Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist

<i>B</i>. <i>pseudomallei</i> is internalized into amoebae but could not resist digestion.

<p>CLSM micrographs show the internalized <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A-C), <i>Acanthamoeba</i> sp. (D-F) and isolate A-ST39-E1 (G-I) at 0, 3 and 6 h after kanamycin treatment. Orange fluorescence represents CellTracker^™ Orange-<i>B</i>. <i>pseudomallei</i> and green fluorescence indicates the amoebae stained with FITC-ConA for visualization.</p

Numbers of <i>Acanthamoeba</i> sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with <i>B</i>. <i>pseudomallei</i> (▲) or <i>E</i>. <i>coli</i> (positive control) (■) or deprived of bacteria as a negative control (●).

<p>Graphs and figures show no significant differences between amoebae fed on <i>B</i>. <i>pseudomallei</i> and <i>E</i>. <i>coli</i>. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (<i>p</i> ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.</p

Specific primers for the 18s rRNA gene of amoebae.

<p>Specific primers for the 18s rRNA gene of amoebae.</p

Intracellular survival through time of <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A), <i>Acanthamoeba</i> sp. (B) and isolate A-ST39-E1 (C).

<p>Time zero represents 3 hours after <i>B</i>. <i>pseudomallei</i> feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * <i>p</i> < 0. 0001 using ANOVA.</p