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Molecular Characterization and Functional Analysis of a Gene BnMicEmUP Upregulated in Embryogenic Brassica napus Microspore Cultures

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Title: Molecular Characterization and Functional Analysis of a Gene BnMicEmUP Upregulated in Embryogenic Brassica napus Microspore Cultures
Author: Shahmir, Fariba
Department: Department of Plant Agriculture
Program: Plant Agriculture
Advisor: Pauls, K. Peter
Abstract: Microspores of Brassica napus can be diverted from pollen development to embryo development by isolating and culturing them at a slightly elevated temperature (32ºC). Embryo development in microspore cultures closely resembles zygotic embryo development making it possible to use this system to identify genes involved in zygotic embryogenesis. A previous study comparing the transcriptomes of three–day-old sorted embryogenic and pollen-like (non-embryogenic) cells of B. napus microspore cultures identified 120 transcripts that were up-regulated at least two fold in embryogenic microspores, including a fragment of a gene homologous to the unidentified gene AT1G74730. The current study characterized AT1G74730 homologs in B. napus and tested their function by over-expressing it and reducing its expression by RNA silencing in B. napus and Arabidopsis. The complete genes were isolated and sequenced from B. napus by PCR and RT-PCR using primers designed from the AT1G74730 sequence and named BnMicEmUP (B. napus microspore embryogenesis up- regulated gene). Four forms of BnMicEmUP mRNA, BnMicEmUP1 (766bp) BnMicEmUP2 (744 bp), BnMicEmUP3 (742bp) and BnMicEmUP4 (685bp) and three forms of genomic DNA (711bp, 806bp and 864bp) were identified.BnMicEmUP2,3 was up-regulated more than seven-fold compared to non-induced cultures by day 3 in embryogenic microspore cultures. BnMicEmUP1,3 is expressed in leaf, root, stem and embryo cells. To investigate the function of BnMicEmUP, Arabidopsis transgenic plants with silencing constructs (pFGc 5941) and overexpressing constructs driven by a 35S promoter (pB1121) or an inducible promoter (pER8) were created. Transgenic Arabidopsis plants, which over-expressed the gene (35S::AtBnMicEmUP1 and XVE::AtBnMicEmUP), had flowering times, biomass production, and reproductive traits that were lower than those for control plants, whereas, RNAi and mutant transgenic plants often exceeded control plant values. Most notably, over expressing lines had a 34 % reduction in seed production relative to knockdown lines. It is proposed that BnMicEmUP (bZIP) is a transcription factor gene with dual targeting, first to the plastid and then, in the nucleus with a role in ABA-mediated transcriptional regulation. The results obtained from this study can be used to improve crop yield; as well as basic information on embryogenic processes leading to a greater understanding of totipotency in plants.
URI: http://hdl.handle.net/10214/7882
Date: 2014-03
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada