Identification Of Four Kinds Complement Component Genes And Expression Analysis In Large Yellow Croaker ( Larimichthys Crocea )

Large yellow croaker(Larimichthys crocea)is a famous economic native species in China Seas.During farming,various diseases can cause heavy losses in Large yellow croaker aquaculture every year.So studies on the immune factors affecting resistance to diseases were carried out to understand their relationship with disease resistance,which will provide the theoretical base for molecular breeding of Large yellow croaker.In this thesis,the Large yellow croaker complement C3,C4,C7 and C9 genes were identificated and characterized,the full length of complement component C3(L.c-C3),complement component C4(L.c-C4),complement component C7(L.c-C7)and complement component C9(L.c-C9)cDNAs were identificated,respectively.The expression of the mRNA was examined by FQRT-PCR.The study's main results were as follows:1.The large yellow croaker full genome sequencing sketches was used for four complement gene cDNAs sequences and related software were used to analyze them: L.crocea four kinds complement component are L.c-C3?L.c-C4?L.c-C7?L.c-C9.CDNA complete length is 4962bp(KJ544508)? 5088bp(KM514922)? 2496bp(KM514923)?1818bp(KM514924),respectively.Encoded amino acids respectively is 1653aa?1695aa?831aa?605aa?2.In this study,real time PCR method was used to investigate the four genes expression patterns in various tissues of healthy fish.The mRNA expressions of L.c-C3,L.c-C4,L.c-C7 and L.c-C9 were all expressed in wide range of adult tissues,with the highest level of expression of these genes detected in the liver and spleen.3.At different developmental stages,the highest expression levels of L.c-C3 gene was not found from fertilized egg to newborn larvae of the large yellow croaker,but L.c-C4 was significantly up-regulated.4.Four Large yellow croaker genes expression patterns in various tissues of healthy fish after challenge with Vibrio alginolyticus bacterium by Real time PCR method.The results showed that,in response to Vibrio alginolyticus injection,L.c-C3,L.c-C4,L.c-C7 and L.c-C9 genes expressions were significantly up-regulated in observed tissues.A.In liver,the expression level of L.c-C3 was high at the post-infection 48 h stage.It was significantly increased at post-infection 12 h,24h and at 48 h post-infection L.c-C3 was highest,then 72 h tend down.Taken together,our data provided a better understanding of the physiological function of complement C3 in vertebrates.In liver,the expression of L.c-C4 quantities wavy rising trends,which at the time of 12 h L.c-C4 expression quantity obviously improved but within 24 h,and fell back,48 h starting to L.c-C4 expression is significantly higher than control group(p<0.05);The highest of L.c-C4 express at 72 h.L.c-C4 express volume begins to shrink,but still significantly higher than control group(p<0.05).In liver,L.c-C7 expression quantity showed a trend of rising in the liver tissue,within 24 h after dissolving algal vibrio infections expression quantity and no significant change(P >0.05),but expressed in 48 hours when the amount has increased significantly(P< 0.05)in one step closer to raise 72 hours.In liver tissue,L.c-C9 expression after rising decreased trend.B.In the spleen,L.c-C3 expression are set to rise after the fall,at 12 h post infection quantity rises abruptly peak(p<0.01),then expressing quantity is lower but still significantly higher than the control group(p<0.05).In spleen tissues L.c-C4 express quantity showed a trend of decline after rising first,soluble v.infection after 0h-12 h.C4express quantity has no obvious change(P>0.05)at 24 h was significantly higher than that of control group(P <0.05),the peak at 48 h,72 h is down but still significantly higher than control group(P < 0.05);In the spleen tissues L.c-C7 expression quantity showed a trend of rising gradually,by 12 h after dissolving infect soluble v.,L.c-C7 expression in spleen tissue volume was significantly higher than control group(P < 0.05);In spleen L.c-C9 expression quantity tissues has a downward trend after rising first,peaked at 24 hours a day.Bacterial challenge experiments showed that the levels of L.c-C3,L.c-C4,L.c-C7 and L.c-C9 genes expression were up-regulated in the liver and spleen of adult large yellow croaker.The results showed that L.c-C3,L.c-C4,L.c-C7 and L.c-C9 genes expression were influenced by bacterial stress and that L.c-C3,L.c-C4,L.c-C7 and L.c-C9 genes may play an important role in immunity mechanisms.This study will further our understanding of the function of L.c-C3,L.c-C4,L.c-C7 and L.c-C9 genes and the molecular mechanism of innate immunity in teleosts.