4种直翅目蝗虫全线粒体基因组测序及直翅目线粒体基因组比较与系统分析

【作者】 张红利；

【导师】 郑哲民；

【作者基本信息】 陕西师范大学 ， 动物学， 2013， 博士

【摘要】 直翅目属于节肢动物门、六足总纲、昆虫纲,是起源比较早的类群之一,已知有2万多种,广泛分布于世界各地,以热带地区种类较多。直翅目由蝗亚目(Caelifera)和螽亚目(Ensifera)两个亚目组成。目前在GenBank中虽然已经积累了47个直翅目物种的全线粒体基因组,但是主要集中于蝗亚目的蝗总科物种,螽亚目物种相对较少。迄今为止,利用比较基因组手段来探讨直翅目线粒体基因组的结构及进化特征的研究甚少;采用不同的数据集划分策略来探讨线粒体DNA(mtDNA)基因在直翅目系统研究中的作用的研究也很少,且大多研究者集中于直翅目内高级阶元的系统关系即两个亚目是否为单系群,但是利用mtDNA基因数据集对直翅目内中级阶元和低级阶元系统发育的研究甚少。直翅目中癞蝗科和槌角蝗科均属于蝗亚目物种。癞蝗科中疙蝗属同短鼻蝗属在形态上的差别微乎甚微,且均分布于我国的荒漠草原地区；而槌角蝗科大足蝗属内西伯利亚蝗同李氏大足蝗的形态上也仅仅依据前后肘脉是否合并来判定,且二者在地域分布上有交叉。这种形态相似且地域相近的物种在线粒体基因组水平上是否也很保守?本研究测定了蝗总科中3种癞蝗和1种槌角蝗的线粒体基因组全序列,并对其进行了注释和分析。在此基础上,联合GenBank中已测直翅目线粒体基因组全序列,采用生物信息学同比较基因组学相结合的方法分析了直翅目内不同阶元物种mtDNA基因的演化模式。在此基础上,基于线粒体蛋白基因密码子3位点及不同种类蛋白和rRNA不同区域构建的线粒体基因数据集对直翅目内不同分类阶元的系统发育关系进行了研究。主要研究结果如下：1.西伯利亚蝗(Gomphocerus sibiricus)、红胫波腿蝗(Asiotmethis zacharjini)、贺兰短鼻蝗(Filchnerella helanshanensis)和红缘疙蝗(Pseudotmethis rubimarginis)的mtDNA总长度分别为15590bp、15660bp、15657bp和15661bp,均编码昆虫线粒体基因组中典型的37个基因,即13个蛋白编码基因,2个rRNA基因和22个tRNA基因。4种蝗虫的mtDNA基因排序及转录方向同蝗亚目中已测物种的一致。2.从整体mtDNA、22个tRNA基因和2个rRNA基因序列计算大足蝗属内西伯利亚蝗同李氏大足蝗之间的P-距离最小,但从13个蛋白基因计算,西伯利亚蝗和西藏大足蝗间的P-距离最小。无论从核苷酸和氨基酸水平变异率还是从非同义/同义置换率的比率来分析,线粒体蛋白基因cox1、cox2、cox3和nad4L在槌角蝗科内最为保守,进化最慢,受到的净化选择功能约束最少,而atp6、atp8和nad6进化速率最快,受到自然选择压的影响较少。在4个槌角蝗trnThr的TΨC臂中第3对碱基后存在2个碱基A的插入,此特征是否为槌角蝗科的共源性状还有待证明。rrnS在西伯利亚蝗和李氏大足蝗中几乎完全一致。然而在同属的西藏大足蝗物种中rrnS结构域Ⅰ和Ⅱ却存在较大的变异区域。3.无论从线粒体全基因组整体序列还是从13个蛋白基因、22个tRNA基因或2个rRNA基因计算,4个癞蝗物种中,贺兰短鼻蝗同红缘疙蝗的P-距离均为最近。