蚜虫与其内共生菌Buchnera之间的系统进化关系研究
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摘要
几乎所有的蚜虫体内都含有一种内共生菌Buchnera，这种共生菌在分类上属于和Escherichia coli非常相近的γ-proteobacterium。该共生菌存在于蚜虫腹部的专为内共生菌而特化的细胞含菌细胞(mycetocytes或bacteriocytes)的胞质中，该共生菌随亲代垂直传递给后代。蚜虫与Buchnera互利共生；Buchnera充分供给蚜虫不能从外界获得的各种营养物质，从而满足蚜虫生存和繁殖的需要；同样，Buchnera离开蚜虫也不能存活。国外学者通过分子生物学方法对一些蚜虫体内的Buchnera进行了深入研究，发现共生菌全序列基因组大大缩小，但保留了多数必需氨基酸合成的基因。
正是由于Buchnera对蚜虫的重要作用，国内外学者对Buchnera开展了相关研究。国外学者对Buchnera的研究，涉及了Uroleucon sp., Acryrthosiphon pisum, Diuraphisnoxia(Kurdj)，Schizaphis graminum等有限的蚜虫，这些研究通过分析这些蚜虫Buchnera及其这些蚜虫自身的系统进化，探讨了这些蚜虫Buchnera与其寄主之间的系统进化关系。但是这些研究未曾涉及桃蚜、豆蚜、棉蚜。国内学者对这三种蚜虫Buchnera的研究很少，仅仅涉及了桃蚜的Buchnera groEL基因和16srDNA基因。这三种蚜虫遍及中国乃至世界各地，属于多食性害虫，危害白菜、黄瓜、西红柿、茄子、豆科植物等多种蔬菜和农作物。鉴于Buchnera与蚜虫的特殊关系，对这些蚜虫体内的Buchnera开展研究非常必要。本研究首先分别探讨了这三种蚜虫及其内共生菌Buchnera的系统进化特征，然后综合分析了这三种蚜虫与其内共生菌Buchnera的系统进化关系。
一、试验材料的采集和饲养
我们选择典型的危害严重的三种蔬菜蚜虫：桃蚜、豆蚜、棉蚜为研究对象。在实验室内分别用萝卜、菜豆、黄瓜进行饲养；各种群分别置于50×50×50的正方体铁架外罩100目双层沙网内，饲养条件为20±2℃，相对湿度60-70％。
二、测定三种蚜虫的mtDNA COⅠ／COⅡ、12S／16SrRNA以及核基因EFlalpha的序列，分析三种蚜虫的系统进化
在这部分工作中，我们通过以前的研究中采用的引物扩增了并测定了桃蚜、豆蚜、棉蚜的线粒体基因COⅠ／COⅡ、12S／16S rRNA以及核基因EFalpha的序列。每个地理种群随机抽取3～5个样本进行序列测定，以它们的一致序列为准。利用“BLAST”工具(NCBI站点)对测定的序列进行分析、DNA序列检索；用GenDoc软件进行序列同源性比较；用BioEdit软件进行序列编辑；用ClustalⅩ软件进行序列比对(alignment)；比对结果输入MEGA2.0软件，采用距离法计算各样品间的遗传距离，并基于Kimura-2-Parameter模型，用邻接法(Neighbor-Joining，NJ)构建系统发育树，通过自展1000次检验获得系统树分支的置信度。
比较测定的这些基因序列，结果发现，四个地理种群的桃蚜的COⅠ／COⅡ、12S／16SrRNA和EFlalpha基因序列差异非常小，相似性高达99％。而桃蚜和棉蚜的COⅠ／COⅡ、EFlalpha的这些基因序列相似性为90％左右；桃蚜和豆蚜的COⅠ／COⅡ、EFlalpha基因序列的相似性分别为近85％、近90％，豆蚜和棉蚜的COⅠ／COⅡ、EFlalpha的相似性分别为过85％、近93％。但是，两种蚜虫的12S／16SrRNA基因序列的相似性则基本达95％。
分析这些序列构建的系统发育树，我们可以发现，桃蚜与麦双尾蚜的亲缘关系最近。豆蚜和棉蚜的亲缘关系最近，它们又与麦二叉蚜关系最近。
三、测定并分析Buchnera的trpB、dnaN、trpEG的序列，分析三种蚜虫Buchnera的系统进化
