论文标题:直翅目昆虫的线粒体谱系基因组学研究
Phylogenomic Study of Orthoptera Mitochondrion
论文作者
论文导师 黄原,论文学位 硕士,论文专业 动物学
论文单位 陕西师范大学,点击次数 119,论文页数 85页File Size5977K
2007-05-01论文网 http://www.lw23.com/lunwen_86158137/
mitochondrial genome;; Phylogenomics;; phylogeny;; Orthoptera;; Tettigonia chinensis;; Conocephalus gladiatus
直翅目属于节肢动物门、六足总纲、昆虫纲，是一类较常见的昆虫，包括蝗虫、蟋蟀、螽蟖、蝼蛄等昆虫，已知有20000种以上，广泛分布于世界各地，以热带地区种类较多。目前针对直翅目的系统发育关系尚未形成统一的系统。 本研究采用了基于长PCR的二次PCR策略，采用PCR直接测序和克隆测序相结合的方法，对螽蟖总科的两种昆虫中华螽蟖和长瓣草螽的线粒体基因组部分序列进行测定和组装，并进行了注释。通过联合GenBank收录的9种已测昆虫的线粒体基因组数据和本实验室2种已测的直翅目昆虫中华稻蝗和优雅蝈螽，采用谱系基因组学研究方法，对直翅目昆虫系统发育关系进行了研究。主要结论如下： 1、所测得的两种直翅目昆虫的线粒体基因组长度分别为中华螽蟖14974bp，长瓣草螽14591bp。中华螽蟖的所测线粒体部分包括13种蛋白质基因、21种tRNA基因(未测出tRNA~(Ile))、2种rRNA基因。长瓣草螽的所测线粒体部分包括13种蛋白质基因、19种tRNA基因(未测出tRNA~(Ile)、tRNA~(Met)、tRNA~(Gln))、2种rRNA基因。两种螽蟖的控制区都未能成功扩增。2、所测两种螽蟖的线粒体基因在基因顺序上与东方蝼蛄一致，即tRNA~(Lys(K))和tRNA~(Asp(D))基因排序为KD，而与飞蝗不同。中华螽蟖线粒体基因在16对基因之间存在总长度达88bp的重叠，基因间的间隔区长达296bp。长瓣草螽在15对基因之间存在总长度达128bp的重叠，tRNA~(Gly)几乎全部位于COⅢ基因内部，基因间有38bp的序列间隔区。 3、中华螽蟖和长瓣草螽线粒体基因组的蛋白编码基因、rRNA基因和tRNA基因均存在很强的碱基A+T含量偏向性，最高的两个基因为tRNA~(Glu)和tRNA~(Asp)。蛋白质基因序列的A+T含量在密码子的1、2、3位及整体水平都具有明显的A+T碱基偏向性，这与线粒体基因组具有高A+T偏向性的特征是一致的；蛋白质编码基因的A+T含量在第3位密码子处都是最高的，这线粒体具有GC→AT突变压，以及密码子第3位点存在的高变异率规律是十分相符的。 4、中华螽蟖和长瓣草螽的13种蛋白质基因都使用ATN作为起始密码子，除ND5基因以单独的T作为终止密码子外，其余的蛋白质编码基因都以完整的三联密码子TAA或TAG作为终止密码子。蛋白质基因的氨基酸组成中Leu、Ser、Ile、Phe的含量很高。 5、除tRNA~(Ser(AGN))缺失了D-臂外，其余的tRNA基因二级结构都可以折叠成典型的三叶草结构，反密码子和多数六足动物一致，多为GNN或UNN。反密码子环和氨基酸接受臂非常保守，其它臂环的变异较大。在tRNA二级结构的4个臂上均存在不同程度的碱基错配，错配类型多为G-U配对。2个rRNA基因较保守。 6、对13种昆虫线粒体的蛋白质编码基因(PCG)数据集、蛋白质数据集、tRNA数据集，应用谱系基因组学方法进行直翅目昆虫系统发育研究表明，蝗亚目具有单系性，本研究所选用的数据对直翅目的系统发育关系缺乏足够解析力，在排除长枝吸引的影响后，只有部分数据能确定直翅目的单系性。而目前大多数学者认为直翅目是一个单系群，因此关于直翅目的单系性问题，有待于基于更多样本量的线粒体基因组数据进行深入探讨。
Orthoptera is one of the largest families among the insecta with more than20000 described species widely distributed throughout the world especially in tropicalzone, such as locust, cricket, long-horned grasshopper, mole cricket and so on. Theclassification of species in Orthopteran has been controversial and complicate. In this paper, we report the mitochondrial genome of Tettigonia chinensis andConocephalus gladiatus determinded by sub-PCR method, which are representativeinsect in Tettigonioidea. Phylogenomic analysis among mitogenomes of T. chinensis and C.gladiatus and other insect orders was accomplished, and the utility of mitogenome forOrthopteran phylogeny construction was briefly discussed. 1. The determined mitochondrial genome sequeces of T. chinensis and C. gladiatus are14,974 bp and 14,591 bp long respectively. T. chinensis mitgenome encodes 13 putativeproteins, 2 rRNAs and 21 tRNAs(excluding tRNA~(Ile)) while the C. gladiatus mitgenomeencodes 13 putative proteins, 2 rRNAs and 19 tRNAs (excluding tRNA~(Ile), tRNA~(Met),tRNA~(Gln)) without A+T-rich region. 2. The arrangement of mitochondrial coding genes matches almost with those ofGryllotalpa orientalis. A total of 88 bp overlap regions among T. chinensis mt genomesare found in sixteen locations with a total of 296 bp of intergenic spacer. C. gladiatus hasa 128bp-overlap region and a 38bp-intergenic spacer while the tRNA~(Gly) almost locatedin COⅢentirely. 3. The average A+T content of the T. chinensis and C. gladiatus mitochondrial genomeprotein coding sequence, rRNA and tRNA gene was corresponding well to the A+T biasgenerally observed in insect mitochondrial genomes. The strongest A+T blase is foundat the tRNA~(Glu) and tRNA~(Asp) and at third codon positions which are under the lowpurifying selection. 4. Common Met start codons could be assigned to nine and six of the proteincoding genes in T. chinensis and C. gladiatus mitogenome respectively. Common stopcodons (TAA and TAG) could be assigned to most of the putative protein sequences.Only the ND5 genes terminate into a single T residue. Leucine, isoleucine, serine,phenylalanineare are the most frequently used amino acids in the mt protein-coding genes. 5. All of tRNAs showed typical clover leaf secondary structures and theiranticodons are similar to those found inother Hexapoda animals, tRNAs possessinvariable aminoacyl stem, anticodon loop, and anticodon stem. Most of the sizevariation among tRNAs stemmed from length variation in the TΨC and DHU arms. TheG-U pair is found in all the tRNA genes. The two rRNA genes are conservative in T.chinensis and C. gladiatus. 6. Phylogenomic analysis of mitochondrion were performed with maximumparsimony, maximum likelihood and Bayesian inference methods with protein-codinggene data set, putative protein data set and tRNA gene data set. Caelifera ismonophyletic in this study. Excluding the long-brnach attraction, a monophyly of theancient insect order Orthoptera was still not recovered. The reason for the lack ofsupport by the mitogenome sequences is not obvious. Whether the paraphyly ormonophyly of the Orthoptera can"t be concluded in the paper. To further ravel thephylogenetic relationships of Orthoptera insects, a larger number of insect orders andmitogenome are required.
|
