Analysis Of Key Genes Involved In Kappaphycus Alvarezii Photosynthesis And Trentepohlia Jolithus Carotenoids Synthetic Based On RNA-Seq

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.