| Peanut (Arachis hypogaea L.) is one of the. most important oil crops in the world. The cultivated peanut is an allotetraploid species (AABB,2n=4X=40), which originates from a hybridization event of AA and BB wild type species followed by chromosome duplication. The large genome size (2,800 Mb) which is 20 times the size of Arabidopsis thaliana genome,5 times the size of rice (oryza sativa) and 2.5 times the size of the soybean (Glycine max) genome makes it a great challenge for peanut genomic study. The genomics research of peanut is far behind model plants such as rice and Arabidopsis thaliana. Individual gene cloning such as diseases resistance genes, abiotic tolerance genes and genes controling important agronomic traits is also very limited. Full-length cDNA library construction and EST sequencing is an efficient way for gene cloning from peanut. Microarray analysis is a powerful tool for gene discovery and global gene expression profiling. However, the unavailability of peanut commercial genechip makes high-throughput gene expression analysis impossible in peanut.In this study Luhua-14 immature seeds were used to make a full-length cDNA library and subsequently large scale EST sequencing was carried out using this library. Seeds from 20 days after pegging (DAP) to 60 DAP were collected for cDNA library construction. Random colonies were picked for sequencing and a total of 17,0005'EST sequences were produced. These sequences represented 734 contigs and 2759 singletons. Totally,8569 ESTs were submittied to NCBI GenBank. Among these ESTs,12.5% were annotated as metabolic related, and 4.6% encoded transcription or post-transcription factors. ESTs encoding storage protein and enzymes related to protein degradation accounted for 28.8% and formed the largest group of the annotated ESTs. ESTs encoding stress responsive proteins and pathogen-related proteins accounted for 5.6%. Sequences encoding unknown proteins or proteins with on hit found accounted for 20.1% and 13.9%, respectively.In order to have a primary study of the expression of tese ESTs in different tissues and seeds at different developmental stages we desined a peanut cDNA genechip using part of these ESTs. A total number of 5066 sequences were selected to make the(?)cDNA microarray. Expression analysis revealed that these sequences showed diverse expression patterns in seed, leaf, stem, root, flower and gynophore of peanut. There were 277 ESTs showed preferential expression in seed to compare with leaf, stem, flower and gynophore. Most of these genes were storage protein genes, LEA protein genes, oleosin genes and genes involved in oil metabolism. One third of these upregulated genes showed a 10 times higher expression level in seed than in other tissues. In addition, some important transcription factor genes including LEC1 and B3 domain genes were also on the list of upregulated genes. Further more, we analyzed the gene expression pattern during seed development. Genes that specifically up regulated (> 2 folds) at 15,25,35,45 DAP were identified. For example,550 ESTs specifically upregualted at 15 DAP,175 ESTs upregulated at 25 DAP,65 ESTs upregulated at 35 DAP, and 17 ESTs upregulated at 45 DAP. Expression patterns of some selected genes were verified by semi-quantitative RT-PCR. The potential value of these genes and promoters in the peanut gene engineering study was discussed.Late Embryogenesis Abundant (LEA) protein belongs to a large protein family comprising various groups of proteins. LEA proteins accumulate during the late stages of seed development and also response to drought, salinity, cold stress and exogenous application of abscisic acid in vegetative tissues. In order to isolate peanut LEA proteins genes, randomly selected clones were sequenced and analyzed from our peanut cDNA library. Nineteen LEA proteins encoding genes were identified that could be classified into eight distinct groups. The expression of these genes in different tissues and seeds with different developmental stages were analyzed by semi-quantitative RT-PCR. There was only little overlap between the expression of these genes in vegetative tissues and in seeds. The expressing levels of these genes were generally higher in seeds. The response of these genes to ABA, low temperature, high salinity and PEG treatment was also studied. In vegetative tissues, these genes showed different expression patterns on the treatments. The work provided valuable information to understand the roles of LEA protein in peanut growth and development.The innovations of this thesis were shown as below:1. By construction of a peanut cDNA library and large scale EST sequencing, this study cloned a large number of genes that involved in stress tolerance, fatty acid metabolism, storage proteins as well as genes with important regulatoary roles in peanut seed development.2. For the first time, we designed a cDNA genechip using the ESTs generated from the cDNA library. Expression of these sequences in different peanut tissues and seeds with different developmental stages was analyzed using this genechip. From this study a large number of genes with tissue or developmental specificity were identified from peanut. 3. A total of 19 members of LEA protein genes were identified from peanut. The expression patterns of these genes were analyzed in different tissues and seeds with different development stages. The response of these genes to ABA, low temperature, high salinity and PEG treatment was also studied.4. AhDHNl gene was cloned and its expression pattern in different development stages was analyzed. The plant expression construct was made and transformed into Arabidopsis and tobacco for functional study.
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