IOCAS-IR  > 海洋生态与环境科学重点实验室
甲藻维生素B12依赖性营养的分子机理研究及有害甲藻褐色马格里夫藻种内遗传多样性研究
林思恒
Subtype博士
Thesis Advisor唐赢中
2020-08-28
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士学位
Keyword甲藻 维生素b12 依赖性营养 褐色马格里夫藻 种内遗传多样性
Abstract

甲藻是引起有害藻华的主要种类(75%以上藻华事件由甲藻引起),但长期以来对甲藻营养生态学的研究主要集中于常见的无机营养盐如氮和磷等,很少关注微量营养元素如维生素。但是实际上90%以上的甲藻都是维生素B12的营养依赖型(auxotrophs)。故探索甲藻对维生素B12依赖性营养的遗传学基础对甲藻生物学及其有害藻华生态学具有重要意义。已有研究证明某些藻类对维生素B12的依赖性营养主要取决于二种甲硫氨酸合成酶基因的有无:以B12为辅酶的METH基因和不以B12为辅酶的METE基因。为了揭示甲藻维生素B12依赖性营养的遗传学基础,本研究使用cDNA末端快速扩增技术(RACE)获得14种甲藻的相关基因全长,并结合实时荧光定量PCR技术比较其中10种甲藻的METHMETE基因在不同维生素B12浓度和添加方式下的表达模式,对其基因功能进行判断。通过整理分析已公开的甲藻转录组和基因组信息,建立甲藻cDNA文库,以获得的14种甲藻的甲硫氨酸合成酶基因METHMETEcDNA全长为基础比对获得共61种甲藻的METHMETE基因信息并进行系统进化分析,明确地解释了甲藻维生素B12依赖性营养在基因水平的原因,从而确认了甲藻维生素B12依赖性营养的分子机理。主要实验结果简述如下:

114种甲藻的METHMETE基因全长克隆

对实验室培养的14种甲藻的共38METHMETE基因进行了全长克隆,发现所有14种甲藻均具有完整的METH,且与其它模式生物的METH功能域一致,但只有12种甲藻存在METE,且无证据表明另外2种甲藻存在METE。使用CD-search进行功能域预测发现,存在于该12种甲藻的所有METE功能域均不完整,即缺少N端结构域,因此推测它们可能不能行使甲硫氨酸合成的功能。

2)甲藻的维生素B12依赖性营养的证明

通过维生素B12营养限制和再添加实验以及直接供给甲硫氨酸的生理实验,发现8种甲藻的生长取决于维生素B12的有无,添加维生素B12可以使受营养限制的甲藻恢复生长,再次确认了8种甲藻对维生素B12的依赖性营养,并且不能靠添加甲硫氨酸缓解维生素B12不足导致的生长限制。

3甲藻METHMETE基因表达与维生素B12浓度的响应关系

根据10种甲藻的生理实验结果和qRT-PCR实验结果,METHMETE基因对B12浓度响应的表达模式可总结如下:在受到低浓度维生素B12营养限制后,METH的表达量与维生素B12浓度存在负反馈调节机制,即当受到低浓度维生素B12限制时,METH的表达量上升,但恢复维生素B12浓度后METH表达量下降;METE基因在维生素B12充足时表达未表现出明显下调,但在低浓度维生素B12限制时也没有出现高表达(即代替METH行使甲硫氨酸合成功能),而且在恢复维生素B12至正常培养基浓度后也未见明显抑制或下调。通过与其它兼具METHMETE两个基因且有完整功能的物种相比,本研究中所有甲藻METE基因表达与维生素B12浓度的响应关系佐证了该基因在甲藻中因功能域不完整无法行使功能的推断。

4)甲藻METE基因功能初探

以剧毒卡尔藻(Karlodinium veneficum)为实验对象,通过qRT-PCR发现METHMETE基因在生长周期和培养的光暗周期不同阶段的表达如下:随着甲藻进入指数生长期,METHMETE基因表达量逐渐下降;METHMETE基因在无光照时表达量较高,在有光照时表达量下降。METE呈现的在生长周期和昼夜周期不同阶段的表达动态说明其可能参与了甲藻的其它生化过程,特别是与细胞增殖有关的反应。

