【作者】 王帅宇；

【导师】 雷仲仁；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 斑潜蝇隶属于双翅目(Diptera)潜蝇科(Agromyzidae)斑潜蝇属(Liriomyza)。其中南美斑潜蝇(Liriomyza huidobrensis)、美洲斑潜蝇(L. sativae)和三叶斑潜蝇(L. trifolii)是全世界最危险、发生最严重的三种检疫性害虫。这三种斑潜蝇的入侵给我国农业生产和对外贸易带来重大损失,其暴发为害时甚至造成作物绝产。斑潜蝇具有个体小,近缘种形态极其相似,种内存在高度遗传分化,甚至存在隐种,短期内可以迅速爆发成灾等特点。因此,加深对其遗传背景的认识,弄清其遗传发育关系,对其检验检疫及防控具有重要的意义。同时,斑潜蝇作为入侵害虫,对其传入途径和地理分布与遗传多样性的了解,可为其持续控制提供更多的科学依据。本文对三种斑潜蝇的线粒体全基因组序列进行了测定分析,并联合其它双翅目种类的线粒体基因组数据对双翅目的系统发育关系进行了更全面的探讨。同时对三叶斑潜蝇及南美斑潜蝇的不同种群遗传分化进行了研究,主要研究结果如下:1.联合核内rDNA-ITS2序列及核外mtDNA-COI、COII序列对三叶斑潜蝇不同种群的遗传分化研究表明:该种不同种群已产生一定程度的分化,分为美国种群分支和亚欧种群分支。首次对国内三叶斑潜蝇的不同种群分化进行研究,结果表明国内的种群分化水平还很低。并采用分子数据首次推测了我国三叶斑潜蝇的入侵来源。初步认为三叶斑潜蝇是从欧洲传入我国的。2.联合本研究及Genbank中的mtDNA-COII数据分析结果显示南美斑潜蝇的不同种群分化也发生了一定的遗传分化,也主要分为两支,美国种群独立为一支,而南美洲、中美洲、欧洲及亚洲种群聚为一支。并对国内不同种群分化进行了更全面的分析,结果显示目前国内不同种群仍基本无遗传分化。结合分子数据推测南美斑潜蝇是由南美洲传入欧洲,再从欧洲传入我国的。3.采用L-PCR技术和二次PCR扩增方法,首次获得三种重要入侵斑潜蝇的线粒体全基因组序列。三叶斑潜蝇、美洲斑潜蝇及南美斑潜蝇的线粒体全基因组长度分别为16141 bp、16149 bp和16238 bp。三种斑潜蝇线粒体基因组基因排列次序与Drosphila yakuba相似。均由13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个A+T丰富区组成(三叶斑潜蝇和美洲斑潜蝇的A+T丰富区中还分别包含额外的tRNA基因)。各种线粒体基因组碱基组成均有明显的A+T偏好性,A+T含量分别为78.06%、78.10%及78.23%。4.首次发现ATCA作为昆虫线粒体基因组蛋白质编码基因的四联起始密码子,三种斑潜蝇的COI基因均采用该密码子,三叶斑潜蝇和美洲斑潜蝇的ND1基因起始密码子为GTG。而三种斑潜蝇的大多数蛋白质编码基因均采用ATN作为起始密码子。三种斑潜蝇除CYTb、ND4基因及三叶斑潜蝇和美洲斑潜蝇的ND5基因使用不完全终止密码子T,其它的终止密码子为TAA或TAG。其氨基酸密码子的使用有明显的偏向性、第三位点为A、T的密码子使用频率远高于G、C。三种斑潜蝇中亮氨酸(Leu)含量最高,在15%-16%之间。半胱氨酸(Cys)含量最低,约为1%。5.三种斑潜蝇tRNA基因的二级结构多为典型的三叶草结构,仅tRNASer(AGN)缺失DHU臂。而且多数tRNA的二级结构中存在碱基错配,并且大多数为G-U弱配对。6.在三叶斑潜蝇和美洲斑潜蝇的A+T丰富区发现额外tRNA基因的存在。其中三叶斑潜蝇中包括tRNAThr和tRNALeu(UUR),tRNALeu(UUR)在反密码子环后第2-50bp位置存在内含子;而tRNAThr中有一个可变环茎区,由10个碱基配对形成。位于美洲斑潜蝇A+T丰富区的额外tRNAIle,发现其使用非典型的反密码子TAT,tRNAIle也包含一个可变环茎区,由10个碱基配对形成。三种斑潜蝇A+T丰富区的N链和J链中都发现了与线粒体基因组复制起始相关的Poly-T结构。7.联合双翅目31种昆虫线粒体基因组数据,对双翅目系统发育关系进行了更全面的分析,绝大部分系统发育树中,支持双翅目、短角亚目、芒角亚目和蚊总科的单系性,同形态分类结果一致。而长角亚目及短角亚目中家蝇次目内部的聚类关系未完全解决,仅在BI树中,不仅短角亚目所有种类聚为一支,而且长角亚目所有种类优先聚类,强烈支持长角亚目的单系性。本研究结果丰富了对三种斑潜蝇及整个双翅目昆虫系统发育关系的认识,初步弄清了三叶斑潜蝇与南美斑潜蝇在我国的传入途径与遗传分化多样性。同时为构建快速的分子鉴定体系提供了可靠的实验数据和理论依椐,为检疫性诊断及可持续控制体系的制定具有重要的参考价值。更多还原

【Abstract】 Liriomyza huidobrensis, L. sativae and L. trifolii are the three dangerous and most serious damage quarantine pest, which belong to Diptera, Agromyzidae. They had caused great losses and even to unproductive in agriculture production and export trade when they break out. The leafminers have the characters of small size, very similar morphology in closely related species, high degree of genetic differentiation even containing cryptic species, and it can also outbreak to be disaster in short time. Therefore, it is very important to study their genetic background and make clear their phylogenetic relationship for the quarantine and intergrated control. Meanwhile, as invasion pests, studies on their invasive pathway, geographic distribution and genetic diversity of knowledge, can provide proofs for more scientific basis for sustainable control. In this paper, the complete mitochondrial genomes of three leafminers were sequcened and phylogeny relationships of Diptera were reconstructed using the genebank data and ours. We also compared the geographic populations’differentiation in L. trifolii and L. huidobrensis. The main results were summarized as follows:1. Analysis of nuclear rDNA-ITS2 sequences, and mtDNA-COI, COII sequences of L. trifolii suggested that genetic differentiation appeared in different populations, The results showed that the different populations had separated into two clades: one was the United States clade, and the other was the Asia-Europe clade, while the low level differentiation were showed in domestic populations. According to our molecular data and the quarantine survey, for the first