The Global Transcriptomeof Pyropia Haitanensis Anddigital Gene Expression Profileanalysis Under High-temperature Stress

Pyropia haitanensis is an economically important marine crop in southern China. Over the past several years, high temperatures that have been associated with global warming, markedly affected the cultivation of P. haitanensis and reduced its yield remarkably. Although several high temperature-tolerance strains of P.haitanensis have been selected, the knowledge of the mechanisms of response to high temperature stress is rather limited so far. Studing and revealing the molecular mechanisms of high temperature stress tolerance are great significance for guidance the stress-resistant varieties breeding in P. haitanensis.In this study, the whole transcriptome of P. haitanensis was first sequenced by Solexa technology. Then, the gene expression pro?ling of P. haitanensis under high temperature(29℃) for 0h, 3h, 6h and 12 h were analyzed by digital gene expression(DGE) technology, and based on the data of DGE, a number of candidate genes related with high-temperature tolerance were selected. The main results are as follows:1. High-throughput sequencing was first used to analyze the global transcriptome of P. haitanensis. Approximately 103 million 90 bp paired-end reads were generated using an Illumina HiSeq 2000. De novo assembly with paired-end information yielded 24,575 unigenes with an average length of 645 bp. Based on sequence similarity searches with known proteins, a total of 16,377(66.64%) genes were identified. Of these annotated unigenes, 5,471 and 9,168 unigenes were assigned to gene ontology and clusters of orthologous groups, respectively. Searching against the KEGG database indicated that 12,167(49.51%)unigenes mapped to 124 KEGG pathways. And from the database of these unigenes, a lot of heat shock protein genes and antioxidant system-related genes were selected.2. DGE technology was used to analyze the gene expression pro?ling of P. haitanensis under high temperature(29℃) for 0h, 3h, 6h and 12 h, respectively. Compared with control(0h), the expressed levels of 2,076, 1,927 and 1,298 unigenes changed significantly under high temperature stress for 3h, 6h and 12 h, respectively. Gene function classification results show that these differentially expressed genes involved in protein biosynthesis and degradation, carbon metabolism, photosynthesis and stress response. Pathway significant enrichment analysis found that the expressed levels of genes related with protein biosynthesis, photosynthesis and carbon fixation pathway were first down-regulated and increased at later times; Correspondingly, the expressed levels of heat shock protein family genes were first up-regulated and then diminished. So the molecular process of P. haitanensis response to high temperature stress was: in the early period of high temperature stress, the rates of photosynthesis, protein synthesis, glucose metabolism, amino acid metabolism in P. haitanensis were slowed down due to the inhibition of functional protein activity; Then the physiological state stimulated the expression of heat shock protein to stabilize the activity of functional protein and raise the expression levels of genes related with each metabolic pathway; At last, the expressed levels of heat shock protein were down-regulated accordingly.These results suggest that heat shock protein genes play important roles in high temperature tolerance of P. haitanensis.3. Eleven heat shock protein genes(one HSP100, two HSP90, five HSP70, one HSP60, one HSP40 and one sHSP)were cloned by rapid amplification of cDNA ends(RACE)technology. Sequence and expressed level analysis during high temperature stress were also carried out on all of these genes. The gene expression analysis showed that the expressed levels of almost all of the heat shock proteins were up-regulated under high temperature stress early, and then diminished. Thereinto, the changed of sHSP was the most significant. These results were accorded with the DGE.4. To select an appropriate internal control gene for gene expression studies in P.haitanensis, the absolute expression abundance of six housekeeping genes(18S, UBC, ACT, TUB, EF2, and GAPDH)were examined by qRT-PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative CT method and by two different software packages: geNorm and NormFinder. The results of the three means are concordant, the most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, we propose that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC.These results made the foundations for the study of functional genomics and molecular mechanism of high temperature tolerance in P. haitanensis.