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Cloning, Expression and Functional Analysis of a Metallothionein cDNA from Chimonanthus Praecox (L.) Link

Author: QinPingWei
Tutor: LiMingYang
School: Southwestern University
Course: Floriculture
Keywords: Metallothionein Cloning prokaryotic expression Heavy metal stress
CLC: S685.99
Type: Master's thesis
Year: 2010
Downloads: 85
Quote: 1
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Abstract

Metallothioneins(MT)are low-molecular-weight proteins combine with heavy metals, which are rich in Cys and widely distributed in the biosphere.Rcently, as many genes encoding metallothionein have been cloned, metallothionein has been studied deeply. Many experiments demonstrated that metallothionein played an important role in carring and storing trace element, scavenging of free radicals and detoxifying of heavy metals. Several physiological processes like preventing cancer from growing delying aging seem to benefit by metallothionein. According to the the wax plum cDNA library to clone chimonanthus metallothionein gene and construct the prokaryotic expression vector of CpMT. The protein was purified by His-Bind protein retrieve box. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.1. Cloning of CpMT from Ch. praecox flowerIn this paper, a MT gene, designed as CpMetallothionein (CpMT), was obtained by sequencing the randomly selected clones, on the basis of cDNA library construction of Chimonanthus praecox (L.) Link. The bioinformation analysis showed that the cDNA sequence had a length of 1 083 bp, containing ORF of 240 bp which encoded 79 amino acids.2. Structural characteristics analysis and function prediction of CpMT proteinThe structure characteristics of CpMT protein were analyzed with bioinformatic method. The biggest open reading frame (ORF) contained a predicted protein of 79 amino acids. The molecular weight was about 7 765.95 D. The isoelectric point was 4.67. It had 6 phosphorylation sites. Structural characteristics analysis of CpMT protein by NCBI showed that the Conserved regions had some similarities with the MTs. That codes for a protein with MT-like proteins domain, belonged to MT-like proteins family.3.The construction of prokaryotic expression vector of CpMT and the optimization of expression systemAccording to characteristics of the target gene and vector synthetic primer.we inserted CpMT into prokaryotic expression vector pET-32a, and then transformed to competent cells Origami2. We optimized expression system by adjustment of inducing temperature, conditions of IPTG and inducible time. The fusion protein was proved to be expressed in both soluble and insoluble by SDS-PAGE. We got the recombinant protein finally. SDS-PAGE shown that, expressed product contain a 29 KD target protein.4. CpMT preliminary analysis of gene functionAt first,we did transformed bacteria heavy metal tolerance experiment. The results showed that transformed bacteria was better in heavy metal tolerance than control bacteria. In order to further verify its function, we constructed a plant expression vector PC2301-CpMT, and transformed to tobacco, and got transgenic plants idengtifing by both GUS and PCR. We have done heavy metal stress on the transgenic positive strains and control strains, the transgenic plants with the control strains had significant differences. Preliminary descripted that the CpMT may have the function to against heavy metals.

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