Cloning And Molecular Characterization Expression Of Gene Related To Reproductive Development From The Chinese Mitten Crab,eriocheir Sinensis

Ontigenesis of germ cells is an important subject in developmental biology, and a complex developmental regulation and activity of multiple genes involved in the process of time and space. The study of primordial germ cell origin, migration,differentiation and other aspects of research are the focus of reproductive biology research direction. Unfortunately, compared with other models organisms, Decapodas research about the molecular mechanism of reproductive are relatively weaker.Therefore, identifying and discovering plenty of reproductive-related genes the crustacean resources, has great significance and potential application value. Regarding Eriocheir sinensis as the research object, this thesis studied on the vasa, piwi, nanos,PL10 gene which are closely related with reproduction and development of Eriocheir sinensis through reviewing relevant literature. This study is divided into four parts as follow:1、Expression analysis and expression patterns of vasa gene of Eriocheir sinensisThe vasa gene is one of the main components of germ plasm, and specifically expressed in the gonads, often as a molecular marker tracer gonad formation. This research use Real-time quantitative PCR technology to study vasa gene expression in Eriocheir sinensis embryonic and larval stages of development. Research showed that vasa m RNA expression levels during embryonic development in the larval stage of development relative expression level is higher, suggesting that vasa m RNA probably played a major role in embryonic development. Results of in situ hybridization in testis and ovaries showed that the vasa m RNA was distributed in chromatin of spermatocyte and in nuclear cup of sperm cells as well as in cytoplasm of the oocyte. Whole-mount in situ hybridization showed that vasa m RNA expression during embryonic and larval development are concentrated, from which we can predicted an initial trajectory of Eriocheir sinensis gonads.2、Cloning and characterization expression of piwi gene of Eriocheir sinensisThe piwi gene has the functions of maintaining germline stem cell and regulating cell division in Drosophila melanogaster. However, research in piwi genetic of Crustaceans is very little. In the study we first cloned Eriocheir sinensis piwi full length c DNA sequence by using the rapid amplification of c DNA end(RACE) method.Analysis of the nucleotide sequence revealed that the c DNA of piwi comprises 3253bp(Gen Bank registration number: KR061909) consists of 26 bp 5 ’UTR, 179 bp 3’ UTR,2868 bp open reading frame, encoding 955 amino acids. Multiple sequence alignment and homology analysis showed that Eriocheir sinensis piwi gene and Portunus trituberculatus piwi gene had 70% homology. Transmembrane domain analysis showed that the PIWI protein is hydrophilic structure. Real-time quantitative PCR analysis showed that piwi m RNA were expressed in blood, muscles, heart, thoracicganglia, gill,hepatopancrea, intestine, ovary, testis. However, the expression in gonads was significantly higher than that of other tissues. Especially, with the change of testis development, the amount of expression is gradually decreased and the amount of expression became the highest in spermatocytes period; the amount of piwi m RNA expression in the ovary also fluctuated with the development of ovaries and became the highest in the period of the oocytes; in the embryonic and larval development phase, the amount of piwi m RNA expression became the highest in megalopa. Results of in situ hybridization in testis and ovaries showed that piwi m RNA in spermatocyte distribution in chromatin, mainly distributed in nuclear cup in the sperm cells, located in the cytoplasm in the oocyte. This study found the whole-mount in situ hybridization to piwi m RNA positive indicator of gonad primordium in the heart period was obvious. In M.zoea I period, it was distributed in head cuirass and abdominal junction. As development was focused on the back of the head cuirass, in M. zoea II period, it had migrated to head cuirass. In M. zoea III period it was more concentrated, completely in an area which was located in the back of the head cuirass.3、Cloning and characterization expression of nanos gene of Eriocheir sinensisNanos m RNA and vasa m RNA is the most important components of P grainules in the original composition of gonad. This study first cloned Eriocheir sinensis nanos full length c DNA sequence by using the rapid amplification of c DNA end(RACE) method.Analysis of the nucleotide sequence revealed the c DNA of nanos comprises 2886 bp(Gen Bank registration number: KR061910) consists of 59 bp 5 ’UTR, 1543 bp 3’ UTR,1284 bp open reading frame, encoding 427 amino acids. Transmembrane domain analysis showed that the NANOS protein is hydrophilic structure. Real-time PCR analysis showed that nanos m RNA were expressed in blood, muscle, heart, thoracic ganglia, gills, hepatopancrea, intestine, ovary, testis. nanos m RNA expressed the lowest in the blood, the highest in gonads, higher levels in testis and ovary. Ovary nanos gene in ovarian development of large growth(logarithmic growth phase) peaked early, with the maturing ovarian development, there is a tendency to flatten decline; in the testis during development, in spermatocytes expression of July highest, gradually reduced to a sharp rise again early spermatogenesis, decreased rapidly after the formation of sperm.In the embryonic and larval development period, the expression was the highest in the larval stage and megalopa period, extremely significant different with the other phases.Results of testis and ovaries in situ hybridization showed that the nanos m RNA in spermatocyte distribution in chromatin in the sperm cells was mainly distributed in the cup nuclear, positioning in the cytoplasm in the oocyte. This study also found that the whole-mount in situ hybridization to nanos m RNA positive indicator of gonad primordium upfront was more noticeable in the membrane.In M. zoea stage I nanos positive signals in head cuirass and abdominal junction, as development was focused on the back of the head cuirass. To M. zoea II period when it was fully distributed in head cuirass, M. zoea III period was more concentrated, completely in an area which is located in the back of the head cuirass.4、Cloning and characterization expression of PL10 gene of Eriocheir sinensisPL10 gene is the ancestral of vasa gene.In the study the cloning PL10 gene c DNA is1671 bp(Genbank registration number: KR061911),encoding 566 amino acids. The deduced amino acid sequence of PL10 has high identity with the reported PL10 sequences of other species in the Gen Bank,sharing 85%, 84%, 84% indentity with Exopalaemonmodestus,Macrobranchium nipponense,Fenneropenaeu schinensis respectively, Phylogenetic analysis proves that PL10 gene is highly conserved in evolution.Real-time quantitative PCR analysis showed that PL10 m RNA are expressed in all tissues, but the expression of differences, blood, thoracicganglia, express low levels of gill tissue,hepatopancrea,testis and ovary express in the high levels. Each developmental stage of Eriocheir sinensis in the gonad PL10 m RNA showed that reached a peak period of sperm formation in December.PL10 gene involved in the onsetof sperm; In ovarian PL10 m RNA expression in the early stage of the logarithmic growth have edged up, logarithmic phase after a downward trend. Experiments show that PL10 gene expression in the embryos was higher than the larval stage.This study found that in embryonic development periods and m. zoea stage, the vasa and nanos have similar expression patterns, which can prove that their functions be same in the process; PL10 gene expression in the early stages of embryonic development of quantity is higher than the vasa and nanos, which showed that Description PL10 functions were not consistent with the vasa and nanos. Through the above research and analysis, the access to reproductive-related genes piwi, nanos and PL10 c DNA sequences, enriched the Eriocheir sinensis genetic resources for solving breed process of precocious puberty, and also provided basic data for research on individuals which have unified specification. Compared with other model organisms,the molecular mechanism of crustaceans reproductive physiology research is relatively weaker. The experiment of Eriocheir sinensis reproductive related gene research may provide theoretical basis for Eriocheir sinensis genetic breeding improvement.