ABSTRACT
PTEN, a tumor suppressor gene, suppresses cell survival, growth, apoptosis, cell migration and DNA damage repair by inhibiting the PI3K/AKT signaling pathway. In this study, the full-length Litopenaeus vannamei PTEN (LvPTEN) cDNA was obtained, containing a 5'UTR of 59bp, an ORF of 1269bp and a 3'UTR of 146bp besides the poly (A) tail. The PTEN gene encoded a protein of 422 amino acids with an estimated molecular mass of 48.3 KDa and a predicted isoelectric point (pI) of 7.6. Subcellular localization analysis revealed that LvPTEN was distributed in both cytoplasm and nucleus, and the tissue distribution patterns showed that LvPTEN was ubiquitously expressed in all the examined tissues. Vibrio alginolyticus challenge induced upregulation of LvPTEN expression. Moreover, RNAi knock-down of LvPTEN in vivo significantly increased the expression of LvAKT mRNA, while reducing that of the downstream apoptosis genes LvP53 and LvCaspase3. LvPTEN knock-down also caused a sharp increase in cumulative mortality, bacterial numbers, and DNA damage in the hemolymph of L. vannamei following V. alginolyticus challenge, together with a sharp decrease in the total hemocyte count (THC). These results suggested that LvPTEN may participate in apoptosis via the PI3K/AKT signaling pathway in L. vannamei, and play an important role in shrimp innate immunity.
Subject(s)
Immunity, Innate/immunology , PTEN Phosphohydrolase/genetics , Penaeidae/immunology , Vibrio Infections/immunology , Vibrio alginolyticus/immunology , Amino Acid Sequence , Animals , Apoptosis/immunology , Base Sequence , Caspase 3/biosynthesis , Cloning, Molecular , DNA Damage/genetics , DNA, Complementary/genetics , Hemocytes/immunology , Hemolymph/cytology , Hemolymph/microbiology , PTEN Phosphohydrolase/immunology , Penaeidae/genetics , Penaeidae/microbiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/biosynthesis , RNA Interference , RNA, Small Interfering/genetics , Sequence Alignment , Tumor Suppressor Protein p53/biosynthesis , Vibrio Infections/microbiologyABSTRACT
We have prepared four diastereomers of TT dinucleotides containing an isoxazoline moiety and four dodecanucleotides incorporating these TT dinucleotides in the middle of the DNA sequence. We also have determined the melting temperatures of these modified oligonucleotides (Tm values) by measuring change in UV absorbance.