首先，我们通过Buchnera的基因探讨了这三种蚜虫体内共生菌Buchnera的系统进化。具体来说，我们根据GenBank登录的Uroleucon sp.、Acryrthosiphon pisum、Rhopalosiphum padi以及Schizaphis graminum等已知蚜虫的Buchnera trpB、dnaN、trpEG的基因片段序列，利用Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign)软件设计引物，扩增了并测定了桃蚜、豆蚜、棉蚜内共生菌Buchnera的trpB、dnaN、trpEG的基因片段序列。每个地理种群随机抽取3～5个样本进行序列测定，以它们的一致序列为准。利用“BLAST”工具(NCBI站点)对测定的序列进行分析、DNA序列检索；用GenDoc软件进行序列同源性比较；用BioEdit软件进行序列编辑；用ClustalⅩ软件进行序列比对(alignment)；比对结果输入MEGA2.0软件，采用距离法计算各样品间的遗传距离，并基于Kimura-2Parameter模型，用邻接法(Neighbor-Joining，NJ)构建系统发育树，通过自展1000次检验获得系统树分支的置信度。
比较获得的这些基因序列，结果发现，四个地理种群的桃蚜的trpB、dnaN、trpEG基因序列差异很小；而桃蚜与豆蚜、桃蚜与棉蚜之间的trpB、dnaN、trpEG基因序列差异非常明显，豆蚜与棉蚜的这三段序列差异相对小一些。
由trpB，dnaN、trpEG的基因片段序列构建的系统树可以发现，四个地理种群桃蚜的trpB、dnaN基因序列均处于同一支序，它们又与麦双尾蚜的相应基因序列形成一个分支，即桃蚜和麦双尾蚜的Buchnera的亲缘关系最近；并且桃蚜Buchnera的两个染色体基因trpB、dnaN的系统进化与其质粒基因trpEG的进化关系一致，即桃蚜的质粒基因trpEG可能随其Buchnera垂直传递。
而豆蚜和棉蚜的trpB、dnaN基因序列总是处于一个支序，关系最近，但是豆蚜和棉蚜所在的这一亚支序的在系统树中的所处的分支变化不定，因此，我们只能认为豆蚜和棉蚜的Buchnera的亲缘关系最近，至于两者又与已知的其它何种蚜虫的Buchnera的亲缘关系最近不能确定。豆蚜和棉蚜的trpEG基因序列处于紧紧相邻的分支，并且这两个相邻的分支所处的大分支与trpB、dnaN基因序列所处的分支也不一样，所以它们的Buchnera trpEG基因是否随Buchnera垂直传递，则不能十分确定。
四、蚜虫与其内共生菌Buchnera之间的系统进化关系分析
蚜虫与其内共生菌Buchnera之间的系统进化关系研究很少，本研究通过比较桃蚜、豆蚜、棉蚜及其内共生菌Buchnera的系统进化特征，揭示了这三种蚜虫与其内共生菌Buchnera的系统进化关系。我们发现，桃蚜与其Buchnera的进化关系一致均与麦双尾蚜的亲缘关系最近，即桃蚜与其Buchnera是协同进化的。豆蚜、棉蚜与其内共生菌Buchnera可能不是严格协同进化的。所以我们认为不同蚜虫与其内共生菌Buchnera的进化关系可能不同，这一结论与前人的研究也是吻合的。这一工作不仅弥补了这三种蚜虫与其内共生菌Buchnera之间的进化关系空白，同时有助于我们在了解这些蚜虫Buchnera的系统进化特征的基础上，进一步全面地了解了这三种蚜虫的内共生菌Buchnera。
综上所述，本研究首次对桃蚜、豆蚜、棉蚜内共生菌Buchnera的系统进化进行了报道，并初步探讨和分析了这三种蚜虫与其内共生菌Buchnera的系统进化关系，该工作对于我们进一步研究这些蚜虫Buchnera的其它特性以及这些蚜虫的次生共生菌都有重要的指导意义。
Almost all aphids maintain an endosymbiotic association with Buchnera aphidicola, aγ-proteobacterium closely related to Escherichia coll. The endosymbiont, harbored in host-derived vesicles within specialized cells known as bacteriocytes, is transmitted maternally, from mother aphid to progeny. The relationship between Buchnera and the aphid host is mutualistic because aphid needs B. aphidicola for its normal growth and reproduction whereas B. aphidicola cannot live outside the aphid. Molecular study found that Buchnera's genome is lessen, but the most amino acids synthetic genes are preserving.
