Molecular Cloning, Expression And Immune Function Of Vitellogenin In Scallop Patinopecten Yessoensis

Vitellogenin (Vg), the major precursor of the egg-yolk proteins, is firstdiscovered in Cecropia moth, which has been shown to be present in all metazoanranging from the placozoa to the bilateria. The primary function of Vg is to form yolkprotein which provides nutrients for the developing embryos and larvae. However,many other biological functions, such as temporal division of labor and foragingspecialization, regulation of hormonal dynamics and change in gustatoryresponsiveness were also reported. In particular, Vg has recently been reported topossess a novel function linking with host immune defense in fish. However, if Vgfrom invertebrate species is also an immune-competent molecule remains open,although amphioxus Vg has been shown to have hemagglutinating and antibacterialactivities. The Japanese scallop, Patinopecten yessoensis (Bivalve: Pectinidae), is animportant marine economic bivalve, which was introduced into China in the1980s,and has now become one of the most popular farming mollusk in China.However, theindustry has been facing the challenge of massive death of adult scallop and earlydeath of larvae in recent years. The body’s immune defense of P. yessoensis mainlyrely on nonspecific immunity, so studies on immune factor and mechanism of P.yessoensis have important scientific significance. Study as such will certainly providenew insights into understanding on the biological role of Vg in molluscs.This study focuses mainly on the Vg immune in P. yessoensis. First of all, wecloned the full-length cDNA of Vg gene in P. yessoensis (termed PyVg), and studiedthe expression patterns in tissues and different development stage, and after bacterialvibrio anguillarum challenge; And then, successfully expressed PyVg domainsDUF1943and VWD in vitro useing prokaryotic expression system and studied theircombination ability to bacteria and pathogen-associated molecular patterns (PAMPs)lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Finally, isolated, purified thenative Vg protein from ovary of P. yessoensis and analysed its bacteriostasis.The PyVg gene cDNA sequence was cloned by using homology-based cloning strategy and rapid-amplification of cDNA ends (RACE) technology. The cDNAcontained an open reading frame (ORF) of6888bp, encoding a polypeptide of2295amino acid protein, which had an N-terminal signal peptide followed by the matureVg. The mature Vg had the domains Vitellogenin_N, DUF143(domain of unknownfunction) and VWD (von Willebrand factor type D domain) as well as the consensuscleavage site (R-X-R/K-R) and conserved motif "KTIGNAG" and "CGLC" that areconservative in invertebrates and vertebrates. The PyVg amino acid sequence showedhigh similarity to other close relatives of scallops, such as Chlamys Farreri and C.nobilis, and also similarity to other mollusk, fish. The high similarity areas weremainly located in the N-terminal region, followed by C-terminal region.According to the results of semi-quantitative RT-PCR, PyVg mainly expressd inovary and hepatopancreas of female P. yessoensis, with stronger expression in theovary, and no expression in other organizations of female and male individual. Resultsof qRT-PCR showed that the expression of PyVg was the minimum in May, graduallyincreased from August, accumulated in high speed in October, sharply rised to thepeak in February next year, then came a fall in March and April. Its expression profilein ovary well reflects the annual cycle of vitellogenesis. qRT-PCR was employed todetect the response model of PyVg to V. anguillarum stimulation, the results showedthat after stimulation, the amount of Vg expression in ovarian tissue gradually rised,expression levels already had significant differences with the control group in4h, theamount continues to increase in8h, and reached the peak in12h, more than70timeswith the control group. After that, expression fell sharply, and fell to the control levelat48h. This means that bacterial challenge caused a significant change in PyVgexpression, hinting an involvement of PyVg in the acute phase response in P.yessoensis.Using prokaryotic expression system, the domains DUF1943and VWD weresuccessfully recombinanted and expressed from PyVg gene. This two recombinantproteins’abilities of bacteriostatic action and combination with bacteria, LPS and LPAwere studied. The results showed that the recombinant proteins were able to combinewith gram-positive bacteria S. aureus and gram-negative bacteria E. coli and V.anguillarum, also combine with LTA and LPS on their wall. But those proteins couldnot inhibit the growth of bacteria. Therefore, the domains DUF1943and VWD inPyVg may play an important role of immunologic function as pattern recognitionreceptors.Native PyVg was purified from P. yessoensis ovary during the proliferation period by using the methods of DEAE-32anion exchange column chromatographyand Sephacryl S-100glucan gel filtration chromatography. The bacteriostatic activityon gram-positive bacteria Staphylococcus aureus and gram-negative bacteriumEscherichia coli, V. anguillarum of PyVg were studied. The results showed that thepurified native PyVg displayed a broad-spectrum antibacterial activity against bothGram-negative (E. coli and V. anguillarum) and positive bacteria (S. aureus).This is the first report on the immune-relevant activity of Vg from molluscs,opening a new angle to understand the roles of Vg and its derived yolk proteins.The second part of the research is about the maternal immunity of C-type lectinin scallop P. yessoensis. Maternal transfer of immune factors from mother to eggs hasbeen reported in many aquatic species, and these factors played important immuneroles in developing embryos and larvae. However, the study on maternal immunity ofshellfish remains lacking. In this study, maternal transfer and bacteriostasis of aC-type lectin in P. yessoensis were analyzed. We detected the mRNA expressionpattern of the C-type lectin gene in ovary and early developing larvae using thereal-time quantitative PCR method, and tested the antibacterial activity of maternalC-type lectin in eggs by colony-forming unit (CFU) assay. The results of qRT-PCRshowed that the expression of C-type lectin gene in ovary after bacterial challengewas time-dependent, it was up-regulated gradually from2h after injection of bacteriaand reached the peak at8h which was6.2times compared to the control group, andthen dropped progressively to the original level. On the other hand, the fact thatmRNA was found both in the eggs and larvae indicated that it could be transferredfrom mother to offspring. The mRNA expression in bacterial challenge group wassignificantly higher than those in normal group except for the36h point. The CFUassay revealed that the cytoplasm of eggs with protein concentration of200μg/ml and400μg/ml had lethal effect on the bacteria Vibrio anguillarum, and the functionweakened significantly once the antibody of C-type lectin was added, which indicatedthe C-type lectin from mother had antibacterial activity. It is concluded that thematernal C-type lectin can transfer from mother to the offspring and has antibacterialactivity.