Isolation and temporal endospermal expression of γ-kafirin gene of grain sorghum (Sorghum bicolor L. moench) var. M 35-1 for introgression analysis of transgene

A gamma-kafirin gene of Sorghum bicolor L var. M35-1 was isolated using a PCR based approach by designing specific primers. The primers gave highly reproducible amplification. The amplicons were then cloned and sequenced. Nucleotide sequences were subjected to the homology search and a comparative analysis was done with other prolamins. Amplified γ-kafirin gene (accession no. AY566298 and AY566299) showed 99% homology with a γ-kafirin gene of Sorghum vulgare. Compared to Sorghum vulgare, only 3 extra bases were present in Sorghum bicolor at position 40 nucleotide bases downstream of transcription initiation site. These sequences were related with prolamins of other genera, i.e. coix and maize with 84% sequence homology. The deduced protein sequence of γ-kafirin gene of S. bicolor (accession no. AAS73290) gave significant similarity of 99%, 79% and 77% with gamma-kafirin protein of S. vulgare, γ-zein and γ-coixin proteins, respectively. All cysteine residues of γ-kafirin were highly conserved. Probable secondary structure of gamma-kafirin protein was predicted using the online PSIPRED server. Study of temporal expression of the γ-kafirin gene is needed for analysing the expression pattern of introgressed gene(s) driven by its promoter. Temporal expression of gamma-kafirin gene in endosperm studied by semi quantitative RT–PCR with specifically designed primers showed that γ-kafirin expression started at 7 DAP (days after pollination). There was statistically a non-significant increase in expression up to 14 DAP, thereafter expression increased significantly (at the 5% level) and reached a maximum at 21 DAP. Expression of γ-kafirin started decreasing after 21 DAP and a very low expression was detected at 28 DAP.