Because of the importance of Buchnera, many authors studied this symbiosis. Previous studies about Buchnera of some aphids by foreign authors only conferred some limited aphids, such as the genus Uroleucon sp., Acryrthosiphon pisum, Diuraphis noxia (Kurdj), Schizaphis graminum etc, but no Myzus persicae, Aphis craccivora and Aphis gossypii. And domestic studies about Buchnera of the three aphids were also few, and this study only conferred the two genes (groEL and 16srDNA) of Buchnera in M. persicae. The three aphids, which are abundant throughout China as well as worldwide, are polyphagous herbivores attacking many vegetables and crops such as cabbages, cucumber, eggplant, tomato, cucurbits, bean and so on. Because of the special relationships between aphids and the Buchnera, the studies on the Buchnera of the three aphids are necessary. Firstly, we explored the phylogenies of the three aphids, M. persieae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera respectively. Then we synthetically analysed phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera.
1. The collection and rearing of experimental material
We collected three aphids M. persicae, A. craccivora and A. gossypii, which are important pests. Those three aphids were then reared in the laboratory on radish, bean, cucumber respectively. For each population, at least two double-deck gauze cages (around 50cm×50cm×50cm) were setto rear the three aphids. And they were kept in a laboratory under controlled environmental conditions (20±2℃, 60-70% R.H., 16L-8D).
2. The sequencing of mtDNA COⅠ/COⅡ, 12S/16SrRNA and nuclear gene EFalpha in the three aphids, and the phylogenetic analysis of the three aphids
In this part of work, we amplified COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in four populations of M. persicae, Nanjing population A. craccivora and A. gossypii. Three to five individual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.
DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2 Parameter method. Phylogenefic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.
Comparing the obtained sequences, we found that the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes sequences from the four populations M. persicae had very minor differences, and the similarities between the different two populations reached to 99%. And the similarities Of the COⅠ/COⅡ, EFalpha genes between M. persicae and A. gossypii were about 90%, those of the two genes between M. persicae and A. craccivora were near 85%、near 90% respectively, while the similarities of those genes between A. gossypii and A. craccivora were hyper-85%、near 93% respectively. But the similarities of the 12S/16SrRNA genes sequences in different two aphids reached to 95%.
Through the phylogenetic trees constructed by the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in M. persicae, we found that the COⅠ/COⅡand 12S/16SrRNA gene sequences in M. persicae were more closely related to those in D. noxia, while the EFalpha genes of M. persicae and D. noxia located in two neighboring branches, and the these genes sequences in A. gossypii and A. craccivora were all in the same clades. So we concluded that M. persicae and D. noxia were more close, and A. gossypii and A. craccivora were more close.
3. The sequencing of trpB, dnaN, trpEG in Buchnera, and the phylogenetic analysis of Buchnera in the three aphids
Firstly, we designed primers based on the nucleotide sequences from the currently known trpB, dnaN and trpEG sequences in Buchnera (isolated from Uroleucon. sp, A. pisum, D. noxia, S. graminum and R. padi), and amplified and sequenced these genes sequences of Buchnera from the three aphids, M. persicae, A. craccivora and A. gossypii. Three to five indiyidual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.
DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship, was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2-Parameter method. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.
Comparing the obtained sequences, we found that the trpB, dnaN and trpEG sequences in Buchnera from the four populations M. persicae had very minor differences, but these sequences between M. persicae and A. craccivora, M. persicae and A. gossypii had major differences, and these differences were little minor between A. craccivora and A. gossypii.
Through the phylogenetic trees constructed by this genes (trpB, dnaN and trpEG) in Buchnera, we found that the trpB, dnaN gene sequences in M. persicae were in the same clade with those in D. noxia, that is to say the Buchnera in M. persicae is more similar to the endosymbionts of D. noxia. Besides, the phylogenies of the trpB, dnaN genes of the Buchnera in M. persicae were accordant with that of the trpEG genes, so the plasmid genes trpEG genes of the Buchnera in M. persicae may be vertical transmission with Buchnera. And these genes sequences in A. gossypii and A. craccivora were all in the same clades, but the near clade to the sub-clade of the two aphids was variational. SO we only confirmed that the Buchnera in A. gossypii and A. craccivora were more close, but the conclusions that the Buchnera in these two aphids was close to that in which aphids and the transmission of the the plasmid genes trpEG of the Buchnera in these aphids could not be affirmed.
4. Analysis of phylogenetic relationships between the aphids and their endosymbiotic bacterium Buchnera
Studies about the phylogenetic relationships between aphids and their endosymbiotic bacterium Buchnera are few. In our present study, we explore phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, by comparing the characteristics of the phylogenies of those aphids and their Buchnera. We found that M. persicae and D. noxia were coevolutional, but we cannot conclude the coevolutional relationships between the two aphids, A. gossypii and A. craccivora, and their Buchnera. So we conclude that phylogenetic relationships between different aphids and their endosymbiotic bacterium Buchnera may be different, and this conclusion is accordant with previous study. This work not only fill a gap on phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, but also help us detailedly know the three aphids' Buchnera.
As described above, these results first exposed the phylogeny of Buchnera in the three aphids, M. persicae, A. craccivora and A. gossypii. And these works also explored phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera. These works will be helpful for exploring other characteristics of those aphids' Buchnera and studying those aphids'secondary endosymbiosis.
