凡纳滨对虾系统发生地位及适应性进化分析
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摘要
甲壳动物是节肢动物门下仅次于六足亚门包含物种最丰富的一类物种,凡纳滨对虾(Litopenaeus vannamei)则隶属于甲壳亚门的十足目,是目前海水虾类中产量最高的单一海水经济养殖物种。目前,大量的研究工作都主要集中在水产养殖、病害防治和遗传育种等方面,对于对虾的系统发生问题以及进化过程中的各种生物学问题却少有研究。
     随着二代测序技术的不断发展,各种虾类的基因组和转录组数据也不断释放,这为我们对对虾的系统发生学和进化生物学问题的研究提供了有力的数据支持。本研究论文基于海量基因组和转录组数据,对凡纳滨对虾在节肢动物门下的系统发生和分化时间,以及在甲壳动物亚门下的系统发生进行了深入分析,并对凡纳滨对虾进化过程中的适应性进化进行的深入研究。论文包括以下三个部分:
     1.凡纳滨对虾的系统发生地位研究
     我们从九种节肢动物的全基因序列中挑选出了19,982个基因家族,并从中找到了85个单拷贝基因用于节肢动物的系统发生分析。我们构建了一棵非常保守的系统发生树,修正了传统分子系统发生树之间产生的争议,说明这85个单拷贝基因可以作为很好的分子标记用于节肢动物系统发生分析。通过对分化时间的估计发现,甲壳动物的分化时期很早,大约在3.95亿年左右,而且从甲壳动物祖先分化形成水溞和对虾的过程中发生了大量基因家族的扩增和缺失,说明水溞与对虾之间存在巨大差异。凡纳滨对虾和斑节对虾的分化时间也较早,而且也存在大量的基因家族的扩增和缺失,说明两种对虾之间存在着较大差异,这进一步支持了《枝鳃虾类科属一世界对虾总科和樱虾总科虾类》专著中将对虾属的6个亚属提升至属级阶元的结论。此外,我们还发现两种对虾之间基因家族的扩增和缺失基本呈现一种“此消彼长”的关系,推测其与各自环境适应有关。其中细胞组成结构方面的基因家族的扩增对斑节对虾进化过程中环境适应非常重要,而神经系统方面基因家族在凡纳滨对虾的进化中相对扩增比较显著。
     我们开发了一种基于短串序列(K-strings)构建后生动物系统发生树的方法,找到了23,223个对构建进化树贡献最大的关键进化元件(key K-strings)。该方法构建系统发生树不但能节省计算时间和空间,还能提升构树准确性,在物种分类方面优于其他方法。利用这种方法,我们构建了甲壳动物的系统发生树,结果显示凡纳滨对虾与美对虾属物种关系最近,而与对虾属斑节对虾关系相对较远,推测对虾各属的分化时间约在0.95亿年左右。此外,我们还提取到了427个甲壳动物关键进化元件key K-strings。这些key K-strings都位于其蛋白质序列的保守区,说明这些序列在漫长进化历史过程中,受到了进化选择压力而保留下来,并对物种的系统发生起着重要作用。
     2.凡纳滨对虾的水平基因转移研究
     水平基因转移事件的发生往往会使宿主获取新的性状和功能,以帮助宿主更好的适应新的环境。目前,越来越多的原核生物转移到真核生物的水平转移基因被报导,其中发现得最多的是在节肢动物中。通过对凡纳滨对虾的水平基因转移的研究,可以帮助我们了解对虾进化过程中的环境适应性机制。
     我们通过一系列方法对对虾全基因组范围内的水平基因转移事件进行了研究,最终找到了14个水平转移基因,其中有12个是细菌起源的,另外两个是真菌起源的。通过对这些基因的结构进行分析,我们发现两个真菌起源的水平转移基因上的内含子在转移到对虾基因组上后被替换成了对虾特异性的内含子,而两个细菌起源的水平转移基因中则发现有对虾特异性的内含子的插入。此外,我们还发现有三个大片段的外源序列插入到了对虾基因组上,其中的一个是一个完整的转座子序列,而另外两个则是以操纵子的形式同时转移到对虾基因组上的,而且其上面的非编码区序列在转移到对虾基因组的过程中都被去除了,由此可以推测成熟的mRNA才是这些水平转移基因发生转移的基质。此外,大部分的水平转移基因都受到了强烈的负选择压力,推测他们在对虾基因组上都是具有一定功能的。通过对14个水平转移基因进行功能预测发现,其中有6个基因与能量代谢有关,还有4个基因与防御机制有关。而且大部分的水平转移基因在对虾的幼体发育不同时期展现出了差异基因表达,因此它们在对虾的生长发育过程中起着重要的作用。基于这14个基因功能方面的各种表现,我们推测这些水平转移基因可能有利于提升对虾的能量代谢和防御机制。
     3、凡纳滨对虾适应性进化研究
     在自然选择的作用力下,不同的虾会各自进化出适应周边环境的适应性机制。这些适应性机制直观地以新表型或新的生物性状的形式展现出来,但其本质还是分子水平上的遗传物质在随着选择压力的作用而产生的变化。
     通过对转录组数据进行处理,我们获得了六种虾(凡纳滨对虾L. vannamei、中国明对虾Fenneropenaeus chinensis、斑节对虾Penaeus monodon、罗氏沼虾Macrobrachium rosenbergii、凯氏长额虾Pandalus latirostris以及狭螯螯虾Astacusleptodactylu)平均约12,000条去冗余的编码基因序列,并进一步从中挑选出了1,669组直系同源基因。利用这些直系同源基因我们成功构建了一棵保守的系统发生树,该系统发生树与传统的形态学分类以及分子标记系统发生学分析的结果一致。我们分别以三种对虾和两种真虾作为前景枝,探讨对虾和真虾在进化过程中的适应性机制差异;还分别以四种海水虾和两种淡水虾分别作为前景枝,探讨其在各自的水环境下的适应性机制。
     我们分别在对虾和真虾中找到了231个和110个正选择基因。基因富集的结果显示对虾的正选择基因主要富集在跟核糖体合成、生物膜的组成以及应激反应等相关的功能中。正选择基因在核糖体合成及蛋白质翻译过程上的富集,推测对虾在蛋白质合成上受到较强的正选择作用;而在生物膜的组成上的富集,推测其可能是随蛋白质合成方面基因的正选择作用而产生的一种共同适应性进化的过程。对虾的正选择基因在应激反应相关的功能中富集正好与关于对虾拥有最快的生物信号冲动传导速度的观点不谋而合,说明对虾在面对应激因子刺激过程中逐渐产生出了较强的应激反应适应性。真虾的正选择基因则主要富集在甲基化相关的分类中,推测真虾可能通过甲基化修饰来提高其环境适应性。
