天牛科基因条形码构建及分子快速鉴定技术研究
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摘要
脱氧核糖核酸（DNA）条形码是指用短的、标准的DNA序列作为物种标记来鉴定物种的一种新技术，它是传统形态学分类的有效补充。目前，天牛科昆虫DNA条形码的研究和应用尚处于探索阶段，筛选候选片段、进而确定通用条形码是当前天牛科条形码研究的首要任务。本文以天牛科昆虫线粒体DNA细胞色素c氧化酶I亚基（COI）全序列为对象进行基因分析以寻找可以成为基因条形码的片段，同时针对实验室多年保存标本DNA难以提取的问题，对天牛科昆虫标本的保存与提取方法进行了探讨，最后对实验多年保存天牛科标本进行目标基因测序与基因网站GenBank下载的COI序列一同构建天牛科的基因条形码。研究主要结果如下：
1.昆虫幼虫标本的保存及DNA的提取一直是一个难点，本文以三种保存方式保存一年以上的松墨天牛幼虫为实验材料，采用传统酚-氯仿提取方法、TaKaRaDNA快速提取试剂盒及金麦格动物细胞组织/细胞基因组DNA磁珠提取试剂盒进行DNA提取，对不同提取方法所获取的DNA纯度与质量进行分析比较，确定了短期内的天牛幼虫活体保存DNA保存效果最佳，而磁珠法提取DNA试剂盒提取DNA纯度与质量最好。
2．馆藏标本都已经成为遗传学研究中新的样品来源。成功提取这些标本DNA的报道也很多。但是无论采取何种提取方法，获得的DNA含量极低、片段较短、难以进行PCR扩增。本研究首次采用磁珠DNA提取法对多年保存的昆虫标本进行微量DNA提取。通过对实验结果和所测基因片段进行分析，发现磁珠DNA提取法对于多年保存，DNA微量且已出现断裂的标本具有良好的提取效果，完全满足后续实验需要。但未能从福尔马林浸渍保存标本中获得有效DNA。
3.天牛线粒体COI全基因约1545bp，不可能全部用作构建基因条形码，哪一段既可以代表天牛科昆虫的基因特征，又可以有效区分各种天牛还尚无定论。本研究将实验室测得与网上下载的天牛科五个亚科24种天牛的线粒体DNACOI全序列进行基因分析，并利用这些信息初步构建了天牛科5亚科的系统发育树，结果显示出幽天牛亚科与花天牛亚科的亲缘关系较近，其次是沟胫天牛亚科，再次是天牛亚科，最后是锯天牛亚科，但各亚科间的进化关系仍需进一步研究。而应用氨基酸序列来分析和研究天牛科昆虫的系统发育关系更能够反应物种进化的规律，得出的结论也相对更为科学。
4.将天牛线粒体COI全基因序列建树结果与目前最常用的两段COI基因片段建树结果进行比较发现，位于全序列77-580bp位置的基因片段更能反映出天牛科线粒体COI全序列特点，更适合成为代替全序列的天牛科基因条形码。
5.在本研究理论支持下对天牛科5个亚科160种天牛进行了基因条形码构建，应用BioEdit软件进行序列比对，结果显示此483个位点中没有插入、缺失及终止密码子出现，同时发现这些种类在该基因片段具有明显的多态性，可有效区分这些种类。
6.为了在检疫实践中不受天牛生活周期、发育虫态和残缺虫体材料的限制，本研究通过对
8对引物的筛选，筛选出了两对引物可以分别将暗褐断眼天牛Tetropium fuscum、窝梗天牛Arhopalus foveicollis与幽天牛亚科其它14种天牛在凝胶电泳图谱中区分开来。为天牛科的分子快速鉴定技术提供了研究基础。
DNA bar coding is a new technique for species-level identification by using a short andstandard DNA sequence as species labels. It is an effective complement for traditional taxonomy.At present, the research and applications on DNA bar coding of Cerambycidae are still inexploration stage, so the primary task is to select candidate genes or DNA regions and thenidentify a general DNA bar coding. The gene analysis can be targeted at the complete sequencesof Cerambycidae mtDNACOI to seek the fragment building the DNA bar coding.In order tosolve the difficulties of extracting DNA from the specimen preserved by laboratory for manyyears，we also discussed the methods of extracting DNA from insect specimen. Finally, the DNAbar coding is built by COI sequences from the GenBank and the specimens of Cerambycidaepreserved in laborary. The main results are as follows:
1. There is always a difficulty in the preservation of larva samples and the extraction of DNA.This research shows three ways to preserve Monochamus alternatus larva of more than one yearas experimental materials. The extraction of DNA could be accomplished by using traditionalmethods of Phenol-chloroform extraction with TaKaRa Kit and GenMagBio Kit. It can beconfirmed that the preserved effects of DNA of living longicorn larvae in a short time are the bestway by analysing and comparing the purity and quality of DNA obtained by different extractionmethods, and GenMagBio Kit is better way than other two methods in the purity and quality ofDNA.
