| High temperature stress is one of the major abiotic stresses in the world that severely restricts crop production. The Yangzi River Valley is the most important rapeseed oil production area in China, but this area usually suffers transitory or long period high temperature of over30℃at seed filling stage, which could result in heat-forced maturity, decreased oilseed yield and quality. However, the inheritance and molecular mechanisms of anti-heat-forced maturity in oilseed rape are still largely unknown. Heat resistance genes including heat stress transcription factors were well studied in model plant Arabidopsis, but how to apply the theoretical knowledge to genetically improve heat resistance of crops effectively is poorly understood. To address these issues, three major works were conducted in this study:1) Global transcription profiles in siliques of Brassica napus from20days after flowering after heat stress was analyzed using a95k Brassica60-mer GeneChip. The newly identified transcripts further enriched the reservoir of heat-responsive genes.2) Using double and triple mutants, functional analysis and potential regulation network of HsfAs and HsfBs were conducted in Arabidopsis by phenotypic analysis, gene expression and protein interaction.3) Functions of an unknown gene of H38(AT4G23493) that was discovered to be a potential heat regulator by microarray survey, were systematically studied from the aspects of gene expression, subcellular localization, physiological and biochemical studies and growth and developmental regulation analysis. The main conclusions of this study achieved are as follows:1. We detected1248up-regulated and898down-regulated genes in the heat stressed rapeseed siliques;925up-regulated and581down-regulated were detected in the silique walls (SW), and837up-regulated and383down-regulated genes in the seeds. The alteration of the transcripts may provide clues for revealing the mechanism for heat-resistance in the oilseed rape at the seed filling stage.2.40.9%(511) of the up-regulated genes are present in the seeds and silique walls simultaneously, however, only66genes were decreased in both organs; and the average fold-change of the up-regulated genes (2.1to72.8) was significantly larger than that of the down-regulated ones (2.0to5.6). Notably, stress-related genes and transcription factors were significantly enriched in both organs. Many of the up-regulated genes are known to be heat-marker genes, including13Hsfs and91Hsps, and DREB2a, ROF2, GolSl, MBFlc, and others such as CYP707A4and panthenol oxidized cytochrome bd enzyme which were not identified in heat response previously.3.411up-regulated and514down-regulated genes were detected in the silique walls, but not affected in seeds. Among them, some of the genes were involved in the major metabolic pathways of photosynthesis and transport (water, sugar and ion) and a cluster of21genes that are involving in glucosinolates metabolism were simultaneously reduced in heat stressed silique walls. All these altered expression may directly related to the decreased yield and quality of rapeseed after heat stress.4.325up-regulated and314down-regulated genes were altered in seeds and whose expression did not change significantly in the silique walls. Genes related to seed storage proteins, phytohormones, lip id metabolism and other important metabolic pathways were changed. Similar to the silique walls, we have detected as many as12genes that involved in the anthocyanin biosynthetic pathway were suppressed in heat stressed seeds.5.1/3of all the differentially expressed genes encodes protein with unknown functions (484in silique walls,398in seeds),502of which can be found their homologous sequences in Arabidopsis, while181were rapeseed specific genes. Therefore, there may be many unknown and species-specific genes that regulate plant thermotolerance.6. According to the microarray data,8genes were selected for further studies. Mutants of homozygous genes in Arabidopsis were obtained and confirmed by PCR and gene expression analysis. Further, thermotolerance assays were carried out at the stage of seed germination, young seedling and mature plants (before flowering) of each mutant; and h25, h29and h38were found to be heat-sensitive in at least one of the heat-treatment conditions. 7. From the phenotypic and expression analysis with the double mutants and single mutants of hsfA2and hsfA3under long-time recovery from heat stress showed that HsfA3was downstream of HsfA2and synergistically regulated the expression of HSP18.1and Hsp25.3-P. Moreover, HsfA2and HsfA3could strongly interact though their oligomerization domains by yeast two hybrid approach. Function analysis and regulatory study of three HsfBs were performed by phenotypic analysis after heat stress, and gene expression analysis of heat resistant symbol genes and potential target genes using double and triple mutants.8. H38was identified as a conserved gene with unknown function in the plant kingdom. In Arabidopsis, H38was mainly expressed in the mature and germination seeds and encodes the protein that was located in mitochondrial. H38was significantly induced after heat stress in seedlings and developing siliques by histochemical staining and qRT-PCR analysis, Mutant of H38was sensitive to heat stress at seedlings stage; while it accumulates more anthocyanins after heat stress at the early stage of flowering.9. Co-expression analysis revealed that expression of H38was intimately correlated to that of GolSl, SKI, heat shock protein AT5G37670and AT2G19310. The co-expression genes together with some representative Hsfs/Hsps and mitochondrial localized heat-related genes were studied by qRT-PCR, which provides important information of the regulatory network of H38.10. The tolerance of seed germination under glucose or/and ABA treatment was significantly enhanced in h38mutant; what is more the floral organs and seeds of h38were significant larger than the wild type which was mainly resulted from the increased cell size by cytology observations. |
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