Characterization of the Histone Modification Protein Component, Sgf73 for Boundary Formation and Analysis of High Erucic acid Transgene in Double Haploid Rapeseed Population

The eukaryotic nucleus is organized into structurally and functionally distinct domains that are often located in close proximity to one another. The euchromatin form of DNA is transcriptionally active and maintains an open chromatin structure via the actions of promoter, enhancers, and locus control region sequences. By contrast, heterochromatin is composed of a condensed chromatin structure that is generally transcriptionally repressed. Chromatin barriers prevent silenced chromatin domains from spreading into active domains. In S. cerevisiae telomeres, HML, HMR and ribosomal DNA (rDNA) are transcriptionally silent regions. The Sir (Silent Information Regulator) protein complex, consisting of Sir2, Sir3 and Sir4, plays a key role in transcriptional repression. The regulation of gene expression is crucial for proper development and survival of all organisms; chromatin structure is a central regulator of gene expression. Histone modifications, such as acetylation and ubiquitination, play a crucial role to facilitate a number of cellular events, including gene regulation. Histones acetylation is largely associated with open chromatin structure to support the entry of transcriptional machinery to genomic loci for activation, whereas ubiquitination of histone has been linked to gene activation and repression both. The budding yeast Saccharomyces cerevisiae contains active and inactive chromatin separated by boundary domains. Previously, we used genome-wide screening to identify 55 boundary-related genes. In this study, firstly I have focused on Sgf73, a boundary protein that is a component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) complexes. These complexes have histone acetyltransferase (HAT) and histone deubiquitinase activity, and Sgf73 is one of the factors necessary to anchor the deubiquitination module. Domain analysis of Sgf73 was performed and the minimum region (373–402 aa) essential for boundary function was identified. This minimum region does not include the domain involved in anchoring the deubiquitination module, suggesting that the histone deubiquitinase activity of Sgf73 is not important for its boundary function. Next, Sgf73-mediated boundary function was analyzed in disruption strains in which different protein subunits of the SAGA/SLIK/ADA complexes were deleted. Deletion of ada2, ada3 or gcn5 (a HAT module component) caused complete loss of the boundary function of Sgf73. The importance of SAGA or SLIK complex binding to the boundary function of Sgf73 was also analyzed. Western blot analysis detected both the full-length and truncated forms of Spt7, suggesting that SAGA and SLIK complex formation is important for the boundary function of Sgf73. Secondly, in the present study, 90 DH lines derived from F1 plants of the cross Ld-LPAAT- Bn-fae1 over expressing transgenic rapeseed plants with high erucic acid and low polyunsaturated fatty acid (HELP) plant material were analyzed. Erucic acid (22:1) is a major component in the seed oil of wild type rapeseed. But this erucic acid is toxic for consumption. However, from the last decade, high erucic acid rapeseed (HEAR) cultivars have regained interest for industrial purposes. Here, I study the inheritance of erucic acid content in the segregating transgenic DH population and evaluate the variation and heritability for different phenological and quality traits such as: days to flowering and maturity, oil, protein and different fatty acids content in the DH population. Large variation was found for erucic acid content in DH population varied from 34.6% to 59.1%. Genetic variance components were large and significant for all traits. The segregation pattern of erucic acid content showed 1:3:3:1 separation suggesting erucic acid content was controlled by the alleles of three loci in the DH population. Transgene Ld-LPAAT-Bn-fae1 showed negative effect (-2.7%) to change the erucic acid content in the DH population. This result suggesting that there was no effect of Bn-fae1 for increasing the erucic acid content or the absence of β-ketoacyl-CoA synthase (KCS) activities required for initiating fatty acid elongation from 18:1 to 22:1. On the other hand PUFA (Poly Unsaturated Fatty Acid) had effect to increase the erucic acid content up to 3.7%. In presence of transgene Ld-LPAAT showed limited activities to produce trierucin (8% instead of probable 20%) in the selected best DH line. The selected DH lines showed increase in erucic acid content (59.1%) compared to parental lines TNKAT (46.1%) and HELP (50.4%). Highest amount of erucic acid might be achieved due to introgression of PUFA alleles with those of the strong alleles of the indigenous erucic acid loci, because transgene showed negative effect on erucic acid. There was no adverse effect of the high erucic content on other phenological traits in the selected best DH line. In conclusion the result of this study showed that erucic acid is inherited by alleles of three loci. The variation of erucic acid has been achieved due to the combination of alleles of two indigenous erucic acid loci with alleles of PUFA. Low in PUFA content could contribute markedly to increase erucic acid than the transgene construct present in DH lines. The selected DH line having 59% erucic acid could be source material for crossing to other high erucic acid transgenic line to increase the erucic acid content