Engineering the AEG1 promoter from cotton to develop male sterile lines

Abstract

This work reports on modifying the Upstream Regulatory Module (URM, 1.5 Kb region upstream of the open reading frame) of Anther Expressing Gene 1 (AEG1) from cotton to achieve anther specific activity. AEG1 was identified in a previous study aimed to isolate a promoter with tapetum specific activity. Such a promoter could then be used to express barnase and barstar genes for developing male sterile and restorer lines for hybrid seed production in cotton. The AEG1 URM was observed to be active in tapetum as well as in roots making it unusable to drive the expression of barnase gene. Analysis of the URM showed the presence of several root specific motifs. Two modified AEG1 URMs were developed, by removing or mutating these motifs and its activity checked in tobacco. The activity of one of the modified URMs, AEG1(∆Bmut) was restricted to the anther tissue as observed using the reporter gene ß-glucuronidase. The study also demonstrates that male sterile lines could be developed in tobacco using the AEG1(∆Bmut) URM to express the barnase gene. This work thus shows the possibility of engineering promoters to achieve tissue specificity and to develop male sterile lines in cotton.Tapetum specific promoters are key to develop barnase and barstar gene based male sterile and restorer lines for production of hybrid seeds (Mariani et al., 1992). The Nicotiana tabacum (tobacco) TA29 promoter has been used to develop male sterile and restorer lines in crops like Brassica (Jagannath et al., 2001(Jagannath et al., , 2002. In order to expand the technology for cotton, our laboratory identified the Anther Expressing Gene 1 (AEG1, Paritosh et al., 2018), which from a microarray-based comparative analysis of the transcriptome from different tissues was found to express in anthers from Gossypium hirsutum. In situ hybridization showed presence of AEG1 transcripts in the tapetum.Cotton transgenic lines carrying 1.5 Kb region upstream of the open reading frame (URM, Upstream Regulatory Module) to drive an intron containing ß-glucuronidase gene (Gi) as reporter confirmed GUS activity in tapetum. However, GUS activity was also