线粒体蛋白基因cox1、cox2、cytb和atp6在癞蝗科内最为保守,而nad5和nad3的进化速率相对比较快。4.在槌角蝗科和癞蝗科两个类群中,nad6和atp8都显示了非常高的AT%,在癞蝗中nad4L的A+T含量也非常高,而3个cox基因的A+T含量都为最低。除ATP8为中度的A-偏斜外,分布于J链的每个线粒体蛋白基因几乎不存在AT-偏斜性。然而由N链编码的4个蛋白基因都有显著的T-偏斜值。在4个槌角蝗和4个癞蝗物种的A+T丰富区中均找到了茎环结构,但是该二级结构在红拟棒角蝗中同其他3个槌角蝗物种中差别较大；宽纹蠢蝗同其他3个癞蝗的茎环结构差异也比较大。而且在癞蝗物种中发现了位于茎环J链序列内部的T簇。5.昆虫纲所选6个目物种的A+T含量集中在75%～80%之间；AT-skew集中于0～0.05之间,GC-skew基本为负值(C>G),且集中于-0.3～0之间。其中,直翅目物种的A+T含量和AT-skew分布均比较分散,但二者似乎存在正相关性。直翅目碱基组成异质性分析表明,A+T含量相似、AT百分比接近平均值或亲缘关系较近的物种间ID值较小；线粒体蛋白不同密码子位点的差异指数由低到高顺序为：第2位点<第1位点<第3位点。6.线粒体13个蛋白编码基因中,cox1和cox3在直翅目大多数物种中的A+T含量普遍偏低,而atp8, nad4L和nad6的A+T含量基本偏高。在碱基偏斜方面,N-链蛋白基因的AT偏斜均为显著的T偏斜。J-链蛋白基因在螽亚目物种中基本为T偏斜,而在蝗亚目多数物种中为A偏斜。对GC偏斜而言,N链蛋白基因均为正值,而J链蛋白基因则均为负值。基于JC和K2P两种模型对直翅目内两个亚目13个线粒体蛋白基因的平均遗传距离进行分析后发现,在两个亚目中cox2变异程度都为最小,在蝗亚目中nad5变异最大,而在螽亚目中nad1变异最大。13个蛋白基因无论从核苷酸序列还是氨基酸序列的差异性进行分析,cytb基因变异程度都为最小,其次为3个cox基因,而nad6基因变异度最大。通常,氨基酸序列比核酸序列应该更加保守。然而,在13个线粒体蛋白基因中,仅在cox1基因中发现氨基酸序列相似度高于核酸序列,多数蛋白基因的氨基酸序列差异性高于或等于核酸序列,在nad6和atp8中尤为明显。7.22种线粒体tRNA基因在蝗总科各科的保守位点比例表明tRNA基因的核酸保守性有链间偏向性。16种位于J-链的tRNAs在蝗总科中核酸一致度均超过50%,另外,核酸保守度很高的trnLeuUUR, trnAsn和trnLys基因均位于J-链上。tRNA家族在癞蝗科内最为保守,其次为槌角蝗科。rrnS的3个结构域中,结构域Ⅰ在去除5’端后最为保守,其次为结构域Ⅲ,结构域Ⅱ进化速率最快。在rrnL的6个结构域中,结构域Ⅳ和Ⅴ最为保守,而结构域Ⅰ和Ⅱ进化最快。8. PCG1、PCG2、COX、COX+cytb和rRNA (C)这类保守基因或区段主要适用于分析直翅目内高级阶元的系统发育关系,如亚目及总科阶元。进化中等的PCG23及NADH基因则适用于从亚科至亚目任意一个阶元的系统关系研究,且建树方法也至关重要。rRNA (V)这种可变位点较多的区段比较适用于对亚科级阶元的系统关系研究。但是像ATP这种进化速率超快且总长度比较短的基因似乎不太适用于系统关系研究。更多还原

