Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.
Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as whole grains and sesame and flax seeds, have anticancer activity and can act as phytoestrogens. The lignans secoisolariciresinol and matairesinol can be converted in the mammalian proximal colon into enterolactone and enterodiol, respectively, which reduce the risk of breast and colon cancer. To establish an efficient bioconversion system to generate matairesinol from pinoresinol, the genes encoding pinoresinol-lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, were expressed in Escherichia coli and purified. The two genes, termed plr-PpH and sdh-PpH, were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 min showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of the mixture of rPLR and rSDH. The percent conversion of (؉)-pinoresinol to matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and rSDH. However, conversion of (؉)-pinoresinol by fusion protein SDH-PLR stopped at the intermediate product, secoisolariciresinol. In vivo, (؉)-pinoresinol was completely converted to matairesinol by living recombinant E. coli expressing PLR-SDH without addition of cofactors.
With the development of Building Information Modeling (BIM) technology, there has been an increased application of BIM-based modeling and energy analysis tools in building design. Although there are several popular energy analysis tools available, there is currently no effective way for users to verify the accuracy and reliability of their results and to compare the differences between different analysis tools. Therefore, the Computer-Aided Engineering Group at Department of Civil Engineering, National Taiwan University is developing a test bed for researchers and practitioners to share verification data, with both the BIM model of a test-case building and the actual measurement data of the energy performance of the building, which can be used for verification and comparison of various building energy analysis tools. This paper discusses the design and implementation of the test bed. The test bed consists of three major parts: (1) the repository of the BIM model of the test-case building with measured energy data; (2) user interfaces for users to upload and retrieve test-case information and model data in an organized and convenient way; and (3) utilities for users to convert data between different formats used by different energy analysis tools and to evaluate the energy analysis tools based on comparisons of analysis results with actual measurement data.
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