Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei.pdf
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1、Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei IV Originality Statement The present dissertation is output of my research results, obtained by myself under guidance of project director. To the best of my knowledge, the thesis does not contai
2、n any text written by others or materials used for degree, certificate by other educational organizations, unless properly referenced and acknowledged. Signature of Candidate: Date: Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei V Ontogenesi
3、s of main organs and cloning and expression of two immune- related genes in Litopenaeus vannamei Abstract White leg shrimp Litopenaeus vannamei is a prime aquaculture species, and it covers 90% of western hemisphere culture and is also extensively farmed in Asia. The estimated annual production from
4、 Asia during 2002 was 316000 mt. In China, exotic species (L. vannamei) had introduced during 1988 for experimental purposes and by 1998 is being successfully cultured in commercial scale; over 1000 hatcheries are in operation. The 300000 tons and 210000 tons harvested during 2003 in marine and fres
5、hwater farming respectively. To give more understanding for the white leg shrimp, we have investigated the genesis of important systems and organ, including digestive system, nervous system and haematopoetic tissue, furthermore dealed with two important immune related genes. Our investigation on ont
6、ogenesis of digestive system in Litopenaeus vannamei, revealed that in non-feeding stages the yolk mass is the indigenous source of food and until the fifth nauplius (N5) stage retained in foregut cavity, absorption is gradual in nauplius (N) stages while disappearance occurs at N6 stage. The anteri
7、or midgut caecum generates at N3 and it reaches maximum size in Z3; thereafter in successive stages it reduces in size and becomes a single structure in post- larval stage (P3-P4), named the anterior diverticulum. The lateral midgut caecum first appears at N6 stage and lengthens along both sides of
8、the midgut and then ramifies to form lobes as larval development. Finally, these lobes become a compact structure, the hepatopancreas. The midgut lumen is quite apparent in N stages and only becomes wider in successive stages; it is without a cuticle lining and there is no difference among all inves
9、tigated stages. The foregut in N stages is a simple tube and pyloric and cardiac chamber Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei VI occurs in the third mysis (M3) stage. In the cardiac chamber of post- larval stages, folds were noted
10、and number of that increased gradually. The gastric mill is absent in all investigated larval stages (P1-P10). The pyloric chamber is smaller than the cardiac chamber and possesses a gland filter, which first appears in M3 stage and forms a more complete shape in forthcoming stages. The hindgut glan
11、d first appears in P2 and in successive stages it shows more protuberance and complexity. The hindgut or rectum is a simple tube. Cells of the digestive system in all feeding stages show the presence of acid mucins and general protein, while the inner margins of the foregut and connective tissues of
12、 the gland filter are positive for collegen. The nervous system plays pivotal role in co-ordination of body activities and coping with the task of controlling the behavioral activities during larval development. In present study, we give a basic description in the early stage morphology of nervous s
13、ystem in L. vannamei by classical histological techniques. Results revealed that neuropil first emerged in N1 stage and the front part of nervous system started getting structural shape in N3 where the ganglions were coalesced in front part of head. The complete structure of central nervous system i
14、ncluding proto-, deuto- and tritocerebrum was observed in N6 stage. The structural expansion of nervous system in successive stages was continued and protocerebrum differentiated into anterior and posterior median protocerebrum at Z3 stage. The optic ganglions, sub-esophageal ganglions and pleon gan
15、glions were distinct in zoea stage. Furthermore, we discuss the relativity between the development of olfactory lobes and the voluntary movement of larva. It is believed that haematopoietic tissue (HPT) is the source of haemocytes production in crustaceans and defensive role of haemocytes is progres
16、sing but investigations on structure of HPT are limited hence this study has been designed in which we investigated the genesis and morphology of haematopoietic tissue (HPT) and types of haemocyte based on its morphology and cytochemical feature using histological and histochemistical methods. The r
17、esults revealed that HPT in L. vannamei is not visible histologically in mysis and early two post larvae (P1 the obtained results are as below respectively. Cyclophilins are ubiquitous proteins with an enzymatic activity of peptidyl-prolyl cis-trans isomerase (PPlase), which play important roles in
18、a variety of stress responses. In present investigation a Cyclophilin A gene has been cloned from white leg shrimp (Litopenaeus vannamei) by rapid amplification of cDNA (RACE) and anchored PCR method. The full length of the shrimp L. vannamei CypA cDNA (LvCypA) is 855 bp, containing 495 bp of open r
19、eading frame (ORF), encoding 164 amino acids with an estimated molecular mass of 17.6 kDa. The LvCypA has high homology with that of other known organisms and it has four - strands. The real time PCR analysis revealed that its expression is ubiquitous and expression level is higher in muscle, gill,
20、lymphoid organ and hepatopancrease respectively. The tissues challenged with Vibrio angullarium revealed that in hepatopancrease it was the lowest at 6 h and highest in 48 h. The results revealed that the LvCypA might involve in potential role of innate immunity. The proliferating cell nuclear antig
21、en (PCNA) has important role in nucleic acid metabolism and its immune responses have also been reported. In present Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei VIII investigation the PCNA was cloned from L. vannamei (LvPCNA). The full le
22、ngth cDNA sequence of LvPCNA encodes 261 amino acids showing high identity with PCNAs reported in other species. Tissue distribution of the LvPCNA revealed that its expression is obvious in gill and muscle. In order to understand the response of bacterial challenge, mRNA level of LvPCNA in hepatopan
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