Gene Cloning And Structure Prediction Of The Selenoprotein In The Silkworm

Selenium is an essential biological trace element. Excessive or inadequate selenium intake will lead to physiological disorder or diseases. The biological function of selenium is mainly exerted through selenoproteins. The silkworm genome sequencing and annotation have been accomplished in 2004, and the researchers are paying more attention to selenium and selenoproteins in the silkworm now. Selenium-rich silkworms were screened out and the distribution of selenium in vivo was studied in this paper. Furthermore, a selenoprotein gene Bmb022955 predicted previously via bioinformatic methods was confirmed through gene cloning and sequence analysis.To screen selenium-rich silkworms and to study the distribution of selenium in different kinds of silkworms, we measure the selenium contents of 41 kinds of silkworm by hydride generation atomic fluorescence spectrometry (AFS). Meanwhile, we also measure the contents of selenium in the organs of the silkworm Dazao p50, including its head, silk gland, testis, midgutand, fat body and epidermis when the silkworms were reared to 5 ages 3rd days. Results showed that the selenium content was 182.8255±0.9840μg/Kg in the silkworm p50. Different silkworms had different capacities in enriching selenium, among them Chinese silkworms had higher capacity than Japanese ones, and endemic species had higher capacities than mutational species. The selenium content in the testis is highest among all organs measured, suggesting that selenium plays an important role in the propagation of silkworms. The result is according with previously reports on a sperm selenoprotein found in mammals.To experimentally testify the silkworm selenoproteins predicted previously by bioinformatic methods, the selenoprotein gene Bmb022955 was cloned in this paper by reverse transcriptate polymerase chain reaction (RT-PCR) for the reversed transcription and amplification of the gene. The amplified segments were inserted into the pMD20-T vector and the recombinants were transformed, screened and sequenced before they were spliced and used for gene analysis. Results showed that the gene contained only a TGA codon in the open reading frame (ORF) that was terminated with a TAA stop codon. The TAA codon was immediately downstream of the TGA. Relationship between the expression sequence tag (EST) and the genomic sequences were studied by the online software Sim 4, showing that there were two exons and one intron in the gene. One exon had the size of 33 bp, another had 373 bp. The size of the intron was 113 bp. The gene contained a selenocysteine insertion sequences (SECIS) element in its 3′-untranslated region (3'-UTR) at the size of 102 bp. Sequence alignments via the program basic local aligment search tool (BLAST) revealed that the gene we cloned is a new one that have not been embodied in the national center biotechnology information (NCBI) databases up to the present.To infer the biological function of selenoprotein gene Bmb022955, a series of software were used to predict the selenoprotein structures and domains. Results showed that the C- and N-terminal are hydrophobic. There are 68 amino acids formingα-helices, 19 amino acids formingβ-sheets, 45 amino acids forming irregular coils. The selenoprotein can't be a transmembrane protein, because there is not a trans-membrane region, and there is not signal peptide in the selenoprotein.This paper not only screened out the highest selenium-rich silkworm, but also figured out the distribution of selenium in the silkworm. Besides, the most important result is that we clone a gene that meets the requirements for a selenoprotein, which establishes strong bases for further work.