The introduced cry1F(synpro) gene and its encoded protein
The cry1F(synpro) gene in the GM cotton event is a synthetic gene, combining parts of three different cry genes isolated from Bt. The first portion of the synthetic cry1F(synpro) gene is derived from the native cry1Fa2 gene of Bt var aizawai strain PS81I (nucleotides 1 – 1810, encoding the first 603 amino acid residues). The cry1Fa2 gene was described by Feitelson in 1993 (GenBank accession number AAA22347). The remainder of the gene, encoding the carboxy-terminal portion of the protein is derived from parts of the cry1Ca3 (nucleotides 1811-1917, encoding the amino acid sequence up to residue 639) and cry1Ab1 (nucleotides 1918-3447, encoding the remaining amino acid residues) genes as described for Cry1Ac(synpro). The active core toxin is made up of the cry1F sequence, together with a small portion of the cry1Ca3 gene sequence (see Figure 3) (Narva et al. 2001a). The coding sequence of the chimeric cry1F(synpro) gene has been further modified to achieve optimal expression in plants, without affecting the predicted protein sequence.
Figure Structure of synthetic cry1F(synpro) gene
The cry1F(synpro) gene encodes the full length protoxin, a protein of approximately 130 kDa (Gao et al. 2006). The core protein is an approximately 65 kDa protein.
The synthetic cry1F(synpro) gene encodes a protein toxin, Cry1F(synpro), which is very similar to the native Cry1F protein.
As described for other Cry1 toxins (Bravo et al. 2007), specific proteases cleave off the carboxyl-terminal domain of Cry1F(synpro), as well as approximately 25-30 amino acids from the amino-terminal end, leaving an active protease-resistant core of approximately 600 amino acids. The applicant has stated that putative protease cleavage sites for Cry1F(synpro) are located at R28 or R31 (N-terminal) and R612 or K615. The second site thus may be within the sequence encoded by cry1Ca3, which is predicted to encode 8 or 11 amino acid residues of domain III of Cry1F(synpro). As noted above (Section 5.2.1), domain III is implicated in determining the range of susceptible organisms affected by Cry proteins.
Toxicity/allergenicity of the Cry proteins encoded by the introduced genes
General information on toxicity of the native cry1Ac and cry 1F genes, and the synthetic genes is presented here, with more specific information on the toxicity of the WideStrike™ plant material in Section .
Equivalence of microbially produced proteins
In order to carry out the toxicity, biochemical and insecticidal studies, the applicant modified bacteria (Pseudomonas fluorescens strain MR872) to produce Cry1Ac(synpro) and Cry1F(synpro). These proteins were then compared to those produced in planta. It was concluded from a number of experiments that in planta and microbially produced proteins were biochemically equivalent. These experiments included Western blot and SDS-PAGE analysis, amino-terminal sequencing, glycosylation analysis, peptide mass fingerprinting and matrix-assisted laser desorption ionisation time-of-flight mass spectroscopy.
The predicted amino acid sequences of the bacterially-derived and plant-derived Cry1F(synpro) proteins are identical for the first part of the Cry1F core toxin sequence (amino acids 1-603). For the remaining sequence, the plant derived sequence corresponds to Cry1Ca3 and Cry1Ab1 as expected (Figure 2). However, there are four amino acid differences for the microbially derived sequence compared to the plant derived Cry1F(synpro) protein: an F604L substitution, resulting from a codon change to enable cloning of the chimeric carboxy-terminal part of the protoxin, and Y608S, I624S and I629L within the Cry1Ca3 portion of the carboxy-terminal domain (Gao et al. 2001). These changes render the microbial sequence identical to native Cry1F for this region. Two of the changes (F604L and Y608S) lie within the predicted carboxy-terminal domain of the core toxin, the region thought to be involved in receptor binding.
The applicant provided a study which investigated the equivalency of plant-produced versus microbially-produced Cry1F(synpro) (Herman 2001a), whereby the latter was investigated in a separate study. Three lepidopteran pests important for cotton growing in the USA were included, H. virescence, S. exigua and H. zea. None of these pests are significant in any of the Australian cotton growing regions. Mortality and insect-weight data were collected after six days for the plant-produced protein and after seven days for the microbe-produced protein. Insect weights in the negative controls differed across the studies and it is unknown how the data were adjusted for the different studies.