Preliminary Study Of The MicroRNA Transcriptomes In Two Generations Of Porphyra Haitanensis

Porphyra haitanensis is one of the most important marine crops in south China whichpossesses two heteromorphic life-history generations. Several studies have been carried on thegene different expressed of two generations and found that the expression of a majority of geneshave generation-relative. However, the knowledge about how these generation-relative genesexpression is regulated is rather limited so far. MicroRNAs (miRNAs) are a new class ofendogenous small RNAs that paly essential regulatory role in gene expression. So aiming atgaining insight into the characteristics and expression patterns of miRNAs and investigating itsroles in difference generations, next generation high-throughput sequencing technology was usedto large-scale identify miRNAs of P.haitanensis.In this study, the conchocelis and thallus of strain Z-61of P.haitanensis were used asexperimental materials. By high-throughput sequencing and bioinformatic analysis, miRNAs ofP. haitanensis were identified and different expressed profile of miRNA between the thallus andconchicelis generations was constructed. The main results are as follows:(1)5methods were used to isolating vthe total RNA from thallus of P.haitanensis whichcontained rich polysaccharides and polyphenol，and results showed that the total RNA whichisolated by CTAB method and spin column kit method had good quality and high purity, thesubstance which can restrain the active of reverse transcription enzyme also was cleared.(2) By Solexa sequencing, a total of1,927,426unique sRNA sequences from the thalluslibrary and1,903,844unique sRNA sequences from the conchocelis library were obtained.Among all the unique sRNA sequences, only9.68%sequences were common sequences ofthallus and conchocelis phases, and other sequence were all specific sequences of thallus orconchocelis.(3) After Blastern searches and further sequence analysis,588known miRNAs and40novelmiRNAs were identified. Among them,197known miRNAs were belonged to175miRNAfamilies, and all the miRNAs families had been shown to be conserved in a variety of species,such as P.haitanensis and Arabidopsis thaliana shared63conserved miRNAs families. ButP.haitanensis with Chlamydomonas Reinhardtii and Ectocarpus siliculosus only shared3and1conserved miRNAs families, respectively.(4) After convered the reads of each known miRNA into reads per million (rpm), the different expressed profile of known miRNAs between the two generations was constructed. Among allthe588known miRNAs,238known miRNAs were removed for their normalized expressionwas lower than1rpm either in thallus or in conchocelis, and the reminder350known miRNAscan be divided into three groups: one is that the normalized expressions of miRNAs (included113miRNAs) were significantly higher in the thallus than in conchocelis, the other is that thenormalized expressions of miRNAs (included186miRNAs) were significantly lower in thethallus than in conchocelis, another is that the normalized expressions of miRNAs (included51miRNAs,) were no significantly difference in the two generations, and each has61knownmiRNAs were expressed exclusively in thallus generation or in conchocelis generation.(5) To comfirm the expression difference of known miRNAs between the thallus andconchocelis,15known miRNAs were selected randomly to analyze by qRT-PCR. Resultsshowed that all the15known miRNAs had the same expressed pattern in the two generations asthat from sequencing data, but the relative expressed level of9known miRNAs had differencewith the sequencing data.(6) At last, a total of6752putative target sites of246differentially expressed miRNAs wereidentified by aligning miRNA sequences to ESTs and unigenes of Porphyra. Furthermore, all theobtained miRNA target sequences was annotated against the COG database and KEGG pathwayanalysis, the functional category are all relative with the metabolism of polysaccharides, aminoacid and nucleotide.