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题名: 五种肢孔派(Podotremata)蟹类线粒体基因组测定及短尾类系统发生研究
作者: 师国慧
学位类别: 硕士
答辩日期: 2015-04-23
授予单位: 中国科学院大学
授予地点: 北京
导师: 崔朝霞
关键词: 线粒体基因组 ; 短尾类 ; 肢孔派 ; 系统发育基因组学
学科分类: 主要研究方向::海洋生物学
学位专业: 生物工程
中文摘要:  短尾次目(Brachyura),是真正的蟹类,数量大,形态多样,生境复杂。其系统进化一直是甲壳动物系统学研究的热点。尽管短尾类本身作为单系群已经得到大多数甲壳动物学家的认可,但短尾类内部各个类群之间的的系统发育关系仍有许多有争议,尤其是肢孔派(Podotremata)的进化地位。肢孔派进化地位的确立有助于了解早期短尾类的系统进化、真短尾类起源。
后生动物线粒体基因具有分子结构简单、进化速度快、母系遗传和容易获取等优点,广泛用于系统发育和种群遗传研究。相比于单个基因和几个基因的简单联合,线粒体基因组能够提供从基因排序和DNA序列变异两个方面提供进化信息。因此,线粒体基因组全序列已经成为探讨后生动物深层次的系统发育关系的理想材料。
本研究通过Long-PCR和步移测序的方法确定和注释了毛刺贝绵蟹(Dynomene pilumnoides)、东方人面蟹(Homola orientalis)、大摩罗蟹(Moloha majora)、东方乌氏蟹(Umalia orientalis)和短额琵琶蟹(Lyreidus brevifrons)线粒体基因组。利用比较基因组学和生物信息学等手段分析短尾类线粒体基因组的特征。联合GenBank数据库中已有的短尾类和歪尾类,以及本实验室已测定的马来似人面蟹线粒体基因组序列,以7种海姑虾下目物种为外群,利用蛋白编码基因的核苷酸和氨基酸两个数据库集,基于贝叶斯和最大似然法重建了短尾类系统发育关系。主要结果如下:
(1)毛刺贝绵蟹、大摩罗蟹、东方人面蟹、东方乌氏蟹和短额琵琶蟹的线粒体基因组长度分别为16,475 bp、15,910 bp、16,082 bp、15,466 bp和16,112 bp。与其他后生动物一样,每个低等蟹线粒体基因组均包含经典的37个基因(13个蛋白编码基因、2个rRNA基因和22个tRNA基因)。
(2)与大多数短尾类一样,大摩罗蟹、东方人面蟹和东方乌氏蟹线粒体基因组中仅有一个非编码区。但是,短额琵琶蟹和毛刺贝绵蟹有多个非编码区。与原始泛甲壳动物线粒体原始基因排序相比,大摩罗蟹、东方人面蟹、东方乌氏蟹和短额琵琶蟹中位于rrnStrnI之间的非编码区为其假定控制区。然而,通过与干练平壳绵蟹和脊足武田绵蟹控制区序列同源性分析,认为毛刺贝绵蟹中最长的位于trnL1trnQ之间的非编码区为其假定控制区;其控制区位置发生易位。
(3)大摩罗蟹、东方人面蟹和马来似人面蟹线粒体控制区中均存在重复单元和重复次数均不同重复序列区。正是由于重复序列的出现,人面蟹的控制区长度差异明显。值得一提的是,东方人面蟹和马来似人面蟹中的重复单元序列相似性高达95%以上。
(4)与泛甲壳动物线粒体基因原始序列相比,5种低等蟹线粒体基因中trnH均转移trnEtrnF之间,该特征为短尾类动物所“共享”。除trnH外,在毛刺贝绵蟹线中,还存在2个基因/区域的重排(trnQCR)。这种新型的线粒体基因排序可能是由于部分序列发生重复并伴随着随机的不完全删除造成:trnQ基因从trnItrnM基因之间移至CRrrnL之间;CR位于trnQtrnL1之间。
(5)在5种低等蟹线粒体13个蛋白编码基因中,绝大多数的基因是以经典的ATN为起始密码子。部分ATP8nad1 基因的起始密码子为GTG。与真短尾类不同,低等蟹cox1基因均是以ACG为起始密码子。在5种低等蟹蛋白编码基因中,使用最频繁的氨基酸为Leu、Phe、Met和Ile。在二倍和四倍兼并密码子中,NNA和NNT的概率要远远大于NNC和NNG;与编码蛋白基因中第三位密码子呈现出强烈的AT偏好性一致。
(6)通过分析未矫正Pi、两两基因之间序列的相似性、非同义替换和同义替换的比率(Ka/Ks)等进化参数发现,nad5nad4nad1进化速率快,cox1cox2cytbcox3基因保守。因此,nad5nad4nad1可以作为备选标记用于接下来研究低等蟹的系统发育。
(7)除trnS2trnI,5种低等蟹tRNA基因均可折叠形成经典的四叶草结构。与大多数后生生物一样,东方乌氏蟹、毛刺贝绵蟹和大摩罗蟹trnS2基因二级结构缺失了二氢尿嘧啶(DHU)臂;而在短额琵琶蟹和大摩罗蟹中分别存在2 bp和1 bp的DHU臂。trnI仅在短额琵琶蟹中,缺少DHU臂,这种现象在短尾类中首次出现。5种低等蟹tRNA基因的长度、编码极性,以及反密码子序列均与已公布的短尾类线粒体基因组一致。
(8)在5种低等蟹中rRNA基因均由轻链编码,其基因长度和相对位置保守。7种低等蟹线粒体基因组中,rrnL的相似性高于rrnS
 (9) 基于线粒体基因组13个蛋白编码基因的核酸和氨基酸序列方分别使用最大似然法(ML)和贝叶斯法(BI),以7种海姑虾为外群,构建6种歪尾类和33种短尾类之间的系统发育关系,分析认为:歪尾类和短尾类为互为单系群,形成姐妹群体。毛刺贝绵蟹和3种人面蟹亲缘关系更近,位于短尾类系统发育树的底部,支持绵蟹亚派的分类系统。东方人面蟹、大摩罗蟹和马来似人面蟹聚为一枝,支持人面蟹的单系发生。短额琵琶蟹、东方乌氏蟹和旭蟹聚为一枝,支持蛙蟹总科为单系发生。3种蛙蟹与真短尾类聚互为姐妹群体,支持肢孔派为并系发生。
英文摘要:  Brachyura is one of the most species-rich taxa within the Decapoda,occurring in marine, freshwater, and terrestrial habitats. The phylogenetic relationships among Brachyura have been contentious for more than a century. Though Brachyura itself is widely regarded as a monophyletic grouping, the relationships among the various groups of the Brachyura are still controversial, in particular, the status of Podotremata. The phylogenetic position of Podotremata is important not only for uncovering the pattern of early brachyuran evolution, but also as a prerequisite to understanding eubrachyuran diversification. 