5)甲藻和非甲藻METHMETE基因的系统进化分析

基于构建的甲藻cDNA文库,以获得的14种甲藻的METHMETE基因为基础进行比对检索,确认了目前可获得的绝大多数甲藻物种的METHMETE基因信息。整理分析的共61甲藻中,所有甲藻均含有METH基因且功能域完整。通过与其它物种的METH基因进行系统进化分析,构建的系统进化树不仅较好地反映了物种间的分类学亲缘关系,并且甲藻METH基因的功能与其它物种特别是METH基因功能已被确认的种类相同。而在所有61种甲藻cDNA文库中只有43种甲藻中可以比对获得METE基因(其余18种甲藻未发现),并且所有获得的METE基因其功能域均不完整,无N端结构域。通过与来自其它门类物种已证明具有甲硫氨酸合成功能的METE基因进行系统进化分析并构建系统进化树表明,所有种类的METE基因根据功能域的完整与否在进化树上形成不同分枝,证明甲藻中存在的METE基因因功能域不完整无法完成甲硫氨酸合成,从而在基因结构水平解释了绝大多数甚至全部甲藻对维生素B12的依赖性营养。

基于上述实验结果,我们从基因水平上完整地解释了绝大多数受试甲藻都是维生素B12的营养依赖型的遗传学基础,即因为缺少METE或者METE基因功能域不完整使得甲藻必须依赖以维生素B12为辅酶的METH合成甲硫氨酸。我们据此提出假说认为所有的甲藻都是维生素B12营养依赖型。了解甲藻维生素B12依赖性营养的遗传学基础和分子机理,很大程度地深化了我们对维生素B12及其依赖性营养在藻华特别是甲藻藻华发生中重要作用的认识,从而将为进一步探索有害藻华爆发机理和防控原理提供基础性依据。

上述主要研究之外,本学位论文还包括对一种常见的有害藻华甲藻褐色马格里夫藻(Margalefidinium fulvescens)的中国胶州湾种群的种内遗传多样性研究。核糖体大亚基DNALSU rDNA)序列被越来越多地用于推断各类物种的系统发育关系和物种鉴定,因为相较于rDNA的其它亚基LSU rDNA含有一个高可变的D2区而更适合于鉴别近亲种或鉴别种内遗传变异。早前的研究中发现某些甲藻存在较高的种内遗传多样性,甚至是个体内(即单细胞内)的遗传多样性。一般认为这种遗传多样性的原因是甲藻基因组内rDNA基因具有很高拷贝数导致。但是,rDNA的拷贝数在甲藻物种之间存在巨大差异,因此需要对每一重要物种的种内遗传多样性进行单独研究。褐色马格里夫藻是一种产毒的常见有害藻华甲藻,在美国、加拿大、韩国等多个海域多次发生藻华且造成鱼类死亡事件。自2015年以来,本课题组在中国胶州湾几次发现其已接近藻华细胞密度。为了更好对这一中国海域新发现的藻华原因种进行常规监测、分析其与世界其它种群的亲缘关系和追溯其可能的来源,本研究分别对野外接近藻华密度样品和通过单细胞分离建立的实验室纯培养进行了LSU rDNA测序和系统遗传学分析,发现褐色马格里夫藻胶州湾种群在种群内和个体内均存在较高遗传多样性。本研究对7个野外样品和11个分离的单细胞纯培养构建序列文库,扩增LSU rDNAD1–D6区序列(1435个碱基),经过克隆测序共获得的2341条序列,并对其进行系统遗传学分析。在获得的所有序列中,只有一条在所有样品中均被检测出,该序列被认为是该物种大量rDNA拷贝中丰度最高从而具有代表性的序列。本研究发现褐色马格里夫藻相较其它研究过的甲藻物种(如红色赤潮藻Akashiwo sanguinea和链状亚历山大藻Alexandrium catenella),其LSU rDNA存在更高的种群内和个体内的遗传多样性,序列具有更多的多样性位点数量和更高的序列多样性。不同野外样品和不同单细胞纯培养内每条序列之间的平均核苷酸差异分别为6.43个碱基和4.42个碱基,其中与该物种参考序列差异最大的序列存在132个碱基差异,序列相似度仅90%。而以往环境基因组研究(或称宏基因组、DNA宏条形码)中被注释为相同物种的序列相似度判断阈值通常需达到97%以上。由于7个野外样品和11个纯培养样品之间整体的遗传多样性程度相当,结合分子方差分析(AMOVA)的结果,可以判断褐色马格里夫藻单细胞内的rDNA拷贝间的多样性导致了个体内遗传多样性并进一步导致了种群内的遗传多样性。因此不同地理种群间可能存在更高的种群间多样性。本工作为未来对该藻华原因种不同地理种群间的全面比较提供了数据和理论基础,并对以基于数据库中参考序列间比较为主要方式的甲藻物种鉴定和建立新的甲藻分类群都具有重要的意义。

