| Bangiaceae is one of the representatives of red algae species, including the important economic peices Pyropia haitanensis and Bangia fuscopurpurea. Py. haitanensis were cultured widely along the coasts of South China, accounting for 75% of the total cultivation of Pyropia in our country, and the B. fuscopurpurea contains a high percent of highly unsaturated fatty acids EPA, which can up to about 50% of its total fatty acid content. At the same time, the two species were located in intertidal rangions, so they are also considered to be research models for studying the molecular mechanisms for different stress tolerance and the physiological ecology. At present, extensive research on cytology, genetics and so on have been performed in these two species. However, genome, functional genome information and related relations still ambiguous and the molecular investigations of their adaption to the intertidal adversity is still lack systematic study.In this paper, based on the research of the Py.haitanensis genome and functional genome, we have understanded the characteristics of its genomic structure. And the the transcriptome of B.fuscopurpurea demonstrate the EPA synthesis pathway. The complete plastid genome makes us understand the phylogenetic relationships and divergent time of Rhodophyra.We used the NGS sequencing technology combined with bioinformatics method to analyze the genome characteristics of Py. haitanensis, and using single molecular real time sequencing technology to do a comprehensive and systematic study of transcriptomic characteristics of Py.haitanensis. We also analyze the transcriptome of B. fuscopurpurea and sequence the complete plastid genome of B. fuscopurpurea using next-generation sequencing technology and Sanger sequencing The major findings are as follows:1. The genome characteristics of Py. haitanensisThis study obtained high-quality nuclei from Py. haitanensis and then estimated the size of nuclear genome by using flow cytometry. The experimental results show the nuclear genome size was 117.84±11.22 Mb. Using next-generation sequencing technology combined with bioinformatics methods, we get rid of the high copies of organelles DNA and exogenous microorganisms DNA.The sequences were assembled into cotings with N50 of 10.527bp, N90 of 2,625bp.The total assembly reached to 29.8Mb. The average GC content is 65.85%. The repetitive sequence of Py.haitanensis includes 971 reverse transcription transposons,360 DNA transposons. 11,253 SSR were received, including 144 kinds of motifs. The composition of the repetitive sequence showed that Py. haitanensis prefers high GC.2. Transcriptomic characteristics of Py.haitanensis based on single molecular real time sequencing technologySingle molecular real time sequencing technology was used to analyze the comprehensive transcriptome of Py. haitanensis. A total of 123.645 full length reads were generated from three differert size cDNA library constructed for 12 different RNA of Py.hatanensis at different stages and different stress treatments. And then clustering these reads, we obtained 17.383 high quality consensus isoforms with the average length of 1.339 bp.The average depth of sequencing-12 fold. For the gene content, lots of candidate genes related to resistance were founded.The resluts show 12 germin-like proteins clustered together as a seperated clade, belong to different clusters with the existing five kinds of germin-like proteins and germin proteins, it may exist the similar response mechanism of drought in higher plants. Also we found that it may be exsited a xanthophyll cycle pathway responsing to the excess irradiation of high luminous intensity. In addtion,9,1940 alternative splicing location and 3,274 alternative splices were predicted based on genome sequence. Aamong these,we found four types of alternative splicing factor (SC35.RS40, RS31 and RS2Z33) and predited their possible variable isoformes, which can increase the complexity of the transcript and help Py.haitanensis to adapt to environmental stress and adversity.3. Aanlysis of transcriptome of B.fuscopurpureaIn order to study the characteristics of B. fuscopurpurea. the cDNA library was constructed for mixed samples from different stages and different stress conditions and then sequenced by Hieseq2000 sequencing platform. Aotal of 71,069,330 raw reads were obtained, the total base is about 7.11 G, the average GC content is 63.99%. Then clean data were assembled into 41.935 unique contigs, average length of 533 bp, N50 of 645 bp. There are many EPA synthesis pathways in B. fuscopurpurea:a classic n-6 pathway, a classic n-3 pathway and the connected pathways of n-3 and n-6, the other pathyway. This may explain the reason of high content of the EPA. The MAPK pathways of animal P38 lightning, JNK and type of ERK5 MAPK signaling system and the types of yeast were all exist in B. fitscopurpurea which may respond to a variety of stress factor, and play an important role in adapting the harsh environment of intertidal zone.The transcrptome of B, fitscopurpurea help us build the EPA synthesis pathway and lay he foundation for the lipid synthesis.4. Aanlysis of complete plastid genome of B. fuscopurpureaThe B.fuscopurpurea plastome was a typical circular DNA molecule of 196,913bp in size. Overall, the GC content was 33.5%. The quadripartite structure consisted of a 151 Kb LSC region and 36 Kb SSC regions, separated by two direct, non-identical repeats. The two repeats encode ribosomal 16s rRNA,23s rRNA,5s rRNA, and two tRNA genes (trnI,trnA). The plastid genome included 207 protein-coding genes,37 tRNAs and 6 rRNAs, which represented 73.01%,2.07% and 4.88% of the total sequence, respectively. The gene-coding regions occupied 79.96% of the entire genome with 14 cases of overlapping genes (psbC-psbD, atpD-atpF, ycf24-ycf16, rps19-rpl2, rpl23-rpl4, carA-ORF238 and rpl24-rpl14). These features demonstrate that it is a high capacity, compact genome. We estimated divergence times within the Bangiales species using the plastome consensus sequences. The time constraints were chosen according to the divergence time between P.yezoensis and Graclilaria tenuistipitata (about 900-1000 Ma) which was calculated by eight fossils. Our results showed that B. fuscopurpurea diverged from Pyropia ～435 Ma and Porphyra ～361 Ma. Within the genus Pyropia, the plastid DNA of six species shared a common ancestor around 287 Ma. |
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