Isolation And Functional Analysis Of A CDNA Encoding Ferrochelatase 1 From Peanut (Arachis Hypogaea L.) Under Salt Stress

Peanut{Arachis hypogaea L.) is one of the most important oil crops in China and even the world. It possesses highly nutritional and economic value, due to its large amount of unsaturated fatty acids and proteins. The development of industry, together with improper irrigation practices and misusage of chemical fertilizers, aggravates the water and soil pollution. As a result, the secondary salinizated soil area is continuesly expanding. The planting area of the dry-farming crop, peanut is inevitably affected, leading to low yields and poor quality of nuts. Therefore, how to keep the stable and high production and good quality of peanut become the major target of breeding and also an challenging technical problems. To reach the goal and accelerate the progress of peanut variety improvement, the attention is gradually paid to the study of the molecular mechanism of salt resistance and development of salt tolerant peanut varieties based on the current peanut cultivars by gene engineering.Ferrochelatase plays a vital role in catalyzing the synthesis of heme. Heme is not only an important prosthetic group of respiratory complexes and peroxidase, but also a probable signal molecule which responds to environmental adversity such as drought stress. The purpose of this study is to clone the gene encoding peanut ferrochelatase 1 and explore its function under salt stress. AtFCl gene (GI:145358447) was used as a probe to blast the peanut EST database on NCBI website. Two homologous EST fragments were obtained and assembled. Based on this imfomation, the full-length cDNA of ferrochelatase 1 was isolated from salt-sressed peanut leaves by RT-PCR and RACE. Subsequently, biological information of the nucleotide sequence and putative protein was predicted. The subcellular localization of AhFC1 protein and the gene expression profiles under stresses was also analysed. Finally, the probable function of AhFC1 under salt stress was explored by introducing the gene into wild tobacco. The main results are listed as follows:(1) From salt-stressed peanut leaves, the cDNA encoding ferrochelatase 1 was isolated and named as AhFCl, which has been submitted to NCBI with the Genbank NO. KU560625. The full-length cDNA is 1965 bp, and contains an open reading frame of 1449 bp encoding a putative protein of 482 amino acids. There is a 157 bp 5’ UTR and a 359 bp 3’UTR.(2) Multiple methods were applied to predict the domain of AhFCl protein. It was found that the putative protein has highly conserved ferrochelatase_N and_C domains, in which several active sites are distributed. There is also a transmembrane region in its carboxyl terminal domain. It is a memeber of chelatase II family, whose enzymatic activity is independent of ATP, and it localizes in chloroplast. Phylogentic analysis indicated that AhFC1 is most homologous with the ferrochelatase 1 in Cucumis sativus.(3) Expression profiles analysis revealed that the expression of AhFCl was induced by drought stress, which hited the peak after 6 hours and then decreased slowly. Salt stress also resulted in the higher expression of AhFC1. Compared with that in response to drought stress, the expression of AhFCl increased smoothly and took more time to hit the maximum.(4) When exposed to salt stress, the germination of transgenic tobacco seeds is obviously higher than that of wild tobacco seeds. Although the content of heme in the leaves of both the trangenic tobacco and wild tobacco decreased, it was significantly higher in trangenic tobacco. Compared with wild tobacco, transgenic tobacco leaves contained higher amount of chlorophyll. ROS content, and MDA were evidently lower than that in wild tobacco. By contrast, peroxidases such as catalase and ascorbate peroxidase which have heme as prosthetic group, showed higher enzymatic activity than those in wild type. Even though the activity of SOD in the trangenic and wild tobacco was induced by salt stress, there is no significant difference between them. These findings suggest that increases in heme levels and ROS scavenging IV capacity allievate salt induced oxidative stress and enhance salt resistance in transgenic tobacco.