Due to maternal inheritance, lack of the genetic recombination, and relatively rapid evolutionary rate, the mitochondrial DNA sequence has been widely used for phylogenetic and population genetic studies of animals. Compared with a signle or a few genes, the complete mitogenome sequence can provide more genetic information and thus has gained increasing popularity in reconstruction of deeper phylogenetic relationships within metazoans.
The complete mitochondrial genomes of Dynomene pilumnoides, Homola orientalis, Moloha majora, Umalia orientalis and Lyreidus brevifrons were determined by Long- PCR and primer walking sequencing. The date was combined with the other 28 species of brachyuran and six species in Anomura. Seven Thalassinidea species as outgroups were used to reconstruct the phylogenetic relationship of Meiura. Main results of the study were as follows:
 (1) The mitogenome sizes are 16,475 bp in D. pilumnoides, 15,903 bp in M. majora, 16,082 bp in H. orientalis, 15,466 bp in U. orientalis and 16,112 bp L. brevifrons, respectively. Each of mitogenomes contains 37 genes, including 13 PCGs, 2 rRNA genes and 22 tRNA genes.
(2) The mitogenomes of H. orientalis, M. majora and U. orientalis have only one large non-coding region (NCR), which loact between rrnS and trnI. In L. brevifrons, there are two NCRs; one is locted between rrnS and trnI, and the other between trnA and nad3. Comparing to the Pancrustacean ground pattern, the NCR locating between rrnS and trnI is supposed to be putative control region. However, several NCRs are existence in D. pilumnoides mitogenome, and the largest NCR locating between trnL1 and trnQ is the putative control region.
(3) The control regions of these three Homoloidea crabs are characterized by the large repeat region with different repeat length and copy number, accounting for the size variance. The highly levels of sequence identity among repeat units appear in two kinds of Homolidaes, H. orientalis and H. malayens.
(4) Except for D. pilumnoides, the primitive crab gene order is identical to the published marine brachyurans and differ fom the pancrustacean ground pattern by only the position of trnH. D. pilumnoides mitogenome exhibits rearrangements and a novel pattern with positional translocation of two genes/regions (trnQ and CR). The novel gene order could have resulted from one-step occurrence of this process: tandem duplication occurring in the region between trnI and trnM, followed by deletions of redundant genes. Multiple deletions of redundant genes seem to be incomplete, because the short non-coding sequences remain at the deleted region.
(5) In five primitive crabs, except for cox1, the other protein genes are all used the typical start codons ATN, the ATP8 and nad1 in U. orientalis and L. brevifrons with GTG. The start codon for cox1 is ATG in all reported Eubrachyura mitogenomes, while a common start codon ACG is found in the Podotremata. The Leu, Phe, Met and Ile are the most used amino acids, and the UUA, GUA, UCU, CCU and GCU were the most used codons.
(6) The values of uncorrected nucleotide diversity (Pi), Ka/Ks and the similarities between the same gene indicated that nad5, nad4 and nad1 evolved faster while cox1, cox2, cytb, cox3 evolved much slowly. nad5, nad4 and nad1 are considered to be more reliable molecular marker for the further population studies among Podotremata.
(7) Most (20 of 22) tRNAs are folded into a normal clover-leaf secondary structure, except for trnS-AGN and trnI. As in other brachyuran crabs, trnS-AGN lacks DHU stem arm in D. pilumnoides U. orientalis and M, majora mitogenome, while it is folded into a clover-leaf structure with short DHU arm in H. orientalis and L. brevifrons. Moreover, it is also worth noted in L. brevifrons that trnI of the mitogenome lacks a DHU stem arm while the other reported brachyuran species all have a standard cloverleaf structure for trnI.
(8) In the primitive crab mitogenomes, lrRNA and srRNA are separated by the trnV and are both coded on the L-strand.
(9) The phylogenetic relationships indicate that Homoloidea is a sister group to Dynomenidae locating the basal position of phylogenetic tree, and Raninidae is a sister group to Eubrachyura, indicating that Podotremata is paraphyletic and Dromiacea separates from a primitive brachyuran line at earlier stage than Raninoidea.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.qdio.ac.cn/handle/337002/22793
Appears in Collections:实验海洋生物学重点实验室_学位论文

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作者单位: 中国科学院海洋研究所

Recommended Citation:
师国慧. 五种肢孔派(Podotremata)蟹类线粒体基因组测定及短尾类系统发生研究[D]. 北京. 中国科学院大学. 2015.
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