【作者】 刘志萍；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2012， 博士

【摘要】 毒隐翅虫亚族Paederina隶属于鞘翅目Coleoptera,隐翅虫科Staphylinidae,母隐翅虫亚科Paederinae,分布于除南极洲外的世界各地,是农业害虫的重要天敌。毒隐翅虫素(Pederin)是由聚酮合酶(polyketide synthase, PKS)合成的一种昆虫内毒素,具有强烈的抗肿瘤、抗病毒活性,是目前治疗肿瘤的“候选药物”之一,研究其生物合成一直是该领域的热点之一。目前对毒隐翅虫亚族昆虫的分类学研究多以传统的外部形态为主,而关于其分子系统学的研究鲜见报道,而且亚族下各分类单元之间的系统发育关系也不清楚。因此本论文在传统分类学研究的基础上,依据线粒体COⅠ、COⅡ基因序列,分析了中国毒隐翅虫亚族的系统发育关系。此外,针对PKS的保守区域：酮基合酶(Ketosynthase, KS)设计简并引物,扩增了约1000bp的KS基因片段,推测了该基因的功能,完善了Pederin的生物合成路径。经过近四年的研究,获得以下主要结果：1.分类学研究及区系分析本论文共记述中国毒隐翅虫亚族Paederina4属45(亚)种,其中包括1新种：勐养毒隐翅虫Paederus mengyangensis sp. nov.,1云南省新纪录种：黑足毒隐翅虫Paederus melampus Erichson,1840;补充了其它43(亚)种的外部形态特征描述；依据基毒隐翅虫Paederus coxalis Fauvel,1895的触角后部各节和胫节黑色、阳茎原始等特征将其归入原毒隐翅虫亚属Paederus(Eopaederus) Scheerpeltz,1957。分析讨论了毒隐翅虫亚族在中国动物地理区划中的分布情况。结果表明毒隐翅虫亚族在我国以单纯的东洋界成分为51.11%,占绝对优势。在七个动物地理区划中,各区占有的种类数差异较大,以华中区种类最为丰富,分布25种,占我国已知种类的55.56%,其次为华南区和西南区。华南区单区分布种最多,为12种,占我国已知种类的26.67%。中国特有种较为丰富,达26种,占我国已知种类的57.78%,华中区分布17种,华北区为7种,华南区为6种、西南区5种、青藏区为6种和蒙新区3种。地区特有种共16种,其中华中区和华南区各6种,西南区、华北区、蒙新区和青藏区各1种,东北区无地区特有种。2.分子系统学研究利用线粒体COⅠ、COⅡ基因的通用引物,从14种毒隐翅虫中分别扩增出了约800bp的COI基因和约700bp的COII基因。将扩增产物测序并手动切除引物后经BLAST比对,发现COI基因与GenBank中亲缘关系最近的序列同源性为82%-96%；COII基因与亲缘关系最近的序列同源性为78%-82%,可确定扩增产物即为目的片段。利用PAUP*4.0beta10对COⅠ、COⅡ基因进行同质化检验,P=0.89,表明二者为同质性样品,可进行联合分析。本论文基于COⅠ、COⅡ和COⅠ+COⅡ基因对毒隐翅虫属昆虫进行了分子系统学研究。经系统发育信号检测,发现COⅠ、COⅡ基因中当遗传距离小于0.88时,转换与颠换同遗传距离有很好的线性关系；当遗传距离大于0.88时,颠换趋于饱和,而转换与遗传距离仍有很好的线性关系；COⅠ+COⅡ基因中转换与颠换未达到饱和,均与遗传距离呈很好的线性关系,表明本数据拥有较强的系统发育信息,而非随机数据。对COⅠ、COⅡ、COⅠ+COⅡ基因进行碱基序列组成分析,发现T、C、A、G碱基平均含量分别为35.9%-37.1%、15.3%-15.5%、32.4%-33.6%、14.8%-15.1%,密码子在第一、二、三位上的A+T平均含量为64.5%-68.2%、66.9%-75.8%、68.5%-73.2%,表明密码子使用频率存在明显的偏A+T性,此偏倚性在密码子各位点基本保持一致；COⅠ、COⅡ、COⅠ+COⅡ基因颠换数均高于转换数,S/V在0.43-0.60间,密码子不同位点处转换与颠换比例基本一致。对COⅠ、COⅡ、COⅠ+COⅡ基因分别采用相邻连接法(NJ)、最小进化法(ME)、最大简约法(MP)构建系统发育树,显示COⅠ+COⅡ基因与COI基因构建的树基本一致,本论文主要采用置信度最高的联合基因COⅠ+COⅡ构建的系统发育树进行分析。以毒隐翅虫族的一种Homaeotarsus pimeri和前角隐翅虫亚科的一种Aleochara brevipennis Gravenhorst,1806为外群,发现背齿毒隐翅虫亚属Paederus (Gnathopaederus) Chapin,1927为单系群；矛毒隐翅虫亚属Paederus (Harpopaederus) Scheerpeltz,1957可能为单系群；原毒隐翅虫亚属P.(Eopaederus) Scheerpeltz,1957和异毒隐翅虫亚属Paederus (Heteropaederus) Scheerpeltz,1957在COⅠ、COⅡ、 COⅠ+COⅡ基因的NJ、ME、MP树中均聚为一支,而且二者在外部形态上非常相近,分子系统学与外部形态学研究均证明这两个亚属亲缘关系最近,为姐妹群；多刺毒隐翅虫Paederus describendus Willers,2001与毒隐翅虫指名亚属Paederus (Paederus) Fabricius,1775亲缘关系最近,但从外部形态上看多刺毒隐翅虫无后翅,与毒隐翅虫指名亚属具后翅的特征不符,故不能归入该亚属；COⅠ、COⅡ、COⅠ+COⅡ基因所构建的三种系统发育树均表明,基毒隐翅虫P. coxalis Fauvel,1895应归入原毒隐翅虫亚属P.(Eopaederus) Scheerpeltz,1957,与形态分类结果一致；黑足毒隐翅虫P. melampus Erichson,1840和塔毒隐翅虫Paederus tamulus Erichson,1840外部形态非常相似,亲缘关系较近。3.酮基合酶的研究本论文设计了酮基合酶(KS)基因的简并引物,对毒隐翅虫属11个样本进行扩增,结果仅从4个不同采集地的雌性梭毒隐翅虫Paederus fuscipes Curtis,1826中扩增出了约1000bp的片段,命名为pedKS基因,而雄虫及其它种类毒隐翅虫没有测出,这一结果证明了只有梭毒隐翅虫雌虫产生毒素,而雄虫及其它种类毒隐翅虫不产生毒素。经BLAST比对,发现pedKS基因与假单胞杆菌Pseudomonas的KS基因片段同源性达76%-86%。从NCBI下载有代表性的KS基因构建NJ系统发育树,显示pedKS基因与FAD-依赖型氧化还原酶(FAD-dependent oxidoreductase)基因亲缘关系较近。在荧光假单胞杆菌Pseudomonas fluorescens中与pedKS同源的基因经氧化还原作用合成吡啶六元环,故推测pedKS可能也经氧化还原作用参与了Pederin中吡喃六元环的合成。更多还原

