Isolation And Characterization Of Pericarp-abundant Expression Genes And Promoters In Arachis Hypogaea

Aflatoxin contamination is serious harm to human and animal health, which impacts the development of the peanut industry. It is considered that fostering anti-aflatoxin peanut varieties is the most effective way to solve the aflatoxin contamination of peanut. Fostering anti-aflatoxin peanut varieties has broad prospects by means of genetic engineering, but transgenic security concerns have been affecting the development of transgenic breeding. To solve this problem, we put forward cloning peanut pericarp specific promoter and induced different promoter in the international arena for the first time, which can drive disease resistance gene transferred to peanut expression or induced expression only in its pericarp. In this work, we have established a large-scale peanut cDNA Microarray, screening peanut pericarp-specific and induced by Aspergillus flavus genes.3 rapid amplification of cDNA ends (3’RACE) and 5’RACE were used to isolate thespecific genes cDNA flanking fragments. The promoters region of these were isolated by Flinking PCR; and the promoter sequences were analysed by Bioinformatic analysis. The results are as follows:1、A series of peanut pericarp genes were screened form peanut cDNA Microarray, and seven pericarp abundant expression genes and a aflatoxin induced gene were isolated by analysis of tissue expression pattern of these genes with semi-quantitative RT-PCR.2、RACE were used to isolate seven pericarp abundant expression genes cDNA flanking fragments from the Peanut pericarp cDNA library of our lab. The gene AhLACl which encoded a laccase protein expressed specific in the pericarp and independent of a variety of hormone-induced expression. A 2,231bp promoter region of AhLACl was isolated from peanut genomic DNA. Bioinformatic analysis showed that AhLACl promoter contained the Frequent elements:TATA-box and GATA-box. Movever, some motifs like RY, GCN4, Sknl which which were associated with pericarp expression were found. We predict it a the promoter pericarp/seed-specific promoter.3、Rich pericarp expression gene was not expressed in peanut seed kernel. Bioinformatics analysis showed that the protein encoded by AhPLEC1 is one of a legume lectin family. A 1,674bp promoter region of AhPLEC1 was isolated from peanut genomic DNA. Analysis revealed that sequence contained the basic components of the promoter, and there is still a dark-induced response element ACGTG, injury induced response element AGATCCAA, etc. so we predict the promoter is a peanut pericarp/seed-specific promoter, which may be related to the development and the growth of peanut pericarp.4、We cloned AhOMT1 full length cDNA used the aflatoxin induced cDNA template in the pericarp of peanut,which was expressed abundantly induced by aflatoxin;this gene expression abundance is most indced by aflatoxin,but almost no expression in various organs of the peanut.it showed that the gene belongs to the methyltransferase after analyzed functional domain of amino acid coded by this gene.the 849bp upstream regulatory sequences of this gene was isolated from peanut genomic DNA, combined with database analysis, found that the promoter contains fungal elicitor responsive element Box-W as well as two defense response element. We predicted the promoter was aflatoxin-induced or fungal Elicitor-specific promoter.The above results laid the foundation for the further study the relationship of peanut pericarp specific genes and pericarp development, aflatoxin induced gene and Aspergillus flavus and pericarp specific promoter, and also for aflatoxin inducible promoter applied for resistance of peanut aflatoxin breeding.