Study On The Molecular Difference Of The Mantle Of Pacific Oyster Crassostrea Gigas

The Pacific oyster(Crassostrea gigas),is an oyster native to the Pacific coast of Asia,it belongs to the Mollusca,Bivalvia,Ostreoida.Ostreidae,Crassostrea,C.gigas.Oyster meat is delicious and nutritious.It can be processed into frozen oyster shell,oyster extract,oyster health products,oyster sauce and so on.The biggest feature of the oyster is that the left shell can firmly attach to the surface of body,while the right shell is unable.We infer that the left shell should have some special material that does not exist in the right shell.The oyster shell is formed by mantle biomineralization.The difference between the right and left shell also means the difference between the left and right mantle molecules.We focus on the molecular differences between mantles,attempt to find out the left shell attachment mechanism implemented by which special adhesive protein.Addationally,oyster has an early life stage as planktonic larvae before fixing on some place,its attachment is completed in water,which means the adhesive protein must play a key role in this process.This provides a theoretical support for the next step of separation and extraction of the protein,and also provides a way to increase the added value of oyster shell.The molecular mechanism of functional difference between left and right mantle is still not clear at present.In this study,a comparative analysis of the differences between the left and right oyster mantle protein,gene expression,gene methylation difference,by taking the intersection,screened and oyster attachment related genes,through analysis,that oyster left shell may have a close relationship with the attachment of TGF-beta signaling pathway;through the study of two kinds of potassium ion channel inhibitor TEA(voltage-gated potassium channel inhibitor)and glibenclamide(inward rectifier potassium channel inhibitors)effect on the attachment of oyster larvae,found that voltage-gated potassium channels attached to the oyster larvae may play a more important role;in addition,we cloned in the oyster mantle around in the expression of calmodulin difference protein gene(cgCaM),analyzes its characteristics and evolution characteristics of expression and functional significance.The results of this study provide some clues for the study of the symmetrical organ function differentiation of the symmetrical animals,and also provide important reference for the production of the oyster.Specific results are as follows:1.The extraction and determination of the protein and the right and left shell protein of the oyster were completed.The total proteins(including soluble and insoluble matrix proteins)were isolated and extracted.According to the completed oyster shell protein group,the left and right shells and the protein groups of the attachment spots were identified by comparative analysis.The 26 and 22 proteins were found in the left and right shells by comparing with the known proteins of the 248 oysters.Comparative analysis of the expression of the protein in the left and right shells,a total of 3 differentially expressed proteins were found.The left and right shell by using the difference of oyster shell protein has been found in the data for the protein expression,therefore,only in the 248 protein,comparison of peptides,found 6 common protein changes in protein data of left and right shell.Compared with the right shell,3 proteins(EGF,Hsp70,SOD)were found in the left shell of Crassostrea gigas.2.The oyster mantle mRNA expression was analyzed by RNA-seq comparative method,a total of 18457 genes with the GO annotation information about mantle tissue within the gene are more concentrated in the cell process,metabolic process,biological regulation,stress response and other biological process in left and right mantle.Comparison of about mantle gene FPKM and analysis of gene expression difference distribution showed that there are some differences related to cellular expression of oyster in protein around the mantle,the mantle around there are certain differences in membrane transport and guidance molecules related to cell adhesion and transcription process,which may lead to the appearance of and one of the reasons for the differences appear on both sides of the shell.Analysis of differentially expressed genes,in which there was a significant down-regulation of the expression of 5 peroxidase in the left mantle.The low expression of POD(peroxidase)in the left mantle may indicate that the right shell of the oyster plays an important role in the oxidative stress of non-specific immune defense.The differential expression of KCP and Temptin in the left and right mantle may be the key factors for the regulation of the left and right shells of Crassostrea gigas.3.Through the MethylRAD technology,the difference of genomic DNA methylation in the mantle of Crassostrea gigas was analyzed and compared.A total of 2286 and 2522 differentially expressed genes were found at the CCGG and CCWGG locus,respectivley.The differentially methylated genes shows that the related to possible neural signal transmission.transcription,adjusting RNA and protein are significant differences,the TGF-beta signaling pathway key genes(PITX2 and BMP4)are significant differences in methylation levels,proves that the mantle oyster may have developmental differences and resulting difference function and function on both sides.Integration of RNA-seq and methylation analysis results,which showed that many TGF-(transforming growth factor-beta beta beta,TGF-)expression of signaling pathway related genes,may indicate that although long oysters are bivalves,but in the long term natural selection,the asymmetric development around the mantle,and evolved a different function.Especially the left shell can be attached to the function.Through quantitative analysis,compared to the right set of membrane expression in oyster left in the mantle of CMP and upregulation of COL may explain the formation of structural differences in the mantle around.However,the down-regulation of PITX2 and KCP in the left mantle suggests that the difference in the development of asymmetric regulation of the left and right of the Crassostrea gigas may be the cause of the different shapes and functions of the left and right shells.4.Two potassium channel proteins were found in the differential transcriptome of the mantle.To identify which of them is more important for the determination of the adhesion of oyster.We finished the potassium ion concentration effects of attachment research and metamorphosis rate of oyster,exposure test through different potassium ion,the results showed relatively oyster larvae abnormal rate will decrease with the increase of potassium ion concentration.By using two kinds of potassium channel inhibitors(TEA voltage-gated potassium channel inhibitors)and glibenclamide(inward rectifier potassium channel inhibitor)effect of potassium channel on the attachment of oyster larvae,we found that the inhibitory effect of TEA on larval attachment is more obvious,that voltage-gated potassium channels attached to oyster larvae may play a more important role.5.The calcium regulatory protein gene was found in left and right mantle transcriptome gene difference analysis.To characterize sequence and functional characteristics of oyster calmodulin gene can help us understand the molecular mechanism of the formation of animal soft shell.In order to obtain specific information of oyster calmodulin,we cloned the oyster calmodulin gene(cgCaM)full-length cDNA sequence of the ORF encoding 113 amino acids,containing 3 EF-hand calcium binding domain,the expression level of the mRNA is the highest in the mantle,suggesting that cgCaM gene may play an important role in the formation of long oyster shell,phylogenetic analysis showed that the common ancestor of Gastropoda and Bivalvia may have at least 3 CaM gene.We also found that the degree of calcification in high species contain more EF-hand calcium binding domain,and participated in the comparison of several soft animal,oyster has the thick shell,oyster genome also contains the maximum number of EF-hand calcium binding domain,which further suggests that the link between CaM and biomineralization..