titipan keduahahahaha

Discussion

We present here new methods for analysing gene expression in transformed plants that we feel will be of general utility. The beta-glucuronidase gene from E. coli has been expressed at high levels in transformed tobacco plants with no obvious ill effects on plant growth or reproduction. The ability to quantitate gene expression through the routine use of enzyme kinetics greatly enhances the precision and resolution of the questions that we can ask. It should be emphasized that the determination of rates of enzyme activity eliminates the vagaries inherent in CAT, NPTII and luciferase assays, and allows accurate determination of a quantity of chimaeric gene product, even over an intrinsically fluorescent background. The fluorometric assay is very specific, extremely sensitive, inexpensive and rapid. Minute quantities of tissue can be assayed with confidence; recently we have measured GUS levels in isolated single cells of transformed plants (R.A. Jefferson et al., in preparation).

Beta-Glucuronidase is very stable in extracts and in cells, with a half-life in living mesophyll protoplasts of – 50 h (unpublished data). Because of this, we feel it is reasonable to interpret GUS levels as indicative of the integral of transcription and translation, rather than the rate. In addition, GUS is not completely inactivated by SDS- PAGE, can tolerate large amino-terminal fusions without loss of enzyme activity (Jefferson et al., 1986, 1987) and can be transported across chloroplast membranes with high efficiency (T.A.Kavanagh et al., in preparation). We feel, therefore, that the system will also be very useful in studying the transport and targeting of proteins, not only in plants, but in other systems that lack intrinsic 3-glucuronidase activity, such as Saccharomyces cerevisiae and Drosophila melanogaster (Jefferson, 1985, 1986).

We have used a commercially available histochemical substrate to demonstrate GUS activity in transformed plant tissue. Other substrates are available and give excellent results (e.g. Jefferson et al., 1987). We emphasize that meaningful interpretation of results of histological analysis in terms of extent of chimaeric gene activity, whether by in situ hybridization methods or by histochemistry, as presented here, is not a trivial or straight-forward matter. There are numerous variables that must be dealt with (reviewed in Pearse, 1972). However, with these cautions, histochemical methods can be very powerful for resolving differences in gene expression between individual cells and cell-types within tissue.

We have observed a distinctly non-uniform distribution of GUS activity in stem sections of several CaMV-GUS transformed plants. Different cell-types within plants are expected to have differing metabolic activity with corresponding differences in rates of transcription and translation, and our results may reflect such a difference. Alternatively, since many of the cells of the phloem have very small cross-sectional areas, the intense dye deposition we see in these regions may simply reflect the greater cell number per unit area. The localization that we observe may also be due to a real difference in the level of expression of the CaMV 35S promoter between cell types. Recently, Nagata et al. (1987) have argued that the CaMV 35S promoter is preferentially active in cells during the S phase of the cell cycle. If this is true, then the pattern of GUS staining that we observe may reflect cell division activity in these cells. This observation is consistent with the proposed role of the 35S transcript of CaMV in viral replication (Pfeiffer and Hohn, 1983). It is also interesting that the other class of plant DNA viruses, the geminiviruses, replicates in the phloem parenchyma (Kim et al., 1978). We conclude therefore that it is no longer adequate to describe the 35S promoter as ‘constitutive’ solely by the criteria of expression in all plant organs, when there may be a strong dependence of transcription on cell-type or cell cycle. This question is being investigated further.

The distribution of GUS activity in the stem sections of plants transformed with rbcS -GUS genes is consistent with data that indicate a requirement for mature chloroplasts for maximal transcription of chimaeric rbcS genes (e.g. Simpson et al., 1986b). Cortical parenchymal cells in the stem contain varying numbers of chloroplasts, while those in the pith and epidermis of the stem rarely contain chloroplasts.

Different cell-types present in each organ contribute differently to the patterns of gene expression and each organ consists of different proportions of these cell-types. We have undertaken to mininimize this effect on quantitative analysis of extracts by suitable choice of a denominator. The parameter that needs to be studied with gene fusions is most often the expression of the gene fusion in each cell. When preparing homogenates from plant organs, the number of cells that contribute to the extract will vary, as will the protein content of each cell and cell-type. The DNA content of the extract will reflect the number of cells that were lysed (Labarca and Paigen, 1980) whereas the traditional denominator, protein concentration, will not. For example, a single leaf mesophyll cell contains much more protein than a single epidermal cell or root cortical cell (R.A.Jefferson et al., in preparation). However, each will have the same nucleus with the same potential to express the integrated gene fusion.

