Total RNA were extracted with good quality and quantity to produce cDNA(fig 1). The number of bands between five temperature treatments were compared. The -5ï¿½ï¿½C treatment had made more band than another treatments (fig2). It could be implied that between five cold temperatures the -5ï¿½ï¿½C is the best temperature in gene expression profile in Viola. wittrockiana under the cold stress. In the reverse transcription reaction three types of Oligo-dT primers anchored with C, G or A were used. The results showed among these three primers, oligo-dT C and G had more bands than oligo-dT A (fig3). Also the result for eight arbitrary primers used in this study showed in figï¿½ï¿½4. According to this figure, the most efficient primer (number of bands) that has created more bands was HAP30 and the weakest one was HAP25 (fig4). The result of this study was similar to the results of Caamal et al. (2007), which have investigated the differences in gene expression of banana plants under cold conditions. They showed that the combination of primers (HAP25 to HAP32), Oligo-dTG and HAP30 and HAP31 made many more bands than other combination of primers.
mRNA differential display
According to the figures produced by poly acryl amide gel electrophoresis, three anchored primer combinations amongst the eight arbitrary primers produced abundant different bands of amplified cDNA and obtained only fifty one differential bands from the Viola.wittrockiana (data not shown). From this number (fifty one), six differential bands were selected for cloning, sequencing, and analysis. The clones, obtained from each band on the gel, were examined. These clones were randomly selected from each transformation, and the sequence of the cloned insert in each of the clones was determined. It is clear that all of the cold-responsive genes are not affected to the same degree (fig5). Among the six selected sequences, three sequences showed the highest expression at below 0ï¿½ï¿½C (Fig5:a:b:c), two sequences showed the highest expression at above 0ï¿½ï¿½C (Fig5:d:f) and finality only one sequence, showed the highest expression at 0ï¿½ï¿½C (Fig5:e).
Sequence analysis of differentially displayed cDNA fragments
Sequences analysis of the produced cDNAs from Viola.wittrockiana led to identification of six different fragments named as MHV1, MHV2, MHV3, MHV4, MHV5 and MHV6 for the first time. The sequences have been recorded in the EST database of NCBI.
The results of this analysis were summarized in table3. The MHV1, MHV3, MHV4 and MHV5 had no significant homology with the published DNA and protein sequences. It can imply that the four displayed sequences (MHV1, MHV3, MHV4, MHV5) were identified and recorded for the first time. But MHV2 and MHV6 showed significant homology to Populus alba chloroplast DNA, total genomic DNA(Accession number :AP008956.1) and total genomic and plastid DNA of Viola seoulensis(Accession number :KP749924.1), respectively. MHV2 sequence also showed 98% similarity to hybrid cultivar of Popolus tree (Populus tremula x Populus alba) under drought stress condition (EST registered in NCBI with accession number: CU227874.1). The Homology of sequences were evaluated using DNA STAR software. This sequence similarity between Viola and previously documented Populous DNAs, can suggest that MHV2 is a sequence related to the gene encoding the 23S rRNA in Viola chloroplast. Presence of ribosome subunits can be related to protein or enzyme production. Since the bands of MHV2 sequence showed a higher appearance at lower temperature (fig5:b), Perhaps, it can be said that a particular protein or group of proteins were produced underï¿½ï¿½ the cold conditions in chloroplast. In a study on tobacco plant under cold stress conditions, it is shown that presence of Rpl33 is important in cold acclimation. Rpl33 is a ribosomal protein that is created in the chloroplast. It was found that when Rpl33 is nuked out, it doesn’t affect the plant viability and growth under standard conditions. Whereas, exposure of the mutant tobacco plants to low temperature stress, it caused severe damages ( Rogalski et al. 2008). There is a report about the 23S rRNA that has been submitted a sequence (EST: GR942609.1) which is very similar to 23S rRNA gene expressed in chloroplast of Cowpea under drought conditions (Coetzer and et al. 2010). Since drought, salinity and temperature stresses are often associated and may have similar effects and the plant cell cause similar damages on plants (Wang et al. 2003). Drought, salinity, and low temperature stress impose an osmotic stress that can lead to turgidity loss. Cell membrane may become disorganized, the proteins may undergo loss of activity or get denatured. Often excess reactive oxygen level of species (ROS) are produced and led to oxidative damage. As a consequence, inhibition of photosynthesis, metabolic mal functions, and damage of cellular structures contribute to growth delay, reduced fertility, and premature senescence)Krasensky and Jonak 2012(. Generally, the complex and interactive relationships among different pathways are involved in the molecular regulation of cold acclimation.
The inferred DNA sequence of MHV6 shows similarity to trnI-GAU gene that encoded the tRNA-Ile. tRNA-Ile is one of tRNAs involved in translation and carries the amino acid isoleucine to the ribosome. This sequence was present in all of the five temperature treatments, but it’s appearance at +5ï¿½ï¿½C was much stronger (fig5:f),with the assumption that the presence of tRNA-Ile , necessarily lead to the production of isoleucine. Mayer et al.(1990) showed that the rate of isoleucine synthesis were increased up to 6-fold in cowpea plant under heat shock conditions. Also It has been found that compound of Jasmonoyl- isoleucine accumulation is needed for abscisic acid (ABA ) build-up in roots of Arabidopsis thlliana under water stress conditions (Ollas et al. 2015). ABA is a growth regulator which is known to be responded to osmotic and cold stresses in plants(Ishitani et al.ï¿½ï¿½1997). As previously mentioned, stresses such as drought, salinity and temperature are often happening in concert and may cause similar effects on plants (Wang et al. 2003). In another study on the Bacillus subtilis it was shown that the isoleucine has playï¿½ï¿½ a major role in cold resistance and the absence of isoleucine reduced the synthesis of anteiso-branched fatty acids(Klevin et al. 1999). The correct physical state of membrane lipids is required for optimal membrane structure and function. Temperature markedly affects membrane lipid composition, and changes in lipid composition are thought to occur in order to maintain an appropriate amount of the liquid crystalline state. Bacillus cells respond to a decrease in the ambient growth temperature by increasing the proportion of low-melting-point fatty acids of their membrane lipids (Cybulski et al. 2002,Mansilla et al. 2004). The changes in plasma membrane fluidity have been suggested as the basis for understanding the responses of low temperature. Perhaps, certain lipids in the plasma membrane of V.wittrockiana cells may have a slightly higher degree of saturation, which may result in increased cell membrane leakage during cold stress. The importance of membrane lipid fluidity for plant cold tolerance is well known (Ishitani et al. 1998, Miquel et al. 1993,Murata et al. 1992), But it is not clear if there is any relationship between membrane fluidity and gene expression under low temperature condition in higher plants. Mechanism of genes expression under chilling stress is not fully understood. More information is required for factors involved in the cold resistance to get a better understanding. In the case of cold-hardy plants, like Viola, there is little information about the molecular processes associated with cold acclimation. This information could allow one day, to block specific pathways that currently result in production of cold stress-tolerant transgenic crops. Our experiments clearly showed that there are significant differences between the gene expression patterns in the control temperature (25’ï¿½C) and the low temperature treatments (ï¿½ï¿½25’ï¿½C).ï¿½ï¿½Some of the bands showed the higher appearance at a certain temperature. Based on the similar experimental conditions, it can be said that the differential bands at the certain temperature, are related to the genes that are directly or indirectly involved in the chilling resistance. But more genes should be isolated with complete set of experiments, in order to get a more clear understanding of the molecular mechanisms of chilling resistance in Viola and other related species.
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