Alternative splicing, expression patterns and promoter characters of vasa-like gene from the silkworm, Bombyx mori.
VASA is considered to be one of the most reliable molecular marker of germ cells. In order to study the Bombyx mori                         vasa-like gene (Bmvlg), the cDNAs of Bmvlg were cloned and sequenced, and the results showed that the Bmvlg gene from the fifth instar larval testes had four alternative splicing isoforms. The open reading frame (ORF) of the longest isoform was composed of 1,806 nucleotides encoding 601 amino acid residues and contained some known conserved domains. The other three isoforms had complete ORF, suggesting that the Bmvlg gene had several alternative splicing forms, completely different from that of Drosophila                         melanogaster. The results of sequencing demonstrated that the Bmvlg gene promoter had several elements conserved in eukaryotic and gonadal tissue-specific promoters. To detect the specificity of the Bmvlg promoter, a transient expression vector pSK-vlg-DsRed-polyA with a red fluorescent protein gene (DsRed), controlled by the Bmvlg promoter and a vector pIZT/V5-His-vlg-DsRed containing a Bmvlg fused with DsRed driven by the Bmvlg promoter, was constructed, respectively. Red fluorescence could be observed in some transfected BmN cells derived from silkworm ovaries and in the eggs injected with the vector pSK-vlg-DsRed-polyA, but red fluorescence could not be detected in the tissues of silkworm larva, after the transient expression vector was injected into blood, suggesting the Bmvlg promoter had gonadal tissue specificity. The transcription levels of Bmvlg in gonads of the fourth and fifth instar larvae were determined by fluorescent quantitative PCR, and the results revealed that the expression level of the Bmvlg gene in testes was slightly higher than that in ovaries. The expression levels of Bmvlg were lower in the fourth instar larva than that in the fifth instar larvae. Moreover, subcellular localization experiments showed that Bmvlg mainly existed in cytoplasm. These results provided new clues for understanding the function of the Bmvlg gene.
School of Biology and Basic Medical Science, Soochow University, No.199 Ren-ai Road, Suzhou, 215123, Jiangsu, People's Republic of China.
Guangli
G
Cao
Yaoyao
Y
Zhang
Renyu
R
Xue
Yuexiong
Y
Zhu
Yuhong
Y
Wei
Xiaojian
X
Zheng
Chengliang
C
Gong
D017398:Alternative Splicing / Q000235:genetics*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D017124:Conserved Sequence / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017344:Genes, Insect / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000166:cytology / Q000201:enzymology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D014158:Transcription, Genetic
D019476:Insect Proteins; D053487:DEAD-box RNA Helicases