Synthetic plant genes and method for preparation

Abstract

A method for modifying structural gene sequences to enhance the expression of the protein product is disclosed. Also disclosed are novel structural genes which encode insecticidal proteins of B.t.k. HD-1, B.t.k. HD-73, B.t. tenebrionis, B.t. entomocidus, 2 protein of B.t.k. HD-1, and the coat protein of potato leaf roll virus.

Description

[0001] The present invention relates to genetic engineering and more particularly to plant transformation in which a plant is transformed to express a heterologous gene.[0002] 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.[0003] 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. S...

AI Technical Summary

Benefits of technology

[0041] All reports in the field have noted the lower than expected expression of B.t. genes in plants. In general, insecticidal efficacy has been measured using insects very sensitive to B.t. toxin such as tobacco hornworm. Although it has been possible to obtain plants totally protected against tobacco hornworm, it is important to note that hornworm is up to 500 fold more sensitive to B.t. toxin than some agronomically important insect pests such as beet armyworm. It is therefore of interest to obtain transgenic plants that are protected against all important lepidopteran pests (or against Colorado potato beetle in the case of B.t. tenebrionis), and in addition to have a level of B.t. expression that provides an additional safety margin over and above the efficacious protection level. It is also important to devise plant genes which function reproducibly from species to species, so that insect resistant plants can be obtained in a predictable fashion.

[0044] Messenger RNA levels are determined by the rate of synthesis and rate of degradation. It is the balance between these two that determines the steady state level of mRNA. The rate of synthesis has been maximized by the use of the CaMV 35S promoter, a strong constitutive plant expressible promoter. The use of other plant promoters such as nopaline synthase (NOS), mannopine synthase (MAS) and ribulose bisphosphatecarboxylase small subunit (RUBISCO) have not led to dramatic changes in the levels of B.t. toxin protein expression indicating that the effects determining B.t. toxin protein levels are promoter independent. These data imply that the coding sequences of DNA genes encoding B.t. toxin proteins are somehow responsible for the poor expression level, and that this effect is manifested by a low level of accumulated stable mRNA.

Problems solved by technology

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).

In fact, it was suggested that expression of the full length gene is toxic to tobacco callus (Barton et al., 1987).

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