Plant viral leaders influence expression of a reporter gene in tobacco

Abstract

In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.

Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter

“…The latter could be important for greater message stability or translation efficiency. The first 60 nucleotides of the CaMV leader (from + 1 to the first ATG) stimulate expression of a downstream gene by about 2-fold [14,17]. Similar effect has been reported for the untranslated leader of the rice tungro bacilliform virus promoter [11 ].…”

Untitled

“…Previous studies have shown that activity of the CaMV 35S promoter was enhanced two- to threefold with the addition of the translational enhancer TMV omega element in stably transformed Arabidopsis thaliana plants [29] as well as in transiently transformed tobacco protoplasts [30]. …”

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N -methyltransferase

“…The PEAMT coding sequence was amplified by PCR, using Pfu polymerase (Stratagene) with the forward primer 5Ј-AGCTTCTAGAT T T T TACA ACA AT TACCA ACA ACA-ACA A ACA ACA A ACA ACAT TACA AT TACTAT T TAC-A AT TACA A A A ATGGCCGCT TCAGCTATGG-3Ј, the reverse primer 5Ј-ACGTGGATCCTCACAT T T TCT TG-GCAATG-3Ј, and pREP3-PEAMT (12) as template. The forward primer includes the tobacco mosaic virus ⍀ sequence (18) and alters the sequence context of the start codon as described (19). The reaction product was ligated to pFMV as an XbaI͞ BamHI fragment to yield pFMV-PEAMT.…”

“…The spinach PEAMT coding sequence (12), preceded by the ⍀ translational enhancer (18) and the figwort mosaic virus 34S promoter (17), was cloned into an Agrobacterium binary vector with a glyphosate-resistance marker. This construct was used to transform tobacco plants that were coexpressing spinach CMO (19) and beet BADH (20) and so contained a transgenic GlyBet pathway in their chloroplasts.…”