Figure 3.

Expression of cryIC genes in E. coli, Arabidopsis, alfalfa and tobacco. (A) Schematic map of plant transformation vectors. The s-cryIC gene was cloned in an optimized gene expression cassette in pNS6 between promoter (pCaMV35S) and polyadenylylation sequences (pA35S) from the 35S RNA gene of CaMV. The CaMV 35S promoter (18) contains four repeats of the upstream enhancer region (−90 to −418; marked by open boxes). The same CaMV 35S expression cassette is carried by pAEN4, a vector used for transient expression of b-cryIC and s-cryIC genes in Arabidopsis protoplasts. In addition to s-cryIC, vector pNS7 contains a phosphinothricine acetyltransferase gene (pat) under the control of a mannopine synthase (mas) 1′ promoter, and a chitinase AII (chiAII) gene driven by the mas 2′ promoter. The s-cryIC gene of pNS7 was exchanged for the bacterial b-cryIC gene in pGIF1. The structure of pGIF1 is otherwise identical with that of pNS7. ori_T and ori_V, Conjugational transfer and vegetative replication origins of plasmid RK2; LB and RB, the left and right 25 bp border repeats of the T-DNA, respectively; ori_pBR, replication origin of pBR322; Ap^R, bacterial ampicillin resistance gene; pg5, promoter of gene 5; pnos, nopaline synthase promoter; hpt, hygromycin phosphotransferase gene; pA4 and pA7, polyadenylylation signal sequences from the T-DNA encoded genes 4 and 7, respectively; pA_ocs, polyadenylylation signal sequence of the octopine synthase gene. Open arrowheads label plant promoters, and filled boxes mark plant polyadenylylation signal sequences. (B) Expression of b-cryIC and s-cryIC genes in E. coli and Arabidopsis. (Left) The b-cryIC and s-cryIC genes were cloned, respectively, in vectors pET-11a and pET-11d (24), and their expression in E. coli was monitored by immunoblotting with (+) or without (−) isopropyl β-thiogalactopyranoside (IPTG) induction, using a polyclonal anti-CryIC antibody. The lanes contain equal amounts of protein samples (15 μg) from E. coli extracts separated by SDS/PAGE. (Right) Arabidopsis protoplasts were transformed by polyethylene glycol-mediated DNA uptake with pAEN4 (1), and pAEN4-derived vectors carrying the b-cryIC (2) and s-cryIC (3) genes. After transient expression for 48 hr, samples containing 25 μg of soluble protein extract from protoplasts were separated by SDS/PAGE and subjected to immunoblotting. To estimate the amount of CryIC toxin in plant samples, purified CryIC protein of 86 kDa (carrying amino acid residues 1–756) was used as a standard (2 and 20 ng). (C) Screening for CryIC expression in alfalfa calli, carrying the transferred DNA of plant transformation vectors pNS6 and pNS7. Each lane contains 25 μg of soluble proteins from calli. For comparison, Arabidopsis protoplast extract (A.th), shown in lane 3 of B, was loaded as a standard, in addition to control protein extracts prepared from callus tissues of wild-type (wt) nontransformed alfalfa. (D) Screening for CryIC accumulation in leaf tissues of transgenic alfalfa and tobacco plants. Soluble proteins (50 μg) were prepared from NS6 (lanes 1 and 3–6) and NS7 (lane 2) alfalfa transformants, as well as from transgenic tobaccos carrying the NS7 s-cryIC gene construct (lower lanes 1–6). (E) Screening for transcripts of transgenes in leaves of soil-grown alfalfa plants carrying the transferred DNA of pGIF1, pNS6, and pNS7 vectors (three lanes each for NS6 and NS7 reflect three independent transgenic plants). Each lane in the three identical blots contains 20 μg of total RNA. The blots were hybridized, respectively, with s-cryIC, b-cryIC, and chiAII probes labeled to similar specific activity. Although several GIF1 transgenic plants expressing the chiAII gene were found during this screening (data not shown), no expression of the b-cryIC gene was detected in any of the GIF1 transformants. (The positive hybridizations with the b-cryIC probe are due to the partial homology between the synthetic and natural cryIC genes and the difference in the intensity of hybridizations with the s-cryIC and b-cryIC probes reflects differences between these cryIC sequences.)