Study On The Sequencing And Analysis Of The Transcriptome Of Early-stage Embryos And The Cloning And Expression Of POU3Gene From Shrimp Marsupenaeus Japonicus

The shrimp Marsupenaeus japonicus, also known as Kuruma prawn, is one of theeconomically important shrimp species of marine aquaculture, which has relatively higherdisease-resistance than other penaeus shrimp species, thus widely farmed by aquaculturists.However, the genomic and transcriptomic information as well as the genetics backgroundof M. japonicus are lacking, and works on the genetics breeding are not well developed,and the isolation of functional genes and the molecular breeding techniques are limited.Therefore, in the present study, the main purposes are to analyze the transcriptome ofearly-stage embryos of M. japonicus using the new generation of high-throughputsequencing technique of Illumina, and to find out the functional genes involved in thegrowth, reproduction, metabolism and disease resistance of M. japonicus through theassembly of high quality sequences, gene annotation and classification. And also, we wouldclone the open reading frame (ORF) of the transcription factor gene of POU III fromM. japonicus (i.e. Mj-POU3) and analyze its tissue-specific expression profile by RT-PCR.The obtained results of the transcriptome analysis indicated that,(1) there are108,320,302clean reads were obtained, and67183transcripts and46661unigenes aresuccessfully joined and assembled. The average length for these transcripts is about1216bp, and for the unigenes is910bp. All the obtained unigenes are blasted in the public databanks of NT, NR, SwissProt and PFAM, then classified by GO and KOG annotation, andthe interactions among unigenes and the biochemical metabolic parthways involved areinvestigated by KEGG pathway mapping;(2) Among the annotated genes,52growthfactors and their receptors, and11genes from MAPK and TGFβ signal transductionpathways are identified. A serial of candidate genes involved in the growth, reproduction, metabolism and immunization of shrimp are also identified, providing valuable geneticresource for gene function study and molecular breeding practice.The transcription factor of Mj-POU3, a member of POU gene family, plays a key role inthe regulation of growth and development by transcriptional activation of DNA.（1）In thisstudy, the ORF of Mj-POU3is successfully isolated from the early-stage embryos of M.japonicus. It is1194bp in size, encoding397amino acids. The amino sequences ofMj-POU3is very conservative with two functional domains of POU-S (POU specific) andPOU-H (POU homologous)，and Mj-POU3protein share more than62%homology withinvertebrate and over55%homology with vertebrate, indentifying that Mj-POU3was amember of the POU family.（2）The same size of POU3gene (1194bp) was amplifiedfrom the genomic DNA of the early-stage embryos of M. japonicus, suggesting thatMj-POU3gene have no introns（.3）The mRNA expression profile of Mj-POU3in differenttissues of the early-stage embryo, muscle and ovary were also examined by RT-PCR. It wasfound that, the highest expression level of Mj-POU3were detected in the ovary tissues,then the embryo, but not in the muscle, inferring that Mj-POU3plays an important role inthe development of embryos and oocytes.