Molecular Cloning, Bioinformatics Analysis and Overexpression of the Tyrosine Aminotransferase Gene in Rehmannia Glutinosa

Rehmannia glutinosa is an important medicinal plant producing many bioactive compounds such as catalpol, acteoside and so on. Tyrosine aminotransferase (TAT) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis. To confirm its role for acteoside accumulation, we isolated a full-length cDNA from Rehmannia glutinosa Libosch. Sequence analysis indicated that it contained a 1266 bp open reading frame, encoding a TAT of 421 amino acid residues. Multiple sequence alignment revealed that the homology of RgTAT amino acid sequence to that of Sesamum indicum(XP_011100354.1) was the highest (89.94%). Evolutionary tree showed that Sesamum indicum TAT and RgTAT were grouped together. Quantitative real-time PCR analysis indicated that the expression of RgTAT in leaves was much higher than in roots and stems,and that the expression levels of RgTAT in the tuberous roots, stems and leaves of high-acteoside cultivar BJ-3 were higher than in that of low-acteoside cultivar Wen85-5. A plant expression vector was constructed containing the RgTAT and hygromycin resistance gene (Hyg). Transgenic Rehmannia glutinosa Libosch overexpressing RgTAT was obtained via an Agrobacterium tumefaciens-mediated transformation system, in which Hyg expression was confirmed by PCR. RgTAT expression in transgenic plantlets measured by real-time quantitative PCR was 7.72 ± 0.17 times greater than its expression in the untransformed plantlets. Moreover, HPLC analysis indicated that enhanced RgTAT expression corresponded to significantly increased acteoside for transgenic plantlets. Our results elucidate the role of RgTAT in the acteoside biosynthesis in Rehmannia glutinosa.

Downregulation of high-affinity potassium and sodium symporter gene, EcHKT1;1, in Eucalyptus roots enhances salt tolerance

Engineering for restricted root Na+ uptake could potentially enhance salt tolerance in Eucalyptus. High-affinity K+ transporters (HKTs) have been implicated in Na+ uptake from the external medium as in the case of TaHKT2;1 or in the unloading of Na+ from xylem like in AtHKT1;1. To rapidly determine the in planta role of EcHKT1:1, composite transgenics in which EcHKT1:1 was specifically downregulated via RNAi in the roots were generated. Compared to the controls that failed to survive at 350 mM NaCl, 33 % of the composite transgenic plantlets generated using the EcHKT1;1 silencing construct were able to tolerate up to 400 mM NaCl. In these composite transgenics, EcHKT1;1 downregulation ranged from 37 % to 74 %. The average shoot to root ratio of sodium was 4.9 folds lower than the controls indicating restricted translocation of Na+ to the shoots. Relative expression analysis in the leaves of two non-transgenic genotypes contrasting for their salt tolerance also showed downregulated EcHKT1;1 expression in the tolerant clone. The study thus determined that EcHKT1;1 is a major gene determining Na+ transport from the roots to shoots. This study also demonstrated the utility of the composite transgenic approach for screening genes conferring salt tolerance in tree species.

Functional Identification of EjGIF1 in Arabidopsis and Preliminary Analysis of Its Regulatory Mechanisms in the Formation of Triploid Loquat Leaf Heterosis

Although several results have been obtained in triploid loquat heterosis (i.e., leaf size of triploid loquat) studies in the past years, the underlying mechanisms of the heterosis are still largely unknown, especially the regulation effects of one specific gene on the corresponding morphology heterosis. In this study, we sought to further illustrate the regulatory mechanisms of one specific gene on the leaf size heterosis of triploid loquats. A leaf size development-related gene (EjGIF1) and its promoter were successfully cloned. Ectopic expression of EjGIF1 in Arabidopsis showed that the leaf size of transgenic plantlets was larger than that of WTs, and the transgenic plantlets had more leaves than WTs. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression level of EjGIF1 showed an AHP expression pattern in most of the hybrids, and this was consistent with our previous phenotype observations. Structure analysis of EjGIF1 promoter showed that there were significantly more light-responsive elements than other elements. To further ascertain the regulatory mechanisms of EjGIF1 on triploid loquat heterosis, the methylation levels of EjGIF1 promoter in different ploidy loquats were analyzed by using bisulfite sequencing. Surprisingly, the total methylation levels of EjGIF1 promoter in triploid showed a decreasing trend compared with the mid-parent value (MPV), and this was also consistent with the qRT-PCR results of EjGIF1. Taken together, our results suggested that EjGIF1 played an important role in promoting leaf size development of loquat, and demethylation of EjGIF1 promoter in triploid loquats caused EjGIF1 to exhibit over-dominance expression pattern and then further to promote leaf heterosis formation. In conclusion, EjGIF1 played an important role in the formation of triploid loquat leaf size heterosis.

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

Background Recent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. Results Abiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. Conclusions The present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

Agrobacterium-mediated Introduction of Athb-7, Water Stress Gene, into Solanum spp.

A genetic transformation in Solanum spp. was performed using Agrobacterium tumefaciens: C58:pGV2260:Athb-7. Athb-7 gene, known to be related to water stress and ABA level, one of Arabidopsis thaliana homeobox genes was inserted into pBin-Hyg-Tx. Explants were placed on callus induction medium for 14 days, and then transferred on shoot induction medium. Shoot primordium appeared on callus surface after 2 weeks of culture. About 6 weeks later, 100 putatively transgenic plants were obtained, and DNA was extracted from each plant for PCR analysis. Twenty out of 100 putatively transgenic plantlets turned to be positive, having a band of 800 bp in M.W. corresponding to the hygromycin gene. Both PCR and genomic Southern hybridizations using HPTII and Athb-7 genes as probes showed that these genes were inserted into plant genome.