2. Specimens preserved have become new sources in genetics study. Reports on extracting DNAfrom old specimens successfully were abundant. But no matter which methods they used, theDNA content obtained was very low, the fragment was quite short and the PCR amplification wasdifficult to carry out. In this paper we had accomplished the trace extracting of insects specimenspersevered for many years with MagGenBio Kit for the first time. The analysis of gene fragmentshowed that MagGenBio Kit had a good extracting effect on the specimens which were preservedwith trace amounts of DNA appearing ruptures for many years. MagGenBio Kit meets the needof follow-up experiments completely. But it was failed to obtain a valid DNA from formalinimpregnated specimens.
3.The whole gene of Cerambycidae mitochondria COI is about1545bp, the entire of whichcould not be used as gene bar coding. We don’t know which fragment can reflect the genetic characters of Cerambycidae and effectively distinguish various longicorn beetles at the same time.This research analyzed the the whole sequences of mtDNA COI in24species belong to fivesubfamilies of Cerambycidae, and built phylogenisis tree of five subfamilies. It had a conclusionthat the genetic relationship of Aseminae and Lepturinae is closest, the second is Lamiinae, thethird is Cerambycinae and finally is Prioninae, yet the evolutionary relationship between differentsubfamilies needs further research. The phylogenesis relationship analyzed by using amino acidsequences could reflect the traits of species evolution more clearly, and the conclusion is morescientific.
4. Comparing the results of whole sequences tree of longicorn beetles with two common-usedCOI gene fragment tree, we found that the gene located from seventy-seven to five hundredeighty bp could reflect the characteristic of mitochondria COI more exactly, so it was moresuitable to be gene barcoding for replacing whole sequences of longicorn gene.
5. Under the theory of this experiment,the gene bar codings of160species of longcorn beetlesbrlonging to five subfamilies of Cerambycidae had been built, and the sequence werealignmented in Bioedit. The results showed that there didn’t exist insertion, deletion or terminatorcodon in this four hundred eighty-three locus. There was an obvious polymorphism on thefragment among one hundred sixty species of longcorn beetles and which could be used fordistinguishing these kinds effectively.
6. In order to be free from the restriction of longicorn beetles cyclogeny, insect stages anddamaged, this research screened out two pairs of primer, which could distinguish Tetropiumfuscum, Arhopalus foveicollis from the other fourteen kinds of longicorn beetles of Aseminae inphotograph of Gel electrophoresis. This study could pay a foundation for deeply study of ormolecular rapid identification technology.
1.昆虫幼虫标本的保存及DNA的提取一直是一个难点，本文以三种保存方式保存一年以上的松墨天牛幼虫为实验材料，采用传统酚-氯仿提取方法、TaKaRaDNA快速提取试剂盒及金麦格动物细胞组织/细胞基因组DNA磁珠提取试剂盒进行DNA提取，对不同提取方法所获取的DNA纯度与质量进行分析比较，确定了短期内的天牛幼虫活体保存DNA保存效果最佳，而磁珠法提取DNA试剂盒提取DNA纯度与质量最好。
2．馆藏标本都已经成为遗传学研究中新的样品来源。成功提取这些标本DNA的报道也很多。但是无论采取何种提取方法，获得的DNA含量极低、片段较短、难以进行PCR扩增。本研究首次采用磁珠DNA提取法对多年保存的昆虫标本进行微量DNA提取。通过对实验结果和所测基因片段进行分析，发现磁珠DNA提取法对于多年保存，DNA微量且已出现断裂的标本具有良好的提取效果，完全满足后续实验需要。但未能从福尔马林浸渍保存标本中获得有效DNA。
3.天牛线粒体COI全基因约1545bp，不可能全部用作构建基因条形码，哪一段既可以代表天牛科昆虫的基因特征，又可以有效区分各种天牛还尚无定论。本研究将实验室测得与网上下载的天牛科五个亚科24种天牛的线粒体DNACOI全序列进行基因分析，并利用这些信息初步构建了天牛科5亚科的系统发育树，结果显示出幽天牛亚科与花天牛亚科的亲缘关系较近，其次是沟胫天牛亚科，再次是天牛亚科，最后是锯天牛亚科，但各亚科间的进化关系仍需进一步研究。而应用氨基酸序列来分析和研究天牛科昆虫的系统发育关系更能够反应物种进化的规律，得出的结论也相对更为科学。
4.将天牛线粒体COI全基因序列建树结果与目前最常用的两段COI基因片段建树结果进行比较发现，位于全序列77-580bp位置的基因片段更能反映出天牛科线粒体COI全序列特点，更适合成为代替全序列的天牛科基因条形码。
5.在本研究理论支持下对天牛科5个亚科160种天牛进行了基因条形码构建，应用BioEdit软件进行序列比对，结果显示此483个位点中没有插入、缺失及终止密码子出现，同时发现这些种类在该基因片段具有明显的多态性，可有效区分这些种类。
6.为了在检疫实践中不受天牛生活周期、发育虫态和残缺虫体材料的限制，本研究通过对
8对引物的筛选，筛选出了两对引物可以分别将暗褐断眼天牛Tetropium fuscum、窝梗天牛Arhopalus foveicollis与幽天牛亚科其它14种天牛在凝胶电泳图谱中区分开来。为天牛科的分子快速鉴定技术提供了研究基础。
