Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes.
Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 μm. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.
Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan; JST, ERATO, Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Yoko
Y
Mizuta
Tetsuya
T
Higashiyama
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005964:Glucosyltransferases / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D053205:Pollen Tube / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D011494:Protein Kinases / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects
D029681:Arabidopsis Proteins; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; C585562:CalS5 protein, Arabidopsis; D005964:Glucosyltransferases; C546340:ANXUR1 protein, Arabidopsis; C546341:ANXUR2 protein, Arabidopsis; D011494:Protein Kinases; D019204:GTP-Binding Proteins; C501663:ROP1 protein, Arabidopsis
Arabidopsis thaliana; antisense oligodeoxynucleotides; gene inhibition; pollen tube; sexual reproduction; technical advance