【Abstract】 Paederina is a big subtribe in Paederinae (Coleoptera:Staphylinidae) all over the world except Antarctica. Many species in the subtribe are natural enemies of the important agricultural pests. Pederin, a polyketides extracted from Paederus Curtis, is a non-protein insect toxin. Like other polyketides, it displays potent and selective bioactivities that trigger biomedical interests. Up to date, taxonomy on Paederina has focused on the external morphology while related molecular systematic studies are rarely reported and phylogenetic relationships among taxa in Paederina are unknown. In the current study, on the basis of traditional morphological characters, the phylogenetic relationship among taxa in Chinese Paederina are analyzed via COI and COII genes sequences. Besides, one about1000bp fragment of Ketosynthase (KS) was amplified applying degenerate primers designed based on the conservative regions of PKS. This gene functions were speculated in biosynthetic pathway of Pederin. After about4years’ work, the main results are as follows:1. Study on Systematics and fauna of PaederinaFour genera and45species (subspecies) from Paederina in China were described, including a new species:Paederus mengyangensis sp. nov. and a new recorded species in Yunnan Province:Paederus melampus Erichson,1840. Paederus coxalis Fauvel,1895is classified to Paederus (Eopaederus) Scheerpeltz,1957based on black retral antennae and tibia, primitive aedeagus.The distribution of Paederina in Chinese zoogeographical regions was analysed in detail and the Oriental species dominate the Palaearctic species in Paederina of China. Different species possess different distribution regions and different zoogeographical regions have different species. Endemisms at species level of Paederina are abundant in China amounting to26species, taking up57.58%of the fauna of China. There are16species distributing in one region.2. Molecular systematics of PaederinaOne COI gene of about800bp and one COII gene of about700bp were amplified from14species of Paederina using universal primers, respectively. BLAST alignment indicated that COI gene is homologous with GenBank’s closest sequence, between82%-96%, and for COII gene the rate accounts for78%-82%. The COI and COII genes are homogeneity tested with PAUP*4beta10. The value of P (0.89) showed that two samples can be homogeneous adopting to conjoint analysis. This paper discussed the phylogenetic relationship among taxa in Chinese Paederina based on molecular systematics based on COⅠ and COⅡ genes.After phylogenetic signal detection, both transition and transversion exhibit a good linear relationship with genetic distance when the value of genetic distance is less than0.88in COⅠ and COⅡ genes. Transversion tends saturated and transition with genetic distance still has a good linear