基于转录组测序的长心卡帕藻光合作用和 乔利橘色藻类胡萝卜素合成关键基因分析

IOCAS-IR > 海洋生物技术研发中心

	基于转录组测序的长心卡帕藻光合作用和乔利橘色藻类胡萝卜素合成关键基因分析
	李倩倩1
学位类型	博士
导师	刘建国
	2015-05-15
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	转录组长心卡帕藻乔利橘色藻光合作用类胡萝卜素合成
摘要	本文以生长于热带海域的大型藻类长心卡帕藻（Kappaphycus alvarezii）和低温高原的陆生微藻乔利橘色藻（Trentepohlia jolithus）为对象，利用转录组测序研究手段探讨它们的基因注释和代谢途径。一方面，详述了长心卡帕藻在不同温度条件下的转录组测序结果，深入研究了其假定光合作用基因及其参与的代谢过程。另一方面，分析了乔利橘色藻的转录组测序数据并讨论了其碳固定和类胡萝卜素的合成过程。长心卡帕藻是一种不抗低温的热带大型经济红藻，也是提取卡帕型卡拉胶的重要原料，目前对该藻在不同温条件下分子水平的基因表达变化情况所知甚少。本研究将该藻分别在高温（HT，34℃）、低温（LT，17℃）和正常温度（CK，26℃）下处理24小时后，通过Illumina HiSeq™2000双端测序获得约1.08亿bp原始reads，将3个样品的Clean Reads共同拼接后注释得到11,537条非冗余unigene，最后利用Blast算法，通过与不同公共数据库中的基因进行同源比对，分别将9,920（85.98%）和10,952（91.81%）条unigene注释到GO Slim和EggNOG分类数据库中。同时，通过KEGG代谢通路分析将5,015条unigene聚类到六大类生化途径、295条KEGG途径中。研究发现参与长心卡帕藻光合作用和天线蛋白表达的基因分别有48和23个，且缺失普通绿色植物中参与锰簇合物结构形成的PsbP基因，而存于该藻的PsbU基因很可能代替之以行使功能。两两比较不同温度条件下的基因表达情况，共筛选出677个差异表达基因。与常温环境相比，低温和高温条件下差异表达的基因数目分别为245和183个，其中低温下上调（下调）表达的有59（186）个，高温下上调（下调）表达的有20（163）个。在此基础上，重点讨论了光合作用参与基因的温度适应机制：低温条件下，光系统II中3个基因（PsbO、PsbQ、PsbR）和光系统I的5个基因（PsaD、PsaF、PsaG、PsaH、PsaK）出现下调表达，未发现上调表达基因；高温条件下，上调和下调表达的基因数目分别为9个和4个（PsbA、PsbC、PsaB、atpF1A）。高低温胁迫条件下，细胞色素b6/f复合体和光合电子传递相关基因依然稳定表达，表明其不受温度胁迫的影响。与此同时，不同温度条件下叶绿素荧光动力学曲线中I和K点的快速上升印证了温度胁迫会导致光损伤。较之低温胁迫，高温胁迫下基因差异表达数目更多，叶绿素荧光动力学曲线的变化幅度更剧烈，表明高温对长心卡帕藻所造成的生理学损伤更大。本部分研究中大量光合作用和天线蛋白等相关基因的识别筛选，将促进长心卡帕藻的温度适应机制的分子生物学研究，对筛选新变种与扩张养殖区域具有重要意义。乔利橘色藻是一种不抗高温的高海拔地区陆生微藻，大量积累β-胡萝卜素是其重要特征之一。目前针对该藻的研究主要集中在形态学、分类学、系统发育学和其地理分布与繁殖等方面，分子生物学相关研究相对稀少。本研究首先基于Illumina HiSeq™2000测序得到了41,328条unigene；然后根据NR注释结果，将22,018（53.28%）条unigene聚类到52个GO分类中，其中涵盖了161,451个子功能分类；接着通过KEGG注释分析将26,217（63.44%）条unigene归属到128条生化途径之中；最后，在乔利橘色藻转录组中分别检测到5,798个SSRs和131,478个SNP位点。在以上基因注释与信息挖掘基础上，发现C3、C4、脂肪酸及类胡萝卜素合成代谢相关基因分别有139、157、122和99个。乔利橘色藻C固定途径分析中，C3、C4两种途径中所涉及的基因都有所体现，表明其C固定系统的复杂性，这种高效的固碳方式对其在高光、低温、干旱环境下生存十分有利。类胡萝卜素合成通路分析发现，该藻缺失了甲醛戊酸激酶（MVK），导致其不能通过甲醛戊酸途径（MVA path way）合成异戊二烯焦磷酸（IPP）和二甲烯丙基焦磷酸（DMAPP）。相比之下，所有编码非甲醛戊酸途径（MEP/DOXP path way）相关基因中都有所体现，使之成为类胡萝卜素合成底物的来源。此外，将乔利橘色藻类胡萝卜素合成路径与其他微藻对比发现，该藻转录组序列注释基因中没有胡萝卜素β酮醇酶（CrtO），这很可能是其不能合成虾青素的重要原因。本研究是对橘色藻目转录组测序的首次报道，丰富了石莼纲的分子生物学数据库。在完成基因功能注释的同时，碳固定、脂肪酸合成代谢及类胡萝卜素合成通路的分析对确定乔利橘色藻的资源开发方向具有重要意义。
其他摘要	In this thesis, we discussed two algae including Kappaphycus alvarezii and Trentepohlia jolithus with different characteristics. Kappaphycus alvarezii is generally found in the tropical oceans. While, Trentepohlia jolithus typically grow under chilling or cold alpine environment. Based on RNA-Seq results, we aimed to investigate their gene annotation and metabolic pathways analysis. We divide the whole in two parts. The first part focused on de novo sequencing and comparative of Kappaphycus alvarezii (Solieriaceae, Rhodophyta) under high and low temperatures, to discover putative genes associated with photosynthesis. The second work focused on de novo transcriptome analysis of an aerial microalga Trentepohlia jolithus, pathway description and gene discovery for carbon fixation and carotenoid biosynthesis was carried out to fulfill our discussion. Kappaphycus alvarezii is well known for its commercial utility. It is an important raw material for the extraction of phycocolloid, κ-carrageenan. Changes at molecular level under high temperature (HT) and low temperatures (LT) are poorly understood in seaweeds. This study aims to characterize the transcriptome and identify the differently expressed unigenes at 24 h after HT and LT treatment compared to its control by using Illumina Hiseq2000 sequencing. After removal of low-quality sequences and assembly, approximately 108 million RNA-Seq reads were obtained. Further blast analysis showed that a total of 9,920 unigenes (85.98%) and 10,952 (91.81%) were functionally annotated and assigned each unigene to a set of GO Slims and eggNOG orthology, respectively. KEGG analysis showed that 5,015 genes were classified into six main categories and assigned to 295 KEGG pathways. Through comparison among them, 677 significantly differentially expressed genes with ≥ 2-fold change in expression levels were identified. In this context, forty-eight non-redundant unigenes were identified with close identity to the principal molecules involved in photosynthesis, including photosystem II, photosystem I, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase, twenty-three non redundant unigenes showed similarity to the principal molecules of the photosynthesis antenna-protein pathway, including AP, PE, and the light-harvesting chlorophyll protein complex (LHC). With