JIA-2018-09

1948 XU Li-ming et al. Journal of Integrative Agriculture 2018, 17(9): 1946–1958 proper AlCl 3 concentrations for the experiment. The Al treatment of the preliminary test was 0, 20, 40, 60, 80, and 100 μmol L –1 AlCl 3 , respectively. Then, 60 μmol L –1 AlCl 3 was selected to treat maize seedlings for the later analysis. At least 20 seedlings were used at every concentration and three replicates were carried out for the experiment. AlCl 3 treatment After the proper AlCl 3 concentration was decided, 5–10 seedlings after adaptation period in CaCl 2 solution were treated with 200 μmol L –1 CaCl 2 +0 μmol L –1 AlCl 3 (control) and 200 μmol L –1 CaCl 2 +60 μmol L –1 AlCl 3 (Al treatment) for 1 and 6 h, respectively, and the pH was adjusted to 4.0 with HCl. Three replicates were set for the experiment. Net growth length (NGL) The original growth length of root just before treatment and the final growth length after treated for 1 and 6 h were measured respectively, then NGL was calculated as follows: NGL=Final growth length–Original growth length. After treated for 1 and 6 h, root tips (used for microarrays) were collected, and frozen in liquid nitrogen respectively. The treated seedlings of three replicates were all collected and stored at –80°C. 2.2. RNA extraction and cDNA synthesis Total RNAwas extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to themanufacturer’s protocol. Additional on-column DNase digestion was performed three times during RNA purification using the RNase-free DNase Set (Qiagen, Germany). The concentration and quality of each RNA sample were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Quantified RNA was reverse transcribed into cDNA with the superscript the first-strand synthesis system and the oligo(dT) primers (Invitrogen, Carlsbad, CA). 2.3. Microarray hybridization and data analysis The cRNAof each sample was obtained by complementation with the first-strand cDNA and labelled with biotin Cy3 (Al-treated samples) and Cy5 (the control samples), respectively. AQiagen RNeasy Mini Kit (Qiagen) was used to purify the fluorescent cRNA probes. An equal amount of cRNA probes were mixed for all of experiment groups and used for hybridisation on an Agilent Maize Whole Genome Microarray (Agilent) following the instructions. And one- channel chip hybridizations were performed by ShanghaiBio Corporation (SBC) (Shanghai, China) with the Agilent Genechip Maize GenomeArray (Agilent Technologies). The chip hybridization results were scanned using an Agilent Dual Laser DNA Microarray Scanner (GeneChip Scanner 3000, Affymetrix, USA) and Command Console Software 3.1 (Affymetrix) with default settings. The raw data were normalized by the MAS 5.0 algorithm with GeneSpring Software 11.0 (Agilent). Genes with more than 2-fold difference ( P <0.01) between the control and Al-treated maize seedlings were selected using the SBC Analysis System (http://sas.ebioservice. com/). To avoid false positives, the q -value, the minimum false discovery rate at which the test may be called significant, was selected at P <0.05. Gene Ontology (GO) terms which could specifically describe genes character and their regulation mechanisms were performed by AgriGO (http://bioinfo.cau.edu.cn/agriGO/ ). The annotation information was obtained from GenBank (http://www.ncbi . nlm.nih.gov/genbank/). 2.4. Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was conducted with TaKaRa ExTaq RT PCR Kit and SYBR green dye (TaKaRa, China) and a DNA Engine Opticon 2 Machine (MJ Research, Waltham, MA). Primers (Appendix A) were designed to amplify 150–250 bp. The efficiency of the primers set was calculated by performing qRT-PCR on several dilutions of first-strand cDNAs, and they were similar. The specificity of each primer set was checked by sequencing PCR products. Amplification profiles were as follows: 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s, and a final melting-curve 70–95°C. The melting-curve was used to check the specificity of the amplified fragment. Each sample was performed in triplicate. The maize genes Actin2-like and Ubiquitin , were used as internal controls to normalize all data. Relative expression levels of candidate genes were calculated using the 2 −ΔΔC T method. 3. Results and discussion 3.1. AlCl 3 concentration in the present study In Fig. 1, NGL of root decreased with AlCl 3 concentration increasing after treated for 6 h, but NGL was similar under different AlCl 3 concentration treatments for 1 h. The root growth length was reduced about 40% with 60 μmol L –1 AlCl 3 treatment for 6 h. In addition, NGL under 100 μmol L –1 AlCl 3 treatment decreased the same as that under 60 μmol L –1 AlCl 3 treatment for 6 h, suggesting 100 μmol L –1 AlCl 3 treatment for 6 h may active a tolerance response in maize. Since the maize transcriptome has no or a little change in adaption to Al environment, 60 μmol L –1 other than 100 μmol L –1 AlCl 3 could induce a seriously disorder transcriptome in maize. In this study, 60 μmol L –1 AlCl 3 was the best choice for microarray analysis because it induced acutely Al toxicity symptome. Some similar results have been reported in Medicago truncatula cultivar response