Molecular Characterization And Physiological Function Of Metal-responsive Transcription Factor-1 Gene OcMTF-1 From Oxya Chinensis (Orthoptera: Acridoidea)

Oxya chinensis (Orthoptera:Acridoidea) is widely distributed in rice planting area in our country, and it is one of major agricultural pests in rice growing areas. With the heavy metal pollution of agricultural ecological system, the living environment and feeding of O. chinensis are also affected by heavy metal pollution, heavy metals of the environment can accumulate in the body through the food chain and affect its physiological metabolism. The metal-responsive transcription factor-1 (MTF-1) is a pluripotent transcriptional regulator, which plays an important role in the body response to heavy metals and hypoxia or oxidative stress. In this study, the full-length OcMTF-1 cDNA was cloned and verified, then we analyzed its roles in the activation of MT transcription when response to Cd2+stress. The results provided a foundation for further research on the mechanism of gene regulation of OcMTF-1.1. Molecular characterization and expression analysis of OcMTF-1 from O. chinensisThe cDNA fragment of MTF-1 was obtained from the transcriptome database of O. chinensis, then its full-length cDNA from O. chinensis was cloned using RACE methods and named OcMTF-1 (GenBank number: KM017748). Its length is 1834 bp, with a 1527 bp open reading frame encoding a 508-amino acid polypeptide. The structure prediction, sequence alignment and phylogenetic analysis of the coding product of this gene were performed using different bioinformatics softwares. It has six C2H2-type zinc finger regions only and no other activating domains were identified. Multiple alignments revealed that the zinc finger domain is conserved at the N terminus of OcMTF-1 and MTF-1 proteins from other species. Based on the cloned mRNA nucleotide sequences of OcMTF-1, specific primers were designed to analyze its developmental and tissue-specific expression patterns using RT-qPCR. The results showed that the expression level of OcMTF-1 was the highest in the Malpighian tubules, moderate in the hindgut and fat body, and the lowest in other tissues. OcMTF-1 was expressed at both the 1st to 5th instar nymphal stages and adult stage.2. Functional analysis of OcMTF-1 from O. chinensisThe 2nd instar nymphs of Oxya chinensis was feed with Cd contaminated wheat seedlings for the chronic intoxication, The expression characteristics of OcMTF-1, OcMTl and OcMT2 after induction of chronic cadmium was analysed by RT-qPCR. The results showed that the OcMT1 mRNA expression increased relative to control by about 10-fold. Expression of OcMTF-1 and OcMT2 showed no significant changes compared with the control. The result was identified with digital gene expression profiling (DGE) of O. chinensis.We performed RNA interference experiments to investigate biological function of OcMTF-1 in insect development. RNA interference of OcMTF-1 showed that when the 4th instar nymphs of day 4 was injected by OcMTF-1-specific dsRNA, the transcript levels of OcMTF-1 could be repressed by 72.3% and 73.6% at 24 h and 48 h, respectively, as compared with the control group. The low expression of OcMTF-1 did not influenced the growth and development of nymphs.Compared with the control group, the expression quantity of OcMTl was upregulated in acute cadmium induced, However, the expression of OcMT1 was significantly decreased when the OcMTF-1 silencing before cadmium treatment. This indicated that the expression of OcMT1 is regulated by MTF-1. Whether only injection of cadmium solution or OcMTF-1 silencing before induced by cadmium, the expression level of OcMT2 had no obvious changes compared with the control. We conclude that the expression of OcMT2 is not completely dependent on MTF-1.To further understanding the roles of OcMTF-1 on the regulation of OcMT1 expression under heavy metal exposure, OcMTF-1 and OcMTl expression level under Cd2+exposure was investigated via RT-qPCR. The expression level of OcMTF-1 had no obvious changes compared with the control, however a significant increase in OcMT1 expression was observed after 12 h of Cd2+ exposure.3. Cloning of MT promoter of O. chinensisAccording to the genome sequence characteristics of LmMT1, we cloned a intron of OcMT1 gene from O. chinensis using PCR methods. Its length is 1977 bp, after the 321st base of OcMT1 cDNA. LmMTl and OcMTl contain only one intron. This is different from the introns of other MT genes. The results provided a foundation for further research on OcMTl promoter cloning and the mechanism of gene regulation of OcMTF-1.