Correlation of Cry1Ac mRNA and protein abundance in transgenic Gossypium hirsutum plant.

Transcription and translation in eukaryotes are distinct processes of the molecular cascade leading to protein production from genetic material. However, establishing correlation between mRNA expression and protein abundance, the end results of the two processes of central dogma, remains a challenge. For transgenic plants, such correlation between mRNA and protein expression serves as a guide to design the transgene, in particular the choices of promoter and codon usage to ensure stable expression of the target protein in relevant tissues under various stress conditions. To elucidate level of mRNA-protein correlation in a commercial transgenic cotton plant Gossypium hirsutum, Bollgard II® (MON15985), we present the results of Cry1Ac protein expression correlating with corresponding mRNA levels. Protein was quantitated using a home-grown validated ELISA assay with a monoclonal-polyclonal antibody pair, whereas mRNA level was detected by a real-time quantitative PCR assay using standardized reference genes. Our results indicate that protein and mRNA levels are highly correlated in the leaves, but not in squares and stem. The correlations seem to be consistent between young and mature leaves and increase over time of harvesting of samples from months 1-3. These findings demonstrate that transcript level measurement could serve as a proxy to protein abundance for this commercially important cotton species, particularly for leaf tissues which are the most vulnerable organs to cotton bollworms and other pathogens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02828-2.

Anti-tumor effects of Artemisia nilagirica extract on MDA-MB-231 breast cancer cells: deciphering the biochemical and biomechanical properties via TGF-ß upregulation.

PURPOSE: Artemisia nilagirica (AN), which is known to have antimicrobial, antioxidant, antiulcer, and anti-asthmatic properties, has been recently shown to have anti-cancer activity. However, the mechanism responsible for the anti-cancer property and its effect on cellular properties and functions are not known. MATERIAL AND METHODS: We have characterized the biochemical and biomechanical properties of MDA-MB-231 cells treated with the methanolic extract from AN. RESULTS: We show that AN-treatment decreases cell-eccentricity, increases expression of actin and microtubules, and do not affect cell-area. Increased expression of cytoskeletal proteins is known to change the mechanical properties of the cells, which was confirmed using micropipette aspiration and Atomic Force Microscopy. We identified the upregulation of the tumorigenic pathway (TGF-ß) leading to activation of Rho-A as the molecular mechanism responsible for actin upregulation. Since the initial stages of TGF-ß upregulation are known to suppress tumor growth by activating apoptosis, we hypothesized that the mechanism of cell death due to AN-treatment is through TGF-ß activation. We have validated this hypothesis by partially recuing cell death through inhibition of TGF-ß using Alk-5. CONCLUSION: In summary, our study reveals the mechanism of action of Artemisia nilagirica using a synergy between biochemical and biomechanical techniques.

Genome wide search to identify reference genes candidates for gene expression analysis in Gossypium hirsutum.

BACKGROUND: Cotton is one of the most important commercial crops as the source of natural fiber, oil and fodder. To protect it from harmful pest populations number of newer transgenic lines have been developed. For quick expression checks in successful agriculture qPCR (quantitative polymerase chain reaction) have become extremely popular. The selection of appropriate reference genes plays a critical role in the outcome of such experiments as the method quantifies expression of the target gene in comparison with the reference. Traditionally most commonly used reference genes are the "house-keeping genes", involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes for every experimental platform. This study presents a data science driven unbiased genome-wide search for the selection of reference genes by assessing variation of > 50,000 genes in a publicly available RNA-seq dataset of cotton species Gossypium hirsutum. RESULT: Five genes (TMN5, TBL6, UTR5B, AT1g65240 and CYP76B6) identified by data-science driven analysis, along with two commonly used reference genes found in literature (PP2A1 and UBQ14) were taken through qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaluated using four algorithms - geNorm, BestKeeper, NormFinder and RefFinder. CONCLUSION: Based on the results we recommend the usage of TMN5 and TBL6 as the optimal candidate reference genes in qPCR experiments with normal and transgenic cotton plant tissues. AT1g65240 and PP2A1 can also be used if expression study includes squares. This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes over the selection based on function alone.

Chitinase from Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora.

The tea mosquito bug (TMB), Helopeltis spp. (Hemiptera: Miridae) is an insidious pest that poses a significant economical threat to tea plantations. Pseudomonas cultures are being used extensively for pest management which, however, resulting in a low mortality rate of insects and which has prompted us to search for a new microbial metabolite for TMB control. A chitinase purified from P. fluorescens and partially characterized by our group showed insecticidal activity against TMB. The mode of action behind chitinase toxicity is the enzymatic hydrolysis of chitin, which is a common constituent of the insect exoskeleton and gut lining of the peritrophic membrane. A chitinase-secreting strain MP-13 was characterized based on 16S rRNA sequencing and validated as Pseudomonas fluorescens. In the present study, purified chitinase (0.048 units/ml) enzyme from P. fluorescens MP-13 revealed 100% TMB mortality under in-vitro conditions. The results of this study can be utilized for future crop improvement programs and integrated pest management strategies.

Molecular characterization of tea mosquito bug from tea growing regions of India.

The tea mosquito bug, Helopeltis (Hemiptera: Miridae), is an insidious pest that poses a significant economical threat to tea plantations. As a basic first step to control this pest is authentic identification, but the inability to determine morphological characters of Helopeltis species makes this process very difficult. DNA barcoding is a reliable alternative to traditional morphological identification of this pest. Since tea is cultivated in different parts of the country, an attempt was made to molecular characterization of Helopeltis. This is the first report on molecular identification and diversity characterization of Helopeltis collected from tea growing regions of southern and north India, using cytochrome c oxidase subunit I (COI) gene of mitochondrial (mt) DNA. Beginning with the molecular identification of this pest is essential to start an effective pest management strategy, and will provide basic information for diffusion pattern, population dynamics and chemical application.