Table 1.
List of primers used in this study.
Figure 1.
Nucleotide and deduced amino acid sequences of OcMT1 and OcMT2 cDNAs from Oxya chinensis.
The deduced amino acid sequence is shown below the nucleotide sequence. Blue letters indicate the start codon (ATG), and an asterisk (*) indicates the stop codon. The putative polyadenylation signal sequence (AATAAA) is underlined. The numbers on the right refer to the amino acid residues. The cysteines (C) are highlighted in red. The deduced amino acid sequences of OcMT1 and OcMT2 are shown, with the cysteine residues arranged as C-C, C-X-C and C-X-X-C motifs, in which X can be any amino acid other than cysteine.
Figure 2.
Multiple sequence alignments of the deduced amino acid sequences of OcMT1 and OcMT2 with other known homologues species using the GeneDoc software.
Amino acid residues are shaded in color. Conserved and identical cysteine amino acids are highlighted in red and blue, respectively. The species and the GenBank accession numbers are as follows: Oxya chinensis (OcMT1 KJ153014 and OcMT2 KJ153015), Drosophila melanogaster (DmMTB: NP524413, DmMTC: NP650882, DmMTD: NP788695, and DmMTE: NP001189254) Musca domestica (MdMT2: AEO50699), Anopheles gambiae (AgMT1: AAX86006 and AgMT2: AAX86007), Chironomus riparius (CrMT: ADZ54163), Tabanus yao (TyMT1: ABX80078 and TyMT2: AAX860079).
Figure 3.
Tissue-specific transcript expressions of OcMT1 and OcMT2 in adults on day 3.
The β-actin gene was used as an internal control. The tissues include the brain (BR), optic lobe (OL), muscle (MU), foregut (FG), midgut (MG), hindgut (HG), gastric caeca (GC), Malpighian tubule (MT), fat body (FB), testis (TE) and ovary (OV). The different letters on the error bars indicate significant differences in the expression of the same gene in different tissues. The data are expressed as the means ± SE of three biological replicates. The relative expressions of OcMT1 and OcMT2 were calculated using 2−ΔΔCt. The vertical bars represent the mean ± SE. (P<0.05, Tukey's HSD test; n = 3).
Figure 4.
Analysis of stage-dependent expressions of OcMT1 and OcMT2 in O. chinensis by RT-qPCR.
The same letters on the error bars indicate no significant differences in the expression of the same gene in the seven developmental stages. The relative expression of OcMT gene was calculated using 2−ΔΔCt. The vertical bars represent the mean ± SE. (P<0.05, Tukey's HSD test; n = 3).
Figure 5.
Efficiency of RNAi of OcMT1 and OcMT2.
Insects were injected with OcMT1 and OcMT2 dsRNA, and the control group was injected with dsGFP. The expression of OcMT1 and OcMT2 in the whole body was detected by RT-qPCR after 12, 24, 48 h of treatment. The expression levels of OcMT1 and OcMT2 mRNA for the control groups were set as 100%. An asterisk (*) on the error bars indicates significant differences (P<0.05, n = 3).
Figure 6.
Mortalities of O. chinensis injected with different heavy metals after OcMT1 and OcMT2 gene silencing by RNAi.
A: OcMT1 RNAi; B: OcMT2 RNAi. Insects were injected with OcMT1 and OcMT2 dsRNA, and the control groups were injected with the same amount of dsGFP. After 24 h, at least 100 insects were randomly selected, and 4 µL of a concentration gradient of CdCl2 (0.87, 1.74, 2.61, 3.48, 4.35 mM), CuCl2 (8.79, 11.73, 14.67, 17.61, 20.55 mM) and ZnSO4 (15.65, 19.13, 22.61, 26.09, 29.57 mM) solution were injected. The control groups were injected with distilled water. Mortalities were recorded at 48 h after the injections of the metal solutions. An asterisk (*) on the error bars indicates significant differences (P<0.05, Tukey's HSD test; n = 3).
Figure 7.
SDS-PAGE analysis of His and two His-OcMT fusion proteins expressed in E. coli BL21 (DE3) cells.
Protein samples were separated by 15% SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, medium range molecular weight marker; Lane 2, E. coli BL21 with pET-28a-OcMT2 cell lysate induced with IPTG; Lane 3, E. coli BL21 with pET-28a-OcMT2 without IPTG; Lane 4, E. coli BL21 with pET-28a; Lane 5, E. coli BL21 with pET-28a-OcMT1 cell lysate induced with IPTG; Lane 6, E. coli BL21 with pET-28a-OcMT1 without IPTG induction.
Figure 8.
Cadmium tolerance of E. coli BL21 cells expressing OcMT1 and OcMT2.
A: OcMT1, B: OcMT2. Bacterial growth curve of E. coli cells transformed with pET-28a, pET-28a-OcMT and pET-28a-OcMT-IPTG. pET-28a is an “empty” vector; pET-28a-OcMT group is transformed with the OcMT1 or OcMT2 gene without IPTG; pET-28a-OcMT-IPTG group is transformed with the OcMT1 or OcMT2 gene with IPTG. Five microliters of CdCl2 was added into medium when bacteria were grown to OD600 = 0.6. All bacteria were grown for 11 h. Concentration gradient of CdCl2 were 0, 0.82, 1.74, 3.27 mM.