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The cah genes (wild-type and mutants) were amplified using gene-specific
primers and recombinant plasmids, pQ-cah (wild-type), pQ-cahMutK and pQ-
cahMutL (mutant- type) were ~ O n s t ~ c t e d using E. coli expression vector, pQE-60.
The cah genes were overexpressed by IPTG induction of E. coli carrying wild-type
pQ-cah as well as mutants, pQ-cahMutK and pQ-cahMutL as His-tag fusion protein
and the Cah enzyme was purified using ~ i " column. Crude enzyme preporations of
pQ-cah, pQ-cahMutK and pQ-cahMutL showed similar level of cyanamide hydratnse
activity (1090, 1060 and 1000 Ulmg) respectively and purified enzyme exhibited
5-
fold higher activity than the crude enzyme preparation. SDS-PAGE and Western blot
analyses of the purified enzyme preparations with cah antibody (1:1000 dilution)
confirmed the presence of 27.7 kDa Cah protein.
The mutated cah gene devoid of HindIII site was successfully cloned along
with Ubil promoter and nos terminator into pCAMBIA 1300 vector and the
recombinant plasmid, pCAMBIA-cah was constructed. The gene cassettes harboring
cah marker, chitinase and LTP were constructed by three-fragment ligation of the
dephosphorylated vector (pCAMBI.4-cah) digested with HindIII. Ubil promoter
(HindlII-BamH1 fragment) and Bar2chi or Ltp 3FI gene (BamH1-Hind111 fragment).
Freeze-thaw method facilitated the transformation of pCAMBIA-cah-Ltp 3F1
into Agrobacterium LBA4404. Agrobacrerium-mediated gene transfer (leaf-disc
method) was followed for gene transformation in tobacco. Untransformed leaf disc
when cultured on tobacco shoot selection media with cyanamide failed to regenerate.
Co-cultivated leaf discs, produced shoots after 2 weeks on MS medium amended with
5
mM
cyanamide and putative transformants formed roots by 2 weeks.
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