DNA bar coding is a new technique for species-level identification by using a short andstandard DNA sequence as species labels. It is an effective complement for traditional taxonomy.At present, the research and applications on DNA bar coding of Cerambycidae are still inexploration stage, so the primary task is to select candidate genes or DNA regions and thenidentify a general DNA bar coding. The gene analysis can be targeted at the complete sequencesof Cerambycidae mtDNACOI to seek the fragment building the DNA bar coding.In order tosolve the difficulties of extracting DNA from the specimen preserved by laboratory for manyyears，we also discussed the methods of extracting DNA from insect specimen. Finally, the DNAbar coding is built by COI sequences from the GenBank and the specimens of Cerambycidaepreserved in laborary. The main results are as follows:
1. There is always a difficulty in the preservation of larva samples and the extraction of DNA.This research shows three ways to preserve Monochamus alternatus larva of more than one yearas experimental materials. The extraction of DNA could be accomplished by using traditionalmethods of Phenol-chloroform extraction with TaKaRa Kit and GenMagBio Kit. It can beconfirmed that the preserved effects of DNA of living longicorn larvae in a short time are the bestway by analysing and comparing the purity and quality of DNA obtained by different extractionmethods, and GenMagBio Kit is better way than other two methods in the purity and quality ofDNA.
2. Specimens preserved have become new sources in genetics study. Reports on extracting DNAfrom old specimens successfully were abundant. But no matter which methods they used, theDNA content obtained was very low, the fragment was quite short and the PCR amplification wasdifficult to carry out. In this paper we had accomplished the trace extracting of insects specimenspersevered for many years with MagGenBio Kit for the first time. The analysis of gene fragmentshowed that MagGenBio Kit had a good extracting effect on the specimens which were preservedwith trace amounts of DNA appearing ruptures for many years. MagGenBio Kit meets the needof follow-up experiments completely. But it was failed to obtain a valid DNA from formalinimpregnated specimens.
3.The whole gene of Cerambycidae mitochondria COI is about1545bp, the entire of whichcould not be used as gene bar coding. We don’t know which fragment can reflect the genetic characters of Cerambycidae and effectively distinguish various longicorn beetles at the same time.This research analyzed the the whole sequences of mtDNA COI in24species belong to fivesubfamilies of Cerambycidae, and built phylogenisis tree of five subfamilies. It had a conclusionthat the genetic relationship of Aseminae and Lepturinae is closest, the second is Lamiinae, thethird is Cerambycinae and finally is Prioninae, yet the evolutionary relationship between differentsubfamilies needs further research. The phylogenesis relationship analyzed by using amino acidsequences could reflect the traits of species evolution more clearly, and the conclusion is morescientific.
4. Comparing the results of whole sequences tree of longicorn beetles with two common-usedCOI gene fragment tree, we found that the gene located from seventy-seven to five hundredeighty bp could reflect the characteristic of mitochondria COI more exactly, so it was moresuitable to be gene barcoding for replacing whole sequences of longicorn gene.
5. Under the theory of this experiment,the gene bar codings of160species of longcorn beetlesbrlonging to five subfamilies of Cerambycidae had been built, and the sequence werealignmented in Bioedit. The results showed that there didn’t exist insertion, deletion or terminatorcodon in this four hundred eighty-three locus. There was an obvious polymorphism on thefragment among one hundred sixty species of longcorn beetles and which could be used fordistinguishing these kinds effectively.
6. In order to be free from the restriction of longicorn beetles cyclogeny, insect stages anddamaged, this research screened out two pairs of primer, which could distinguish Tetropiumfuscum, Arhopalus foveicollis from the other fourteen kinds of longicorn beetles of Aseminae inphotograph of Gel electrophoresis. This study could pay a foundation for deeply study of ormolecular rapid identification technology.
引文
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