Synthetic plant genes and method for preparation

#The present invention relates to genetic engineering and more particularly to plant transformation in which a plant is transformed to express a heterologous gene.

#Although great progress has been made in recent years with respect to transgenic plants which express foreign proteins such as herbicide resistant enzymes and viral coat proteins, very little is known about the major factors affecting expression of foreign genes in plants. Several potential factors could be responsible in varying degrees for the level of protein expression from a particular coding sequence. The level of a particular mRNA in the cell is certainly a critical factor.

#The potential causes of low steady state levels of mRNA due to the nature of the coding sequence are many. First, full length RNA synthesis might not occur at a high frequency. This could, for example, be caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription. Second, full length RNA could be produced but then processed (splicing, polyA addition) in the nucleus in a fashion that creates a nonfunctional mRNA. If the RNA is properly synthesized, terminated and polyadenylated, it then can move to the cytoplasm for translation. In the cytoplasm, mRNAs have distinct half lives that are determined by their sequences and by the cell type in which they are expressed. Some RNAs are very short-lived and some are much more long-lived. In addition, there is an effect, whose magnitude is uncertain, of translational efficiency on mRNA half-life. In addition, every RNA molecule folds into a particular structure, or perhaps family of structures, which is determined by its sequence. The particular structure of any RNA might lead to greater or lesser stability in the cytoplasm. Structure per se is probably also a determinant of mRNA processing in the nucleus. Unfortunately, it is impossible to predict, and nearly impossible to determine, the structure of any RNA (except for tRNA) in vitro or in vivo. However, it is likely that dramatically changing the sequence of an RNA will have a large effect on its folded structure. It is likely that structure per se or particular structural features also have a role in determining RNA stability.

#FIG. 1 illustrates the steps employed in modifying a wild-type gene to increase expression efficiency in plants.

#FIG. 2 illustrates a comparison of the changes in the modified B.t.k. HD-1 sequence of Example 1 (lower line) versus the wild-type sequence of B.t.k. HD-1 which encodes the crystal protein toxin (upper line).

#FIG. 3 illustrates a comparison of the changes in the synthetic B.t.k. HD-1 sequence of Example 2 (lower line) versus the wild-type sequence of B.t.k. HD-1 which encodes the crystal protein toxin (upper line).

#The present invention provides a method for preparing synthetic plant genes which genes express their protein product at levels significantly higher than the wild-type genes which were commonly employed in plant transformation heretofore. In another aspect, the present invention also provides novel synthetic plant genes which encode non-plant proteins. For brevity and clarity of description, the present invention will be primarily described with respect to the preparation of synthetic plant genes which encode the crystal protein toxin of Bacillus thuringiensis (B.t.). Suitable B.t. subspecies include, but are not limited to, B.t. kurstaki HD-1, B.t. kurstaki HD-73, B.t. sotto, B.t. berliner, B.t. thuringiensis, B.t. tolworthi, B.t. dendrolimus, B.t. alesti, B.t. galleriae, B.t. aizawai, B.t. subtoxicus, B.t. entomocidus, B.t. tenebrionis and B.t. san diego. However, those skilled in the art will recognize and it should be understood that the present method may be used to prepare synthetic plant genes which encode non-plant proteins other than the crystal protein toxin of B.t. as well as plant proteins (see for instance, Example 9).

#The expression of B.t. genes in plants is problematic. Although the expression of B.t. genes in plants at insecticidal levels has been reported, this accomplishment has not been straightforward. In particular, the expression of a full-length lepidopteran specific B.t. gene (comprising DNA from a B.t.k. isolate) has been reported to be unsuccessful in yielding insecticidal levels of expression in some plant species (Vaeck et al., 1987 and Barton et al., 1987).

#It has been reported that expression of the full-length gene from B.t.k. HD-1 was detectable in tomato plants but that truncated genes led to a higher frequency of insecticidal plants with an overall higher level of expression. Truncated genes of B.t. berliner also led to a higher frequency of insecticidal plants in tobacco (Vaeck et al., 1987). On the other hand, insecticidal plants were provided from lettuce transformants using a full-length gene.