| Sinonovacula constricta is one of the most economiclly important shellfish in China,it's necessary to study the function of growth related gene,which may help to lay the foundation in molecular breeding.This study obtained a partial sequence from the transcriptome library of S.constricta,cloned the full length EGFR cDNA by RACE and found a alternative splicingin coding sequence.The author compared the expression difference and proliferating ability between two isoforms,and study the function of EGFR in the progress of body growth.1.cDNA clone and analysis of Sc-EGFRsThe full-length Sc-EGFR cDNA sequence was obtained from S.constricta and two splice isoforms named Sc-EGFR-1a(Accession number: MF958947)and Sc-EGFR-1b(Accession number: MF958948)were also isolated.The 5,962 bp full-length cDNA of Sc-EGFR-1a had a 1,152 bp 5?-UTR,a 226 bp 3?-UTR with a polyA tail and 4,581 bp ORF that encoded a protein of 1,527 amino acids.The mature Sc-EGFR-1a had a deduced molecular mass of 176.33 kDa and a theoretical pI of 5.69.Sc-EGFR-1b had a deduced molecular mass of 168.37 kDa and a theoretical pI of 5.70.Sc-EGFR-1a possessed a delete of 27 amino acids in the C-terminus changed the phosphorylation sites and may have influenced downstream kinase activities.2.Expression of Sc-EGFR-1a and Sc-EGFR-1bThe expression profile of Sc-EGFR-1a and Sc-EGFR-1b were studied,including different developmental stages and tissues.Both Sc-EGFR-1a and Sc-EGFR-1b were detecTab.in the early embryo stages,and the levels increased up to the middle-late stages.Sc-EGFR-1a and Sc-EGFR-1b had a widespread tissue distribution.Both isoforms were highly expressed in the siphon.High levels of the Sc-EGFR-1a transcripts were also found in the foot,gut,and liver,while high levels of the Sc-EGFR-1b transcripts were also found in the liver,gill,and hemolymph.3.Responses of Sc-EGFR-1a and Sc-EGFR-1b to the proliferation of cellsIn vitro,the proliferating cell nuclear antigen indicator and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that cells transfected with Sc-EGFR-1a and Sc-EGFR-1b exhibited different proliferation patterns.The study shows levels of PCNA revealed a significant up-regulation after transfection of Sc-EGFR-1a,while no significant variation was observed after transfection of Sc-EGFR-1b.Furthermore,cell proliferation was measured using MTT assays to estimate cell population post transfection.Interestingly,cells that overexpressed Sc-EGFR-1a displayed a higher cell population than those that overexpressed Sc-EGFR-1b.4.Effects of Sc-EGFR silencing on the growth traitsIn vivo,The expression of Sc-EGFR in the foot tissue of the treated clams showed a reduction in 2 days and a significant reduction from 5 to 15 days post-injection when compared with the control group.The result indicated that the Sc-EGFR dsRNA was sufficient to silence Sc-EGFR for up to 15 days.Sc-EGFR silencing group showed a significant inhibition of shell length and low survival rate when compared with the control group.5.Polymorphisms of Sc-EGFR and its association with growth traitsIn order to study the correlation between Sc-EGFR gene and growth traits(shell length,shell width,shell height and weight),we analyzed the viriation of EGFR intron 1 sequence from S.constricta by using direct sequencing.There were 17 SNP sites in intron 1: T785 A,A911C,G966 A,G996A,A1068 G,G1092A,A1125 T,T1193A,T1207 G,G1262A,T1281 A,C1293T,C1301 A,C1314T,T1328 C,A1359T and C1371 G,named SNP1-SNP17 respectively.Based on Chi-square test,the 13 sites were fitted to Hardy-Weinberg equilibrium(P > 0.05),and the 10 sites showed moderate polymorphism through polymorphism detection(0.25 < PIC < 0.5).The correlation between SNPs and growth traits(shell length,shell width,shell height,and weight)were further analyzed using a General Linear Model and multiple comparisons,the results showed that despite SNP 7,all SNP sites were significantly associated with shell length,shell width,shell height and weight. |
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