Cloning And Characterization Of Fenneropenaeus Chinensis Metallothionein Gene And Promoter

Metallothioneins (MTs) are low molecular weight metal binding proteins whose synthesis is usually induced by metal ions(such as Zn, Pb, Cu et al). MT promoter is one of the common inducible promoters which can enhance expression of gene 100 times after being induced by weight metals. MT has been the interest point of researcher for its special biological functions and gene modulation mechanism. Thus cloning of MT promoter from F. chinensis will promote research on the gene modulation of F. chinensis and offer a strong support to the transgenic study of F. chinensis. 3 pairs primers were designed according to the homologous sequence between ORF of F. chinensis and other species. The whole cDNA sequence was obtained by PCR which was carried with the above 3 pairs primers, T3 and T7 as primers and with cDNA library as template. Sequence analysis revealed that the cDNA of MT from F. chinensis was highly homologous(78.9%) to American lobster Homarus americanus. Moreover, we cloned a intron of MT gene which can be important to study the gene construction and regulation of MT further. In this experiment, a 628bp fragment containing the proximal promoter region of a F. Chinensis MT was isolated using the "targeted gene walking PCR" method. Sequencing results confirmed that the 628-bp fragment contained TATA box, CAAT box, and the origin code ATG which the common promoters had. Some combining sites of transcription factors (MTF-1, Sp1, Ap-2 etc.) were found after analysis with the software Promoter Prediction. Comparison with published sequences of related MT gene promoters enabled us to identify a MRE(TGTGTGCA) identical to and another (CAGTGCA ) that closely resembled(one mismatch only) to the MREs found in MT promoter of ayu fish(Plecoglossus altivelis). In addition, a box(GGTTTGT) was proximal with a hepatic nuclear factor 5 responsive elements(HNF5REs). According to the author, the boxes play a role in regulating expression of MT gene. This will facilitate the research of gene regulation on the Chinese Shrimp (F. chinensis) and the transgenic experiment in future. The promoter fragment was fused to the report gene EGFP and then the resulting construct is termed as pMT-EGFP. The vector was transfered into the shrimp embryos through electroporation and the hemocyte of primary culture through lipofection. PCR results and detection using the fluorescence microscope indicated respectively that the foreign gene was successfully delivered into the embryos and hemocyte,which expressed the green fluorescence. Moreover, the transcription experiment in vitro supported the above conclusion. Then the MT promoter we isolated proves effective and can be available in the future shrimp transgenic research.