Sub cellular localization and generation of overexpression lines in barley for TaHSFA6b
Chauhan et al. (2013) have shown that TaA6b has all the characteristic features of a class A Hsf such as DNA binding domain, HR A/B region, C-terminal AHA type transactivation domain and a predicted nuclear localization signal. To assess the subcellular localization of TaA6b, we made a C-terminal GFP fusion construct and transiently expressed it inNicotiana benthamiana leaves. In Figure 1, it can be seen that the distribution of GFP is uniform in nucleus and cytoplasm in both control and heat stress (HS) conditions, suggesting that TaA6b protein is localized in both nucleus and cytoplasm regardless of the presence of HS, it should be noted that it is absent from the nucleolus.
Previously, TaHSFA6b has been shown to increase heat stress tolerance in Arabidopsis upon over expression. In the present investigation we have over expressed TaHSFA6b in barley by cloning TaHSFA6b under control of ZmUbi promotors. The transformation was done by biolistic method and a total of 40 lines were generated by using immature embryos of Golden promise as per the protocol described by Harwood and Smedly (2009). Transgenic lines were confirmed by PCR amplification of complete cDNA clone of TaHSFA6band HPT gene (Supplementary Figure S2A and B). Further, PCR product of 4 different lines viz. 1, 2, 6 and 8 were sequenced and they all were found to have the wheat gene i.e TaHSFA6b . Over expression of TaHSFA6b was confirmed in T1 generation by RT-PCR. Most of the transgenic lines were found to constitutively expressTaHSFA6b revealed by RT-PCR (Supplementary Figure S3). For all further analysis, plants were grown in controlled environment and other analysis were done on the plants of T2 generation in 3 different transgenic lines viz. AP2-1, AP2-2 and AP6-2.