relationship, when genetic distance is more than0.88. Both transition and transversion with genetic distance possess good linear relationship in COⅠ+COⅡ gene, indicating that the data implys strong phylogenetic information instead of random data.Analysis of base sequences of COⅠ, COⅠ, COⅠ+COⅡ genes shows that T, C, A, G base average concentration ranges are35.9%-37.1%,15.3%-15.5%,32.4%-33.6%and14.8%-15.1%, respectively. Average content of A+T in different sites of codon is64.5%-68.2%,66.9%-75.8%a nd68.5%-73.2%, respectively. The results suggest that codon in different sites biased towards A+T. Transversion was higher than transition in COⅠ, COⅡ and COⅠ+COⅡ gene, and S/V is0.43-0.60.Phylogenetic trees are built on COⅠ, COⅡ, COⅠ+COⅡ genes using adjacent connection method (NJ), minimum evolution (ME) and maximum parsimony (MP), respectively. Results show that trees of COⅠ+COⅡ gene are the same with those of COⅠ gene. The paper analyses the result using trees of COⅠ+COⅡ genes with high confidence. Homaeotarsus pimeri of Paederini and Aleochara brevipennis Gravenhorst,1806of Aleocharinae are chosen as outgroups. The result shows that Paederus (Gnathopaederus) Chapin,1927is a monophyletic group. Paederus (Harpopaederus) Scheerpeltz,1957might be a paraphyletic group. P.(Eopaederus) Scheerpeltz,1957and Paederus (Heteropaederus) Scheerpeltz,1957unite to a branch in trees of NJ, ME and MP on COⅠ, COⅡ and COⅠ+COⅡ gene, and the two are very similar in external morphology. The molecular phylogeny and morphological studies also prove the two subgenus closest as a sister group. Paederus describendus Willers,2001and Paederus (Paederus) Fabricius,1775are closest relatives, but P. describendus Willers,2001without wings can not be classified to the subgenus with hind wings. Three phylogenetic trees based on COⅠ, COⅡ, COⅠ+COⅡ genes showed that P. coxalis Fauvel,1895should be classified to P.(Eopaederus) Scheerpeltz,1957. The results are consistent with those of morphological classification. Paederus melampus Erichson,1840resembles Paederus tamulus Erichson,1840in external morphology and a close phylogenetic relationship exists between them. 3. Study of KetosynthaseThe degenerate primers of Ketosynthase (KS) gene were designed and in total11samples of Paederus were amplified. Only females of Paederus fuscipes Curtis,1826from4different collections were amplified with a fragment of about1000bp, named pedKS gene. pedKS gene is homologous with the KS gene of Pseudomonas to76%-86%. NJ phylogenetic tree of representative KS gene from NCBI shows that pedKS gene possesses a close phylogenetic relationship with FAD-dependent oxidoreductase gene. Homologous gene in Pseudomonas fluorescens with pedKS synthesizes pyridine six membered rings by redox reaction. It is predicted that pedKS may also participate in the synthesis of pyran six rings in Pederin by oxidation reduction.更多还原