| Abiotic stresses, such as drought, salinity and chillling, negatively influence survival, biomass production and crop yield. Many important crops, such as rice, maize, soybean, cotton and tomato, are sensitive to chilling and are incapable of cold acclimation. Maize is the crop with the largest cultivated area in the northeastern of China. Maize can frequently experience low temperature at the seedling stage in the spring because of the geographical reasons, resulting in yield reduction 20%, especially in the severe chilling injury years. It not only threated stable yeild caused by fluctuations between different years, but also brought down the quality of maize. Chilling stress has become one of the most important abiotic stresses that limits maize yield in this region of China.In the present study, we successfully selected 3 chilling-resistant maize inbred lines from 296 inbred lines by low-temperature germination, low temperature screening at seedling stage, chilling stress-related physiological and biochemical parameters and field identification and proposed a set of effective screening method of germplasm resources of maize chilling-resistant. We used cDNA-AFLP to monitor the gene expression profiles involved in chilling responses in maize by a total of 336 primer combinations. According to the cDNA-AFLP analysis, approximately 10,000 TDFs were displayed. Of these, a total of 441 fragments were verified, about 4%. A total of 30-50 AFLP bands have been detected per combination using autoradiography that ranges from 70 to 600 bp. Based on the temporal patterns of expression, the TDFs were classified as'Induced','Late-Induced','Early Transient-Induced','Up-Regulated','Rapidly Switched-Off','Gradually Switched-Off','Down-Regulated'and'Other expression profile'. 114 of the 441 TDFs were successfully sequenced. Based on the sequence analysis, the 58 TDFs of known functions were involved in metabolism (13, 11.40%), photosynthesis and energy (10, 8.77%), transport (5, 4.39%), signal transduction (11, 9.65%), secondary metabolism (2, 1.75%), defense response (7, 6.14%), transcription (3, 2.63%), cytoskeleton (3, 2.63%) and transposable elements (4, 3.51%), suggesting that maize undergoes a complex adaptive process in response to low temperatures. Seven candidate genes involved in chilling responses were identified by semiquantitative RT-PCR. Three full-length cDNA were isolated through in silico cloning and named as ZmMAPKKK (1266 bp), ZmCLC-D (1302 bp) and ZmRLK (1447 bp), Genbank accession number are GU344734, GU344733 and GU344735. Bioinformatics analysis showed that ZmMAPKKK, ZmCLC-D and ZmRLK had high similarity with the function known genes in other plants, and with the typical conservative region of the gene family. Evolutionary tree analysis showed that the three genes were closest to the gene in the same gene family in rice.According to the real time RT-PCR analysis, the expression of the novel genes appears to be strongly induced in chilling stress, specifically 12 hours after chilling treatments. At their peaks, expression levels of these genes were more than 3-4 folds greater than untreated controls. The plant expression vector pCAMBIA3301-ZmMAPKKK, pCAMBIA3301-ZmCLC-D and pCAMBIA3301-ZmRLK were successfully constructed and transformed into Arabidopsis and maize using agrobacterium tumefaciens mediated method.In this study, we also use the MSAP technique to study the variation of methylation pattern at different chilling treatment time in maize. The results showed that the overall level of methylation was between 30.76% ~ 31.53% at 6 time points. Although the overall level of methylation was similar, the non-methylated sites of chilling treatment were less than the proportion of untreated controls. According to the results, combined with the known methylation and gene expression, we hypothesized that the increase of non-methylated CCGG sites after chilling treatment may contribute to the increase in the expression of many genes, leading to possible chilling response mechanisms in maize. In addition, 4 types of bands were found and showed a different variation. A1, B1, C1 types did not change, the total amplified bands was about 90% of the total site in the treatment group, suggesting that the majority of sites can maintain their normal methylation status in the process of chilling stress. The methylation status at 2 h, 6 h and 12 h was relatively stable, about 12%. However, the level of DNA methylation reduced at 24 h, only 6%. The level of methylation increased and reached to 10%. A total of 211 fragments were recovered and sequenced, sequence analysis showed that only 28 of the sequence homology to the known genes. These genes are involved in multiple processes of plant growth and development. In addition, we found three sequences were highly homologous to transposons and retrotransposon. Chilling stress might induce the activation of maize transposon, which methylation changes occur to regulate gene expression, to adapt to environmental stress.In the present study, we constructed 4 small RNA libraries (common temperature, chilling treatments for 2 h, 6 h and 12 h), sequenced by high-throughput Solexa sequencing technology, and analyzed the regulation mechanism of chilling adaptation by miRNA under chilling stress in maize. 48,495,408 fragments were sequenced in 4 small RNA libraries, most of them are 21 nt and 24 nt, of the total 28.23% and 35.55%. Numbers of 21,853,812, specieses of 2,360 candidate miRNA were identified by direct cloning method. Expression analysis of known miRNA among different libraries showed that 56 known miRNA were significant or extremely significant, 38 known miRNA were up-regulated (67.86%), while 18 known miRNA were down-regulated (32.14%). In cluster analysis, miRNAs who have similar pattern of differential expression in different libraries were clustered together. All the 82 differentially expressed miRNA were clustered in one after 4 rounds of cluster. There were 5 sub-clusters generated after the second last cluster. A total of More than 2360 novel miRNAs and 11,309 target genes were predicted. These target genes involved in all aspects of plants, including plant growth and development (cellulose synthase, chlorophyll binding protein, early flowering protein,etc.), stress response (defensins, lectins, heat shock proteins, etc.), Signal transduction (calmodulin, serine / threonine protein kinase receptor, mitogen-activated protein kinase source, etc.), metabolic (such as glucose transporter protein, glutamine synthetase, ATP enzymes, etc.), transcription (translation initiation factor protein, transcriptional coactivator protein, etc.), and nutrient absorption (potassium transporter protein, magnesium transporter protein, chloride ion channel protein, etc.), antioxidants (catalase, ascorbic acid oxidase enzymes). This indicates that the target genes regulated by miRNA and a variety of physiological and biochemical changed to adapt to adverse conditions under chilling stress in maize.In this study, a large number of experiments showed that there is a complex mechanism response to low temperature in maize. This study widened the understanding of maize response to chilling stress on the transcriptional level, methylation variation and post-transcriptional levels, and provided important information for the study of complex regulative networks and related miRNAs, it has important theoretical and application value for improving the chilling resistance traits of maize by the usage of chilling resistant genes and microRNA. |
