Transcriptome Analysis And Reproduction Related Genes Cloning, Expression Pattern In Chinese Mitten Crab

The mitten crab (Eriocheir sinensis) (Henri Milne Edwards 1854), which belongs to Crustacea, Decapoda, Grapsidae, Eriocheir, is a unique species of crab that is native to China. This species is a traditional savory food and therefore comprises one of the economical aquatic species of China. The farming of E. sinensis has been developing rapidly in China over the last two decades and reached its highest yield of 4.0 x 105 tonnes in 2005. With the development of intensive culture, various diseases caused by bacteria, viruses, or rickettsia-like organisms and the sexual precocity, individual small, germplasm degradation are frequent in cultured species populations and have caused catastrophic losses. It has become a major bottleneck in the development of aquaculture industry. Understanding the immune defence mechanisms of the species; characterizing of immune effectors, and the study of reproductive biology, especially the molecular mechanism of reproductive are believed to be helpful in health management and disease control in crab aquaculture, and provide a theoretical to the reproductive control of the artificial crab breeding process.In the present study, we presented the methodology and results of transcriptome analysis, targeting immune-related genes and reproduction-related genes in the Chinese mitten crab and conducted experiments to further clone the reproduction-related gene by reverse transcript-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods from Chinese mitten crab, compare its sequence with other related proteins. We evaluated the expression of the reproduction-related gene in various tissues by Real-time quantitative reverse transcription-PCR (qRT-PCR), to explore the regulation of reproductive function of these genes during the development. The study is to provide a basis for Chinese mitten crab reproductive mechanisms information, but also provide an important reference for further research on the mechanism of the male reproductive in Chinese mitten crabs. The main results are as follows:1 Due to its popularity as a traditional food, intensive harvesting of the mitten crab (Eriocheir sinensis) is common, and has lead to an increase in disease incidence, resulting in catastrophic losses to crab aquaculture. The hepatopancreas of E. sinensis is not only an important digestive organ but also an indispensable immune organ. We constructed a non-normalized cDNA library from the hepatopancreas of E. sinensis and acquired 3,297 high-quality expressed sequence tags representing 1,178 unigenes. More than half of these unigenes were novel genes for this species; the remaining had homologs in public databases, which is of great importance for future functional research. We also investigated the association of these genes with immune processes for insight into one of the main functions of the hepatopancreas besides metabolism. Despite the relatively low sampling scalar of our cDNA library, we were able to demonstrate several important properties of the hepatopancreatic transcriptome and identified numerous genes that were closely associated with immune responses. These results might serve as the basis for an in-depth genomics study of E. sinensis, including transcriptome analysis, physical mapping, and whole genome sequencing.2 Beads with Oligo(dT) are used to isolate poly(A) mRNA after total RNA is collected from accessory sex gland of the Chinese mitten crab. Fragmentation buffer is added for interrupting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer is used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments are connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeqTM 2000. Transcriptome de novo assembly is carried out with short reads assembling program-SOAPdenovo. A total of 33,221,284 read including total nucleotides 2,657,702,720 nt, which generated 91.06% successful reads. Of these, the average G+C content was 55.19%. These reads were assembled by software consisting of 85,913 contigs, of which 40,977 (47.70%) contigs with the mass of lengths ranging from 100-200nt length. The total length of all the contig is 18,750,722. Functional annotation by Unigene, of which 1713 genes annotated to Article General function prediction only, comment on the Translation, ribosomal structure and biogenesis were 952 genes, associated with the Transcription has 869 Unigene. About Posttranslational modification, protein turnover, chaperones, and Replication, recombination and repair genes were 802 and 789. KEGG is deciphering the genome's database. The accessory sex gland of Chinese mitten crab with 1704 genes (14.99%) in Metabolic pathways, 675 genes involved in Regulation of actin cytoskeleton. Spliceosome has 564 (4.96%) genes involved. Get the original small RNA of fq data, to its connection to low-quality reads, to deal with pollution, access to clean sequence 7939380. Statistics of small RNA (sRNA) species and quantity, and the length distribution of small RNA to do statistics, in general, the length of the small RNA interval 18-30nt, through the length distribution of the peak we can determine the type of small RNA, miRNA concentrated in 21 or 22nt, siRNA concentration in the 24nt, piRNA concentrated in the 30nt. Total Total sRNAs are 7939380, of which miRNA are 382,650 (4.82%), rRNA with 214,013 (2.70%), snRNA with 2573 (0.03%), snoRNA with 563 (0.01%), tRNA with 432,171 (5.44%), unann with 6,907,410 (87.00%). Total Unique sRNAs are 2268484, of which miRNA are 23,021 (1.01%), rRNA with 33,764 (1.49%), snRNA with 1003 (0.04%), snoRNA with 224 (0.01%), tRNA with 29,425 (1.30%), unann with 2,181,047 (96.15%).3 Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals. In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab(Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5'-untranslated region (5'UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3'UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs. We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.4 Ecdysteroid causes various developmental events associated with larval-larval ecdysis. Ecdysteroid-regulated gene could be a putative element of the metamorphic ecdysone response cascade of crustacean. The ecdysteroid-regulated genes mRNA level correlates well with the haemolymph ecdysteroid titre during the molting process. A full-length cDNA of ecdysteroid-regulated gene from Eriocheir sinensis was cloned by the rapid amplification of cDNA ends (RACE), with primers that designed based on the EST sequence of the cDNA library of hepatopancreas from E. sinensis. The cDNA is 1384 bp in size and consists of a 117-bp 5'-untranslated region (UTR), a 306-bp open reading frame (ORF) and an 841-bp 3'-UTR. The genomic DNA is 3059-bp containing two exons interrupted by one intron. Multiple alignments revealed that a conserved Niemann-Pick type C2 domain was identified in the deduced amino acids sequence of ecdysteroid-regulated gene. The mRNA expression of ecdysteroid-regulated gene in ten tissues and the temporal expression in hepatopancreas of four phases were measured by real-time RT-PCR. Ecdysteroid-regulated gene mRNA transcripts could be detected in all examined tissues, and are higher expressed in hepatopancreas than that in other nine tissues (cranial ganglia, P<0.05; the rest eight tissues, P<0.01). Among the four phases, the gene expression at pre-molt is the highest, down-regulated during inter-molt, up-regulated at post-molt, and continues to increase in hard-shell phase. The mRNA expression pattern of ecdysteroid-regulated gene confirmed that the hepatopancreas is a sensitive indicator for ecdysis phase in various crustaceans, and ecdysteroid-regulated gene would be involved in the process of crustacean molting. Therefore, this study by E. sinensis Ecdysteroid-regulated gene cloning and expression analysis, contribute to clarify the mechanism of molting occurs, and may be helpful for the prevention of related problems.