     通过分别以海水虾和淡水虾作为前景枝,我们分别找到了174个和360个正选择基因。渗透调控是虾类水环境适应过程中非常重要的一个生物学过程。我们从这些正选择基因中找到了海水虾中两个正向调控渗透作用的基因NKA和NKCC,它们都能有效的维持细胞较高的钠钾离子渗透梯度,提高海水虾在高盐环境的适应能力。此外,在淡水虾中也找到了一个负调控渗透作用的基因RhoGDI,它能有效的调整细胞内离子浓度,以维持细胞在淡水环境下的生存。因此,我们认为这些正选择基因中确实存在一些跟渗透调节有着直接关系的基因,它们分别有利于提升海水虾和淡水虾在各自的水环境中的适应性机制。
Except Insects, crustacean is one of the groups with most species in Athropoda.As a species in Crustacea, Pacific white shrimp, Litopenaeus vannamei, is animportant economic breeding species in China, which produce millions of tons everyyear. In present, most of the shrimp researches are focused on aquaculture, diseasecontrol and genetic breeding, while few of them are focused on phylogeny andevolutionary biology.
     As the development of next-generation sequencing technology, a growingnumber of shrimp genomes and transcriptomes are sequenced and published gradually,which may provide good resources for the exhaustive research on shrimpphylogenetic and adaptive evolution analysis. In this study, we performedphylogenetic analysis of arthropods and estimated the divergence time of shrimpbased on the amount of transcriptome data. We also developed a new method toanalyze the phylogeny of shrimp under Crustacea. Besides, novel bioinformaticsmethods were implemented to analyze the adaptive evolution of shrimp. There arethree major sections included in this thesis as follows:
     1. Phylogenetic analysis of L. vannamei
     Through using TreeFam methodology, we extracted19,982gene families fromfull genes of nine arthropods. Then,85single-copy genes were selected from them forthe phylogenetic analysis of Athropoda. The phylogenetic tree constructed bysingle-copy genes was similar with the trees constructed by custom molecularmarkers, and also has high confidence on each branch, which indicated that these85single-copy genes could be used as good molecular marker for the Arthropodaphylogenetic analysis. After estimating the divergence time of each species, we foundthat Crustacea was divergent at about395million years ago. And there were a great many of gene gain and gene lose when the ancestor evolved to shrimp and Daphnia,indicating that there is significant bias between the genomes of Daphnia and shrimps.L. vannamei and Penaeus monodon were also divergent early and a lot of gained orlosed gene families were found between them, which suggest these two Penaeidea arequite different each other. These results rightly supported the opinion of upgrading thesix subgenus of Penaeus to the stage of genus. Furthermore, we found that there aremany GO term with one shrimp gene gain significantly while the other shrimp losesignificantly. Among these GO terms, cellular component related gene families weresignificantly gained in P. monodon, while nervous system related gene gamilies weresignificantly enriched in L. vannamei. The bias of gene gain and gene lose betweentwo shrimps may help them adapting to their new environments, respectively.
