Identification Of Peanut Pericarp Or Testa Specific-Expressed Genes And Its Promoter Cloning

Peanut is one of the important oil and economic crops in the country and seed is the harvesting organ of peanut. Seeds developing in soil are easily afected by diseases and pests from soil, especially Aspergillus flavus, which seriously influences the safety of peanut products. So far, there is a great difficulty in improving peanut with resistantance to A. flavus by traditional breeding method. However, with the rapid development of biotechnology, it now could be effectively solved using transgenic technology, but transgenic plant with nonspecifically expressed antifungal gene could bring about accused safety problems. Based on some obtained genes specially expressed in peanut pericarp or testa, the study focuses on analyzing the expression pattern of these specific-expressed genes in developing pericarp, kernel and testa of peanut and the specificity in different organs of peanut. The aims in the study is to obtain genes abundantly and specifically expressed in peanut pericarp and/or testa, and then to clone the promoters starting from them in order to regulate transformed exogenous resistant genes only expressed in peanut pericarp or testa, but not in kernel. This could make transgenic peanut meet the quirements of safety in the future.1. Thirteen genes were analyzed for their expression patterns in various developing stages of pericarp, testa and kernel, i.e. 10d (days), 20d, 30d, 40d, 50d and 60d after pegging, employing semi-quantitative RT-PCR. Seven of the 13 genes, 8#, 9#, 13#, 16#, 8A4R19G1, PSC11 and 1O10, showed higher expression activities in pericarp and testa than in kernel. Another 6 genes (12#, 14#, 2N22, 4F2, 5C22 and 9A4R19GZ) have no obvious expression difference between pericarp, testa and kernel. Among the 7 specific expression genes, 2 genes (8# and 8A4R19G1) highly and specifically expressed in pericarp with lower or not expression in testa, but not any expression activity of them could be found in kernel. By Blastn analysis, 8# gene could not be found homologous genes with any interested sequences in NCBI. 8A4R19G1 showed homology with aquaporin genes from Gossypium hirsutum, Nicotiana glauca, Vernicia fordii. By evaluating conjecture amino acid sequence, 8A4R19G1 could be main aquaporin genes.2. The expression levels of 7 genes (8#, 9#, 13#, 16#, 8A4R19G1, PSC11 and 1O10) are also checked in different organs such as root, term, leaf, flower and gynophores of peanut, using semi-quantitative RT-PCR. And number 8#, 9#, 13#, 16# and 8A4R19G1 genes were found no trays expression in these organs, but PSC11 and 1O10 showed express higher in root and leaf, but no expression in the rest organs.3. A 607bp of 5-flank upstream sequences of 8A4R19G1 gene was obtained using TaKaRa LA PCR^TM in vitro Cloning Kit, according to the known sequence of 8A4R19G1. Through bioinformatics analysis, the sequence contained transcription start site and some basic promoter elements such as CAAT-box and TATA-box. This sequence can be further used to obtain intact promoter sequence for the promoter function characterization, which could provide basis in solving A. flavus contamination problem safely by transgenic peanut.4. 5' full sequences of PSC11 gene were obtained employing RACE method, and translation initiation site (ATG) is found by "Kozak" rules and ORF Finder analysis. The sequence could be some help for cloning upstream promoter of this gene.Given its economic and nutritional importance, peanut is virtually slower progress made in molecular biology studies. The obtained result could do benefit to genetic engineering study toward removing aflatoxin in peanut in the near future.