Cloning And Functional Analysis Of Peanut (Arachis Hypogaea L.) Seed-specific Promoter

Peanut(Arachis hypogaea L.)is one of the most important oil crops in the world.The promoter is a segment of DNA sequence located at the upstream of the 5’ end of the gene that is specifically recognized and bound by RNA polymerase,and is an important element in regulating the expression characteristics of downstream genes.The study of peanut seed specific promoters is helpful to understand the molecular mechanism of peanut oil synthesis and seed development,which is of great significance to the heredity and improvement of peanut.Identification of seed-specific genes(SDGs)of peanut is a prerequisite for obtaining seed-specific promoters(Arachis hypogaea Seed-specific promoters,Ah SDPs).The main results of the research are as follows:1.In order to explore seed-specific candidate genes,transcriptome sequencing was performed on seven types of tissues in cotyledons and non-cotyledons of peanut pods at different stages.According to the analysis of transcriptome data,the FPKM value of 0.1 was used as the threshold for judging whether the gene was expressed,and finally 291 seed-specific candidate genes were obtained.Forty-two of them were identified by semi-quantitative RT-PCR and labeled as SDG1-SDG42,and 19 of them were further determined to be specifically expressed in seeds.2.Compared the seven candidate genes(SDG1,SDG2,SDG5,SDG9,SDG14,SDG13 and SDG16)with the cultivar peanut Tiffrunner genome through Peanut Base,peanut cultivar Yuhua 9326 ended up with seven corresponding promoters(Ah SDP1,Ah SDP2,Ah SDP5,Ah SDP9,Ah SDP14,Ah SDP13,and Ah SDP16)cloned.The online analysis of Plant Care showed that the cloned promoter contains not only the core regulatory elements TATA-BOX and CAAT-BOX,but also multiple abiotic stress response elements,such as jasmonic acid response elements,drought stress response elements,light response elements and drought-induced MYB binding sites,among which six promoters including Ah SDP2,Ah SDP5,Ah SDP9,Ah SDP14,Ah SDP13 and Ah SDP16 contain cis-acting elements RY-REPEAT related to seed-specific expression.3.The seed specificity of seven promoters was verified by GUS histochemical staining in transgenic arabidopsis.The results showed that Ah SDP5,Ah SDP9,Ah SDP13 and Ah SDP16 were only observed blue in the seeds,which proved that the above promoters were seed-specific promoter.In this study,through comparative transcriptome sequencing and semi-quantitative RT-PCR verification of cotyledons and non-cotyledon tissues in peanut pods,291 seed-specific candidate genes were obtained.Through promoter cloning and functional verification of SDG1,SDG2,SDG5,SDG9,SDG14,SDG13 and SDG16,the results showed that Ah SDP5,Ah SDP9,Ah SDP13 and Ah SDP16 were seed-specific promoters.The peanut seed-specific promoter identified in this work can be used in peanut genetic improvement.