The storage proteins that are contained in the protein bodies of endosperm tissue constitute approximately 60% of the protein in the mature kernel. Approximately 80% of these proteins are zeins encoded by 4 gene subfamilies; the remaining protein body proteins consist mainly of the zein associated proteins (ZAPs) encoded by a number of gene subfamilies (Rubenstein and Geraghty, Adv. in Cereal Sci. Tech. Vol VIII, Y. Pomeranz, ed., AACC, St. Paul, 1986). ZAP gene subfamilies contain few genes. This simplifies their molecular analysis and suggests that these genes contain strong promoter sequences. The ZAP3 subfamily of the maize inbred W22 consists of two genes (Z36A and Z36B) encoding proteins of about Mr 27,000. These genes have been isolated on a single cosmid clone and characterized in our lab (Geraghty, thesis, Univ. of MN, 1985). They constitute a tandem duplication of about 12 kilobases (kb) that is separated by 2.3 kb of unduplicated sequence. The genes are functional since cDNA clones have been isolated for both. The 5' sequences for these genes are virtually identical over a length of 3 kb.
Expression vectors have been constructed to study the biological activity of the untranslated 5' sequences of the ZAP3 genes. We have used site-directed mutagenesis to convert the base sequence of the ZAP3 gene Z36B initiation codon into the recognition sequence for the restriction endonuclease NcoI with no change being made in the gene's 5' untranslated region. The Z36B 5' region, with its presumed transcriptional control sequences, was then ligated to the protein coding regions of two reporter genes, chloramphenicol acetyl transferase (CAT) and beta glucuronidase (GUS). These reporter genes contain the transcriptional termination sequence from the nopaline-synthase (NOS) gene. The CAT gene was also modified to contain an initiator-codon-centered NcoI site; a GUS construct with an NcoI site was already available. The use of NcoI sites in this way allows for the construction of vectors retaining the exact relationship of the Z36B 5' sequences to their native initiation codon.
These vectors were delivered directly into developing endosperm by the use of DNA-coated gold particles accelerated by the high-voltage-induced explosion of a water droplet (McCabe et al.,Bio\Technology 6:923, 1988). Field or greenhouse grown plants of W22 were pollinated, and the entire cob harvested 12 to 20 days after pollination. The cobs were husked and surface sterilized, and endosperm target explants were prepared by excising cob sections containing 4 rows of 6 kernels each. The developing endosperm tissues were made accessible to particle bombardment by slicing off the top 1 mm of each kernel. The cob tissue was then trimmed to allow the explant to lie flat on the bottom of a small petri plate, and held in place with agar solidified nutrient medium.
Our work with the CAT reporter gene has utilized both the Z36B 5' sequence-CAT gene fusions described above and previously available constructs in which the CAT gene is fused to the strong, but non-tissue-specific, cauliflower mosaic virus (CaMV) promoter. Two days after bombardment the upper 2 mm of endosperm tissue was extracted and assayed with a standard CAT assay utilizing a radioactive substrate (14C-chloramphenicol). CAT constructs containing the CaMV 35S promoter yielded detectable enzyme activity; those utilizing the Z36B 5' sequences gave a signal at least ten times as strong. We interpret these results as a good initial indication that these fusions are being expressed in a manner related to their normal tissue specific expression, despite the alien mechanism by which they were introduced.
Preliminary experiments with the GUS reporter gene have utilized the vector pBI221, which contains the GUS gene fused to a CaMV 35S promoter. Endosperm tissue was bombarded with particles coated with this DNA and subsequently exposed to staining with a histological substrate (x-GUS). An insoluble blue product accumulated which allowed the detection of cells in which GUS gene expression occurred. The blue product was seen both in cells of the aleurone layer of the endosperm as well as in cells of the outer regions of the endosperm. The only non-endosperm tissue exposed in this particular experiment, the developing pericarp, did not show GUS expression, perhaps because the strength of its cell walls prevented efficient particle penetration. Expression was also not detected in the inner regions of the endosperm tissue. We do not yet know whether this was due to some unsuitability of this tissue for the CaMV 35S promoter, or whether the thin-walled cells in this area are too weak to withstand bombardment. Each tissue type shows maximal expression of foreign DNA over a narrow range of particle velocities (controlled in this case by adjusting the voltage used to explode the water drop). At too low a voltage, little or no expression is seen, presumably because the tissue's cell walls are not penetrated by the particles. Higher voltages fail to increase (and may even decrease) expression, perhaps because cell damage by the particles offsets any additional penetration.
The GUS activity of endosperm tissue
bombarded with pBI221 DNA was also quantified by mixing an extract prepared
in the same way as for the CAT assay with the substrate analog 4-methylumbelliferyl
glucuronide, and measuring the product on a fluorometer. We expect to use
our Z36B 5'-GUS gene constructs to increase the signal of the fluorometric
assay, as well as to test for the tissue-specific expression of these vectors
via our histological assay -- the x-GUS product should be seen in the cells
of the storage endosperm, but not in the aleurone or other kernel tissue
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