Identification of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance.

Stress proteins such as metallothioneins (MTs) play a key role in cellular protection against environmental stressors. In nature, insects such as houseflies (Musca domestica) are commonly exposed to multiple stressors including heavy metals (e.g. Cadmium, Cd) and high temperatures. In this paper, we identify two novel MT genes from the cDNAs of M. domestica, MdMT1 and MdMT2, which putatively encode 40 and 42 amino acid residues respectively. Expression of the two MTs' mRNAs, which are examined in the fat body, gut, hemocyte, and the epidermis. From our study, we saw that the expression of MdMT1 and MdMT2 are enhanced by Cd and thermal stress. Levels of expression are highest at 10 mM Cd(2+) within a 24-h period, and expressions increase significantly with exposure to 10 mM Cd for 12h. Levels of the mRNAs are up-regulated after heat shock and that of MdMT2 reaches its maximum peak faster than MdMT1. Both of the MT genes might be involved in a transient systemic tolerance response to stressors and they may play important roles in heavy metal and high temperature tolerance in the housefly. To detect whether or not the MTs bind heavy metals, the target genes are cloned into the prokaryotic expression vector pET-DsbA to obtain fusion protein expressed in Escherichia coli BL21 (DE3). Recombinant DsbA-MdMT1 significantly increases tolerance of the host bacteria to Cd(2+), but DsbA-MdMT2 is absent. These differential characteristics will facilitate future investigations into the physiological functions of MTs.