正是由于Buchnera对蚜虫的重要作用，国内外学者对Buchnera开展了相关研究。国外学者对Buchnera的研究，涉及了Uroleucon sp., Acryrthosiphon pisum, Diuraphisnoxia(Kurdj)，Schizaphis graminum等有限的蚜虫，这些研究通过分析这些蚜虫Buchnera及其这些蚜虫自身的系统进化，探讨了这些蚜虫Buchnera与其寄主之间的系统进化关系。但是这些研究未曾涉及桃蚜、豆蚜、棉蚜。国内学者对这三种蚜虫Buchnera的研究很少，仅仅涉及了桃蚜的Buchnera groEL基因和16srDNA基因。这三种蚜虫遍及中国乃至世界各地，属于多食性害虫，危害白菜、黄瓜、西红柿、茄子、豆科植物等多种蔬菜和农作物。鉴于Buchnera与蚜虫的特殊关系，对这些蚜虫体内的Buchnera开展研究非常必要。本研究首先分别探讨了这三种蚜虫及其内共生菌Buchnera的系统进化特征，然后综合分析了这三种蚜虫与其内共生菌Buchnera的系统进化关系。
一、试验材料的采集和饲养
我们选择典型的危害严重的三种蔬菜蚜虫：桃蚜、豆蚜、棉蚜为研究对象。在实验室内分别用萝卜、菜豆、黄瓜进行饲养；各种群分别置于50×50×50的正方体铁架外罩100目双层沙网内，饲养条件为20±2℃，相对湿度60-70％。
二、测定三种蚜虫的mtDNA COⅠ／COⅡ、12S／16SrRNA以及核基因EFlalpha的序列，分析三种蚜虫的系统进化
在这部分工作中，我们通过以前的研究中采用的引物扩增了并测定了桃蚜、豆蚜、棉蚜的线粒体基因COⅠ／COⅡ、12S／16S rRNA以及核基因EFalpha的序列。每个地理种群随机抽取3～5个样本进行序列测定，以它们的一致序列为准。利用“BLAST”工具(NCBI站点)对测定的序列进行分析、DNA序列检索；用GenDoc软件进行序列同源性比较；用BioEdit软件进行序列编辑；用ClustalⅩ软件进行序列比对(alignment)；比对结果输入MEGA2.0软件，采用距离法计算各样品间的遗传距离，并基于Kimura-2-Parameter模型，用邻接法(Neighbor-Joining，NJ)构建系统发育树，通过自展1000次检验获得系统树分支的置信度。
比较测定的这些基因序列，结果发现，四个地理种群的桃蚜的COⅠ／COⅡ、12S／16SrRNA和EFlalpha基因序列差异非常小，相似性高达99％。而桃蚜和棉蚜的COⅠ／COⅡ、EFlalpha的这些基因序列相似性为90％左右；桃蚜和豆蚜的COⅠ／COⅡ、EFlalpha基因序列的相似性分别为近85％、近90％，豆蚜和棉蚜的COⅠ／COⅡ、EFlalpha的相似性分别为过85％、近93％。但是，两种蚜虫的12S／16SrRNA基因序列的相似性则基本达95％。
分析这些序列构建的系统发育树，我们可以发现，桃蚜与麦双尾蚜的亲缘关系最近。豆蚜和棉蚜的亲缘关系最近，它们又与麦二叉蚜关系最近。
三、测定并分析Buchnera的trpB、dnaN、trpEG的序列，分析三种蚜虫Buchnera的系统进化
首先，我们通过Buchnera的基因探讨了这三种蚜虫体内共生菌Buchnera的系统进化。具体来说，我们根据GenBank登录的Uroleucon sp.、Acryrthosiphon pisum、Rhopalosiphum padi以及Schizaphis graminum等已知蚜虫的Buchnera trpB、dnaN、trpEG的基因片段序列，利用Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign)软件设计引物，扩增了并测定了桃蚜、豆蚜、棉蚜内共生菌Buchnera的trpB、dnaN、trpEG的基因片段序列。每个地理种群随机抽取3～5个样本进行序列测定，以它们的一致序列为准。利用“BLAST”工具(NCBI站点)对测定的序列进行分析、DNA序列检索；用GenDoc软件进行序列同源性比较；用BioEdit软件进行序列编辑；用ClustalⅩ软件进行序列比对(alignment)；比对结果输入MEGA2.0软件，采用距离法计算各样品间的遗传距离，并基于Kimura-2Parameter模型，用邻接法(Neighbor-Joining，NJ)构建系统发育树，通过自展1000次检验获得系统树分支的置信度。
比较获得的这些基因序列，结果发现，四个地理种群的桃蚜的trpB、dnaN、trpEG基因序列差异很小；而桃蚜与豆蚜、桃蚜与棉蚜之间的trpB、dnaN、trpEG基因序列差异非常明显，豆蚜与棉蚜的这三段序列差异相对小一些。
由trpB，dnaN、trpEG的基因片段序列构建的系统树可以发现，四个地理种群桃蚜的trpB、dnaN基因序列均处于同一支序，它们又与麦双尾蚜的相应基因序列形成一个分支，即桃蚜和麦双尾蚜的Buchnera的亲缘关系最近；并且桃蚜Buchnera的两个染色体基因trpB、dnaN的系统进化与其质粒基因trpEG的进化关系一致，即桃蚜的质粒基因trpEG可能随其Buchnera垂直传递。
而豆蚜和棉蚜的trpB、dnaN基因序列总是处于一个支序，关系最近，但是豆蚜和棉蚜所在的这一亚支序的在系统树中的所处的分支变化不定，因此，我们只能认为豆蚜和棉蚜的Buchnera的亲缘关系最近，至于两者又与已知的其它何种蚜虫的Buchnera的亲缘关系最近不能确定。豆蚜和棉蚜的trpEG基因序列处于紧紧相邻的分支，并且这两个相邻的分支所处的大分支与trpB、dnaN基因序列所处的分支也不一样，所以它们的Buchnera trpEG基因是否随Buchnera垂直传递，则不能十分确定。
四、蚜虫与其内共生菌Buchnera之间的系统进化关系分析
