Molecular Characterization And Functional Analysis Of Metallothionein Exposured To Metal In Oxya Chinensis (Orthoptera:Acridoidea)

The rapid industrialization and urbanization in developing countries have increased pollution by heavy metals, which is a concern for human health and safety of the environment. The increasing presence of heavy metals in water, soil and air is directly linked to the food chain and transfer to animals through polluted irrigation water. Rice is the largest grain crop and paddy fields are still contaminated with heavy metals in china.Metallothionein (MT) is first isolated in 1957 from horse kidney, subsequently different isoforms are characterized depending on the species. Studies focus on myriad topics from life science, physical chemistry, applied medicine and environmental monitoring. MTs are a low molecular weight cysteine-rich proteins characterized by high affinity for heay metals, including essential (Zn and Cu) and non-essential (Cd and Hg) trace metals. MTs are ubiquitous and have been isolated in a wide variety of organisms from bacteria to humans playing a key role in metal metabolism. Few studies have examined the molecular characteristics of MTs in Diptera (Drosophila melanogaster, Anopheles gambiae) and Collembola (Orchesella cinctd) insects. By contrast, less information is available on MTs in agricultural pest.Oxya chinensis, which is an agricultural pest, feeds on the leaves of gramineous plants, particularly rice, and inhabits rice-growing areas of China. Due to grasshopper behavior in the farmland ecosystems, heavy metals (such as cadmium) in the agricultural environment transfer into the bodies of the grasshoppers through the food chain. In this thesis, the molecular characteristics and functional of metallothionein were analyzed in O. chinensis exposured to metal stress. Acute effects of heavy metals on detoxification enzymes and Histopathological alterations in O. chinensis were studied under laboratory control condition. The present study will help to elucidate the characteristics and functions of MTs in O. chinensis and demonstrate the potential value of heavy metal pollution prevention. The main contents are as follows:1. Analysis of OcMT molecular characteristicsTwo full-length cDNA sequences were obtained from the O. chinensis transcriptome database putatively encoding two different OcMTs, which were named OcMT1 and OcMT2. The full-length cDNA sequence of OcMTl was 552 base pairs (bp) long, with an open reading frame of 123 bp that encoded a 40-amino acid peptide. The full-length cDNA sequence of OcMT2 was 363 bp, with an open reading frame (ORF) of 195 bp that encoded a 64-amino acid peptide. The deduced amino acid sequences of the MTs from O. chinensis were compared with the other insect MTs using the GeneDoc FASTA sequence comparison program. The two OcMTs shared low amino acid sequence similarities but high identities.The cysteines of the deduced OcMT1 and OcMT2 protein sequences were 22.5% and 25%, respectively. All cysteine residues were in the characteristic Cys-Cys, Cys-X-Cys, Cys-X-Y-Cys or Cys-X-Y-Z-Cys configuration similar to that observed in other MTs. Thus the complex M-MT were formed by metal-thiolate clusters of MT molecule and metal ions.2. Tissue and Stage-dependent transcription patterns of OcMTTo determine the expression characteristics of the OcMTl and OcMT2 genes, transcription analysis of OcMT mRNA were detected in the eleven different tissues and at the seven developmental stages. The RT-qPCR analysis indicated that OcMT1 mRNA was widely expressed in all tissues examined and the seven life stages. The highest transcription levels of OcMTl and OcMT2 were detected at the egg stage. The OcMT higher transcription were detected at the 2nd-instar nymphs and adults. The highest transcription of OcMT1 and OcMT2 were detected in the brain and optic lobe, respectively. This may suggest that the OcMTs in insects are different responsive to harmful environmental stresses.3. Analysis of OcMT gene and protien functionsTo evaluate their vital biological functions, an RNA interference analysis of both the OcMTl and OcMT2 was performed by injecting sequence-specific double-stranded RNA(dsRNA) into O. chinensis. After the injections of the dsRNAs, the OcMT1 and OcMT2 transcript levels in the whole bodies of the adults decreased by approximately 63.1% to 70.9% by 48 h post-injection. The mortalities of each group displayed dose-dependent increases of 8.5% to to 16.7% after the silencing of OcMT1 and OcMT2, respectively. Enhanced tolerance to the heavy metal in a recombinant strain expressing an OcMTs has also been demonstrated in E. coli. which may have been due to the chelation of OcMTs protein.4. Effects of the metals on detoxification enzymes activity and morphologyThe effects of the Cd concentration on ACP, AKP, CarE and GST activity were increased generally. Grasshoppers exposed to Cd, midguts, gastric caeca, Malpighian tubule and peritrophic membrane were damage, and morphological changes in the differential haemocyte have been assessed. The diameter of fat body were decreased after Cd and Zn treatment. Accumulation of crystal or flocculent deposits in Malpighian tubules were observed. Studies have demonstrated that several tissues participated in heavy metals detoxification in different forms and protect the insects from metals damage.