Studies On Identification And Molecular Mechanism Of Seed Heat Tolerance In Rapeseed

High temperature has a negative impact on seed preservation. Along with the rise of global temperatures and the frequent appearance of the extreme high temperatures, heat tolerance of seed directly influences the quality of seed after storage, even relates to the development of agricultural production and seed industry. In our study, an identification method was established by studying the effects of different temperature and time on seed preservation, the molecular mechanisms of heat tolerance of seed were systematic researched from the aspects of morphology, genomics, epigenetics and proteomics. The main results were as follows:1. It was found that seed viability declined after storage 3 months at 60℃and there were genotypic differences of seed viability through the study of seed viability change after different temperature and time storage, so the temperature of 60℃ was chosen to be a criterion for evaluating the seed heat tolerance. Seed viability of 300 varieties were monitored after 60℃ storage, the results showed there were genotypic differences of seed heat tolerance among B.napus, B.rapa and B.juncea. The seed heat tolerance of 472 accessions of B. napus existed abundant genetic variation which was analyzed from the ecological type, geographical distribution and breeding time.2. The correlations between the heat tolerance and agronomic and quality traits were analyzed in 472 accessions; the results showed that GR and GI after heat stress were significant positive correlation with PY, DWP, TSW, POY and OC.3. The extent of cell ultrastructure damage was inconsistent in seed heat tolerance genotype P7 and seed heat sensitive genotype Hunongzao under high temperature stress. The changes of radicle and cotyledon ultrastructure in Hunongzao were more obvious than in P7 after high temperature stress. After super high temperature stress, the cell structure of P7 and Hunongzao had been destroyed, but there were more serious in Hunongzao.4. Genome-wide association analysis of seed heat tolerance was carried out by 60 K Brassica Illumina SNP array in 472 accessions. The results showed 4 SNP markers associated with GE were located on linkage group A1, and 3 SNP markers associated with GS were located on linkage group A2, A6 and C7, respectively.5. The level of global DNA methylation under heat stress was analyzed by MSAP. The results showed that the level of DNA methylation decreased and the status of DNA methylation changed after heat stress. Both DNA methylation and demethylation occurred during heat stress, and more DNA demethylation bands were recorded than methylation bands. The GE, GR, hypocotyl length and vigor index were significantly negatively correlated with the number of bands of full-methylated(both bands) at the internal cytosine, but were positively correlated with the number of bands of full-methylated(both bands) at the external cytosine.6. Proteomics analysis of seed under heat stress was carried out by i TRAQ technology, 2515 reliable proteins with quantitative information were identified. Differential expression proteins were selected according to the expression times and P values. 87 differential expression proteins were identified at 40℃ heat-treated seeds, 99 proteins were identified at 70℃, and 88 proteins were identified at 40-70℃. The expression patterns of different proteins were similar after the three heat-treated by hierarchical cluster analysis. Subcellular localization revealed the different proteins were mainly located in cytoplasm, nucleus, chloroplast, mitochondria and plasma membrane. These proteins were classified into 18 categories based on their biological function, the majority was involved in function unknown proteins, followed by those translation, ribosomal structure and biogenesis, and then posttranslational modification, protein turnover, chaperones and carbohydrate transport and metabolism. These proteins were expressed mainly in the metabolic pathway of cellular component and biological process through GO function enrichment analysis. Pathway analysis indicated that those proteins took part in 10 metabolic pathways, among which carbon metabolic, pyruvate metabolism and glycolysis/ gluconeogenesis were significantly affected in different heat-treated seeds.