【Abstract】 Orthoptera is one of the oldest extant insect lineages with more than20thousands described species widely distributed throughout the world but mainly focused on the tropical areas. It is one of the largest and best researched of the hemimetabolous insect orders and consists of two suborders Caelifera and Ensifera. So far, complete47orthopteran mitogenomes were available in the GenBank, of which27for Acridoidea in Caelifera, and the number of complete mitogenomes sequenced were not balanced in the two suborders of Orthoptera. Currently, few studies on Orthoptera focus on using the comparative genomics to analyse the mitogenome sequence divergence and molecular evolution and using different data partitioning strategies in mitogenome to resolve the phylogenetic relationships at various taxonomic levels. Previous studies were limited to the phylogenetic relationships at higher taxonomic levels, and it is needed to pay more attention to the phylogenetic analyses at the middle and low taxonomic levels.based on the mitogenome sequence.Pamphagidae and Gomphoceridae belong to the Caelifera. The two genus Filchnerella and Pseudotmethis in Pamphagidae is so similar in the morphology and both are mainly distributed in the northwestern area of China. Additionally, the difference of G. sibiricus and G. licenti in Gomphoceridae are identified only according to whether the anterior and posterior cubitus connate or not, and the geographical ranges of the two species overlap in some northwestern areas of China. Do the mitogenomes of these taxa similar in the morphology and close in the distributed area have many common important characters?In this study, the four complete mitogenome sequence in Acridoidea were successfully sequenced, annotated and analysed. Additionally, a total of43orthopteran mitogenomes available in the GenBank were selected based on our different analyses. The evolutionary patterns of orthopteran mitogenomes were firstly investigated using the comparative genomics based on the bioinformatics. Additonally, the phylogeny of Orthoptera was analysed based on12datasets from a total of47orthopteran mitogenomes. Followings are the main results: 1. The complete mitogenome sequences of G. sibiricus, A. zacharjini, F. helanshanensis and P. rubimarginis are15,590bp,15,660bp,15,657bp and15,661bp in size, respectively. All four mitogenomes share the same37typical metazoan genes (13protein-coding genes,22transfer RNA genes and2ribosomal RNA genes), and they have identical gene arrangement and orientation with all previously determined caeliferan mitogenomes.2. The average values of P-distance between G. sibiricus and G Licenti are lower than those between other two species in Gomphoceridae based on the whole mtDNA,22tRNAs or2rRNAs, however, that between G. sibiricus and G tibetanus is the lowest based on the13mitogenome protein coding genes. The ratio of cox1, cox2, cox3and nad4L were lower either on the nucleotide and amino acid sequence heteromorphosis or KalKs, which may indicate the four PCGs are highly conserved and evolve slowly. The evolutionary rate of atp6, atp8and nad6is relatively higher, and the three PCGs may under lower selection pressure. Two bulged adenines insertion after the third couplet in the TΨC stem of trnThr are common in the four Gomphoceridae species. Whether it is a molecular synapomorphy to Gomphocerinae may require more data to verify. The rrnS is very consistent between G. licenti and G. sibiricus. However, variable regions in domain Ⅰ and domain Ⅱ are obvious in G. tibetanus.3. The average values of P-distance between F. helanshanensis and P. rubimarginis are lower than those between other two species in Pamphagidae based on the whole mtDNA,13PCGs,22tRNAs and2rRNAs. In the thirteen PCGs of Pamphagidae mitogenomes, the coxl, cox2, cytb and atp6are the slowest evolving genes, while the nad3and nad5have higher evolutionary rate.4. In the two families of Caelifera, Pamphagidae and Gomphoceridae, both nad6and atp8reveal much higher A+T content, Without exception, A+T contents in all three COX genes are lower than the other gene categories. The number of adenine is almost equal to thymine in all PCGs distributed in J-strand except in atp8which has a moderate A-skew value, while each of the other four PCGs (nad5, nad4, nad4L and nadl) coded by N-strand has an obvious T-skew value. The hairpin in A+T-rich region can be predicted in all four species respectively from Gomphoceridae and Pamphagidae. one T-stretch in the majority strand was only found in the A+T-rich region of the four Pamphagidae mitogenomes, however, they are not adjacent to the trnIle but inside the stem-loop sequence in the majority strand.5. We evaluate the nucleotide-compositional behavior of the insecta mitogenome by the three parameters:A+T content (AT%), AT-skew and GC-skew. The A+T contents of the mitogenomes from6orders of insecta range from75%to80%, the AT-skew values range from0to0.05and the GC-skew values ranges from-0.3to0. Among these species, the A+T content and AT-skew in orthopteran mitogenomes are dispersed, but both seems to be positive correlations. The disparity index value is lower among those orthopteran species near in A+T content and near the average A+T content value or among the closely related species. The average ID value are different in three codon positions of PCGs, the highest ID value was observed in the third codon positions, the second codon positions shows the lowest ID value.6. In the13PCGs of orthopteran mitogenomes, the A+T contents of coxl and cox3are the lowest in most species, while atp8, nad4L and nad6reveal higher A+T content than other PCGs. The four PCGs coded by N-strand in Orthoptera have obvious T-skew value. The9PCGs coded by J-strand in Ensifera also have obvious T-skew value, while these PCGs show A-skew in most caeliferan species. Each PCG in J-strand is C-skew, However, each PCG in N-strand is G-skew. We compared overall mean distance of13PCGs in two suborders of Orthoptera based on the JC and K2P model and found the cox2is the slowest evolutionay gene, the nad5is the fastest evolutionary gene in Caelifera, while the nadl is the fastest in Ensifera. We compared the sequence heteromorphosis of13PCGs at the DNA and amino acid level, the cytochrome oxidase subunits and cytochrome b show overall much slower rates of evolution, while the nad6is the fastest evolving protein-coding gene. Generally, the amino acid sequence should be more conserved than the nucleotide sequence. However, the amino acid of all13PCGs show higher divergence than the DNA sequence with the exception of the cox1gene, especially obvious in the nad6and atp8.7. The pattern of nucleotide conservation in tRNA genes was markedly majority strand-biased. Eleven tRNAs showed%INUC>50, only one of them was located on the minority strand. Indeed, trnLeuUUR, trnAsn and trnLys, which showed the highest levels of nucleotide conservation (%INUC≥70), were all located on the majority strand. In the three domains of rrnS in orthopteran mitogenomes, the domain Ⅰ (excluding the5’ half) is the most conserved region, while the domain Ⅱ has many fewer conserved nucleotide strings than other two domains. Similarly, in the rrnL of Orthoptera mitogenomes, Domains Ⅰ and Ⅱ, on average, are less conserved than domains Ⅳ and Ⅴ.8. In terms of the ability to resolve deeper level relationships in Orthoptera, the conserved datasets, PCG1, PCG2, COX, COX+cytb and rRNA(C) may be the best choice, while the genes or regions of intermediate rates might be of better phylogenetic utility for the orthopteran phylogenetic analyses at various taxonomic level, nevertheless, the analytical regimen is essential. Those regions including slightly more variable sites may be useful for the phylogenetic studies at the lower taxonomic level. However, the faster small evolutionary gene such as ATP genes seem to be usefulless in the phylogenetic studies.更多还原