Vicia sativa

Dobosz, Renata, Flis, Łukasz, Bocianowski, Jan & Malewski, Tadeusz, 2023, Effect of Vicia sativa L. on Motility, Mortality and Expression Levels of hsp Genes in J 2 Stage of Meloidogyne hapla, Journal of Nematology 55 (1), pp. 1-17 : 4-5

Effect of V. Sativa seeds on the expression levels of HSp genes in J 2 stage

ExPOSURe Of J2 STage TO V. SaTiva Seed diffUSaTeS: Only the diffusate from non-sterilized vetch seeds of the Ina cultivar in water was used in the study. The Ina cultivar is characterized by more than twice the content of cyanogenic compounds per gram of dry seeds compared to the Jaga cultivar. Three separate experiments were performed. They were carried out at a temperature of 21 ± 1° C. Each experiment was repeated three times. 200 specimens of the J2 stage of M. HaPla were used for each run. In the first experiment, the nematodes were placed in Petri dishes in a diffusate for 24 hours. Each of the three Petri dishes with the diffusate contained 200 specimens of the J2 stage specimens. After this time, the immobile (paralysed) nematodes were transferred to Eppendorf tubes and immediately preserved with phenosol ( RNA preservative reagent by A & A Biotechnology RNA) and frozen at -80° C, until obtaining the isolation of total RNA. In the second experiment, nematodes (200 specimens x 3 repetitions) were placed in a common vetch seed diffusate for 24 h and then transferred to water for 24 h. In this experiment, 24 hours after being transferred from the diffusate to water, almost all the nematodes regained the ability to move (approximately 8% of the specimens remained stationary in each run of the experiment). The specimens were then preserved with phenosol and immediately frozen at -80 ° C until RNA isolation. In the third experiment, J 2 specimens (200 specimens × 3 repetitions) were left in the water for 24 hours, the control sample. The control sample was then preserved and frozen at -80 ° C.

RNA exTRaCTiOn: The isolation of total RNA from the three experiments described above was performed by the modified phenol-chloroform method with the use of the A & A Biotechnology kit ( Chomczynski and Sacchi, 1987). RNA extraction was performed in the pre-PCR room using disposable DNase- and RNasefree pipette tips with filters and test tubes. All stages of the experiment were performed at a temperature of approx. 4° C. The obtained RNA was stored at -80° C until further analysis. The quantity and quality of the obtained RNA was determined using the NanoDrop 1000 Spectrophotometer vs. 3.7 (Thermo Scientific). RNA integrity was assessed by electrophoresis using the RiboRuler High Range RNA Ladder (Thermo Scientific).

CDNA SynTHeSiS: DNase digestion of the obtained RNA and first-strand cDNA synthesis were performed with the iScript ™ gDNA Clear cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the procedure described by the manufacturer. The DNAse reaction mixture, in the amount of 0.5 μl and 1.5 μl of DNAse buffer for each 14 μl of RNA, was used to treat the residual DNA in the isolated RNA at a concentration of 10 ng / μl. The mixture thus prepared was incubated for 5 min at 25° C. It was then incubated for 5 min at 75° C to inactivate DNase and transferred to ice. The RNA prepared in this manner was used for the synthesis of cDNA. However, if longer storage was necessary, the RNA sample was placed at -80° C.

For cDNA synthesis, we used 16 μl of DNase treated RNA and 4 μl of the reaction mixture containing i.a. iScript reverse transcriptase enzyme, RNAase inhibitor, primers oligo (dT), free nucleotides (dNTPs), magnesium chloride (MgCl 2) and polymerase stabilizing substances. The synthesis was carried out in the following steps: priming – 5 min at 25° C; reverse transcription (cDNA synthesis) – 20 min at 46° C; inactivation of the enzyme reverse transcriptase – 1 min at 95° C. The obtained first strand of cDNA was used directly in a QUanTiTaTive POlymeRaSe CHain ReaCTiOn (qPCR) analysis or stored at -20° C.

PRimeR deSign: The primers for the tested genes MH – HSP 90, MH – HSP 1, MH – HSP 60, MH – HSP 43 and MH – HSP 12.3 used in the qPCR reaction were designed using the PRIMER3 vs. 0.4.0 program ( Untergasser et al., 2012). The Merck company performed the synthesis of the designed primers. The reference malate dehydrogenase (MdH) gene and primers for amplifying a fragment of this gene (forward 5´–GAAAGCCAGGGATGACAC–3´, reverse 5´–AGAAAAGCATTGGGACAG–3´) were selected on the basis of research by Wu et al. (2019a). The MdH gene has been shown to be one of the most suitable reference genes for gene expression studies in M. HaPla.

QPCR ReaCTiOn: We analyzed the expression of five HSP genes ( MH – HSP 90, MH – HSP 1, MH – HSP 60, MH – HSP 43, and MH – HSP 12.3). Expression of the 2 ΔΔ Ct method ( Livak and Schmittgen, 2001) was used in calculating the relative ratio, but instead of the value being 2, the correct amplification efficiency was used. We used a noise-resistant iterative nonlinear regression algorithm (qPCR miner) to determine the efficiency of the PCR reaction ( Zhao and Fernald, 2005). The expression levels are indicated as the foldchange normalized to the control (untreated diffusate), normalized to the value of 1.

qPCRs were performed on the 7500 qPCR system (Applied Biosystems, Waltham, MA, USA). Reactions were carried out using SsoAdvanced Universal SYBR Green Supermix kit (Bio-Rad), while cycle threshold (Ct) estimates were obtained using the relative quantification module in the software package. PCR reactions were performed in a final volume of 20 µl containing 1.5 µl of the cDNA sample, 3.0 µl of the primer mix (5 micromoles of each primer), 10 µl of the 2x SsoAdvanced Universal SYBR Green Supermix, and 5.5 µl of H 2 O. The assay included a no template control and each of the test cDNAs from three biological replications. According to the instructions for this kit, after 30 sec. at 95° C, the cycling conditions were as follows: 40 cycles at 95° C for 15 sec. and 59° C for 60 sec. To validate the specificity of amplification, a post-amplification melt-curve analysis was performed. Amplicons were first denatured at 95° C for 60 sec. and then cooled to 72° C, and the temperature was then gradually raised to 95° C. Fluorescence data were recorded continuously during this period, and analyzed subsequently using the Tm calling module in the SDS Software v1.4 software.

STaTiSTiCal analySiS: HSP gene expression values were expressed as the mean fold difference. Statistically significant differences between treated and control samples (p≤0.01 based on T -Student test, using an online tool “Do my qPCR calculation”) are shown ( Tournayre et al., 2019).