Other AbstractDinoflagellates are the major causative organisms of marine Harmful Algal Blooms (HABs; more than 75% HAB events are caused by dinoflagellates). The nutritional studies of dinoflagellates have mainly focused on the inorganic macro-nutrients such as nitrogen and phosphorus, while little attention has been paid to micro-nutrients such as vitamins and trace elements. However, over 90% of dinoflagellates have been proven to be vitamin B12 auxotrophs, it is therefore important to understand the genetic basis of vitamin B12 auxotrophy in dinoflagellates in the framework of the ecology of HABs, especially that of dinoflagellates HABs. Recent studies have shown that vitamin B12 auxotrophy of some algae mainly depend on the presence of two methionine synthase genes: B12-dependent METH, of which vitamin B12 acts as the coenzyme, and B12-independent METE, which can synthesize methionine without the involvement of B12. Therefore, we obtained the full-length sequences of METH and METE for 14 species of dinoflagellates by rapid amplification of cDNA ends (RACE). In order to determine the functions of METH and METE in dinoflagellates, Quantitative real time polymerase chain reaction (qRT-PCR) were used to observe the molecular responses of METH and METE to various levels and re-supplements of vitamin B12 in 10 species of dinoflagellates. Then we constructed cDNA libraries of dinoflagellates by collecting from all publicly available databases of transcriptomes and genomes. Based on the abovementioned full-length sequences of METH and METE obtained from 14 species of dinoflagellates, we retrieved the METH and METE sequences of 61 dinoflagellate species via translated Basic Local Alignment Search Tool (tblastn) and analyzed the phylogenetic relationships among all sequences. Together, our experimental results and phylogenetic analyses revealed the genetic basis of vitamin B12 auxotrophy of dinoflagellates, or provide a mechanistic explanation from the molecular level for this particular trophic mode of dinoflagellates. The major findings of our study are itemized as follows: (1) Full-length cloning of METH and METE in 14 species of dinoflagellates A total of 38 full-length cDNA sequences of METH and METE genes from 14 species of dinoflagellates maintained in the laboratory were cloned. All 14 species have METHs with the same conserved domain as found in other model organisms. However, only 12 of the 14 dinoflagellate species have METEs (note METE is the only annotation for the gene in all current databases), all of which have C-terminal domain only but lack the N-terminal domain, suggesting a loss of their function as a methionine synthase gene. We did not find any evidence for the existence of METE gene in the other two species of dinoflagellates. (2) Re-confirming the vitamin B12 auxotrophy of dinoflagellates Via physiological experiments, we found that the growth of 8 dinoflagellate species depended on the availability and levels of vitamin B12 and directly responded to limitation and supplement of vitamin B12. Re-supplement of vitamin B12 in the cultures of the 8 dinoflagellate species showing growth-limiting effects of lower levels of vitamin B12 could recover the growth of all cultures, while the supplement of methionine could not do so. Based on these results, we reconfirmed these 8 species of dinoflagellates are auxotrophs of vitamin B12. (3) Transcriptional responses of METH and METE to the changing level of vitamin B12 Based on the results of qRT-PCR measurement for 10 species of dinoflagellates, the transcriptional responses of METH and METE to the changing level and re-supplement of vitamin B12 can be briefly summarized as follows: the expression of METH exhibited a negative feedback to the level of vitamin B12, i.e., the expression of METH increased when the concentration of vitamin B12 decreased to limit the growth of dinoflagellates, while the expression of METH decreased after the concentration of vitamin B12 was increased via re-supplementing to the initial level of f/2 medium; The expression of METE, however, exhibited not to respond to the change in vitamin B12 concentration and particularly, did not appear a high expression to take over the function of METH in response to limited levels of vitamin B12. The expression of METE was also not repressed when vitamin B12 was restored to a high level. Judged on the basis of the expression patterns of METH and METE in those algal (e.g. diatoms) species that have METH and METE functioning in fact as methionine synthases genes and the fact that the growth of 8 dinoflagellates were inhibited by low vitamin B12 levels, the METE gene in our dinoflagellates appeared not to function as a B12-independent methionine synthase. (4) Probing the possible functions of METE in dinoflagellates Since the METE gene appeared not to be a real methionine synthase in all dinoflagellates we investigated but exhibited considerable expressions at all levels of vitamin B12, we, in hope of probing the possible function of METE, observed the expression patterns of both METH and METE in the B12-auxotroph Karlodinium veneficum along the growth cycle and different time points of the light-dark cycle via qRT-PCR. The expression levels of METH and METE decreased gradually with the growth of K. veneficum during the entire exponential growth stage, but were higher during the dark cycle (12 h) than that during the light period (12 h). The expression patterns of METE along the growth cycle and light-dark cycle suggest that METE may be involved in some physiological processes rather than methionine synthesis in dinoflagellates. (5) Phylogenetic analyses of METH and METE genes of dinoflagellates and the species from other taxa We constructed cDNA libraries of dinoflagellates by collecting from all publicly available databases of transcriptomes and genomes and then, based