An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

<p>Abstract</p> <p>Background</p> <p>RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (<it>Jatropha curcas </it>L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)⁺RNA purification and cDNA synthesis.</p> <p>Findings</p> <p>A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)⁺RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds.</p> <p>Conclusions</p> <p>This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.</p

Tasco®, a Product of Ascophyllum nodosum, Imparts Thermal Stress Tolerance in Caenorhabditis elegans

Tasco®, a commercial product manufactured from the brown alga Ascophyllum nodosum, has been shown to impart thermal stress tolerance in animals. We investigated the physiological, biochemical and molecular bases of this induced thermal stress tolerance using the invertebrate animal model, Caenorhabiditis elegans. Tasco® water extract (TWE) at 300 μg/mL significantly enhanced thermal stress tolerance as well as extended the life span of C. elegans. The mean survival rate of the model animals under thermal stress (35 °C) treated with 300 μg/mL and 600 μg/mL TWE, respectively, was 68% and 71% higher than the control animals. However, the TWE treatments did not affect the nematode body length, fertility or the cellular localization of daf-16. On the contrary, TWE under thermal stress significantly increased the pharyngeal pumping rate in treated animals compared to the control. Treatment with TWE also showed differential protein expression profiles over control following 2D gel-electrophoresis analysis. Furthermore, TWE significantly altered the expression of at least 40 proteins under thermal stress; among these proteins 34 were up-regulated while six were down-regulated. Mass spectroscopy analysis of the proteins altered by TWE treatment revealed that these proteins were related to heat stress tolerance, energy metabolism and a muscle structure related protein. Among them heat shock proteins, superoxide dismutase, glutathione peroxidase, aldehyde dehydrogenase, saposin-like proteins 20, myosin regulatory light chain 1, cytochrome c oxidase RAS-like, GTP-binding protein RHO A, OS were significantly up-regulated, while eukaryotic translation initiation factor 5A-1 OS, 60S ribosomal protein L18 OS, peroxiredoxin protein 2 were down regulated by TWE treatment. These results were further validated by gene expression and reporter gene expression analyses. Overall results indicate that the water soluble components of Tasco® imparted thermal stress tolerance in the C. elegans by altering stress related biochemical pathways

λ-Carrageenan Suppresses Tomato Chlorotic Dwarf Viroid (TCDVd) Replication and Symptom Expression in Tomatoes

The effect of carrageenans on tomato chlorotic dwarf viroid (TCDVd) replication and symptom expression was studied. Three-week-old tomato plants were spray-treated with iota(ɩ)-, lambda(λ)-, and kappa(κ)-carrageenan at 1 g·L−1 and inoculated with TCDVd after 48 h. The λ-carrageenan significantly suppressed viroid symptom expression after eight weeks of inoculation, only 28% plants showed distinctive bunchy-top symptoms as compared to the 82% in the control group. Viroid concentration was reduced in the infected shoot cuttings incubated in λ-carrageenan amended growth medium. Proteome analysis revealed that 16 tomato proteins were differentially expressed in the λ-carrageenan treated plants. Jasmonic acid related genes, allene oxide synthase (AOS) and lipoxygenase (LOX), were up-regulated in λ-carrageenan treatment during viroid infection. Taken together, our results suggest that λ-carrageenan induced tomato defense against TCDVd, which was partly jasmonic acid (JA) dependent, and that it could be explored in plant protection against viroid infection

A Cultivated Form of a Red Seaweed (Chondrus crispus), Suppresses β-Amyloid-Induced Paralysis in Caenorhabditis elegans

We report here the protective effects of a methanol extract from a cultivated strain of the red seaweed, Chondrus crispus, against β-amyloid-induced toxicity, in a transgenic Caenorhabditis elegans, expressing human Aβ1-42 gene. The methanol extract of C. crispus (CCE), delayed β-amyloid-induced paralysis, whereas the water extract (CCW) was not effective. The CCE treatment did not affect the transcript abundance of amy1; however, Western blot analysis revealed a significant decrease of Aβ species, as compared to untreated worms. The transcript abundance of stress response genes; sod3, hsp16.2 and skn1 increased in CCE-treated worms. Bioassay guided fractionation of the CCE yielded a fraction enriched in monogalactosyl diacylglycerols (MGDG) that significantly delayed the onset of β-amyloid-induced paralysis. Taken together, these results suggested that the cultivated strain of C. crispus, whilst providing dietary nutritional value, may also have significant protective effects against β-amyloid-induced toxicity in C. elegans, partly through reduced β-amyloid species, up-regulation of stress induced genes and reduced accumulation of reactive oxygen species (ROS)

Proteome Analysis of Rice (Oryza sativa L.) Mutants Reveals Differentially Induced Proteins during Brown Planthopper (Nilaparvata lugens) Infestation

Although rice resistance plays an important role in controlling the brown planthopper (BPH), Nilaparvata lugens, not all varieties have the same level of protection against BPH infestation. Understanding the molecular interactions in rice defense response is an important tool to help to reveal unexplained processes that underlie rice resistance to BPH. A proteomics approach was used to explore how wild type IR64 and near-isogenic rice mutants with gain and loss of resistance to BPH respond during infestation. A total of 65 proteins were found markedly altered in wild type IR64 during BPH infestation. Fifty-two proteins associated with 11 functional categories were identified using mass spectrometry. Protein abundance was less altered at 2 and 14 days after infestation (DAI) (T1, T2, respectively), whereas higher protein levels were observed at 28 DAI (T3). This trend diminished at 34 DAI (T4). Comparative analysis of IR64 with mutants showed 22 proteins that may be potentially associated with rice resistance to the brown planthopper (BPH). Ten proteins were altered in susceptible mutant (D1131) whereas abundance of 12 proteins including S-like RNase, Glyoxalase I, EFTu1 and Salt stress root protein “RS1” was differentially changed in resistant mutant (D518). S-like RNase was found in greater quantities in D518 after BPH infestation but remained unchanged in IR64 and decreased in D1131. Taken together, this study shows a noticeable level of protein abundance in the resistant mutant D518 compared to the susceptible mutant D1131 that may be involved in rendering enhanced level of resistance against BPH

Cultivated seaweeds as functional food: Bioactive organic extract of the red alga, Chondrus crispus, increases stress tolerance in the model worm Caenorhabditis elegans

Seaweeds are rich sources of bioactive compounds with potential health benefits. Anti-stress effects of the cultivated red alga, Chondrus crispus, were investigated in the Caenorhabditis elegans model. Methanolic extracts of C. crispus (CCME) enhanced C. elegans tolerance to juglone-induced oxidative stress and increased life span; however water extracts did not show such effects. CCME treatment reduced reactive oxygen species (ROS) in the worms and increased the transcription of stress response genes; sod3, hsp16.2, daf16 and skn1. The chemical profile of CCME revealed the presence of unsaturated fatty acids, pigments, galactolipids, floridoside, isothionic acid, taurine, phenylalanine and l-citruline. Bioassay guided fractionation revealed that fatty acids, lipids and pigments imparted stress tolerance. Taken together, our results suggest that organic fractions of cultivated C. crispus impart oxidative stress tolerance in C. elegans, possibly by altering the stress response pathways.Peer reviewed: YesNRC publication: Ye