Effects Of Ca²⁺ Regulation On Pif Expression And Pearl Formation

China is the main producer of freshwater pearls in the world,accounting for more than 95%of the world’s freshwater pearls,but due to the low ratio of high-quality pearls,the output value is less than 20%of the total global pearls.There are many factors affecting pearl quality,among which calcium ion is one of the environmental limiting factors affecting pearl quality.Therefore,exploring the Ca²⁺concentration suitable for freshwater pearl clam growth and pearl cultivation in water environment is of great significance to understand pearl formation,especially high-quality pearl formation.In our research,taking the high-quality freshwater pearl mussel Hyriopsis schlegelii as the research object.The biomineralization-related gene--Pinctada fucata aragonite-inducing factor（Pif）,which is involved in the formation of nacre,was identified and cloned.With the concentration of Ca²⁺as the key regulatory factor,the amount of nacre deposition,the state of pearl layer formation and the expression level of related genes Pif were determined,and the data obtained were compared and analyzed comprehensively.The Ca²⁺concentration in water with better pearl deposition effect and the nuclear insertion method with better pearl breeding effect were selected,which can provide a basis for improving the quality of pearls.In our research,the full-length c DNA sequence of Pif gene was obtained from Hyriopsis schlegelii for the first time by RACE-PCR technique,named HsPif,which is 3457 b P in length and 3072 b P in open reading frame（ORF）and encodes a total of1023 amino acids.It is speculated that there are two proteins coding Hyriopsis schlegelii Pif97 and Hyriopsis schlegelii Pif80（hereinafter referred to as HsPif97 and HsPif80）.Through bioinformatics analysis,it was found that the molecular weight of HsPif protein was 110.484k D and its isoelectric point was 4.67.it contained a von-Willebrand factor type A domain（VWA）and three chitin-binding domains（Cht BD2）.The results of amino acid composition showed that the content of aspartic acid was the highest and the content of histidine was the lowest;the prediction result of protein secondary structure was consistent with that of tertiary protein structure.Phylogenetic tree analysis showed that HsPif and Hyriopsis cumingii Pif were the most conservative in one branch.The results of tissue fluorescence quantitative PCR analysis showed that HsPif gene was mainly expressed in the mantle（P<0.05）,and in situ hybridization showed that the hybridization reaction mainly occurred in the mantle epithelial cells.In order to explore the role of Pif in the process of pearl sac formation and the early stage of pearl layer,and the effects of three kinds of nucleation methods on pearl formation,we cultured pearls with nucleation.After nucleation surgery,the expression level of Pif gene was detected quantitatively,and the pearls were collected,the deposition amount of pearls was measured and compared.The three methods of nuclear insertion are respectively:the donor mussel and the host mussel are both pool Hyriopsis shlegelii（SS）;the donor mussel is Hyriopsis cumingii,and the host mussel is Hyriopsis schlegelii（SC）.Both donor and host mussel were of Hyriopsis cumingii（CC）.The results showed that the expression level of Pif varied with the development of pearl sac in three different nuclear insertion methods,and the expression level of Pif decreased significantly on the seventh day after nuclear implantation.When the development of pearl sac was completed,the expression level of Pif stably secreted nacre was stably or significantly increased.The measured data of pearls show that the deposition weight and growth thickness of SS and CC pearls are significantly different from those of SC pearls,but there is no significant difference between SS and CC,indicating that the nacre can be better deposited by adapting to each other between the recipient clam and the donor mussel.In order to explore the most suitable concentration of Ca²⁺for nacre deposition in water environment and the relationship between Pif and the formation of pearl layer,three kinds of nuclear-inserted pearl mussels were cultured in water environment with different concentrations of Ca²⁺,and the expression level of Pif in pearl sac and the concentration of Ca²⁺in pearl sac tissue were detected.Then their pearls were collected,the deposition amount of pearl and the formation state of pearl layer were measured,and the different concentrations were compared.The results showed that with the change of culture time,the change of Ca²⁺concentration in pearl sac of three kinds of clam cultured with different Ca²⁺concentration was almost the same,showing a trend of decreasing at first,then increasing,and finally decreasing,SS was cultured in the water environment with Ca²⁺concentration of 0m M,0.5m M,1.25m M,2m M and 3m M,the Pif expression level on the 7th,15th,30th and 60th day was always lower than that on the 0th day.In the culture of SC with five Ca²⁺concentrations,the expression level of Pif on the 7th day,15th day,30th day and 60th day was always lower than that on the 0th day,while in CC,the expression level of Pif on the 7th day decreased significantly or had no change.Then,the analysis of the expression level of Pif and calcium concentration in pearl sac of three kinds of mussels with different concentrations at the same time showed that the high expression of Pif and the higher calcium concentration in pearl sac mostly appeared in1.25m M,2m M and 3m M.The above results show that when the concentration of Ca²⁺in the culture environment is 1.25m M,2m M and 3m M,the concentration of Ca²⁺in the pearl sac increases（P<0.05）,which may increase the deposition rate of pearl.Based on the statistical analysis of the mortality,pearl deposition and crystal scanning images of cultured mussel,it was found that the survival rate of three kinds of mussel was higher in the treatment group with calcium concentration of 1.25m M and 2m M,and the survival rate of SS and CC was 100%in the treatment group of1.25m M and 2m M,the survival rate of SS and CC was 90%and 95%,respectively.The statistical analysis of the pearl deposition amount of three kinds of clam showed that the prevalence rate of pearl deposition rate in the treatment group with Ca²⁺concentration of 1.25m M and 2m M was significantly higher than that in 0m M,0.5m M and 3m M treatment groups（P<0.05）.Compared with the pictures of the surface of the pearl layer scanned by electron microscope,it was found that the regular hexagonal orderly stacking of calcium carbonate crystals appeared in the group of 1.25m M and 2m M calcium ion concentration,but there were some differences in their crystal morphology and density.The above results show that when the concentration of Ca²⁺in the environment is 1.25m M and 2m M,the pearl cultivation performance of the three kinds of mussel is better.