A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants

Abstract

Tobacco (20) to give pl9S-C (Fig. 1A).An 850-bp Sac I-EcoRV fragment from pUC-19S-CAT, comprising the CAT sequence and the 3' proximal 65 bp of the 19S promoter sequence (including all 12 bp of the 19S 5' untranslated region), was cloned in the antisense orientation next to the nos 3' region in a pUC18 derivative to give pACN9. A 1.95-kilobase (kb) Pvu II fragment from pAGS120 (21) containing a chimeric HPT gene was inserted downstream from the antisense CAT-nos 3' sequence in pACN9.The rbcS promoter (-1040 to + 169) from Nicotiana plumbaginifolia, isolated as a 1.2-kb EcoRI-Pvu II fragment from pNPSS8B (a gift from N.-H. Chua), was cloned into the EcoRI and Sma I sites of pBinl9 to give pBrbcS. A 3.3-kb Xba 1-HindlIl fragment containing the antisense CAT-nos 3' fusion and the chimeric HPT gene was cloned into Xba I-and HindIII-linearized pBrbcS to give pRAC-H (Fig. 1B).An EcoRI-blunted HindIII fragment from pUC13/35SCAT (22) containing the CaMV 35S promoter (-941 to + 9) was cloned into the EcoRI and Sma I sites of Bluescript M13+ (Stratagene, San Diego) to give pBS-35S. A 1300-bp Xba IHincII fragment from pACN9 containing the antisense CATnos 3' sequence was cloned into the Xba I and blunted Sac II sites of pBS-35S to give pBS-35S-AC. The HPT coding sequence, isolated as a 1070-bp Xho II fragment from pAGS120, was inserted between the 35S promoter and antisense CAT-nos 3' sequence of pBS-35S-AC to give

Antisense RNA inhibition of ?-glucuronidase gene expression in transgenic tobacco plants

“…Rothstein et al [21] also reported that antisense RNA from a partial NOS sequence was sufficient for successful inhibition. Conversely, an antisense transcript generated by 172 nucleotides of the 5' terminal CAT sequence was found to be less effective in suppressing CAT activity than an antisense transcript from the fulllength CAT sequence [5]. In our study, antisense RNA from the entire GUS coding sequence was found to be effective.…”

“…Previous studies investigating antisense RNA inhibition in transformed plants have consistently relied on a promoter regulating the transcription of the antisense gene which was different from the promoter of the target gene [5,21,23,24,28]. The promoter chosen to drive the antisense transcript was usually stronger than that of the target gene, so that relatively high levels of antisense RNA could be produced.…”

“…More recently, antisense RNA inhibition was shown to stably repress the constitutive expression of the nopaline synthase (NOS) gene [21,23] and the • CAT gene [5] in transgenic tobacco plants. These experimental systems mimic more closely the conditions under which nuclear genes are stably expressed in plants.…”

Interference with brome mosaic virus replication by targeting the minus strand promoter

“…Ahlquist et al (1984) made use of a hybrid arrest strategy to localize the site of BMV replicase recognition by use of cDNAs complementary to the 3' ends of viral RNAs. Recently there have been a number of attempts to apply the antisense strategy both to gene expression in plants (Delauney et at., 1988;teller et al, 1991) and to interfere with virus replication (Rezaian et al, 1988;Cuozzo et al, 1988;Hemenway et al, 1988;Powell et al, 1989).…”

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

“…Delauney etal. [7] demonstrated that fusion of an antisense CAT gene to a hygromycin resistance gene inhibited CAT expression while the antisense CAT alone was ineffective. Northern blot analyses indicated that this was due to an increased stability of the chimeric transcript.…”