| Mesocentrotus nudus is the major economic species in some coastal cities of northern China.There are abundant fatty acid,amino acid,and polysaccharide in their gonads,which greatly spurred people’s attention,therefore they are regarded as nourishing supplements for human beings.The studies to identify the physical and chemical properties of gonads in M.nudus have shown that in the same area,the development stages of the gonads are different in M.nudus of different body sizes.At the same time of reproduction period,the sea urchins with larger shell diameters own gonads with greater maturity,and the contents of polyunsaturated fatty acids in their gonads are also relatively higher.However,at present,the study in molecular mechanism of regulating gametogenesis and fatty acid biosynthesis during the development of gonads is still limited.In this study,high-throughput sequencing technique was performed to obtain the transcriptome information of M.nudus’ gonad for the first time,in order to clarify the molecular mechanism in the different degrees of maturity in M.nudus’ gonads with different body sizes at the same reproduction period.Besides,three genes participated in PUFA biosynthesis were cloned and expressed based on the results of transcriptome sequencing,to illuminate the biosynthesis pathways of polyunsaturated fatty acids in M.nudus’ gonad.The main research contents and results are as follows.1.Gonad transcriptome sequencing and differentially expressed genes screeningIn this research,Illumina HiSeq 2500 sequencing platform was used for the first time to sequence the gonad transcriptome of adult female M.nudus in two different body sizes.The raw reads were filtered and de novo assembly to generate unigenes.Then,bioinformatics methods were performed to functional annotation,differentially expressed genes(DEGs)analysis and molecular marker exploration for all unigenes,some important results have been obtained.Four cDNA libraries was constructed in total for M.nudus,two biological duplications for big and small urchins.Through de novo assemble and removing redundant sequence,a total of 88,581 unigenes was acquired,with a mean length of 1,354 bp,and the length of N50 was 2,633 bp.Thereinto,66,311 unigenes(74.9 %)could be annotated using six major public databases,namely NR,Nt,Swiss-Prot,KEGG,GO,and COG.Compared to the big urchins,353 DEGs were found in small ones(fold change ≥ 2,FDR ≤ 0.001),of which 260 were down-regulated and 93 were up-regulated.The enrichment analyses of GO and KEGG pathways on DEGs showed that the DEGs were gathered in the pathways related to gametogenesis significantly.Then,20 of the 353 DEGs were selected for quantitative real time RT-PCR validation.The results were consistent with the RNA-seq data.32,076 potential SSRs were identified in 22,224 unigenes.In addition,25,870,26,931,29,024,and 31,597 potential SNPs were found in the transcriptome alignment files of big1,big2,small1 and small2,respectively.2.The cloning,sequence analysis and expression analysis of three genes participated in PUFA biosynthesisThree genes participated in polyunsaturated fatty acid biosynthesis were found in the transcriptome sequence of M.nudus,including two fatty acid desaturase genes Fad1 and Fad2,as well as a fatty acid elongase gene ElovlA.Firstly,the sequences of three genes were cloned.Then some bioinformatics softwares were performed to analysis the amino acid sequences,predict the secondary and tertiary structure of protein,as well as blast the sequences between multiple species.Further,fluorescence quantitative PCR technology was performed to detect the expressions of three genes in six tissues.Lastly,the three genes were expressed in Saccharomyces cerevisiae,and western blotting method was used to test the products of the three genes.The main results acquired are as follows.The ORF sequences of Fad1,Fad2 and ElovlA gene were acquired,are 1,386 bp,1,359 bp and 705 bp,respectively.The results of sequence analyses showed that Fad1 and Fad2 protein had the specific motif of fatty acid desaturase,including cytochrome b5 structure in N terminal and three histidine boxes in C terminal;the amino acid sequence of ElovlA also contained a characteristic histidine box and 17 conservative amino acids of fatty acid elongase.The results of multi-sequence alignment between different species showed that the Fad1 gene had the highest sequence similarity with FadC1 and FadC2 gene of Paracentrotus lividus;Fad2 gene shown the highest sequence similarity with Fad6 gene of Strongylocentrotus intermedius and FadA gene of P.lividus.ElovlA gene was converged with the Elovl5 genes of S.intermedius.The results of fluorescence quantitative PCR detection showed that the three genes had higher expressions in gonad,intestine and coelomic fluid,but almostly didn’t expressed in peristomial membrane and tube foot.The recombinant eukaryotic expression vectors of the three genes were constructed,and the expression products of the three genes had been proved by western blotting test.To sum up,the transcriptome sequences of the gonads of female M.nudus in the second developmental stage of reproduction period were acquired,the results of differentially expressed genes analyses implied some pivotal genes which might regulate the development of gonad,whereafter the biosynthesis pathway of PUFA was explored in M.nudus.These studies lay a foundation for further clarifying the molecular mechanism of gametogenesis and nutrient metabolism in the sea urchins,as well as provide a reference for exploring the synthesis pathway of PUFA in invertebrate. |
