| Metallothioneins (MTs) have multiple physiological functions and diverse regulatory activities, which include metal homeostasis, heavy metal detoxification, and antioxidant protection from free radicals. In some species, MT levels are highest after the reproductive period, perhaps due to the metabolic need and biological availability of essential metals, such as zinc and copper.The study of metallothioneins both domestic and abroad was mainly in the extraction, detection and applications of the protein. With the rapid development of molecular biology, MT gene cloning, RNA blot, Genome walking, real-time quantitative PCR technology for quantitative detection and sequencing of MT were come ture. Researches on metallothioneins of crustacean were more concentrated in the extraction and purification of the protein. The molecular research was relatively weak, and among the majority in this study focused on the cDNA acquisition and expression, while analyses on its gene structure and functional areas are scarce.Chinese mitten crabs (Eriocheir sinensis) live mainly in northern Asia, western Korea and China. As early as 1000 years ago, Chinese people have got the living habits of Chinese mitten crabs, especially reproductive migratory habits. In recent years, the Chinese mitten crab is a commercially important species in South-East Asia, and is widely farmed in China. In recent years a number of potential technical issues are gradually exposed in crab aquaculture, therefore, molecular, genetic researches in aquatic have gained more and more attentions, some immune or metabolic-related genes of E. sinensis have been cloned. in our study, MT-1 gene of Chinese mitten crab will provide some theoretical basis for research in genetic and molecular aspects of the immune defense mechanisms, transgenic technology.1 A full length metallothionein-1 (MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon EST analysis of a hepatopancreas cDNA library. The full length MT-1 cDNA isolated from E. sinensis hepatopancreas was 579 bp, with a 180 bp ORF, and 5'and 3'untranslated regions (UTR) of 81 bp and 318 bp, respectively. The ORF encoded a 59 amino acid protein and the cysteine (Cys) proportion of 30.5%(18/59) was observed in array patterns typical of other reported MT cDNA sequences, with five instances of Cys-X-Cys, two instances of Cys-X-X-Cys, and three instances of Cys-X-X-X-Cys.2 Analysis by real-time quantitative RT-PCR supported semi-quantitative findings, the highest expression was in hepatopancreas, approximately 500 times that of testis, in addition to this, with expression levels high in testis and thoracic ganglia, moderate in intestine, and low but detectable expression ovary, gill, stomach, brain, hemolymph, heart and muscle. MT-1 testicular expression was assessed from August to January. Real-time quantitative RT-PCR analysis revealed peak MT-1 expression in August. Expression then sharply decreased in September, with the seasonal low observed in November.3 The cloned MT-1 genomic sequence was 3816 bp, and included a 1735 bp 5'flanking region, three coding exons (25 bp,78 bp,77 bp), and two introns (984 bp, 380 bp) that occurred in a "3+2" patten. Bioinformatic analysis of the 5'upstream region of the MT-1 gene predicted several transcription factor binding sites, including MREs, HNF-1, AP-1, SP1, GATA, USF and HSF.4 Exposure of crabs to Cu2+treatment via water (0-1 mg/L) for 10 days resulted in a dose dependent bell curve response in MT-1 mRNA expression, with expression peaking at 0.1 mg/L Cu2+. A time course experiment (O.1mg/L Cu2+over 7 d) revealed MT-1 mRNA expression sharply peaked on day 5, decreasing gradually with prolonged exposure.Molecular biology and bioinformatics methods were used in this study to analysis the ESTs in E. sinensis hepatopancreas cDNA library. We obtained the full length cDNA sequence of metallothionein-1 gene. On this basis, we use Genome walking technology and real-time quantitative PCR to analysis the gene structure and its expression pattern. |
PDF Full Text Request