蚜虫与其内共生菌Buchnera之间的系统进化关系研究很少，本研究通过比较桃蚜、豆蚜、棉蚜及其内共生菌Buchnera的系统进化特征，揭示了这三种蚜虫与其内共生菌Buchnera的系统进化关系。我们发现，桃蚜与其Buchnera的进化关系一致均与麦双尾蚜的亲缘关系最近，即桃蚜与其Buchnera是协同进化的。豆蚜、棉蚜与其内共生菌Buchnera可能不是严格协同进化的。所以我们认为不同蚜虫与其内共生菌Buchnera的进化关系可能不同，这一结论与前人的研究也是吻合的。这一工作不仅弥补了这三种蚜虫与其内共生菌Buchnera之间的进化关系空白，同时有助于我们在了解这些蚜虫Buchnera的系统进化特征的基础上，进一步全面地了解了这三种蚜虫的内共生菌Buchnera。
综上所述，本研究首次对桃蚜、豆蚜、棉蚜内共生菌Buchnera的系统进化进行了报道，并初步探讨和分析了这三种蚜虫与其内共生菌Buchnera的系统进化关系，该工作对于我们进一步研究这些蚜虫Buchnera的其它特性以及这些蚜虫的次生共生菌都有重要的指导意义。
Almost all aphids maintain an endosymbiotic association with Buchnera aphidicola, aγ-proteobacterium closely related to Escherichia coll. The endosymbiont, harbored in host-derived vesicles within specialized cells known as bacteriocytes, is transmitted maternally, from mother aphid to progeny. The relationship between Buchnera and the aphid host is mutualistic because aphid needs B. aphidicola for its normal growth and reproduction whereas B. aphidicola cannot live outside the aphid. Molecular study found that Buchnera's genome is lessen, but the most amino acids synthetic genes are preserving.
Because of the importance of Buchnera, many authors studied this symbiosis. Previous studies about Buchnera of some aphids by foreign authors only conferred some limited aphids, such as the genus Uroleucon sp., Acryrthosiphon pisum, Diuraphis noxia (Kurdj), Schizaphis graminum etc, but no Myzus persicae, Aphis craccivora and Aphis gossypii. And domestic studies about Buchnera of the three aphids were also few, and this study only conferred the two genes (groEL and 16srDNA) of Buchnera in M. persicae. The three aphids, which are abundant throughout China as well as worldwide, are polyphagous herbivores attacking many vegetables and crops such as cabbages, cucumber, eggplant, tomato, cucurbits, bean and so on. Because of the special relationships between aphids and the Buchnera, the studies on the Buchnera of the three aphids are necessary. Firstly, we explored the phylogenies of the three aphids, M. persieae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera respectively. Then we synthetically analysed phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera.
1. The collection and rearing of experimental material
We collected three aphids M. persicae, A. craccivora and A. gossypii, which are important pests. Those three aphids were then reared in the laboratory on radish, bean, cucumber respectively. For each population, at least two double-deck gauze cages (around 50cm×50cm×50cm) were setto rear the three aphids. And they were kept in a laboratory under controlled environmental conditions (20±2℃, 60-70% R.H., 16L-8D).
