shows they were distributed over chromesome 7BL, 4AL and 7AS.
3 expression of Triticum aestivum histon H3 gene in response to salt treatment
Semi-quantitative RT-PCR assay was performed to detect the expression pattern of the obtained Triticum aestivum histon H3 gene under salt stress,the result showed that the transcription of one histon H3 gene is accumulated after treated by salt.
1小麦组蛋白H3基因的克隆
以拟南芥组蛋白H3的蛋白序列为信息探针,BLAST检索GenBank中的小麦EST数据库,获得了小麦组蛋白H3基因的cDNA序列。为了进一步验证电子克隆序列的正确性,在起始密码子和终止密码子区域设计特异引物,经RT-PCR进行克隆、序列分析验证,最终得到7个小麦组蛋白H3基因且与电子克隆序列相一致。经过分析,这7个基因所编码的小麦组蛋白H3可以分为两类:H3.1型和H3.2型。
2小麦组蛋白H3基因的生物信息学分析
以普通小麦基因组DNA为模板,扩增上述7个基因基因组序列,获得了3个基因的基因组序列,有一个基因含有一个长度为198bp的内含子,该内含子较其它物种类似基因内含子长一些。采用UNIGENE数据库进行的组织表达谱分析表明,克隆的基因组织特异性表达不明显。采用GrainGenes数据库将这7个基因定位于7BL,4AL和7AS染色体上。
3小麦组蛋白H3基因在盐胁迫下表达量分析
采用半定量RT-PCR的方法,对所获小麦组蛋白H3基因在盐胁迫处理下的表达情况进行了分析,获得了一个随盐处理时间的延长表达量增高的组蛋白H3基因。
In order to further undersranding the functions of histon H3,we hoped to abtain tissue specific expression or stress related histon H3 genes.In this paper,we used the EST Blast method to clone Triticum aestivum histon H3 gene,analyzed its expression changes under salt stress using semi-quatitative RT-PCR,and demonstrated its intron pattern,expression profile and in silico mapping through bioimformatic analysis.
1 Cloning and analysis of Triticum aestivum histon H3 gene
Seven Triticum aestivum histon H3 gene was cloned by Blasting the Triticum aestivum EST database with the Arabidopsis thaliana homologous protein histom H3 sequence as a querying probe.To examine the accuracy of in silico cloning cDNA sequences,these sequences were cloned by RT-PCR and sequenced.The result confirmed that the in silico cloning cDNA sequences were accurate,and they can derided into two kind:type H3.1 and type H3.2
2 in silico analysis of Triticum aestivum histon H3 gene
In order to deternine the intron pattern of wheat histon H3 gene,specific primers which were designed for cDNA cloning were also used to amplify genomic DNA, three out of seven genomic sequences of histone H3 genes were abtained,and only one of them had one 198 bp intron,which is a little longer than those homolog genes in other species.In silico expression patterns in different tissues of those seven wheat histon H3 genes were investigated by Unigene database,the results shows that they have no tissue specific expression.Also Graingene database was used to determine their In silico mapping,which shows they were distributed over chromesome 7BL, 4AL and 7AS.
3 expression of Triticum aestivum histon H3 gene in response to salt treatment
Semi-quantitative RT-PCR assay was performed to detect the expression pattern of the obtained Triticum aestivum histon H3 gene under salt stress,the result showed that the transcription of one histon H3 gene is accumulated after treated by salt.
引文
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黄骥(C)。水稻功能基因的电子克隆与盐胁迫诱导cDNA文库的构建。2002,硕士论文
李鑫,章涛。新基因的克隆策略和方法。海峡药学,2004,16(3):16-19
陆佳韵,王秀琴,吴旻。EST及其应用。生命科学,1999,11(4):186-188
骆蒙,贾继增。植物基因组表达序列标签(EST)计划研究进展。生物化学与生物物理进展,2001,28(4):494-497
宋宇轩,曹斌云。表达序列标签(ESTs)及其应用。家畜生态,2004,25(4):154-155
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张庆华,茅矛,陈竺。基因组研究中全长cDNA克隆的策略。生物工程进展,2000,20(4):3-5
张华莉,邓昊,张瑞芳,夏昆,夏家辉。人类TECIB基因的电子克隆。遗传学报,2003,30(4):317-321
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董利。后基因组时代的生物信息学。国外医学生理与病理科学与临床分册,2001,20(4):327-330
何志颖,姚玉成,胡以平。EST技术及其在基因全长cDNA克隆上的应用策略。国外医学遗传学分册,2002,25(2):67-69
贺林。解码生命:人类基因组计划和后基因组计划。科学出版社,2000
黄骥(a),张红生,曹雅君,钱晓茵,杨金水。水稻功能基因的电子克隆策略。中国水稻科学,2002,16(4):295-298
黄骥(b),张红生,曹雅君,王东,王建飞,杨金水。一个新的水稻C_2H_2型锌指蛋白cDNA的克隆与序列分析。南京农业大学学报,2002,25(2):110-112
黄骥(C)。水稻功能基因的电子克隆与盐胁迫诱导cDNA文库的构建。2002,硕士论文
李鑫,章涛。新基因的克隆策略和方法。海峡药学,2004,16(3):16-19
陆佳韵,王秀琴,吴旻。EST及其应用。生命科学,1999,11(4):186-188
骆蒙,贾继增。植物基因组表达序列标签(EST)计划研究进展。生物化学与生物物理进展,2001,28(4):494-497
宋宇轩,曹斌云。表达序列标签(ESTs)及其应用。家畜生态,2004,25(4):154-155
孙亮先,谢金进,黄周英。表达序列标记(EST)研究进展。泉州师范学院学报(自然科学),2002,20(4):75-79
万海伟,杜立新。表达序列标签(EST)在基因组学研究中的应用。生物技术通报,2004,1:135-138
汪以真,林文学,许梓荣。半定量RT-PCR法评定不同生长阶段猪抗菌肽Pr-39基因表达差异。农业生物技术学报,2004,12(1):52-55
徐小平。生物信息学的现状与前景。郧阳医学院学报,2004,23(4):255-256
于浩,刘娣,杨秀芹。基于表达序列标签数据库(dbEST)的电子克隆技术。黑龙江动物科学,2003,(4):19-21
余波。生物信息学的现状与前景。生物学教学,2003,28(10):1-3
张成岗,贺福初。生物信息学方法与实践。科学出版社,2002
张德礼,丁培国,凌伦奖,陈润生,马大龙。人类新基因C17orf32的电子克隆和编码区序列RT-PCR验证。生物化学与生物物理进展,2002,29(4):543-549
张庆华,茅矛,陈竺。基因组研究中全长cDNA克隆的策略。生物工程进展,2000,20(4):3-5
张华莉,邓昊,张瑞芳,夏昆,夏家辉。人类TECIB基因的电子克隆。遗传学报,2003,30(4):317-321
赵国屏。生物信息学。科学出版社,2002
Adams MD,Dubnick M,Kerlavage AR,Moreno R,Kelley JM,Utterback TR,Nagle JW,Fields C and Venter JC.Sequence identification of 2375 human brain genes.Nature,1992,355(13):632-634
Adams MD,Kelley JM,Gocayne JD,Dubnick M,Polymeropoulos MH and Xiao H.Complementary DNA Sequencing:Expressed Sequence Tags and Human Genome Project.Science,1991,252:1651-1656
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