Molecular Cloning And Immune-related Research Of ZSeBP From Chlamys Farreri And G-type Lysozyme From Chlamys Farreri And Argopecten Irradians

Bay Scallop {Argopecten irradians) and Zhikong Scallop {Chlamys farreri) are the most important cultured marine mollusks in China. Since the summer in 1997, a large scale death of C. farreri scallop have been reported in coastal region of north China and have caused catastrophic losses to scallop aquaculture. Although it is still not very clear about the pathogens for the scallop mortality, the immune response of scallop to the pathogen infection plays a key role in the outbreak of disease. Understanding the immune defense mechanisms of scallop may contribute to develop strategies for management of the disease and for long-term sustainability of scallop or mollusk farming. However, most of knowledge on shellfish innate immunity is based on the biological activities, and the molecular features of immune effectors remain largely unknown. New methods need to be combined with the traditional way to outline the scallop defense system. Identification and cloning of the genes involved in scallop immune response will help us to understand the molecular basis of the innate immune response to the pathogen infection and environment stress.1. Molecular cloning and immune-related research of zSeBP from scallop Chlamys farreriSelenium binding proteins (SeBP) represent a family of proteins to be involved in many physiological processes. The cDNA of scallop SeBP (zSeBP) was cloned from Zhikong Scallop Chlamys farreri by expressed sequence tag technique (EST) and RACE techniques. The full length zSeBP cDNA was 1,664 bp, including a 5' untranslated region (UTR) of 33 bp, a 3' UTR of 191 bp, and an ORF of 1440 bp encoding a polypeptide of 480 ammo acids with an estimated molecular mass of 54 kDa and an estimated isoelectric point of 6.5. The deduced amino-acid sequence of zSeBP has a high similarity when aligned with the putative SeBP of the zebrafish Danio rerio (76% similarity; E=0.0), SeBP of the nematode Caenorhabditis elegans (72% similarity; E=e-143), the human SeBP (67% similarity; E=0.0), SeBP of themosquito Anopheles gambiae (65% similarity; E=e-138) and the fruit fly Drosophila melanogaster (68% similarity; E=e-141), and even with SeBP of plant and microorganism. Homology analysis revealed that zSeBP should be the member of SeBP family.The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after stimulated by oxidative stress or microbial challenge. The mRNA transcripts of zSeBP could be detected in all the blank, control and stimulated samples. The expression of zSeBP was up-regulated gradually after stimulation, and reached its maximum level at 6 or 8 hr timepoint, then dropped progressively to the original level. Seminar zSeBP expression profiles were observed between scallop with oxidative stress and infected one. Scallop infected with fungi Candida Lipolytica showed the most apparent elevation of zSeBP expression.Malondialdehyde (MDA), the product of lipid peroxidation, was measured by the colorimetric method. MDA content was higher in infected scallop comparing to control one. Interestingly the C. Lipolytica infected group appeared the modest MDA value.The results indicated that bacteria infection could incense the disorder of oxidation/reduction and result in the increase of MDA. zSeBP was similarly induced and regulated when facing oxidative stress and microbial challenge. Among infected groups, MDA values showed to be negatively related to the expression levels of zSeBP. The relevance of these findings was evaluated in terms of assistant functions of this protein in mediating the anti-oxidation mechanisms and immune response in marine invertebrates.2. Molecular cloning and phylogenetic analysis of a novel invertebrate g-type lysozyme from scallop Chlamys farreri and Argopecten irradiansLysozymes are considered to be essential in nonspecial immunology, and they also play a digestive role in the process of feeding on bacteria in the world of invertebrates. Expressed sequence tagging method was used to identify the partial sequences of lysozymes from Chlamys farreri and Argopecten irradians, then the full length of the g-type lysozyme cDNA was cloned through the approach of Anchored-PCR with the specific primers according to the sequence of the EST. The full length of g-type lysozyme cDNA from A. irradians was 659 bp and C. farreri 829 bp. They allencoded 200 amino acids by the 6O3bp coding sequence. BLAST analysis revealed that the g-type lysozyme gene from A. irrctdians and C. farreri shared high identity with the g-type lysozyme genes from vertebrate organisms in amino acids. Three catalytic residues (Glu 82, Asp 97, Asp 108) known conserved in g-type lysozyme were detected in the cDNA sequence from A. irradians and C. farreri. The results indicated that the cDNA sequence cloned from A. irradians and C. farreri was a member of g-type lysozyme family. In addition to this invertebrate g-type lysozyme, a number of protein sequences of various types were collected to construct a phylogenetic tree. The phylogenetic study indicated that g-type and c-type lysozymes co-existed in both vertebrate and invertebrate animals and they evolved from invertebrate to vertebrate in parallel routes. The results allowed us to extend the present analysis of the molecular evolution of the c-type, g-type and i-type lysozymes.