     We developed a new method for the metazoans phylogenetic analysis based onthe short sequences segments, namely K-strings. We collected23,223key K-stringswhich make significant contribution for the phylogenetic tree construction. Throughusing this new method, we constructed a phylogenetic tree of Crustacea, and foundthat this tree is topological similar with the custom molecular phylogentic tree. L.vannamei was phylogentic close with Farfantepenaeus californiensis, while far fromP. monodon. The divergence time of Penaeidea was estimated at about95millionyears ago. Besides, we collected427key K-strings of Crustacea and map them on theoriginal protein sequences. The results showed that these phylogentic important keyK-strings are totally tend to accumulated at the conserved regions of the proteinsequences, which indicating that these key K-strings can be thought as evolutionaryelements that suffering selective pressure and play important role in crustaceansphylogeny.
     2. Horizontal gene transfer analysis of L. vannamei
     Generally, horizontal gene transfer (HGT) events could help receipients gainsome new phylotypes or functions to support for the receipients adapting to newenvironments. In recent years, a growing number of genes have been reported asbeing horizontally transferred from prokaryotes to eukaryotes, most of them involvingarthropods. As a member of the phylum Arthropoda, L. vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms,which provide a platform for horizontal gene transfer.
     In this section, we analyzed the genome-wide HGT events in L. vannamei.Through homology search and phylogenetic analysis, followed by experimental PCRconfirmation,14genes with HGT event were identified:12of them were transferredfrom bacteria and two from fungi. Structure analysis of these genes showed that theintrons of the two fungi-originated genes were substituted by shrimp DNA fragment,two genes transferred from bacteria had shrimp specific introns inserted in them.Furthermore, around other three bacteria-originated genes, there were three largeDNA segments inserted into the shrimp genome. One segment was a transposon thatfully transferred, and the other two segments contained only coding regions ofbacteria. These structure alterations provide evidence that mature RNA may be thesubstrate for HGT events. Among14HGT genes, most of them were detected to beexposed to negative selection pressure and they appeared to be functional. Functionalprediction of these14genes showed that6of them might be related to energymetabolism, and4others related to defense of the organism. And most of themexpressed differently at five development stage of shrimp laveral, which indicatingthese genes are important for the shrimp development. Considering the functions ofthese14HGT genes, we thought they may helpful for the improvements of shrimpenergy metabolism and defense mechanism.
     3. Adaptive evolution analysis of L. vannamei
     During the long evolutionary history, because of the natural selection, shrimpswill evolve some adaptive mechanisms for the environment adaptation. Generally,these adaptive mechanisms can be directly displyed on the newly generatedphylotypes or biological characters, but it was actually displayed on the geneticmaterials mutations.
     We collected many transcriptomes of six shrimps: L. vannamei, Fenneropenaeuschinensis, P. monodon, Pandalus latirostris, Macrobrachium rosenbergii, Astacusleptodactylus. After removing redundant sequences, we finally obtained average12,000protein-coding genes, and select1,669groups of orthologs from them. Based on these orthologs, we constructed a conserved phylogenetic tree of these six shrimps.The topology of this tree is similar with normal trees constructed by molecularmarkers, and also correspondent with the custom morphological classification. Then,we set the branches of three Penaeidea and two Caridea as foreground branchesrespectively to analyze the bias of adaptive mechanisms between Penaeidea andCaridea. We also set the branches of four seawater shrimps and two freshwatershrimps as foreground branches respectively to analyze the adaptive mechanisms ofdifferent water environments.
     We obtained231and110positive selected genes from Penaeidea and Caridea,respectively. It was found that most of the positive selected genes in Penaeidea areenriched in the functional related GO terms of ribosome biogenesis, membrane andresponse to stimulus. The genes enriched in the ribosome biogenesis and proteintranslation suffer positive selection pressure may be benefit for the protein synthesisin Penaeidea, while the positive selected genes enriched in membrane compositionmay be co-evolved with the genes in protein synthesis. It was interesting to find thatmany positive selected genes are enriched in stimulus response related GO terms inPenaeidea. As Penaeidea have excellent giant nerve fibers to conduct impulses at thevelocity highest among animals, it seems the nervous systems of Penaeidea wereadaptive evolved during the long evolution history. When compared with Penaeidea,the positive selected genes in Caridea are primarily enriched in methylation relatedfunctions, which suggest that Caridea may improve its adaptation through methylationmodification.
     When analyze the adaptive evolution of the shrimps under seawater or freshwater,we obtained174and360positive selected genes among them, respectively. Becauseosmoregulation is one of the most important biological processes in water adaptationof shrimps, we surveyed genes related to osmoregulation among these positiveselected genes. As expected, we found two genes, NKA and NKCC, positive regulatethe osmoregulation in seawater shrimps. Both genes are responsible for establishingelectrochemical gradients across biological membrane, which were considered benefitfor the seawater shrimps adapt to high salinity environments. In contrast, there is a positive selected gene RHOGTP8in freshwater shrimps can negative regulateosmoregulation procedures. RHOGTP8can maintain the intracellular ionconcentration, which may helpful for the freshwater adaptation. Therefore, all theseosmoregulation related positive selected genes are considered to play important rolein the improvements of water environment adaptation mechanisms.
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