Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. High-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B1 than the controls was performed. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. Two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines were found. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins. The objective of this study was to determine the putative involvement of small RNA populations in aflatoxin reduction. Seeds of RNAi-transformed and control peanut plants were used in this study. Transformed peanut lines (288-10 and 288-11) were obtained through Agrobacterium-mediated transformation, by integrating dsRNA targeting five genes involved in aflatoxin biosynthesis (RNAi-5x) as previously reported. The five genes were AFL2G 07223 (aflS or aflJ), AFL2G 07224 (aflR), AFL2G 07228 (aflC/pksA/pksL1), AFL2G 07731 (pes1), and AFL2G 05027 (aflatoxin efflux pump, afl ep); numbers correspond to accessions in A. flavus genome annotation (BROAD Institute, Cambridge MA), other names are in parentheses. Detection and expression of the selectable marker NPTII and the RNAi-5x insert were carried out using single-tube nested PCR (STN-PCR), as described by Gomes et al. [30] with modifications by Arias et al. [22], using external primers PCAPD 5714F: 5'-AGGCTATTCGGCTATGACTG-3' and PCAPD 6446R: 5'-CGTCAAGAAGGCGATAGAAG-3', and internal primers PCAPD 5730F: 5'-ACTGGGCACAACAGACAATC-3' and PCAPD 6249R: 5'-ATATTCGGCAAGCAGGCATC-3'. Briefly, the STN-PCR involves two PCR reactions, performed in one tube, in 20 μl total volume, containing 4 μl of 5x Phire reaction buffer, 1.25 mM dNTPs, 0.4 μl Phire Hot start II DNA polymerase, 10 pmol of each forward and reverse external primers, 1.0 μl DNA template, and 13.4 μl RNase-DNase free water. The amplification conditions consisted of 2 rounds of 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. The PCR products were analyzed by gel electrophoresis and sequencing. The results showed that the RNAi-5x insert was successfully integrated into the peanut genome and was expressed in the transformed lines. The expression of the RNAi-5x insert was confirmed by RT-PCR analysis. Total RNA was extracted from non-inoculated half cotyledons of immature and mature seeds, after 24 and 48 h incubation, using RNeasy Plant Mini Kit (Qiagen, CA) in the Qiacube robot. Complementary DNAs were synthesized using 1 μg of RNA per sample, combining random hexamers and oligo-dT in Superscript III First Strand Synthesis Super Mix (Invitrogen, MA). The RT-PCR analysis showed that the RNAi-5x insert was expressed in the transformed lines, but not in the control line. The expression of the RNAi-5x insert was also confirmed by Western blot analysis. Total protein was extracted from non-inoculated half cotyledons of immature and mature seeds, after 24 and 48 h incubation, using RIPA buffer (Sigma, MO). The protein extracts were analyzed by Western blot using an anti-NPTII antibody. The results showed that the RNAi-5x insert was