Rongda Qu

Transcriptional and post-transcriptional enhancement of gene expression by the 5′ UTR intron of rice rubi3 gene in transgenic rice cells

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5′ UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5′ UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level (∼45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.

Amplicon-plus Targeting Technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants

High-level expression of transgenes is essential for cost-effective production of valuable pharmaceutical proteins in plants. However, transgenic proteins often accumulate in plants at low levels. Low levels of protein accumulation can be caused by many factors including post-transcriptional gene silencing (PTGS) and/or rapid turnover of the transgenic proteins. We have developed an Amplicon-plus Targeting Technology (APTT), by using novel combination of known techniques that appears to overcome both of these factors. By using this technology, we have successfully expressed the highly-labile L1 protein of canine oral papillomavirus (COPV L1) by infecting transgenic tobacco plants expressing a suppressor of post-transcriptional gene silencing (PTGS) with a PVX amplicon carrying a gene encoding L1, and targeting the vaccine protein into the chloroplasts. Further, a scalable “wound-and-agrospray” inoculation method has been developed that will permit high-throughput Agrobacterium inoculation of Nicotiana tabacum, and a spray-only method (named “agrospray”) for use with N. benthamiana to allow large-scale application of this technology. The good yield and short interval from inoculation to harvest characteristic of APTT, combined with the potential for high-throughput achieved by use of the agrospray inoculation protocol, make this system a very promising technology for producing high value recombinant proteins, especially those known to be highly labile, in plants for a wide range of applications including producing vaccines against rapidly evolving pathogens and for the rapid response needed to meet bio-defense emergencies.

Agrobacterium-mediated transformation of common bermudagrass (Cynodon dactylon)

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were obtained after selection. Among them two green independent transgenic plants were recovered. The plants growing in pots looked relatively compact at the beginning, but the ploidy level of the plants, as determined by nuclear DNA content, was not altered.

Overexpression of ubiquitin-like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

Genetic Factors for Enhancement of Nicotine Levels in Cultivated Tobacco

Nicotine has practical applications relating to smoking cessation devices and alternative nicotine products. Genetic manipulation for increasing nicotine content in cultivated tobacco (Nicotiana tabacum L.) may be of value for industrial purposes, including the possibility of enhancing the efficiency of nicotine extraction. Biotechnological approaches have been evaluated in connection with this objective, but field-based results are few. Here, we report characterization of two genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs), NtMYC2a and NtMYC2b from tobacco. Overexpression of NtMYC2a increased leaf nicotine levels in T1 transgenic lines approximately 2.3-fold in greenhouse-grown plants of tobacco cultivar ‘NC 95′. Subsequent field testing of T2 and T3 generations of transgenic NtMYC2a overexpression lines showed nicotine concentrations were 76% and 58% higher than control lines, respectively. These results demonstrated that the increased nicotine trait was stably inherited to the T2 and T3 generations, indicating the important role that NtMYC2a plays in regulating nicotine accumulation in N. tabacum and the great potential of NtMYC2a overexpression in tobacco plants for industrial nicotine production. Collected data in this study also indicated a negative feedback inhibition of nicotine biosynthesis. Further enhancement of nicotine accumulation in tobacco leaf may require modification of the processes of nicotine transport and deposition.

Effects of overexpression of jasmonic acid biosynthesis genes on nicotine accumulation in tobacco

Abstract Nicotine is naturally synthesized in tobacco roots and accumulates in leaves as a defense compound against herbivory attack. Nicotine biosynthesis pathway has been extensively studied with major genes and enzymes being isolated and functionally characterized. However, the molecular regulation of nicotine synthesis has not been fully understood. The phytohormone jasmonic acid (JA) mediates many aspects of plant defense responses including nicotine biosynthesis. In this study, five key genes (AtLOX2, AtAOS, AtAOC2, AtOPR3, AtJAR1) involved in JA biosynthesis from Arabidopsis were individually overexpressed, and a JA‐Ile hydrolysis‐related gene, NtJIH1, was suppressed by RNAi approach, to understand their effects on nicotine accumulation in tobacco. Interestingly, while transgene expression was high, levels of JA‐Ile (the biologically active form of JA) were often significantly reduced. Meanwhile, nicotine content in these transgenic plants did not increase. The research revealed a tightly controlled JA signaling pathway and a complicated regulatory network for nicotine biosynthesis by JA signaling.

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus

BackgroundLarge T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed.ResultsTo gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells.ConclusionsOur data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well.

Cholesterol accumulation by suppression of SMT1 leads to dwarfism and improved drought tolerance in herbaceous plants

Dwarfism and drought tolerance are 2 valuable traits in breeding of many crops. In this study, we report the novel physiological roles of cholesterol in regulation of plant growth and drought tolerance. Compared with the wild type, sterol-C24-methyltransferase 1 (SMT1) gene transcript was greatly reduced in a bermudagrass mutant with dwarfism and enhanced drought tolerance, accompanied with cholesterol accumulation, elevated transcript levels of a small group of genes including SAMDC, and increased concentrations of putrescine (Put), spermidine (Spd), and spermine (Spm). Knock-down of OsSMT1 expression by RNA interference resulted in similar phenotypic changes in transgenic rice. Moreover, exogenously applied cholesterol also led to elevated transcripts of a similar set of genes, higher levels of Put, Spd, and Spm, improved drought tolerance, and reduced plant height in both bermudagrass and rice. We revealed that it is Spm, but not Spd, that is responsible for the height reduction in bermudagrass and rice. In conclusion, we suggest that cholesterol induces expression of SAMDC and leads to dwarfism and elevated drought tolerance in plants as a result of the promoted Spd and Spm synthesis.