Identification And Analysis Of Genes Involved In Regulation Of Oleic Acid Content In Peanut Oilseed

Peanut (Arachis hypogaea) is an important oilseed crop, riching in protein and edible oil. A major determinant of oil quality of peanut is the composition of fatty acids. Peanut oils with higher content of oleic acid are favorable for human being because of their improved oil stability and enhanced nutritional quality. Therefore, one of the major goals for oilseed research is to improve the nutritive value of oilseed by increasing the ratio of O/L. Meanwhile, the two genes encoding the key enzymes, stearoyl–acyl carrier protein (ACP) desaturase (SAD) and delta 12 fatty acid desaturase(Δ¹²FAD or FAD2), involved in regulation of oleic acid content, have been paid an attention by many researchers.SAD catalyzes the desaturation of stearoyl-ACP (18:0-ACP) to form oleoyl-ACP (18:1-ACP), by introducing the first double bond into the fatty acid chain between C9 and C10. It is a key enzyme for the biosynthesis and accumulation of oleic acid, which plays an important role in the determination of the ratio of saturated to unsaturated fatty acids and the physiological function in higher plants. To increase the content of oleic acid in peanut seeds, two types of AhSAD1 gene, named AhSAD1-1 and AhSAD1-2 respectively, were cloned from Luhua 14 by the method of homologous cloning, and then bioinformatic analysis of these genes including multiple sequence alignment, homologous analysis and estimation of protein physicochemical properties and prediction of protein structure were done. The results showed that the amino acid sequences encoded by two AhSAD1 genes are highly homologous to those of SADs from other higher plants. Among the SADs compared, the great majority of them have more than 73% identity with AhSAD1.The actived AhSAD1 molecule is a homodimer, and has a conserved domains,belongs to acyl-ACP desaturase family and ferritin-like family, which is very conservative in evolution.Gene expression patterns analysis of AhSAD1 revealed that the expression level of AhSAD1 was highest in the developing seeds comparison with other tissues or organs, and reached to the peak at 50 days after pegging. The results of gene expression analysis in combination with the analysis of stearic acid content and oleic acid content in different developmental stages of peanut seeds, indicated that these genes play the important roles in regulating the accumulation and composition of fatty acids. Meanwhile, to investigate the functions of the two types of AhSAD1, the two plant overexpression vectors pCAMBIA3301-35S-AhSAD1-1 and pCAMBIA3301-35S-AhSAD1-2 driven by the CaMV35S promoter were constructed. Then, the genes were introduced into tobacco and peanut genomes by Agrobacterium-mediated transformation respectively.FAD2 catalyzes the conversion of oleic acid to form linoleic acid by the desaturation of oleic acid on carbon 12, which is an important enzyme to controls oleic acid and linoleic acid contents as well as O / L ratio.To investigate the genetic basis that controls high oleate levels-phenotype and improve the peanut-breeding efficiency, a large number of ORF region sequences of delta 12 fatty acid desaturase AhFAD2 gene from 13 peanut varieties were sequenced. The results showed that three types of mRNA transcripts AhFAD2A, AhFAD2B and AhFAD2C existed in all peanut varieties. G448A mutation of AhFAD2A gene was found in the peanut varieties of Linguimake, Feilongxiang, Goule, Luhua 14, Huayu 19, Huayu 23, which results in the production of a non-functional or less active AhFAD2A enzyme, and the G/A heterozygous at this locus exaits in Luhua11. Apart from G448A mutation, eight other SNP polymorphism sites were found to exist in the AhFAD2A gene but not in all peanut varieties. AhFAD2B was relatively conserved in all 13 peanut varieties investigated. Meanwhile, the G451T mutation or the frameshift resulted from 7 bp fragments insertion between 674–680 bp in AhFAD2C gene cause a TAA or TGA terminator,which resulting in AhFAD2C can't encode a complete protein,so AhFAD2C is pseudogene. OL1 and OL2 encode the active enzymes of AhFAD2A and AhFAD2B respectively, and ol1 is a mutant allele of AhFAD2A, and ol2 is a recessive allele of AhFAD2B. Therefore, the genotypes of peanut varieties Taishan Pearl, Taishan Sanlirou, Jiangtian, Chico, 05-21063, Baisha 1016; and Linguimake, Feilongxiang, Goule, Luhua 14, Huayu 19, Huayu 23, and Luhua 11 were found to be OL1OL1OL2OL2, ol1ol1OL2OL2, and OL1ol1OL2OL2, respectively. Combined with the measurement of the fatty acid composition of 13 peanut varieties by gas chromatography, it was found that the peanut varieties of Linguimake, Feilongxiang, Goule, Luhua 14, Huayu 19, Huayu 23, whose O/L ratios in seeds are more than 1.5, also exist G448A mutation in AhFAD2A,.which indicating that with the G448A mutation of AhFAD2A gene in peanut was one of the factors that cause relative increment of O/L ratio of peanut oilseed. Meanwhile, the potential function of pseudogene was also discussed in present research.