Abstract
Site‐specific recombinase enzymes function in heterologous cellular environments to initiate strand‐switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium‐mediated transformation of T‐DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.
Highlights
Successful transfer and expression of foreign DNA in plant cells through the process of transformation was achieved over 30 years ago (Bevan, Flavell, & Chilton, 1983; Fraley et al, 1983; HerreraEstrella et al, 1983; Murai et al, 1983)
Due to the size of each sequence, binding sites can be incorporated into transformation vectors, enabling precise modifications through the introduction of a respective recombinase system
Transient expression relies on the use of Agrobacterium or particle bombardment to deliver recombinase coding sequences into host cells containing target sites, leading to a burst of expression
Summary
Successful transfer and expression of foreign DNA in plant cells through the process of transformation was achieved over 30 years ago (Bevan, Flavell, & Chilton, 1983; Fraley et al, 1983; HerreraEstrella et al, 1983; Murai et al, 1983). Recombinases have been used in a number of plant systems including rice (Hoa, Bong, Huq, & Hodges, 2002; Hu et al, 2007; Radhakrishnan and Srivastava, 2005), tobacco (Albert, Dale, Lee, & Ow, 1995; Dale & Ow, 1991), wheat (Srivastava, Anderson, & Ow, 1999), tomato (Stuurman, Vroomen, Nijkamp, & Haaren, 1996), barley (Kapusi, Kempe, Rubtsova, Kumlehn, & Gils, 2012), soybean (Li et al, 2009), Arabidopsis (Hong, Lyznik, Gidoni, & Hodges, 2000; Thomson, Chan, Thilmony, Yau, & Ow, 2010; Vergunst & Hooykaas, 1998), and maize (Anand et al, 2019; Kerbach, Lörz, & Becker, 2005; Lyznik, Rao, & Hodges, 1996; Srivastava & Ow, 2001; Zhang et al, 2003) In these studies, recombinase enzymes have been used for the purpose of selectable marker removal, transgene targeting to predetermined locations, or resolution of multiple transgene concatemers. We have established and demonstrated the functionality of five different recombinase systems in maize: Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase, which can be used as tools to carry out genome modifications
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