Expression And Functional Analysis To The Gene Col-119 Of Caenorhabditis Elegans

As an important structure protein,collagens play an important role in Caenorhabditis elegans development.Collagens are conserved in multicellutar organisms.Celegans has a big collagen family,composed of 165 members.Collagens represent the main structure of C.elegans curticle.Depended on the conserved sequence,the collagens were divided into 5-6 groups,col-119 belongs to Group 3 and study had showed that col-119 had function in fat metabolism.In this study,multiple sequence analysis of 5 groups of worm collagen sequences and 4 human collagen sequences were made through DNAMAN software,finding it had high similarity among group 3 members.And outside group it had higher similarity with Group dpy-2 than Group dpy-7?Group 2 and Group 1.It also had 39.42%similarity with human collagen COL1A1.Then we made a NJ phylogenetic tree by MEAG 4.0,by which we obtained the same resultSecondly,we studied the nematode temporal expression model in different development stage,including egg,L3,L4 and adult,finding it highly expressed in egg and adult stage,and had a significant difference with the larval stage(p<0.01).There also had a significant difference between egg and adult(p<0.05).Thirdly,RNA interference(RNAi)experiment was carried out to inhibite col-119 expression.qRT-PCR tested the effect of RNAi,and result showed the low expression of experimental group compared with control group.There was no difference on brood number between experimental group(264±10)and control group(256±10).But we observed the significant decrease in hatch rate,experimental group(72.2±1%)compared with control group(91.3±1%).Some abnormal eggs during the RNAi experiment appear,sepecialy in the first two days.The abnormal eggs has a round or irregpLar appearance compared with the oval normal egg;however,it didn's had an effect in nematode life span;Red oil O stained nematode and fat-reporter transgene nematode(dhs-3::GFP::N2)was used to validate the fat content,lipid droplet size,shape and density change.We got a decrease result in those parameters.We caculateed the fat content by ImageJ software,got the same resultIn the end,we constructed a reporter vector,confused col-119 upstream 3Kb sequence with pPD95.75-GFP vector.Confused vector and marker vector lin-44::GFP were injected into worms together,showing that GFP express in whole body cuticle,especially in the adult,and it's hard to find GFP signal in larval stages.The GFP express model conformed to the one temporal expression.In conclusion,col-119 was highly expressed in egg and adult stage,and expressed in the whole body.It had an effect in reproduction,embryo development and fat metabolism,but short life span,variation morphology and locomotivity didn't appear in this study,speculating that col-119 may be involved in egg and adult cuticle construction.In this study,we made a comprehensive study in temporal expression model?reproduction?development?fat metabolism and locomotivity,perfecting col-119 gene's expression and function analysis,and had guidance and reference significance.Meanwhile,it had high homologous with human collagens,thus make it a good model to study human collagen disease and forming different collagen mutation model.