| During the cultivation of non-nucleated pearl,both the donor and the host mussel will have an impact on the quality of pearls,and the growth performance of the host mussel after graft operation will also be different.Therefore,in order to explore the difference and correlation on the growth traits of combinations between Hyriopsis cumingii(S)and Cristaria plicata(Z),as well as the difference in pearl production traits of non-nucleated pearl,H.cumingii(S)and C.plicata(Z)were used as both the donor and the host mussel at the same time,and 4 host mussel combinations were obtained.The body weight,shell length,shell height and shell width of each combination were measured,and the four combinations were compared with the control group.The difference in growth traits of the control group and 4 host mussel combinations was analyzed statistically.The quality and quantity of pearls produced by different combinations were measured,and the differences of pearls among different combinations were analyzed.Bone morphogenetic protein(BMP)family plays an important role in bone development,cell proliferation and differentiation,tissue and organogenesis,and is related to growth and development and biomineralization in mollusks.In this experiment,BMP2 and BMP10 genes were screened from the transcriptome of the pearl sac in H.cumingii,and full length of c DNA sequence was obtained by cloning.The function of bone morphogenetic protein gene was preliminary explored through experiments such as sequence analysis,fluorescence quantification,in situ hybridization and RNAi.1.Comparison and analysis of growth performance and pearl production traits of H.cumingii and C.plicata after graft operationGraft operation affects the growth performance of host mussels,the combined growth traits of host mussels of H.cumingii are better than the control group,while the growth of the combination of C.plicata is inhibited.1 year after inserting,the difference was large 1 years after inserting,and the difference was reduced 2 years after inserting.There is a very significant correlation between the growth traits of each combination(P<0.01).Shell length and shell width were the main factors affecting the body weight of H.cumingii,however,there were differences between the H.cumingii pearl rearing group and the control group.The morphological traits of each combination of C.plicata were shell length,but the indirect influencing factors of different combinations were different.The pearl formation percentage of S-S and Z-Z was extremely high,while the pearl formation percentage of heterogeneous pearl mussel inserts combinations was low.The pearls of S-S combination were the roundest,but the pearl size is not the best in each group.The pearls of Z-S combination have a low pearling rate,and the pearls are not round but the pearls produced were larger.The shell length of the S-S group was significantly correlated with the pearl size.The larger the shell length,the larger the pearl,and a significant negative correlation with the roundness of the pearl.The larger the shell length,the rounder the pearl.Body weight is significantly related to pearl size.The higher the body weight,the larger the pearl.The above results indicate that the graft operation changes the growth performance of host mussuls.H.cumingii grows better than the control group after inserting,while the growth performance of C.plicata after inserting is slower than the control group.The pearl production trait of S-S was the best.2.Identification and functional analysis of BMP2 gene in H.cumingiiIn this experiment,BMP2 was isolated and identified from the mantle of H.cumingii.Sequence analysis showed the full length of c DNA sequence is 2889 bp,of which 5’-UTR is 688 bp,3’-UTR is 998 bp,and the open reading frame(ORF)is1203bp,encoding 400 amino acids.BMP2 protein has no transmembrane structure,but the protein has a signal peptide(1-23aa),a TGF-β propeptide domain(29-259aa)and a typical TGF-β domain(299-400aa).TGF-β domain contains 7 cysteine residues and its structure is highly conserved.The gill and adductor muscle expression were highest,followed by the mantle central;The juvenile mussel was highest during the embryonic development stages,and the glochidium and blastocyst stages were the second;In the mantle of different ages of H.cumingii,the expression of BMP2 gene showed a trend of up-regulation with age;The expression of BMP2 gene rised after the shell was broken and reached the highest level after 7 days of shell broken.BMP2 played an important role in the formation of the shell;During the early stage of pearl formation(0h-35d),BMP2 gene was highly expressed at 28 d after insertion.BMP2 participated in the formation of pearls and played an important role in the formation of the nacre.The expression trend of BMP2 in the pearl sac and gills was relatively consistent.It was speculated that there was a certain correlation between the role of BMP2 gene in the gill and the pearl sac during the pearl formation process,which might be related to calcium metabolism;The ISH results showed that BMP2 was strongly expressed in the outer epithelial cells of mantle outer fold and crust groove between out fold and middle folda.The RNA interference results showed that the interference rate of the gills after the injection was 85.7%,and the interference rate of the mantle was 47.8%.The SEM observation result indicaded that the morphology of the new shell prism and nacre changed,and BMP2 was related to the prism and nacre.The above results indicate that BMP2 gene may be involved in the growth and development and play an important role in the biomineralization of H.cumingii.In addition,BMP2 may be also related to the calcium ion absorption and metabolism of the gills.3.Identification and expression analysis of BMP10 gene in H.cumingiiIn this experiment,the full length of BMP10 c DNA sequence is 2477 bp,including 5’-UTR of 471 bp,3’-UTR of 623 bp,and open reading frame(ORF)of1383bp,which encodes 460 amino acids.Hc-BMP10 have no transmembrane structure,its N-terminal contains a signal peptide(1-25aa).The secondary structure of the protein includes a TGF-β propeptide domain(104-300aa)and TGF-β domain(358-460aa).The TGF-β domain contains 7 cysteine residues.BMP10 was expressed highest in the gills,the membrane central is second;The highest expression is the cleavage stage during the embryonic development stages,followed by the juvenile stage;In the mantle of H.cumingii,the expression of BMP10 gene decreases with age;The expression of BMP10 gene increases at 24h-48 h after breaking the shell,which may be related to shell formation;During the early stage of pearl formation(0h-35d),BMP10 gene is highly expressed at 14d-35 d after insertion.BMP10 participates in the formation of pearls and plays an important role in the formation of the nacre;The result of ISH showed that a strong signal was detected in the epidermal cells on the membrane edge and BMP10 was related to the shell prism layer.The above results indicate that the BMP10 gene plays an important role in the growth development and biomineralization of H.cumingii. |
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