| Cotton is one of the important economic crops, cotton leaf senescence is an important stage in the process of leaf development, it strongly affects cotton yield. It is key to clarify the mechanism of cotton leaf senescence and delay cotton leaf senescence for increasing cotton yield. However, at present cotton leaf senescence research mainly focus on physiological and biochemical levels, large-scale gene expression and the regulatory mechanisms of leaf senescence remain unclear. Therefore, it has important theoretical and practical significance to study on cotton leaf senescence-associated gene expression profile. This research used upland cotton CCRI10 (a short-season cotton cultivar with an entire growth period of 106 days) as material, used Solexa/Illumina digital gene expression (DGE) technology to analyze senescence-associated genes (SAGs) in natural cotton leaves. The target is to study the mechanism of cotton leaf senescence. The main results of this research are as follows:1. Based on leaf phenotype and physiological (chlorophyll and malonaldehyde) tests, samples at 20 days (maximal chlorophyll content, considered as 100%) as"M"and samples at 45 days (70% compared to 20 days) as"S"for DGE sequencing were selected. Samples at 20, 45, 50 and 55 days were used for quantitative real-time PCR (qRT-PCR).2. Through the primary analysis, 3,528,857 and 3,598,523 raw tags were obtained from M and S DGE libraries. After removal of useless tags, 111,672 and 142,655 clean tags were obtained, respectively. The"quantity"and"category"distribution of clean tags with the copy number of clean tags were statisticsed. The results showed: When the copy number of clean tags was added, the quantity of clean tags increased, reaching a maximum when the copy number was > 100; however, the category correlated negatively with the copy number when the maximum was between 2 and 5.3. Dana-Farber Cancer Institute cotton transcript database was used as the reference database, the clean tags of two libraries M and S were matched with the reference genes, only clean tags that matched perfectly with a reference gene and that mapped to the sense strand of this reference gene were analyzed. Using these criteria, 25.75% (M) and 28.59% (S) for"quantity"and 19.71% (M) and 18.85% (S) for"category"were separately obtained. The proportion of non-matched clean tags for"category"was > 50%, indicating that the efficiency of annotation was low when the copy number was between 2 and 5.4. Based on the analysis of the differentially expressed genes, we identified 3,218 upregulated genes. At a standard Q-value≤0.05 and corrected p value≤0.05, the pathway and gene ontology (GO) of these genes were analyzed. Two important pathways and 11 genes involved in organ senescence were identified. Simultaneously, multiple hormone-related and regulatory genes among these upregulated genes were discovered, including the key genes involved in the biosynthesis of gibberellin, ethylene, jasmonic acid and abscisic acid.5. 17 genes were selected for qRT-PCR according to their highly expressed levels and potential roles in senescence leaves. Tubulin beta-5 chain was chosen as the internal reference gene. The results showed: the expression of 15 genes were upregulated at 45 days, which was in agreement with the DGE data; 2 genes were downregulated at 45 days, but one of them was upregulated at 55 days, which meaned it was also upregulated during cotton leaf senescence; and the other gene was not real upregulated gene in DGE.6. We analyse the homology between cotton leaves and other 18 species in the leaf senescence database. The results showed: 196 genes that were homologous to genes in Arabidopsis thaliana, 9 genes having orthologs in rice and several genes that were homologous to a single gene in M. truncatula, red goosefoot, tobacco, pea, soybean and tomato were identified. These orthologs were mainly involved in macromolecular degradation, transcriptional regulation, hormone metabolism, nutrient salvage and defense.7. Treated cotton leaves with 30μg·μL-1 and 100μg·μL-1, and analysed the influence of leaf senescence-associated genes under gibberellin treatment. The results showed: two different concentration gibberellin could inhibit the expression of NAP and SAG18, but could promote the expression of ACS6, which was the key genes to synthesis ethylene after 6 hours treatment, we speculated gibberellin may involve cotton leaf senescence through the synergetic action with ethylene. |
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