Expression Patterns Of GPC-1 And GPC-2 Transcription Factors And Transcriptome Analysis Of The Flag Leaf During Grain Filling In Wheat

Gpc-1(Grain protein content)(NAM-A1, NAM-B1, NAM-D1) and Gpc-2(NAM-B2, NAM-D2) are the members of the plant-specific NAM(No apical meristem) transcription factors. With highly-elevating expression levels, previous studies of NAM-B1 and its homologues(NAM-A1, NAM-B2, NAM-D1 and NAM-D2)showed that these genes were able to promote the senescence of vegetative tissues and the transport of their mineral elements to the grain in wheat(Triticum aestivum L.)and its relatives. Hence, they were important genetic resources for the nutrition quality improvement of wheat in the future. Massive research works had been carried out not only on the function of Gpc-1 and Gpc-2 but also on their possible regulatory genes. However, little is known about the way they coordinated senescence and substance transport and their interacted genes so far, which was believed to be useful for unraveling their regulatory pathways.This thesis studied on the temporal and spatial expression characteristics of Gpc-1 and Gpc-2 during wheat grain filling in order to aid further exploration of their interacted genes. First, five NAM genes of common wheat cultivar Chinese Spring were separated by homology-based cloning strategy, and their nucleotide sequences were analyzed. Their transcription activities were studied subsequently with a yeast one-hybrid system. For accurate quantitative analysis of Gpc-1 and Gpc-2, the expression stabilities of nine internal control candidates were identified by utilizing three freely available programs, namely geNormPlus, BestKeeper and Normfinder. Applying quantitative real-time PCR(q RT-PCR)and optimal reference genes, the temporal expressions of Gpc-1 and Gpc-2 were monitored in nine different tissues. Their spatial expression patterns were further studied in post-anthesis flag leaf, peduncle and the grain using mRNA in situ hybridization technique. Differentially expressed genes and their related biological processes were analyzed in post-flowering flag leaf through transcriptome analysis, and those influencing senescence were also investigated. Combined with the results from previous and current studies, interacted genes of Gpc-1 and Gpc-2 were preliminarily screened. The main findings are as follows:1. TaNAM-A1, TaNAM-B2, TaNAM-D1, TaNAM-D2 and wild TaNAM-B1 were cloned in Chinese Spring. All five NAM genes contained a NARD respression domain; however, the yeast one-hybrid results showed that full-length Gpc-1 and Gpc-2, in fact, were transcription activation factors.2. The best single and multiple reference genes were varied among software programs and the sample groups. Ta27922 was the optimal single inner control gene. And the multiple ones, especially those selected for individual tissue by geNormPlus, outperformed the single one in terms of the stability.3. The divergent expression dynamics of five NAM genes were observed among genes and tissues. TaNAM-A1, TaNAM-B1, TaNAM-B2 and TaNAM-D2 initiated their expressions in all tested tissues before anthesis. As the transcript of TaNAM-D1 was only detected since anthesis and increased until the late sampling period, a close relationship between TaNAM-D1 and the grain development, nutrient transport as well as leaf senescence may exist. On the contrary, TaNAM-B2 showed a weak correlation with these processes owing to its universal decrease in terms of expression level from 15 DAA onwards.4. All NAM genes showed the same cell-type specificities, and no transcripts were detected in leaf epidermal cells, pericarp, and the seed coat. Heterogeneity was observed for the transcript distribution in the grain, in which the strong hybridization signals were aggregated in the tissues responsible for the nutrient transport. And their distributions to some extent were consistent with that of some mineral elements in the grain. Gpc-1 and Gpc-2 not only located in some tissues where the PCD occurred during the sampling period, but also were expressed in the tissues where the PCD did not happen. In conclusion, Gpc-1 and Gpc-2 were closely associated with mineral translocation in the grain rather than the PCD.5. Transcriptome anaysis was carried out on the flag leaves which were sampled at irregular time points after anthesis, and 4887 transcripts were significantly differentially expressed. The GO and KEGG enrichment analysis showed that three distinct stages were divided during our sampling period at the molecular level. Over 90% of DEGs showed no significantly differential expressions before 15 DAA, whereas the expression changes of DEGs that related to various metabolisms became active since then. It implied a potential link between the sink demands and the metabolism dynamics of the flag leaf. Leaf senescence was occurred during our sampling period due to the enriched and increased down-regulated photosynthesis-associated DEGs since 15 DAA. By comparing expression trends of DEGs that involved in the chlorophyll, other function units of photosystem and Rubisco metabolisms, it suggested that the first two elements may be the main factors that led to the post-anthesis decline in photosysthesis.6. A total of 1780 DEGs were defined as putative senescence-associated genes, which mainly played roles in metabolisms, transcription and redox regulation, transport, and the signaling of phytohormones. Only NAM-A1、NAM-B2 and NAM-D1 were DEGs, and their expression trends were consistent with the results of qRT-PCR and a previous finding to some degree. Moreover, no obvious correlations were observed between those genes and the regulation of early senescence simply based on their expression dynamics. A large number of DEGs(126) that related to the synthesis, signal transduction and response of CTK、IAA、ET、BR、ABA、JA and SA were identified, and some of them were associated with multiple ones. Complicated and diverse expression patterns were detected among the DEGs that involved in a certain signaling pathway. Therefore, it implied complex cross-talks that existed among different phytohormones after flowering.7. No comparable results were obtained among the putative downstream genes of partial Gpc-1 and Gpc-2 that found in different previous studies. Hence, a rough screening was carried out on the directly-acting genes of these genes according to the correlated expression patterns between the transcription factors and its interacted genes. And the results showed that the possible directly-targeted genes of TaNAM-A1 included a variety of transcription factors and the genes that responsible for the metal ion transport. And the functions of that for TaNAM-B2,TaNAM-D1 and TaNAM-D2 were complicated; however, it still included some senescence- and transport-related genes.