Using this approach, we find that the differential expression of the rbcS-GUS fusion is much more pronounced between immature and mature leaf when we express GUS activity per micro grams of DNA (see Table I). When protein concentration is used as a denominator, the massive induction of GUS activity during leaf maturation is masked by the concomitant induction of proteins involved in photosynthesis.

The observation that the sp. act. of GUS produced by CaMV -GUS fusions is the same in immature and mature leaves when expressed using a protein denominator indicates that the rate of GUS accumulation closely follows the rate of net protein accumulation. The two-fold difference in GUS sp. act. using a DNA denominator illustrates the accumulation of GUS per cell over time. This quantitative analysis, together with our histochemical data, may indicate that the differences between GUS activity in the leaf, stem and root of CaMV -GUS fusion plants could reflect the larger proportion of phloem-associated cells in roots and stems compared to leaves. We feel that the choice of a DNA denominator best reflects the expression per cell and hence is a more accurate reflection of the true regulation of the gene.

Prospects offurther development of the GUS system

There are many important questions arising from the use of currently available gene-transfer techniques in plants that can be addressed with this new technology. Both Agrobacterium-mediated transformation and direct DNA uptake methods result in cells and plants transformed with varying numbers of integrated copies of the foreign DNA and with different sites of integration, resulting in plants expressing different amounts of chimaeric gene product (e.g. Jorgensen et al., 1987; Jones et al., 1987). Previously, analysis of gene expression in transformed plants has been sufficiently laborious to preclude quantitative assays of the large numbers of plants necessary to finally delineate the contributions of local integration sites and copy number to the expression of transformed genes. Using the methods described here, it will be feasible to quantitate the variation that is often ascribed to differing sites and copy numbers of integrations, and obtain statistically significant answers to these questions.

The availability of routine histochemical analysis will greatly facilitate studies of the mechanism of transformation both by Agrobacterium and by direct DNA methods, as well as permitting a more detailed study of developmental regulation. These methods will also allow very rapid and sensitive screening of transformed cells and tissues. Using the indigogenic substrate X-Gluc, we can easily resolve GUS activity from single cells and small cell clusters from suspension cultures.

GUS assay systems lend themselves very well to automation. The existing spectrophotometric and fluorogenic assays, and new assays using fluorogenic substrates that fluoresce maximally at neutral pH (Jefferson, 1985), will allow the use of automatic microtitre plate analysis of very large numbers of samples. The activity of GUS in lysed single cells can be measured with accuracy; using new fluorogenic substrates, we are conducting an analysis of GUS expression in single cells of transformed plants using the fluorescence activated cell sorter (R.A.Jefferson et al., in preparation).

We have also used the GUS fusion system successfully to monitor the transient expression of chimaeric genes introduced into plant cells via electroporation and/or polyethylene glycol treatment (data not shown). We find the sensitivity to be very high, allowing expression to be reliably measured from a very small number of cells (R.A.Jefferson et al., in preparation).

Because of the lack of intrinsic, beta-glucuronidase activity in all plants thus far assayed in our laboratory, and because the synthesis of , Beta-glucuronides can be relatively straightforward, we are pursuing the use of the GUS system to begin ‘fusion genetics’. Due to the complex genomes and long generation times of higher plants, fine scale genetic analysis of complex processes is unfeasible by conventional means. However, by using the GUS system and novel substrates, we may be able to generate positive and negative selections for GUS activity, thereby selecting mutations in the activity of gene fusions, both in planta and in tissue culture.

Finally, new methods and substrates are being developed to allow the GUS system to be used quantitatively and reliably in vivo and in situ.

Materials and Method

Nucleic acid manipulation

DNA manipulations were performed essentially as described (Maniatis et al., 1982). Enzymes were obtained from New England Biolabs, Boehringer or BRL.

Plant transformation and regeneration

Binary vectors containing CaMV-GUS fusions and rbcS-GUS fusions in E.coli MC1022 were mobilized into A.tumefaciens LBA4404 as described (Bevan, 1984). The integrity of the vectors in Agrobacterium was verified by preparing DNA from Agrobacterium immediately before plant transformation using the boiling method of Holmes and Quigley (1981). Leaf discs of N.tabacum, var. Samsun were transformed as described (Horsch et al., 1984) and transformed plants were selected on MS medium (Murashige and Skoog, 1962) containing 100 /g/ml kanamycin. Plants were maintained in axenic culture on MS basal medium, 3% sucrose, 200 Agg/ml carbenicillin and 100 jig/ml kanamycin, at – 2000 lux, 18 h day, 26°C.

