| Loach belongs to Cyprinidae, Loachidae, Loach subfamily, loach, widely distributed in China, Japan, Korea, Russia and India and other places. It is called "water ginseng" and is a high nutritional value of fish. In addition, the loach is an important aquaculture fish in China. The control and management of the propagation of loach is a necessary step in obtaining the sustainable supply of such fish in aquaculture production systems. Therefore, the study of the molecular regulation mechanism of gonadal development and gametogenesis in this species is of great significance in aquaculture and agricultural production.1?The morphological changes of the tissues of the loach gonad were observed by paraffin section, and the period of the five tissues was defined as PGC period, stage II testis, stage II ovary, mature testis and mature ovary. To provide the basis for the establishment of the library.2?In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to gonad development of loach. Illumina sequencing generated 321050936 raw reads. After removing adaptor sequences, ambiguous nucleotides and low-quality sequences,there were 315776696 clean reads remaining. In total, there were 68718 unigenes (48.15%)successfully annotated in at least one of the Nr, Nt, Swiss-Prot, KEGG, GO, KOG and Pfam databases, with 10813 unigenes (7.57%) in all seven databases. In addition, five digital gene expression (DGE) libraries were constructed for the primitive germ cells (PGC), stage ? testis(T2), stage ? ovary (02),adult testis (TA) and adult ovary (OA). The specific expression of many genes in the five samples was also identified. Some genes among five sub-libraries with high differential expression were selected as candidates connected with gonad development.3?The results of qPCT expression were identical to those obtained from the Illumina sequencing data library by verifying the FPKM values in the QRT-PCR and Illumina sequencing data libraries. It also proves that the measured data of the five transcriptome libraries are reliable and accurate and can provide a basis for future experiments.4?The primers were designed according to the data of the library, and the primers were designed according to the Wnt4 gene sequence of other bony fish. The full length sequence of the gene was obtained by combining RACE. The results showed that the 5 'untranslated region(5'-UTR) 429 bp and the 3' untranslated region (3'-UTR) contained polytetrain 1092 bp, open reading frame 1056bp, encoding 351 amino acids. The molecular weight was 39181 daltons using ProtParam, the theoretical isoelectric point was 8.68, a total of 20 amino acids, 22 amino acid signal peptide sequence, a transmembrane region. The secondary structure of WNT4 protein was predicted. It was found that the a screw accounted for 32.39%, the proportion of ? was 8.24%, the random curl was 39.77% and the extension chain was 19.6%. The amino acid sequence is highly conserved in bony fish and is less conserved than amphibians, birds and mammals. The similarity between the loach and the same type of zebrafish was 96.3%, which was the closest to evolution.In summary, we obtained the five transcriptome libraries of gonadal gland during the critical period of gonadal gland development, and the informational analysis of Wnt4 gene was provided,which provided the basis for the study of gonadal differentiation and decision mechanism. And the specific mechanism of action and gonadal development of genes related to the need for in-depth study. |
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