2. The sequencing of mtDNA COⅠ/COⅡ, 12S/16SrRNA and nuclear gene EFalpha in the three aphids, and the phylogenetic analysis of the three aphids
In this part of work, we amplified COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in four populations of M. persicae, Nanjing population A. craccivora and A. gossypii. Three to five individual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.
DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2 Parameter method. Phylogenefic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.
Comparing the obtained sequences, we found that the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes sequences from the four populations M. persicae had very minor differences, and the similarities between the different two populations reached to 99%. And the similarities Of the COⅠ/COⅡ, EFalpha genes between M. persicae and A. gossypii were about 90%, those of the two genes between M. persicae and A. craccivora were near 85%、near 90% respectively, while the similarities of those genes between A. gossypii and A. craccivora were hyper-85%、near 93% respectively. But the similarities of the 12S/16SrRNA genes sequences in different two aphids reached to 95%.
Through the phylogenetic trees constructed by the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in M. persicae, we found that the COⅠ/COⅡand 12S/16SrRNA gene sequences in M. persicae were more closely related to those in D. noxia, while the EFalpha genes of M. persicae and D. noxia located in two neighboring branches, and the these genes sequences in A. gossypii and A. craccivora were all in the same clades. So we concluded that M. persicae and D. noxia were more close, and A. gossypii and A. craccivora were more close.
3. The sequencing of trpB, dnaN, trpEG in Buchnera, and the phylogenetic analysis of Buchnera in the three aphids
Firstly, we designed primers based on the nucleotide sequences from the currently known trpB, dnaN and trpEG sequences in Buchnera (isolated from Uroleucon. sp, A. pisum, D. noxia, S. graminum and R. padi), and amplified and sequenced these genes sequences of Buchnera from the three aphids, M. persicae, A. craccivora and A. gossypii. Three to five indiyidual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.
DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship, was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2-Parameter method. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.
Comparing the obtained sequences, we found that the trpB, dnaN and trpEG sequences in Buchnera from the four populations M. persicae had very minor differences, but these sequences between M. persicae and A. craccivora, M. persicae and A. gossypii had major differences, and these differences were little minor between A. craccivora and A. gossypii.
Through the phylogenetic trees constructed by this genes (trpB, dnaN and trpEG) in Buchnera, we found that the trpB, dnaN gene sequences in M. persicae were in the same clade with those in D. noxia, that is to say the Buchnera in M. persicae is more similar to the endosymbionts of D. noxia. Besides, the phylogenies of the trpB, dnaN genes of the Buchnera in M. persicae were accordant with that of the trpEG genes, so the plasmid genes trpEG genes of the Buchnera in M. persicae may be vertical transmission with Buchnera. And these genes sequences in A. gossypii and A. craccivora were all in the same clades, but the near clade to the sub-clade of the two aphids was variational. SO we only confirmed that the Buchnera in A. gossypii and A. craccivora were more close, but the conclusions that the Buchnera in these two aphids was close to that in which aphids and the transmission of the the plasmid genes trpEG of the Buchnera in these aphids could not be affirmed.
4. Analysis of phylogenetic relationships between the aphids and their endosymbiotic bacterium Buchnera
Studies about the phylogenetic relationships between aphids and their endosymbiotic bacterium Buchnera are few. In our present study, we explore phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, by comparing the characteristics of the phylogenies of those aphids and their Buchnera. We found that M. persicae and D. noxia were coevolutional, but we cannot conclude the coevolutional relationships between the two aphids, A. gossypii and A. craccivora, and their Buchnera. So we conclude that phylogenetic relationships between different aphids and their endosymbiotic bacterium Buchnera may be different, and this conclusion is accordant with previous study. This work not only fill a gap on phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, but also help us detailedly know the three aphids' Buchnera.
As described above, these results first exposed the phylogeny of Buchnera in the three aphids, M. persicae, A. craccivora and A. gossypii. And these works also explored phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera. These works will be helpful for exploring other characteristics of those aphids' Buchnera and studying those aphids'secondary endosymbiosis.
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