Gene expression profiles in promoted-growth rice seedlings that germinated from the seeds implanted by low-energy N+ beam

摘要

Dry seeds of rice cultivar Xindao-18 (Oryza sativa L. ssp. japonica) were used in ion implantation. After implantation with the ion beam, all seeds were planted on sterile medium with 0.8% agar (Sigma) in a climate chamber in the dark at 28°C. After the seeds had been incubated for 96?h, the seedlings were divided into two groups: one group was used for the RNA isolation, the other group was used in the investigation of the simplified seed vigor index from the 10-day-old seedlings grown in a climate chamber during a 12-h dark/12-h light cycle at 28°C. The low-energy ion beam implantation of seeds was performed with the Ion Beam Bioengineering Facility (UIL.0.512, TNV, Russia). For implantation, the germs of seeds (62 days after harvesting) were positioned facing upward toward the incoming ion beam, while endosperms of seeds were downwardly immersed into the polyfoam that was fixed in the sample disks and then put into the facility's target chamber. When the vacuum of the target chamber was below 10?2 Pa, the seeds in the sample disk were implanted with low-energy (40 KeV) N+ with the ion fluences: 3?×?1017 N+/cm2, 6?×?1017 N+/cm2, 9?×?1017 N+/cm2. Controls included seeds that were in normal surroundings and a vacuum without ion implantation for 264?min, the same as the implantation with fluence 6?×?1017 N+/cm2. After implantation, all seeds were planted on sterile medium with 0.8% agar (Sigma) in a climate chamber under dark conditions at 28°C. For each fluence of ion-beam implantation, three independent biological replicates were used. Two hundred seeds were implanted in each replicate. The germination rate (from 100 seeds) was investigated from the 7-day-old seedlings, and all the seedlings that were used to investigate the germination rate (from 100 seeds) were oven dried at 80°C for 12 h to investigate the dry weight (g). Total mixed RNAs from the promoted growth rice seedlings were isolated using RNA plant reagents (Tiangen Biotech) and purified by use of the RNeasy Plant Kit (Qiagen). The yield and purity of RNA were determined spectrophotometrically (Nanodrop ND1000). Thirty uniformrice seedlings were used to extract the total RNA to construct the RNA pool of a biological replicate. Therefore, at least 15 total RNA pools were constructed, including three control samples, three samples treated with a vacuum, and nine samples implanted by ion implantation. The Agilent Microarray hybridization (Agilent-015241 Rice Gene Expression Microarray) and raw data analysis were carried out by the ShanghaiBio Company Ltd, including the procedures for cDNA and cRNA synthesis, cRNA Cy3 fluorescence labeling (GE healthcare PA13105), hybridization (Agilent G2545A), washing, scanning (Agilent G2565BA Microarray Scanner System), data collection and normalization. This experiment was performed three times, resulting in three biological replication samples for each ion fluence for the significant statistics. The functional cataloging of differentially expressed transcripts was carried out by the web software FunCat 2.1 (http://mips.helmholtz-muenchen.de/proj/funcatDB/search_main_frame.html). One transcript could be classified into more than one catalog group. The results showed that the smaller-fluence implantation of the N+ ion beam (6?×?1017 N+/cm2) enhanced the vigor index (P ?0.05) of the rice seedlings, and the larger-fluence implantation (9?×?1017 N+/cm2) damaged the rice seedlings because of the smaller simplified vigor index compared with the controls (P ?0.05) (Table 1). In order to investigate the key molecules in encouraged rice seedlings responding to ion-beam implantation, we isolated the total mixed RNA from the samples that had undergone the ion-beam implantation with ion fluence: 6?×?1017 N+/cm2 and exposure to the vacuum for microarray. We analyzed genome-wide gene expression profiles of three sample groups including the controls, the vacuum-treated and the ion-implanted, using the Agilent-015241 Rice Gene Expression Microarray, including probes to query 42 476 transcripts (the microarray data was deposited in gene expression omnibus [GEO] with the accession number GSE36152). Based on the analysis of the materials and methods, the resulting analysis revealed that there were great differences (Table 2) in gene expression profiles between the vacuum-treated samples and the ion-implanted samples. More transcripts were differentially expressed underlying the ion implantation than the vacuum exposure, and fold changes of gene expression in ion-implanted samples were more distinctive than in the vacuum-treated samples. In order to have a clear functional categorization of the differentially expressed genes, FunCat was used. FunCat has been est