| Shellfish shell color often exhibits polymorphism,and shell color can be used as a marker color for excellent trait and used in the breeding of improved shellfish species.At present,the research reports on Cyclina sinensis mainly focus on its biological characteristics,morphological structure,reproductive physiology and seedling cultivation.However,there are no reports on the causes and molecular mechanism of shell color formation of C.sinensis and the growth and nutritional differences between purple and white clams.Therefore,this study initially explored the cause of the purple shell of C.sinensis,and learned that the pigment mainly exists in the pearl layer of the purple shell,and carotenoids may be the main component of the pigment in the purple shell.The total carotenoid content in the purple shell is selectively deposited at the outer edge of the mantle,and there is a certain positive linkage between the individual of C.sinensis purple shell and the content of carotenoids;Through the determination of growth indexes and nutritional components between purple and white shell individuals of C.sinensis,the relationship between shell color traits and their growth and nutrition was explored.It was found that there were certain differences in growth and nutrition between purple and white shell individuals,and there was a certain positive correlation between purple shell traits and their growth and nutrition;The full-length c DNA sequence of clam shell color related genes was cloned and identified by race technology,and the tissue distribution of gene expression was analyzed by q RT-PCR technology.It was understood that the related genes may play an important role in the selective transport of carotenoids in the mantle.The specific contents of this study are as follows:（i）In order to explore the cause of the formation of clam purple shell,in this study,the distribution of purple pigment in clam shell was determined by stereoscope,the content of metal elements was measured by inductively coupled plasma atomic emission spectrometer,the Raman spectra of pigments in clam shells were obtained by Raman spectroscopy,the distribution of carotenoids in different tissues was calculated by extracting total carotenoids from different tissues of clam,and a series of experiments to explore the physical and chemical properties of pigments in shell.The results showed that compared with white shells,purple and white areas appeared alternately in purple shells,and purple pigment mainly existed in the nacre at the junction of nacre and prismatic layer of shells;the contents of iron and manganese in purple and white shells were different,but the difference was not statistically significant;the strong peaks of carotenoids(1510-1513,1125-1128,1014-1016,1297-1299 cm-1)appear in the Raman spectrum of the purple part of the purple shell,while no carotenoid peaks are observed in the purple part of the purple shell and the white part of the white shell.Carotenoids may be the main component of the purple pigment in the shell;the total carotenoid content in purple shell was higher than that in white shell.The total carotenoid content in the outer of mantle of purple shell clam was about 4times that in the inner of mantle and 3 times that in the outer of mantle of white shell clam,but the difference was not statistically significant（P>0.05）.Carotenoids were deposited at high levels in the outer margin of the mantle,pipe,foot,and visceral mass.The levels of lycopene,lutein,zeaxanthin,andβ-carotene in both the outer margin of the mantle and shell from purple-shell clams were significantly higher than those in white-shell clams（P<0.01）;the purple pigment in the shell has no obvious connection with the physical structure of the shell.The purple pigment in the shell can be destroyed by strong acid solution and is insoluble in water.The physical and chemical properties of purple pigment are highly consistent with those of carotenoids.The results show that carotenoids may be the main component of purple pigment in the shell,and total carotenoids are selectively transported in the outer membrane and outer edge of the shell.（ii）In order to explore the linkage relationship between shell-color trait of clam C.sinensis and its growth and nutritional components,we measured and analyzed the growth indicators and nutritional components of individuals with different shell colors.The results showed that the wet weight,shell length,wet weight of pipes and gills of the purple-shell individuals were significantly higher than those of the white-shell individuals（P<0.05）,and shell width and wet weight of mantle tissue were extremely significant higher than those of white-shell individuals（P<0.01）.The content of crude fat and crude protein of the edible parts from purple-shell individuals were extremely significant higher than those of the white-shell individuals（P<0.01）.The ratios of essential amino acids to total amino acids in purple-shell and white-shell individuals were 37.46%and 37.02%,respectively,and this ratio of purple-shell individuals was significantly higher than that of white-shell individuals（P<0.05）;the ratios of essential amino acids to non-essential amino acids were 67.72%and 66.60%,and this ratio of purple-shell individuals was significantly higher than that of white-shell individuals（P<0.05）,indicating that the amino acid balance effect in the purple-shell individuals was better than that of white-shell individuals,and they were all high-quality proteins.The monounsaturated fatty acids of purple-and white-shell individuals are mainly palmitic acid,accounting for 9.97%and 9.85%of the total fatty acids,respectively;polyunsaturated fatty acids mainly consist of EPA and DHA,of which EPA accounts for 6.64%and 6.54%of the total fatty acids in purple-and white-shell individuals respectively,and DHA accounts for 8.00%and 8.51%of the total fatty acids respectively.The results showed that there were some differences in growth and nutrition between purple shell and white shell individuals,and there was a positive correlation between purple shell traits and their growth and nutrition,which provided a theoretical basis for using purple shell traits as genetic markers for the breeding of improved C.sinensis.（ⅲ）In this study,the full-length c DNA sequence of clam shell color related gene low density lipoprotein receptor gene（ldlr）was successfully cloned and identified by race technology,and the tissue distribution of ldlr gene expression was analyzed by q RT-PCR.The results showed that the full-length c DNA sequence of ldlr gene was 961 bp,including open reading frame was666 bp and encoding 221 amino acids.The results of phylogenetic tree showed that clam and oyster had a relatively close evolutionary relationship,indicating that their evolutionary status was the most similar.q RT-PCR results showed that the m RNA encoded by ldlr gene was highly expressed at the outer edge of the mantle of C.sinensis.The results show that carotenoids may be selectively transported in the mantle of C.sinensis,and ldlr gene may play an important role in the selective transport of carotenoids in the mantle,but further functional research is needed,which will help to clarify the formation mechanism of C.sinensis shell color. |
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