| Maize(Zea mays L.) is a C4 plant species of gramineous and originated in tropical or subtropical regions. Growth and productivity of maize are severely constrained by chilling stress. Because maize is widely used as forage crop, and is an important raw material for chemical, food, fuel and medicine industry, the planting area of maize ranks first world wide, and it became the first major grain crops in China in 2008. Maize plays an extremely important role in the national food security in our country. The boosting requirement for maize demand of expanding the planting area to the high latitude and high altitude region is increasing. As originated in tropical and subtropical areas, maize showed sensitive to low temperature during the entire growth season. So it has important practical significance to study the molecular mechanism of cold resistance and improve the ability to adapt to cold stress on maize.In this study, the characteristics of two different cold sensitive maize inbred lines were analyzed from the aspects of morphology, cytology, molecular biology and so on. By morphological comparison, damage degree was lighter in W9816 and wilting severely in B73 after 4? cold treatment for 12 h. Cytological evidence showed that B73 stomata were longer than W9816. After cold treatment, B73 accumulated more plastoglobules than W9816 in plastid and the damage of cell membrane is more serious. Study on physiological and biological characterstics of the two maize inbred lines showed that B73 accumulate higher content of MDA and with higher electrolytic leakage than W9816. SOD, CAT and proline content were higher in W9816 than B73. NO content analysis showed that it was increased more rapid after cold treatment in W9816. ABA content analysis showed that it was significantly higher in W9816 than that in B73 after cold treatment. The fatty acid composition analysis on two maize inbred lines showed that linolenic acid(18:3) decreased mildly in W9816 than that in B73, the vaccenic acid(18:1) and linoleic acid(18:2) increased more than that of B73 after cold treatment. The q RT-PCR analysis on laccase, myb31, nnr1 and px3 genes showed that these genes accumulate at higher level in W9816 than that of B73 after cold treatment. In brief, cold-resistance inbred line W9816 and cold-sensitive one B73 shows differernt characteristics in morphology, cytology and molecular biology features.In recent years, a large number of miRNAs have been found in many species, and their expression has an important role in plant in response to biotic and abiotic stress. In this study, the expression profiles of two maize inbred lines in response to cold stress were analyzed by Illumina sequencing. MiRNAome sequencing found that 11 miRNAs were up-regulated after cold treatment: including miR166c/b/d, miR171, miR156, miR398, miR399 e and miR408, etc.. Twenty-six miRNAs include miR159, miR167, miR319, miR393, miR399a/b/c/h and miR166 h were found to be down-regulated after cold treatment. It is interesting to note that 80 miRNAs were differencially expressed between the two maize inbred lines. MiRNAs of higher expression in W9816 include zma-miR164, zma-miR156 h, zma-miR393 and zma-miR398 while zma-miR159, zma-miR156e/k, zma-miR160, zma-miR2118 b and zma-miR399 were found of higher expression level in B73. Twenty-eight target genes of differentially expressed miRNAs in response to cold stress were annotated mainly include MYB transcription factors and kinase. GO analysis indicated that these target genes are involved in auxin signaling pathway, RNA metabolism and DNA transcription process. Low temperature also affects floral organ development, meristem and leaf development. KEGG pathway analysis showed that these target genes involved in the biological process of pentose phosphate metabolism pathway, glucose metabolism, glutamic acid metabolism and ABC transporter metabolism. Sixty-five target genes of differentially expressed miRNAs within the two maize inbred lines were annotated as ARF, SBP and MYB transcription factors, etc.. GO analysis showed that these target genes are involved in plant growth and development, stress response and hormone response. KEGG pathway analysis indicated that these target genes involved in protein processing in endoplasmic reticulum, hormone signal transduction processes, cell autophagy and the splicing pathway.Through the degradome analysis on B73, a total of 158 miRNA target genes were detected, and 160 target sites were identified. The target genes of four differentially expressed miRNAs include miR156, miR159, miR166 and miR171 under cold treatment were detected by degradome sequencing. They may play important roles in the process of cold acclimation. Five target genes of miR171 belong to the GRAS family transcription factors were identified by RLM-RACE. The cleavage site was found to be between 10-11 and 11-12 bases, from the 5' end of the miRNA binding site.Northern blot and q RT-PCR analysis showed that zma-miR156k-5p, zma-miR164a-5p, zma-miR171d-5p and zma-miR398a-3p were up-regulated in responsed to cold stress while zma-miR167a-5p, zma-miR319a-3p and miR-n2 were down-regulated by cold treatment. Intriguingly, zma-miR393b-5p, zma-miR-n8, zma-miR2118 b and zma-miR-n9 showed different expression pattern after cold treatment in two inbred lines.In addition, the effects of maize miRNAs were analyzed in background of Arabidopsis. The ectopic expression of maize miR164, miR171 and miR398 genes in Arabidopsis made the the root length longer than wild type, the germination rate was elevated under low temperature treatment.In conclusion, through the comparative analysis on two different cold-sensitive maize inbred lines, the overall miRNAs and its target genes in response to cold stress were outlined by miRNAome and degradome sequencing. It is a foundmental work to fully explain the molecular mechanism of cold acclimation in maize seedling stage. |
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