time, we deduced the invasion source and broadcast pathway of L. trifolii in our country, the populations of our country most probably were invaded from Europe.2. The results combined our study and Genbank data of mtDNA-COII showed different populations had genetic differentiation and can be divided into two clades, the USA population as one, and South America, Central America, Europe and Asia combined as the other. We also did more comprehensive analysis for the populations of our country and the result showed that there is still no genetic differentiation. Molecular data and Survey findings proved that the population of L. huidobrensis in China was invaded from Europe, which introduced from South America.3. Using the strategy of sub-PCR based on the long PCR, The complete mitochondrial genome of the three leafminers were sequenced and annotated. The genome lengths were 16141 bp, 16149 bp and 16238 bp respectively. The gene numbers and organization were similar to Drosphila yakuba contains one encoding region including 37 genes and one non-coding A+T-rich region, except that three additional tRNA genes (tRNAThr and tRNALeu(UUR), tRNAIle) were found in the A+T-rich region of L.trifolii and L. sativae respectively. The nucleotide compositions of the mitogenome of three leafminers were biased toward adenine and thymine, and the overall A+T contents were 78.06%, 78.1% and 78.23% respectively.4. For the first time, the quadruplet ATCA is used as protein start codon of mitogenome in insect, ND1 in L. trifolii and L. sativae which begins with GTG, the other protein initiation codons are ATN. Ten genes share the typical termination codons TAA and TAG, and ND4, CYTb of three species and ND5 in L. trifolii and L. sativae have incomplete codons of T. The usages of synonymous codons are showed a remarkable bias in the third codon position for A or T than to G or C. The amin acid content of Leu (15%-16%) is the highest and that of Cys (about 1%) is the lowest.5. All of tRNAs in three leafminers form the typical cloverleaf structure except for tRNASer (AGN), which has lost the DHU-arm. There are mismatchs with most of G-U in the secondary structures of majority tRNAs.6. The new additional tRNAs are predicted in A+T region of L.trifolii and L.sativae, the anticodons loop of tRNALeu(UUR) in L. trifolii contains a 50 bp intron located between the anticodon loop and arm. The tRNAThr in L. trifolii has one extra variable arm with a five-base-pair arm between the anticodon loop and the TψC loop. The additional tRNAIle containing one extra five-base-pairs variable arm is also found in the same region of L. sativae. The A+T region in both strands of three leafminers also contains a highly conserved polyT, which is correlated with the origin of mitochondrial genome replication.7. The overall phylogenetic relationships based on mitogenome of 31 Dipterans were reconstructed: the monophyly of Diptera, Brachyceran, Aristocera and Culicoideae are proved, which is consisitent with morphological classification. The relationships of Nematocera and interrelation of Brachycera were not fully resolved. Only in the BI tree, all the species of Nematocera priority clustered into one clade and this result strongly supports the monophyly of Nematocera.These results enrich the theory of phylogenetic relationship in three leafminers and Dipterans, make clear the broadcasting pathways, geodistribution and genetic diversity of L. trifolii and L. huidobrensis initially, and also provide the reliable proofs for building rapid molecular identification system. Our results will also play important role in quarantine diagnosis and sustainable control.更多还原