Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gene
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Date
2019-10
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Publisher
AAU, Jorhat
Abstract
Biosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance
to pod borers is a regulatory requirement and mandatory to document before releasing in the
field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized
for the presence and expression of the transgene in their advanced generations, biosafety
assessments and transcript profile were studied. The homozygous lines were selected for
comparative nutritional equivalence assay. Biochemical estimations of major nutritional
components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that
the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the
seed composition is similar or within the range reported, previously. Seed protein quality was
investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry
(MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage
proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme
system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin
hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic
lines were digested at a similar rate and enzyme-resistant peptides were not observed
in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt
chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The
differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold changeā„2)
frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated)
proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their
expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for
potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be
developmentally regulated or stress-related which was expected because absolute synchronous
growth and development even under a controlled environment are challenging. A few
upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent
dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B,
gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein
12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated
(>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the
nutritional equivalence or transcript profile of transgenic Bt chickpeas.