| Haliotis discus hannai is the important economic marine shellfish in China.It is mainly distributed in Shandong,Liaoning,Fujian.Because of its delicious meat and rich nutrition,it is deeply loved by consumers.However,in recent years,due to the problems such as successive generations of artificial seedlings,inbreeding and deterioration of the marine environment,the abalone germplasm degenerated seriously,the harmful genes showed and the genetic diversity of the breeding population continued to decline.The offspring appeared problems such as slow growth,poor stress resistance and high mortality at the same time.It is urgent to develop new breeding lines to improve the current situations.The egg-color of female individuals of H.discus hannai is very rich.Studies have found that its egg-color may be related to the economic traits such as growth and stress resistance,which provides a new idea for the selective breeding of abalones.The main contents of this study include two parts.A transcriptome sequencing of H.discus hannai with green and gray egg-color was performed using RNA-seq,which can help us identify the Differential Expression Genes(DEGs)and the metabolic pathways that may be related to the formation of egg-color.The Rapid Amplification of c DNA Ends(RACE)experiment was performed to obtain the full-length sequence of the gene GLT1 that may be related to the formation of egg-color.The expression pattern of GLT1 in different tissues of H.discus hannai in different months were analyzed by quantitative real-time PCR(q RT-PCR).On the one hand,the results in this study will provide theoretical basis for further understanding the genetic mechanism of egg-color in H.discus hannai.On the other hand,it can excavate the genes related to formation of egg-color and can provide foundational datas for further exploring the function and the regulatory mechanism of these genes in H.discus hannai.The main findings are as follows.1.Transcriptome analysis of H.discus hannai with two egg-colorsIn this study,a total of six transcription libraries were constructed,including three green-egged individuals(L1,L2,L3)and three gray-egged individuals(H1,H2,H3).44.56 Gb clean reads were obtained.272,310 unigenes were received from 461,162 transcripts with a mean length of 985 bp and N50 of 1,524 bp,respectively.Compared with gray-egged individuals,a total of 185 unigenes were identified as DEGs(P<0.05)in green-egged individuals,including 54 significantly up-regulated and 131 significantly down-regulated genes.Some DEGs were involved in the terms or pathways in GO and KEGG analyses,“flavin-containing compound metabolic process”,“tyrosine metabolic process”,“melanin-concentrating hormone activity”,“melanosome”,“pigment granule”,“Glutathione metabolism”,“cytochrome b6 f complex”,“Nitrogen metabolism”,“Alanine,aspartate and glutamate metabolism”,“pigment metabolic process”,and “beta-Alanine metabolism”,that may be related to the formation of egg-color.9 DEGs were randomly selected for q RT-PCR analysis to confirm the reliability of RNA-seq data.The results showed that the expression levels of 9 DEGs analyzed by q RT-PCR were mainly agreement with the data of RNA-seq,which indicates our RNA-seq data were reliable.2.Cloning and expression analysis of the gene GLT1In this study,the gene GLT1 was cloned.Its full-length c DNA is 7,640 bp,including a 5' untranslated region(UTR)of 381 bp,a 3' UTR of 1,025 bp and an open reading frame(ORF)of 6,234 bp which encodes a peptide of 2,077 amino acids.The estimated PI and molecular mass of the deduced amino acids are 6.32 and 230.02 k Da,respectively.According to the SMART analysis,the deduced amino acids include the following domains,GATase?2,Glu?syn?central,Glu?synthase,GXGXG,Fer4?20,and NAD?binding?8.The results of BLAST analysis showed that the sequence of GLT1 in H.discus hannai had the highest homologous similarity with Mizuhopecten yessoensis(73%),and its similarity with Pomacea canaliculata,Crassostrea gigas and Crassostrea virginica were 72%,71% and 71%,respectively.The multiple sequence alignment revealed that the sequence of GLT1 contained many highly conserved regions in the domain position by comparing with 10 other GLT1 amino acids which are from 8species,and the domain Glu?synthase was the most complete region.The phylogenetic analysis showed that the sequence of GLT1 in H.discus hannai was firstly clustered with P.canaliculata,Biomphalaria glabrata,Aplysia californica,and then clusteredwith M.yessoensis,C.virginica and C.gigas.The expression of GLT1 in different tissues of different months in H.discus hannai were studied by q RT-PCR.Results showed that the m RNA of GLT1 was expressed in muscle,mantle,gonad and gill regardless of male and female individuals,and the expression in muscle was the highest,but there was no expression in hepatopancreas.For gonads,the expression of GLT1 in these five months showed a trend of increasing,decreasing,and gradually stabilizing.The expression in June was significantly higher than that in other months(P<0.05). |
PDF Full Text Request