Identification And Functional Analysis Of Carotenoid Metabolism Related Genes HcStAR-like And HcLPCAT1 In Hyriopsis Cumingii

Since the 1980 s,Hyriopsis cumingii has made a breakthrough in artificial propagation.Due to its high quality of cultured pearls,it has gradually developed into the most widely used freshwater pearl mussel in China.At the same time,China’s freshwater pearl production has also become the highest in the world.However,the quality of freshwater pearls is far behind that of seawater pearls.As an important standard to evaluate the quality of pearls,color is the most intuitive impression of consumers on pearls,which largely determines the market value of pearls.Seawater pearls are rich in color,and black pearls and golden pearls have become market leaders.However,freshwater pearls are mainly white and relatively single in color.In 2017,our laboratory successfully cultivated a new variety "Shen Zi 1" of H.cumingii,realizing the mass production of freshwater purple pearls and enriching the variety of freshwater pearls.Existing studies showed that carotenoids affect the color of pearls,the white,golden and purple varieties(strains)of H.cumingii constructed in the laboratory were used as the research objects,Steroidogenic acute regulatory protein-like and lysophosphaticly lcholine acyltransferase 1 related to carotenoid metabolism were screened.The effects of these two genes on carotenoid metabolism and the color formation of shell and pearl of H.cumingii were studied.The main results were as follows:1.Identification of a HcStAR-like gene in freshwater mussel H.cumingii and its function in carotenoid metabolism and pearl color formation The accumulation of carotenoids in the mantle affects the level of carotenoids in the pearl organic matrix,which influences pearl color.The Steroidogenic Acute Regulatory Protein(St AR)is one key gene in the accumulation of carotenoids.The full-length c DNA sequence of HcStAR-like isolated from H.cumingii contains 994 nucleotides and the open reading frame(ORF)encodes 307 amino acid residues.Gene expression analysis showed that the expression level of HcStAR-like was highest in all tissues of golden(G-)mussels and significantly different from that in white(W-)mussels and purple(P-)mussels(P<0.05).The expression level of HcStAR-like in P-mussels was higher than that in W-mussels,especially the difference was significant in the hepatopancreas(P<0.05).The situ hybridization analysis showed that the positive signal of HcStAR-like located at the outer fold of mantle and the dorsal mantle.After ds RNA interference in G-mussels,and the interference rate reached 79.89%(P<0.05),and,the total carotenoid content(TCC)in RNAi group was significantly lower than that in PBS group and GFP group(P<0.05).2.Identification of a HcLPCAT1 gene in freshwater mussel H.cumingii and its function in carotenoid metabolism and pearl color formation Lysophosphaticly lcholine acyltransferase 1(HcLPCAT1)is an important lipid metabolic enzyme.In order to clarify the function of LPCAT1 gene in carotenoid metabolism of H.cumingii,and to explore the HcLPCAT1 gene correlation with the shell nacre colour of H.cumingii.The full-length c DNA sequence of HcLPCAT1 isolated from H.cumingii contains 1675 nucleotides and the open reading frame(ORF)encodes 431 amino acid residues.HcLPCAT1 belongs to the LPLAT family and has its typical domain.It is predicted that there is a transmembrane domain in its protein but had no signal peptide.Gene expression analysis showed that the expression level of HcLPCAT1 gene was the lowest in all tissues of G-mussel and significantly different from that of W-mussel and P-mussel(P<0.05).The expression of HcLPCAT1 gene in hepatopancreas and foot was significantly different among the W-,G-and P-mussels(P<0.05),and there was no significant difference between the remaining tissues.The expression of HcLPCAT1 in the tissues of P-mussel was higher than that of the corresponding tissues of W-mussel.The situ hybridization analysis showed that the positive signal of HcLPCAT1 located at the outer fold of mantle,dorsal mantle,ventral mantle,the joint of outer fold and middle fold of mantle and part of middle fold.After supplementation with β-carotene,TCC in hepatopancreas,middle mantle and fringe mantle of purple mussel increased extremely significantly(P<0.01),and the expression of HcLPCAT1 gene in hepatopancreas,middle mantle and fringe mantle of purple mussel increased extremely significantly(P<0.01).3.SNP screening of lysophosphaticly lcholine acyltransferase 1 HcLPCAT1 gene and its association analysis with shell color traits in H.cumingii According to the full length of the cloned c DNA of HcLPCAT1,eight SNP sites were screened out in the exon of HcLPCAT1 gene.One of them was moderately polymorphic(0.25<PIC<0.5),and the remaining seven were low polymorphic(PIC<0.25).Six SNP sites were found to be significantly correlated with these parameters L*,a*,b* and d E*.A126 G was moderately polymorphic,and the rest were low polymorphic.H2-1 and H2-2 haplotypes did not appear in the golden population,and rarely appeared in the white population.The frequency of H2-1 and H2-2 haplotypes in the purple population was the highest,which was significantly different from the other two populations(P<0.05),it can be used for breeding of purple population.The frequency of H1-3 and H2-3 was the highest in the golden population,which was significantly higher(P<0.05)than that in the white and purple populations.The frequency of H1-2 and H2-4 in white population was significantly higher than that in purple population and golden population(P<0.05),which could be used as the dominant haplotype of white population.The results showed that HcLPCAT1 gene was involved in the formation of inner shell color of H.cumingii,and could be used as a candidate gene in the process of shell color improvement and breeding of H.cumingii.