Unique Nucleic Acid Promoter Formed from Two or More Promoter Sequences

Non-naturally occurring DNA promoters, include MSGT-PFlt, PFlt-UAS-2X and FSgt-PFlt. The MSGT-PFlt was developed and has expression shown to be equivalent to that of CaMV35S promoter. DNA promoters PFlt-UAS-2X and FSgt-PFlt were developed and tested in transient and transgenic system and found to be stronger than the CaMV35S promoter

Use of the Full Length Transcript (FLT) from Mirabilis Mosaic Caulimovirus to Express Chimeric Genes in Plants

A full-length transcript promoter from mirabilis mosaic caulimovirus (MMV) is identified and its DNA sequence given. The promoter functions as a strong and uniform promoter for chimeric genes inserted into plant cells. This strong promoter function is exhibited by histochemical assay in seeds and floral organs and by reproductive scores of transgenic plants including the promoter. The promoter preferably includes a 3′ untranslated region that may be from the MMV itself or from a heterologous source with respect to the promoter. The promoter is used in a chimeric gene and in methods for transforming plant cells to obtain transgenic plants, plant tissues, plant cells and seeds incorporating the MMV promoter. The MMV FLT promoter shows greater activity (14 to 24 fold) than the CaMV 35S promoter. A modified MMV FLt promoter with duplicated enhancer domains shows greater activity (3 fold) than with a single enhancer domain

Andrographolide: A Novel Antimalarial Diterpene Lactone Compound from Andrographis paniculata and Its Interaction with Curcumin and Artesunate

Andrographolide (AND), the diterpene lactone compound, was purified by HPLC from the methanolic fraction of the plant Andrographis paniculata. The compound was found to have potent antiplasmodial activity when tested in isolation and in combination with curcumin and artesunate against the erythrocytic stages of Plasmodium falciparum in vitro and Plasmodium berghei ANKA in vivo. IC50s for artesunate (AS), andrographolide (AND), and curcumin (CUR) were found to be 0.05, 9.1 and 17.4 μM, respectively. The compound (AND) was found synergistic with curcumin (CUR) and addictively interactive with artesunate (AS). In vivo, andrographolide-curcumin exhibited better antimalarial activity, not only by reducing parasitemia (29%), compared to the control (81%), but also by extending the life span by 2-3 folds. Being nontoxic to the in vivo system this agent can be used as template molecule for designing new derivatives with improved antimalarial properties

Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco(1128) [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco(1128) was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T(1) tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T(0) and T(1) plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant

pSiM24 is a Novel Versatile Gene Expression Vector for Transient Assays as Well as Stable Expression of Foreign Genes in Plants

We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3\u27rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses

Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco

Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach

Anti-malarial activities of Andrographis paniculata and Hedyotis corymbosa extracts and their combination with curcumin

<p>Abstract</p> <p>Background</p> <p>Herbal extracts of <it>Andrographis paniculata </it>(AP) and <it>Hedyotis corymbosa </it>(HC) are known as hepato-protective and fever-reducing drugs since ancient time and they have been used regularly by the people in the south Asian sub-continent. Methanolic extracts of these two plants were tested in vitro on choloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of <it>Plasmodium falciparum </it>for their anti-malarial activity.</p> <p>Methods</p> <p>Growth inhibition was determined using different concentrations of these plant extracts on synchronized <it>P. falciparum </it>cultures at the ring stage. The interactions between these two plant extracts and individually with curcumin were studied in vitro. The performance of these two herbal extracts in isolation and combination were further evaluated in vivo on Balb/c mice infected with <it>Plasmodium berghei </it>ANKA and their efficacy was compared with that of curcumin. The in vivo toxicity of the plant derived compounds as well as their parasite stage-specificity was studied.</p> <p>Results</p> <p>The 50% inhibitory concentration (IC₅₀) of AP (7.2 μg/ml) was found better than HC (10.8 μg/ml). Combination of these two herbal drugs showed substantial enhancement in their anti-malarial activity. Combinatorial effect of each of these with curcumin also revealed anti-malarial effect. Additive interaction between the plant extracts (AP + HC) and their individual synergism with curcumin (AP+CUR, HC+CUR) were evident from this study. Increased in vivo potency was also observed with the combination of plant extracts over the individual extracts and curcumin. Both the plant extracts were found to inhibit the ring stage of the parasite and did not show any in vivo toxicity, whether used in isolation or in combination.</p> <p>Conclusion</p> <p>Both these two plant extracts in combination with curcumin could be an effective, alternative source of herbal anti-malarial drugs.</p

Isolation of total RNA from hard bamboo tissue rich in polyphenols and polysaccharides for gene expression studies

RNA isolation from hard and woody internodal bamboo ( Bambusa balcooa ) tissue is very difficult due to the presence of secondary metabolites, polysaccharides, and polyphenolics. These compounds often co-precipitate with isolated RNA and hinder downstream applications. We have developed an efficient, cost effective and reproducible RNA isolation method from hard tissue of bamboo internode. This protocol includes an additional organic solvent refinement steps to remove endogenous phenolic compounds and acidic phenol (pH 4.2) to critically stabilize RNA in extraction buffer. In addition to these, two 2M Lithium chloride washing steps were introduced to eliminate DNA and polysaccharides contamination. The RNA isolated from the present protocol was found to be superior, when compared to total RNA extracted by other available protocols. The A260/A280 absorption ratio of the isolated RNA was found ranging between 1.89-1.97. The integrity of 28S and 18S rRNA was highly satisfactory when analyzed in agarose denaturing gel. RNA was further used for RT PCR, northern hybridization, cDNA library and subtractive hybridization without any further refinement

Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants