Novel plant transformation vectors containing the superpromoter

Abstract

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2' (mas2') transcriptional activating element plus minimal promoter. We tested a superpromoter-beta-glucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2', and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.

Figure 1.

Maps of the pUC119-based pMSP series of plasmids. pExxxx numbers at the right of each plasmid map indicate Gelvin laboratory stock numbers. amp^r, Plasmid confers resistance to ampicillin upon host bacteria; Aocs, ocs transcriptional activating element; AmasPmas, mas2′-activating and promoter elements; ags-ter, poly(A) addition signal from the agropine synthase gene; ADHin, intron from the maize alcohol dehydrogenase I gene. Restriction endonuclease sites: A, ApaI; B, BamHI; Bc, BclI; Bg, BglII; Bx, BstXI; H, HindIII; N, NotI; Nc, NcoI; P, PstI; RI, EcoRI; SI, SacI; SII, SacII; S, SalI; Sm, SmaI; Sp, SpeI; Xb, XbaI; X, XhoI. Restriction endonuclease sites within parentheses are not unique to the plasmid. The sequence below pMSP-2 shows the TL reading frame following the NcoI site that is at the 3′ end of the TEV TL leader sequence.

Figure 2.

Maps of the T-DNA binary vectors based upon the pMSP series of plasmids. pExxxx numbers at the right of each plasmid map indicate Gelvin laboratory stock numbers. kan^r, Plasmid confers resistance to kanamycin upon host bacteria; Pnos, nos promoter; tAg7, poly(A) addition signal for T-DNA gene 7; hptII, gene conferring resistance to hygromycin; nptII, gene conferring resistance to kanamycin; bar, gene conferring resistance to phosphinothricin/Basta/Bialophos. Other symbols and restriction endonuclease sites are as in the legend to Figure 1. The bar gene contains KpnI and SalI sites; therefore, these sites are not unique to T-DNA binary vectors containing the bar gene (indicated by [K] and [Sl]). A, T-DNA binary vectors based on the pMSP-1 series of plasmids. B, T-DNA binary vectors based upon the pMSP-2 series of plasmids. C, T-DNA binary vectors based on the pMSP-3 series of plasmids.

Figure 3.

GUS activity (pmol/min/mg protein) directed by the superpromoter in various tobacco and maize tissues and organs. L1, L2, etc., Leaf number; MF, maize flower; R, root; R-o, old root section; R-y, young root section; S, stem section. Numbers in parentheses indicate the number of samples analyzed for each tissue or organ.

Figure 4.

Comparison of the relative strengths of various promoters in the leaves and roots of transgenic maize plants. Ubi, Maize ubiquitin promoter; n, number of individual transgenic plants analyzed. The large sd most likely reflects the position effect of T-DNA integration upon transgene activity in independent transgenic events.

Figure 5.

Constructions used for maize BMS protoplast transfection experiments. nos, Nopaline synthase poly(A) addition signal sequence; pUbi, maize ubiquitin promoter.

Figure 6.

Comparison of the relative strengths of various promoters (lacking or containing introns in the gusA gene) in transiently transfected maize BMS protoplasts. Numbers represent the average of four independent experiments with sd. C, Control construction lacking a promoter; mas, mas2′ promoter; 35S, CaMV 35S promoter; sp, superpromoter.

Figure 7.

Effect of the maize ubiquitin intron upon promoter activity in stably transformed maize leaves. n, Number of individual transgenic plants analyzed; −, constructions lacking the maize ubiquitin intron; +, constructions containing the maize ubiquitin intron. The large sd in the data from maize plants containing the maize ubiquitin promoter plus intron construction most likely reflects the position effect of T-DNA integration upon transgene activity in independent transgenic events.