Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species (Arabidopsis and tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

Analysis of MeEf1A6 Gene Promoter Activity with In-vitro and In-vivo using Transient and Stable Expression Techniques in Tobacco Plant (Nicotiana tabacum)

The promoter is a part of the gene that functions in carrying out the gene expression, and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculenta Elongation Factor 1 Alfa - 6) is a promoter derived from cassava plants (Manihot esculenta). In previous studies, the MeEF1A6 promoter was successfully isolated, introduced, and characterized into the pBI121 plasmid, replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in-vivo and in-vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was introduced into Agrobacterium tumefaciens strain AGL1 and LBA4404. The promoter's work was then analyzed by the result of introducing it into the tobacco plant using the transient and stable transformation. The whole part of explants was used for transient study and tested in a minimum of two biological replicates. Sixty sheets of explant leaves that have been cut were used for stable transformation. The promoter work analysis was carried out with the GUS gene expression that integrated with the promoter with histochemical GUS assay. The transient produced a blue color in the roots, stems, and leaves on the whole repetition. The transverse incision in the stem shows the blue color on the epidermis and procambium tissue. Stable transformation using AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 plantlets that were successfully grown. Some plantlets are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants, indicating that the MeEF1A6 promoter has been successfully introduced. The results indicate that the MeEF1A6 promoter could work on plant tissue in roots, stems, leaves, and tissues that connect meristems such as procambium in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter performs work constitutionally as a constitutive promoter.

A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the β-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An efficient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae.Results To improve the expression efficiency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and efficient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green fluorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG efficiently via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP fluorescent proteins in the A. oryzae RIB40ΔpyrG.Conclusion Our work provides a new method that can be applied to other filamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.