on the full-length sequences of METH and METE obtained from the 14 dinoflagellate species in this study, we retrieved the METH and METE sequences of 61 species of dinoflagellates All 61 dinoflagellates contained METH genes that have the same conserved and functional domains, suggesting a genuine function of B12-dependent methionine synthase. The phylogenetic analysis of METH sequences exhibited clustering pattern consistent to the phylogenetic relationships among all species in general. However, METE gene sequences could be obtained from only 43 of the 61 dinoflagellate species, and there was no evidence for the presence of METE gene in the other 18 species. More importantly, all the obtained METE sequences have the C-terminal domain only and lack the N-terminal domain that is required for a genuine function of methionine synthase as demonstrated in non-dinoflagellate taxa (e.g. diatoms). The phylogenetic analysis of all METE sequences together with organisms from other groups that have functional METE exhibited that all METE sequences formed two major clades in accordance with presence or absence of the N-terminal domain. Therefore, METE in dinoflagellates are not functioning as a methionine synthase due to the absence of N-terminal domain. In conclusion, we elucidated the genetic basis of vitamin B12 auxotrophy of dinoflagellates via cloning the full-length sequences and phylogenetic analyses of the methionine synthases genes METH and METE and the transcriptional responses of the two genes to the changing level of vitamin B12 in 10 dinoflagellate cultures. We hypothesize that all dinoflagellates are probably vitamin B12 auxotrophs because of the absence of N-terminal domain in METE needed for the gene to encode a genuine B12-independent methionine synthase, or possibly an absence of the incomplete METE gene in some dinoflagellates, and thus have to rely on the B12-dependent synthase gene METH and consequently the availability of vitamin B12. Revealing the molecular mechanisms of vitamin B12 auxotrophy of dinoflagellates is believed to have greatly deepened our understanding of the role played by vitamin B12 in regulating the growth and consequently the dynamics of HABs, especially that of dinoflagellates. We also anticipate the outputs of this research will provide insights for the forecasting, prevention, and control of HABs. In addition to the work described above, we also assessed the intrapopulational and intraindividual genetic diversity of the naked dinoflagellate Margalefidinium fulvescens in the thesis. Large subunit ribosomal DNA (LSU rDNA) sequences have been increasingly used to infer the phylogeny and species identity of organisms, a few previous studies, however, have observed high intraspecific and even intraindividual variability in LSU rDNA in some dinoflagellate species due to, assumably, large copy numbers of rDNA in dinoflagellates. Since the copy number of LSU rDNA varies tremendously among dinoflagellate species, the intraspecific and intraindividual diversity for a species of particular interest thus needs to be investigated individually. As a toxic and HABs-forming dinoflagellate, Margalefidinium fulvescens has been observed to approach blooming density in Jiaozhou Bay, China since 2015 after numerous blooms having been reported from other countries. In trying to identify the source of this newly observed HABs-forming species in China by sequencing the LSU rDNA for both field samples and clonal cultures, we noticed and thus further investigated high intrapopulational and intraindividual genetic diversities of the dinoflagellate. The D1–D6 region of the LSU rDNA (1,435 bases) was amplified from 7 field samples (pooled cells) and 11 clonal cultures, cloned, sequenced, and analyzed phylogenetically for 2,341 sequences obtained. All the numbers of sequences obtained from each clonal culture were far less than the estimated rDNA copy number in M. fulvescens. In the clone library, only one unique sequence was contained in all samples as the most dominant sequence. We found high intrapopulational and intraindividual genetic diversity in M. fulvescens as reflected in the number of polymorphic sites and unique sequences in the clone library for different field samples and clonal cultures in comparison to other species. The mean number of nucleotide differences of each sequence from different field samples and clonal cultures were 6.43 and 4.42 bases, respectively, with the highest being 132 bases, nearly 10%. The sequences with highest variability may be easily annotated as different species if they were obtained from environmental genomic studies because sequence-based species identification in meta-barcoding studies often use "97% identity" threshold. Based on that the mean and overall intrapopulational genetic diversity calculated for 7 field samples was equivalent to the mean and overall intraindividual variability for 11 clonal cultures in indices of genetic diversity, together with the result of AMOVA analysis, we infer that the variability within individual cells (i.e. variability among LSU rDNA polymorphic copies) caused both the intraindividual and intrapopulational genetic diversities observed in the M. fulvescens population, and a higher interpopulational diversity may exist among different geographic populations. The results provide an insightful basis for such a comprehensive interpopulational comparison and important implications for identifying species and establishing new taxa based on the similarity comparison to reference sequences deposited in databases.
Subject Area海洋科学
MOST Discipline Catalogue理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164773
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
林思恒. 甲藻维生素B12依赖性营养的分子机理研究及有害甲藻褐色马格里夫藻种内遗传多样性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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