The Structure of T-DNA Insertions in Transgenic Tobacco Plants Producing Bovine Interferon-Gamma

Many of the most modern drugs are of a protein nature and are synthesized by transgenic producer organisms. Bacteria, yeast, or animal cell cultures are commonly used, but plants have a number of advantages—minimal biomass unit cost, animal safety (plants are not attacked by mammalian pathogens), the agricultural scale of production, and the ability to produce complex proteins. A disadvantage of plants may be an unstable level of transgene expression, which depends on the transgene structure and its insertion site. We analyzed the structure of T-DNA inserts in transgenic tobacco plants (Nicotiana tabacum L.) belonging to two lines obtained using the same genetic construct but demonstrating different biological activities of the recombinant protein (bovine interferon-gamma). We found that, in one case, T-DNA was integrated into genomic DNA in the region of centromeric repeats, and in the other, into a transcriptionally active region of the genome. It was also found that in one case, the insert has a clustered structure and consists of three copies. Thus, the structure of T-DNA inserts in both lines is not optimal (the optimal structure includes a single copy of the insert located in the active region of the genome). It is desirable to carry out such studies at the early stages of transgenic plants selection.

Functional Analysis of Genes GlaDFR1 and GlaDFR2 Encoding Dihydroflavonol 4-Reductase (DFR) in Gentiana lutea L. Var. Aurantiaca (M. Laínz) M. Laínz

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

Functional characterization of tobacco (Nicotiana benthamiana) serotonin N-acetyltransferases (NbSNAT1 and NbSNAT2)

Nicotiana benthamiana (tobacco) is an important dicotyledonous model plant; however, no serotonin N-acetyltransferases (SNATs) have been characterized in tobacco. In this study, we identified, cloned, and characterized the enzyme kinetics of two SNAT genes from N. benthamiana, NbSNAT1 and NbSNAT2. The substrate affinity (Km) and maximum reaction rate (Vmax) for NbSNAT1 were 579 µM and 136 pkat/mg protein for serotonin, and 945 µM and 298 pkat/mg protein for 5-methoxytryptamine, respectively. Similarly, the Km and Vmax values for NbSNAT2 were 326 µM and 26 pkat/mg protein for serotonin, and 872 µM and 92 pkat/mg protein for 5-methoxytryptamine, respectively. Moreover, we found that NbSNAT1 and NbSNAT2 localized to chloroplasts, similar to SNAT proteins from other plant species. The activities of the NbSNAT proteins were not affected by melatonin feedback inhibition in vitro. Finally, transgenic tobacco plants overexpressing either NbSNAT1 or NbSNAT2 did not exhibit increased melatonin levels, possibly due to the expression of catabolic enzymes. Generating transgenic tobacco plants with downregulated NbSNAT expression would provide further insight into the functional role of melatonin in tobacco plants.

Allium Sativum Leaf Agglutinin (ASAL) Endowed Enhanced Resistance Against Myzus Persicae under Both Constitutive and Phloem Specific Promoters

Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds on more than 400 plant species and transmits more than 100 plant viruses. Aphid infestation is mostly managed by insecticides that cause heavy environmental contamination and insect resistance. Cloning of plant derived insecticidal genes to develop transgenic plants under suitable promoter is a promising technology. In the present study, ASAL (MN820725) was isolated from native garlic and cloned in plant transformation vector, pGA482 through Agrobacterium mediated tobacco transformation. PCR of genomic DNA of transgenic tobacco plants using gene specific primers confirmed the presence of asal gene of 546 bp. To detect the integration of gene Southern blot analysis was conducted that revealed stable integration of asal gene while, gene expression was analyzed through qRT-PCR that showed variable expression of asal gene in transgenic tobacco plants. Efficacy of asal gene was evaluated through aphid bioassay. Aphid bioassay revealed that transgenic tobacco lines LS-17, LS-20, LR-1, and LR-7 exhibited 100% aphid mortality and significantly reduced the aphid population. These findings suggested the potential of ASAL against aphids that can be further used against other notorious sap sucking pests.

CsMYB4a from Camellia sinensis Regulates the Auxin Signaling Pathway by Interacting with CsIAA4

Members of the R2R3-MYB4 subgroup are well-known negative regulatory transcription factors of phenylpropane and lignin pathways. In this study, we found that transgenic tobacco plants overexpressing a R2R3-MYB4 subgroup gene from Camellia sinensis (CsMYB4a) showed inhibited growth that was not regulated by phenylpropane and lignin pathways, and these plants exhibited altered sensitivity to synthetic auxin 1-naphthaleneacetic acid (α-NAA) treatment. An auxin/indole-3-acetic acid 4 (AUX/IAA4) gene from Camellia sinensis (CsIAA4) participating in the regulation of the auxin signal transduction pathway was screened from the yeast two-hybrid library with CsMYB4a as the bait protein, and tobacco plants overexpressing this gene showed a series of auxin-deficiency phenotypes, such as dwarfism, small leaves, reduced lateral roots, and a shorter primary root. CsIAA4 transgenic tobacco plants were less sensitive to exogenous α-NAA than control plants, which was consistent with the findings for CsMYB4a transgenic tobacco plants. The knockout of the endogenous NtIAA4 gene (a CsIAA4 homologous gene) in tobacco plants alleviated growth inhibition in CsMYB4a transgenic tobacco plants. Furthermore, protein-protein interaction experiments proved that domain II of CsIAA4 is the key motif for the interaction between CsIAA4 and CsMYB4a and that the degradation of CsIAA4 is prevented when CsMYB4a interacts with CsIAA4. In summary, our results suggest that CsMYB4a is a multifunctional transcription factor that regulates the auxin signaling pathway, phenylpropane and lignin pathways. This study provides new insights into the multiple functions of R2R3-MYB4 subgroup members as a group of well-known negative regulatory transcription factors.

The Aconitum carmichaelii F3′5′H Gene Overexpression Increases Flavonoid Accumulation in Transgenic Tobacco Plants

Aconitum carmichaelii Debx. is a herbal species that contains many precious bioactive substances, which are alkaloids, flavonoids, steroids, and glycosides. Flavonoids, which are major secondary compounds, play an important role in maintaining redox balance in the cells of the plant body. Many flavonoids have antibacterial, antioxidant, and anticancer properties. However, studies have mainly focused on aconitine, which is a highly toxic group A poison belonging to the alkaloid group, but with little mention of flavonoids. The flavonoids in A. carmichaelii are a group of substances with high content, concentrated in leaves and flowers, including quercetin and kaempferol. F3′5′H (Flavonoid 3′5′-hydroxylase) has been identified as the key enzyme involved in the final steps of flavonoid biosynthesis in plants in general and in A. carmichaelii specifically. This study offers the first report, and demonstrates that the overexpression of the F3′5′H gene from a herbal plant, A. carmichaelii, increases flavonoid content in genetically modified tobacco plants. The A. carmichaelii gene was transformed into tobacco leaf tissue to create transgenic tobacco plants. The AcF3′5′H gene was incorporated into the tobacco genome and was expressed in four transgenic tobacco lines (T01, T03, T05, and T014). The F3′5′H content increased from 20.33% to 32.00% compared with that in non-transformed plants (P < 0.001). Therefore, the flavonoid content of four transgenic tobacco lines increased compared to the WT, from 69.23% to 122.23% (P < 0.001). The results of the successful expression of the AcF3′5′H gene in model tobacco plants are the basis for using the AcF3′5′H gene for improving flavonoid content in other medicinal plants. Thus, the AcF3′5′H gene considered in this work could be a candidate for gene technology to enhance flavonoid accumulation in plants.