Southern blot analysis

DNA was prepared from plants by phenol extraction and ethanol precipitation of plant homogenates, followed by RNase digestion, phenol extraction and isopropanol precipitation. DNA samples (10 ,sg) were digested with restriction endonucleases, electrophoresed in an 0.8% agarose gel and blotted onto nitrocellulose (Maniatis et al., 1982). Filters were hybridized with oligomerprimed, 32P-labelled GUS gene fragments (Feinberg and Vogelstein, 1984) and washed wth 0.2x SSC at 65°C.

Substrates

Substrates used included: 4-methyl umbelliferyl glucuronide (MUG; Sigma M-9130), X-Gluc (Research Organics Inc., Cleveland, OH, USA), resorufin glucuronide (ReG) (Jefferson, 1985; Molecular Probes Inc., Eugene, OR, USA).

Lysis conditions

Tissues were lysed for assays in 50 mM NaH2PO4, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, 10 mM 13-mercaptoethanol (extraction buffer) by freezing with liquid nitrogen and grinding with mortar and pestle with sand or glass beads. Disposable pestles that fit into Eppendorf tubes (Kontes Glass) proved useful for homogenizing small bits of tissue (e.g. leaf). Extracts can be stored at -70°C with no loss of activity for at least 2 months. Storage of extracts in this buffer at -20°C should be avoided, as it seems to inactivate the enzyme.

Fluorometric assay

The fluorogenic reaction is carried out in 1 mM MUG extraction buffer with a reaction volume of 1 ml. The reaction is incubated at 37°C, and 200 ILI aliquots are removed at zero time and at subsequent times and the reaction terminated with the addition of 0.8 ml 0.2 M Na2CO3. The addition of Na2CO3 serves the dual purposes of stopping the enzyme reaction and developing the fluorescence of MU, which is about seven times as intense at alkaline pH. Fluorescence is then measured with excitation at 365 nm, emission at 455 nm on a Kontron SFM 25 spectrofluorimeter, with slit widths set at 10 nm. The resulting slope of MU fluorescence versus time can therefore be measured independently of the intrinsic fluorescence of the extract. The fluorimeter should be calibrated with freshly prepared MU standards of 100 nM and 1 ttM MU in the same buffers. Fluorescence is linear from nearly as low as the machine can measure (usually 1 nM or less) up to 5-10 micro M MU.

A convenient and sensitive qualitative assay can be done by placing the tubes on a long-wave UV light box and observing the blue fluorescence. This assay can be scaled down easily to assay very small volumes (reaction volume 50 d41, terminated with 25 l 1 M Na2CO3 in microtitre dishes or Eppendorf tubes).

If the intrinsic fluorescence of the extract limits sensitivity, it is possible to use other fluorogenic substrates. In particular, ReG has a very high extinction coefficient and quantum efficiency, and its excitation (560 nm) and emission (590 nm) are conveniently in a range where plant tissue does not absorb or fluoresce heavily. In addition, it fluoresces maximally at neutral pH, making it unnecessary to stop the reaction.

DNA concentrations in extracts were determined by measuring the fluorescence enhancement of Hoechst 33258 dye as described by Labarca and Paigen (1980), with the calibrations performed by addition of lambda DNA standards to the extract to eliminate quenching artefacts.

Histochemical assay

Sections were cut by hand from unfixed stems of plants grown in vitro, essentially as described (O’Brien and McCully, 1981), and fixed in 0.3% formaldehyde in 10 mM MES, pH 5.6, 0.3 M mannitol for 45 min at room temperature, followed by several washes in 50 mM NaH2PO4, pH 7.0. All fixatives and substrate solution were introduced into sections with a brief ( 1 min) vacuum infiltration.

Histochemical reactions with the indigogenic substrate, X-Gluc were performed with 1 mM substrate in 50 mM NaH2PO4, pH 7.0 at 37°C for times from 20 min to several hours. After staining, sections were rinsed in 70% ethanol for 5 min, then mounted for microscopy.

Iklan

Tinggalkan Balasan

Isikan data di bawah atau klik salah satu ikon untuk log in:

Logo WordPress.com

You are commenting using your WordPress.com account. Logout / Ubah )

Gambar Twitter

You are commenting using your Twitter account. Logout / Ubah )

Foto Facebook

You are commenting using your Facebook account. Logout / Ubah )

Foto Google+

You are commenting using your Google+ account. Logout / Ubah )

Connecting to %s


%d blogger menyukai ini: