華中農大園藝學院發表文獻《ERF9 of Poncirus trifoliata (L.) Raf. undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S‐transferase U17 gene》，采用LUYOR-3415RG便攜式雙波長熒光蛋白激發光源篩選煙草轉基因陽性葉片。

文獻摘要：

Vector construction and plant transformation

For the PtrERF9 overexpression lines, the full-length PtrERF9 CDS sequence was cloned into the pGWB411 vector according to the gateway recombination technology (Invitrogen). The resulting construct was transformed into tobacco (Nicotiana nudicaulis) and lemon (C. limon) via Agrobacterium. tumefaciens-mediated transformation as previously reported (Fu et al., 2011). And then the transgenic explants were grown on MS (for tobacco; Murashige and Skoog, 1962) or MT (for lemon; Murashige and Tucker, 1969) medium containing 50 lg/mL kanamycin.

Kanamycin-resistant plants were selected which were further confirmed by qPCR assay. The transgenic tobacco lines at T2 generation and lemon lines in the vegetative period were used for phenotype analysis. Similarly, transient expression of PtrERF9 in trifoliate orange was performed according to Acanda et al. (2021) with minor modifications. To this end, full-length cDNA of PtrERF9 was cloned into the pH7WG2D expression vector with a GFP tag. The 35S: PtrERF9-GFP construct was transformed into Agrobacterium strain (EHA105) and then used for injection into the young tender leaves of three-month-old trifoliate orange seedlings. After 12 days of agroinfiltration, the infected leaves were subjected to fluorescence observation using a hand-held lamp providing dual-wavelength fluorescent protein excitation light source (LUYOR-3415RG, USA). The leaves emitting the green fluorescence were used for Western blotting and ChIPqPCR assays.

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