Ripe fruits were collected the same day from the six plants of each line, divided into three biological replicates and independently grinded in liquid nitrogen. Total RNA was extracted from pooled strawberry fruits using a differential 2-butoxyethanol precipitation-based method [ 26 ]. RNA integrity was confirmed by the appearance of ribosomal RNA bands and lack of degradation products after separation in agarose gel electrophoresis and ethidium bromide staining.
More than 30 million reads were generated for each sample. Mapping RNA-seq reads to the reference genome and generation of read counts Raw RNA-seq reads were processed to remove low-quality nucleotides and aligned to the Fragaria vesca reference genome v1. Default parameters of TopHat were used, allowing 40 multiple alignments per read and a maximum of 2 mismatches when mapping reads to the reference.
TopHat builds a database of potential splice junctions and confirms these by comparing the previously unmapped reads against the database of putative junctions. The aligned read files were processed by Cufflinks v2. Reads were assembled into transcripts, their abundance estimated, and tests for differential expression and regulation between the samples were performed.
Cufflinks does not make use of existing gene annotations during assembly of transcripts, but rather assembles a minimum set of transcripts that best describe the reads in the dataset. This approach allows Cufflinks to identify alternative transcription and splicing that are not described by pre-existing gene models [ 28 ]. This classified each transcript as known or novel. Cuffcompare produced a combined. GTF file which was passed to Cuffdiff along with the original alignment.
SAM files produced by TopHat to identify differentially expressed transcripts between the two pools. The significance scores were corrected for multiple testing using the Benjamini-Hochberg correction. The expression testing is done at the level of transcripts, primary transcripts and genes. By tracking changes in the relative abundance of transcripts with a common transcription start site, Cuffdiff can also identify changes in splicing.
Views of individual genes were generated by uploading TopHat-generated files containing the sequence alignment data. The functional clustering tool was used to look for functional enrichment for genes over- and under-expressed more than two-fold between the pools.
A unique list of gene symbols was uploaded via the web interface. Gene Ontology Biological Process was selected as the functional annotation category for this analysis. The transcript contigs most similar to the F.
The metabolism of ricinoleic acid by some Candida strains was investigated by Okui et al. The use of castor oil or castor oil hydrolysate as the substrate is determined by the microorganism s employed in the process. A co-oxidant may be added to the culture medium in order to increase the yield of the process.
The selection of. The microorganisms in the invention may be bacteria, yeast or filamentous fungi. Where the microorganism is used in combination with a lipase and with castor oil, the preferred microorganisms are: Hansenula saturnus, Candida guilliermondii, Candida aikicans, Candida krusei, Candida parakrusei, Candida pseudotropicalis, Candida stellatoidea, Candida tropicalis, Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii or Yarrowia lipolytica, more preferably Candida guilliermondii.
Generally, any type of lipase enzyme may be used to hydrolyze the castor oil, including microbial, pancreatic, fungi or yeast. Wherea lipase is used with the microorganism in the process of the invention, the formation of the enzymatic hydrolysate may be controlled by limiting the amount of lipase used in the process.
This will avoid toxicity resulting from the presence of excessive amounts of hydrolysate. The appropriate amount of lipase required may be conveniently found by experimentation and will depend upon the lipase and microorganism used and the culturing conditions.
The hydrolysis using lipase is most preferably carried out concurrently with the fermentation in the same reaction vessel. However, the hydrolysis may be carried out prior to fermentation if appropriate measures are taken to avoid the toxic effect of the hydrolysate.
When castor oil is used in the invention, the concern for toxicity is eliminated because triglycerides are not toxic to the organisms. Additionally, the use of castor oil and castor oil hydrolysates as the substrate provide co-oxidants to the process which increase efficiency due to the presence of other fatty acids upon hydrolysis of the castor oil. The form in which the microorganisms are used is not critical. They can be used as the culture suspension , i.
The cells or an enzyme extract thereof may be immobilized on a suitable solid support which may then be used to effect the transformations.
A suitable medium is one which contains carbon sources, nitrogen sources, inorganic salts and growth factors. Among the suitable nitrogen sources are, for example, nitrogen-containing organic substances such as peptone, meat extract, yeast extract, corn steep liquor, and casein, urea, amino acids, or nitrogen containing inorganic compounds such as nitrates, nitri les, and inorganic ammonium salts. Among the suitable inorganic salts are, for example, phosphates, magnesium, potassium, calcium, sodium.
However, the process can be performedin avitamin-free medium, for example, when a small amount of yeast extract is added to the medium there is no need for vitamins or trace minerals. The cultivation of the microorganism can be carried out as a stationary culture or as a submersed culture e.
One suitably may work in the pH range of from about 3. The pH may be regulated by the addition of inorganic or organic bases, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, by ion-exchange resins, or by the addition of a buffer such as phosphate or phthalate.
The process in accordance with the invention is conveniently carried out by adding castor oil or castor oil hydrolysate, as the substrate, to the culture medium at the onset of cultivation, as the sole carbon source. Alternatively, the substrate may be added in combination with another carbon source, such as dextrose, either during cultivation, or when cultivation is complete. The amount, level, or concentration of the substrate in the medium may vary.
For example, in the case of hydrolyzed castor oil, levels of from about 0. The reaction time may vary depending on the composition of the culture medium and the substrate concentration. In general, shaking flask cultures require from between about 2h.The following examples serve to illustrate embodiments of the invention as it is now preferred to practice it but in no way are meant to limit the scope thereof. Where the microorganism is used in combination with a lipase and with castor oil, the preferred microorganisms are: Hansenula saturnus, Candida guilliermondii, Candida aikicans, Candida krusei, Candida parakrusei, Candida pseudotropicalis, Candida stellatoidea, Candida tropicalis, Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii or Yarrowia lipolytica, more preferably Candida guilliermondii. The amount, level, or concentration of the substrate in the medium may vary. Considerable time and effort have been expended by microbiologists in the search for better processess for the production of optically active lactones. The parametres derived from EPGs were analysed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues.
The aligned read files were processed by Cufflinks v2. The yields and optical rotations of prepared products are given below. Their odor is more intense compared to the S -isomers. A unique list of gene symbols was uploaded via the web interface. The crude products were purified by preparative column chromatography using silica gel Kieselgel 60, — mesh, Merck with mixtures of hexane-acetone in various ratios as eluents. This bioassay allows to study aphid host preferences under semi-natural conditions.
Nutritional Biochem Corp. The parametres derived from EPGs were analysed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues. SAM files produced by TopHat to identify differentially expressed transcripts between the two pools.
Therefore, looking for new stereoselective methods of synthesis of the optically pure molecules with various biological activities is one of the most developed areas in current chemistry. The bioassays were started at 10—11 a. All lactones originate from their corresponding 4- or 5-hydroxy carboxylic acids, although the precise mechanism by which these substrates are produced remains elusive [ 9 ]. The process in accordance with the invention is conveniently carried out by adding castor oil or castor oil hydrolysate, as the substrate, to the culture medium at the onset of cultivation, as the sole carbon source.
TopHat builds a database of potential splice junctions and confirms these by comparing the previously unmapped reads against the database of putative junctions. Apart from the food industry it is used as a fragrance composition in the cosmetics industry and a flavor in the tobacco industry. Treated and control leaves were placed in a Petri dish and allowed to dry for 1 hour to permit the evaporation of the solvent.
Aphids that were moving or out of any of the leaves were not counted. Biological properties of chiral compounds are often strongly related to the absolute configuration [ 21 ]. Received Sep 15; Accepted Dec The progress of reaction and enantiomeric excesses of the lactones were determined by gas chromatography GC.