Expression Characteristics Of IGFALS Gene Of Sinonovacula Constricta And The Effect Of IGFs System On The Growth And Larval Metamorphosis

Sinonovacula constricta is one of the main economically cultured shellfish in China.It has a short breeding cycle and a fast growth rate.At present,traditional breeding and family selection techniques are mainly used in the selection of good breeds of clam,and research on growth-related genes can provide a certain theoretical reference for further accelerating the breeding process.Insulin-like growth factor(IGF)signaling pathways are involved in regulating physiological processes such as animal development,growth,reproduction,and longevity.This study supplemented the genetic composition and expression characteristics of the clam IGF system through molecular biology techniques,and explored the effect of the IGF system on the growth of larvae by using exogenous insulin.The effects of the IGF system on the growth of juveniles and their effects on larval survival and metamorphosis were investigated using receptor inhibitors.The experimental results are as follows:1.For the first time,two IGFALS genes in the IGF system were identified,named IGFALSa and IGFALSb,respectively.The c DNA sequence of the IGFALSa gene is4809 bp,the length of the ORF is 2991 bp,which encodes 996 amino acids;the c DNA sequence of the IGFALSb gene is 1925 bp,and the length of the ORF is 1641 bp,which encodes 546 amino acids.Both IGFALS genes have signal peptides,do not belong to transmembrane proteins,and both have rich leucine enrichment motifs.The expression patterns of IGFALSa and IGFALSb in different developmental stages and different tissues of the clam were analyzed by fluorescence quantitative technology.The results showed that the expression of both genes increased with the development of larvae,and the expression of IGFALSa was the highest in the stage of Creeping larvae.IGFALSb was highest expressed in the stage of double pipe juvenile.Two IGFALS genes arewidely expressed in various tissues,among which IGFALSa is highly expressed in mantle and gonad tissues,and relatively low in hepatopancreas,hemolymph and other tissues.IGFALSb is mainly expressed in the siphon,and is also expressed in a small amount in the mantle,gonadal,and foot tissues,and only in trace amounts in the hepatopancreas,gills,and hemolymph.2.Using different concentrations of insulin to stimulate juvenile clam,it was found that when the insulin concentration was 0mg / L-10 mg / L,the expression levels of IGFALSa,IGFALSb and their downstream genes IRS increased with the increase of insulin concentration,and the expression level was highest at 10 mg / L.When the concentration reached 20 mg / L,the expression levels of all three genes began to decrease.In addition,the effects of the IGF system on the MAPK pathway and the PI3 K pathway were analyzed.When the insulin concentration reached 10 mg / L,the expression of MPKP1 and PDK1 also increased significantly,and PDK1 fluctuated with the concentration.3.Stimulate juvenile clam with 10 mg / L insulin to investigate the effect of different time of insulin on the expression pattern of genes related to IGF pathway.The results showed that the expression patterns of IGFALSa and IGFALSb were similar.The expression increased with time,and the expression was highest at 8h.As for the downstream genes IRS of the two IGFALS genes,the expression level always increased with the increase of stimulation time,reaching the highest at 72 h.Long-term feeding of juvenile with 10 mg / L insulin showed that the daily growth rate of juveniles in the insulin-fed group was higher than that in the control group.The average daily growth rates of shell length in each group were 4% in the insulin group,3.28% in the blank group,and 3.11% in the Tris-HCL group.The average daily growth rates of shell width in each group were 1.73% in the insulin group,1.34% in the blank group,and 1.33% in the Tris-HCL group.4.The receptor inhibitors in the IGF system were used to stimulate the juvenile clams to further study the effects of the IGF system on growth.Using inhibitors to stimulate juveniles for a long time,the survival rates of each group were 88.42%(blank group),88.5%(DMSO group)and 70.60%(inhibitor group).In addition,the daily growth rate of juveniles in the inhibitor group was significantly lower than that in the control group.The daily growth rates of shell length were 3.28%(blank group),3.30%(DMSO group)and 1.55%(inhibitor group).The daily growth rates were 1.34%(blankgroup),1.41%(DMSO group)and 0.76%(inhibitor group).5.Using different concentrations of inhibitors to stimulate the development of clam larvae,it was found that the inhibitors could affect the survival rate of larvae,and greatly inhibit the metamorphic process of larvae.For the three metamorphosis periods,the increase in inhibitor concentration will significantly reduce the larval metamorphosis rate,and for the two periods of Trochophore-Veliger and Veliger-Umbo larvae,with the inhibition Increasing the concentration of the agent gradually reduced the larval metamorphosis rate,and the lowest rate was when the inhibitor concentration was 1 mg / L.For Umbo larvae-Creeping larvae,the effects of inhibitor concentrations of 0.1 mg / L and 0.5 mg / L on larval metamorphosis rate were not significantly different,while the concentration of 1 mg / L significantly reduced the metamorphosis rate.