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Multiple Cry
genes for avoiding build-up of resistance among insects to Bt
toxins: Learning from Bt-rice
I. Altosaar & M.
Zaidi.
To promote the sustainable use of Bt
crops, Bt cultivars should only be released if they contain two Bt
toxins, both expressed at a high dose in the transgenic plant. If
insects that are able to survive on a plant with one high-dose toxin are
rare, then insects that are able to survive on plants with two high-dose
toxins will be even rarer. If such insects must be homozygous for
resistance alleles for two different genes, and if the frequency of the
allele for resistance of each gene is 10-3, then insects of
the genotype R1R1R2R2 will
occur at a frequency of only 10-12, i.e., 1 out of 1
trillion. Because such insects will be very rare, fewer susceptible
insects will be needed to ensure that resistant insects do not mate with
each other. Fewer refuge fields may then be necessary. Our lab has
synthesized two plant codon optimized coding sequences for the high-dose
expression of Cry1Ab and Cry1Ac. These have been deployed in one-toxin
Bt-rice field trials with success for the time being but more synthetic
sequences are needed to test if pyramiding Cry genes can prevent
resistance from occurring. We are adding three new Cry genes to our
research repertoire, two 1C sequences varying in G-C content (45%, 65%)
and a plant-optimized 2A coding sequence (61%). For example, the
re-synthesis of Cry1C coding sequence, on assembly still contained 16
basepair errors. The clone (pTY185.1) containing the 1.9 kb coding
sequence was cut into two halves, each half subcloned separately into a
cloning vector (pPCR script and pGEM4Z for their convenient restriction
sites). The repair of
mistakes was done by site directed mutagenesis using the “Quick Change
Mutagenesis™” kit from Stratagene. Primer pairs were complementary oligonucleotide sequences
between 25 and 40 bases. They
were designed such that 10-15 bases of correct sequence flanked the
desired mutation site on either side. During the course of this repair
three new errors appeared. These
were the errors incorporated by Pfu Turbo DNA Polymerase used in the
repair process. Luckily, swapping them with the original clone,
pTY185.1, could repair these new errors.
Today, all errors have been removed and their verification
through sequence analysis is in progress. The two halves of the coding
sequence will be assembled together and cloned into an expression
cassette for E. coli (Codon
plusä from Stratagene) and the bioactivity of the
expressed protein will be tested against target podborers and stemborers.
Plant transformation will target these coding sequences to cowpea,
sorghum and tropical maize.
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