regard to PsbP, it may be a subunit lost from the extrinsic part of the oxygen-evolving complex of the Rhodophyta. Another polypeptide (PsbU), which is encoded in the nucleus of the cell, replaces PsbP to stabilize the manganese cluster. In order to identify differentially expressed genes (DEG) between these three samples, we compared them with each other and picked out a total of 677 genes, which were at least 2-fold up- or down-regulated between two samples with p-value smaller than 0.05. Comparing with the CK group, 245 and 183 unigenes were differently expressed for LT and HT, respectively. Overall, three of twelve unigenes involved in PSII (PsbO, PsbQ and PsbR) and five of eight in PSI (PsaD, PsaF, PsaG, PsaH, PsaK) showed down-regulation in K. alvarezii exposed to LT. Among thirteen differently expressed genes under HT, the majority (nine genes) were down-regulated. It is worth noting that the first four genes, including PsbA, PsbC, PsaB and atpF1A (alpha), showed up-regulation only under HT treatment. The cytochrome b6/f complex and photosynthetic electron transport, on the other hand, tended to maintain transcript levels independent of the low/high temperature treatment. In addition, a larger number of differently expressed genes, a greater degree of increase of K-bands and the rapid rise at the I-step were observed under HT as compared with LT. Accordingly it was concluded that HT caused greater damage. Inferences on photosynthesis or metabolism based on transcriptional analysis provide insights into how differential adaptations occurred under HT and LT. HT might evoke the absorption of light energy too much and cause photodamage. These results may also facilitate studies on identification of possible candidates useful for the development of new varieties and expansion breeding area. The second part of the thesis was characterising the Trentepohlia jolithus transcriptomics data. Algae in the order Trentepohliales have a broad geographic distribution and are generally characterized by the presence of abundant β-carotene. The many monographs published to date have mainly focused on their morphology, taxonomy, phylogeny, distribution and reproduction; molecular studies of this order are still rare. High-throughput RNA sequencing (RNA-Seq) technology provides a powerful and efficient method for transcript analysis and gene discovery in Trentepohlia jolithus. Illumina HiSeq™ 2000 sequencing generated 55,007,830 Illumina PE raw reads, which were assembled into 41,328 assembled unigenes. Based on NR annotation, 53.28% of the unigenes (22,018) could be assigned to gene ontology classes with 54 subcategories and 161,451 functional terms. A total of 26,217 (63.44%) assembled unigenes were mapped to 128 KEGG pathways. Furthermore, a set of 5,798 SSRs in 5,206 unigenes and 131,478 putative SNPs were identified. In our study, using BLAST against the KEGG database, most of the genes for the key enzymes related to the C3 (139 unigenes) and C4 (157 unigenes) pathways of carbon fixation were actively transcribed. We also successfully identified 99 unigenes encoding key enzymes involved in carotenogenesis in T. jolithus. Moreover, the fact that all of the C4 photosynthesis genes exist in T. jolithus suggests a complex carbon acquisition and fixation system. This effective mechanism could help T. jolithus cope with high light intensities, low temperatures and arid conditions. There were no identified unique sequences encoding mevalonate kinase (MVK, EC 2.7.1.36), which is required for the MVA pathway. In contrast, all the genes encoding enzymes involved in the MEP/DOXP pathway have been identified. Carotene β-ketolase (CrtO) was not detected in our study and if it is truly missing from the genome, its lack would hinder or preclude the synthesis of astaxanthin by this species. This is the first broad transcriptome survey for T. jolithus, increasing the amount of molecular data available for the class Ulvophyceae. As well as providing resources for functional genomics studies, the functional genes and putative pathways identified here will contribute to a better understanding of carbon fixation and fatty acid and carotenoid biosynthesis in T. jolithus.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22798
专题	海洋生物技术研发中心
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	李倩倩. 基于转录组测序的长心卡帕藻光合作用和乔利橘色藻类胡萝卜素合成关键基因分析[D]. 北京. 中国科学院大学,2015.

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