Isolation And Expression Analysis Of PoFAD₂ Gene In Paeonia Ostii T. Hong & J.X.Zhang

Paeonia ostii（Paeonia ostii T. Hong J. & X. Zhang）is a kind of important new oil crops,which belongs to oil peony of Paeoniaceae. According to the determination, the content of the unsaturated fatty acids（ such as: linoleic acid（LA）, linolenic acid（LNA））, which were obtained by dehydrogenation of oleic acid（OA）, is high in the seeds of Paeonia ostii. In the synthesis of unsaturated fatty acid, the gene of FAD₂（fatty acid desaturase 2）encodings the protein of endoplasmic reticulum, which bounds oleic acid desaturase. It is the limiting enzyme of polyunsaturated fatty acid in the synthesis in plants. It has become one of the hot spots in the field of scientific research to isolate the FAD₂ gene and study its expression pattern in the oil plant. But so far, the research on the FAD₂ gene has not been reported.Therefore, the study of Paeonia ostii FAD₂ has important research significance for further understanding of the gene structure, function and regulation methods. At the same time, the study will lay the foundation for content regulation of unsaturated fatty acid though gene engineering.The test material is the ovule of Paeonia ostii. The full-length cDNA sequence of FAD₂ gene is cloned by RT-PCR and RACE technology. After that, its biology characteristics are analyzed by bioinformation methods. The gene expression is tested by fluorescence quantitative PCR and 2- ΔΔ CT quantitative analysis method. In addition, the fatty acid content in the four stages of seed development is determined. The main results are as follows:（1）Fatty acid desaturase enzyme gene of Paeonia ostii（FAD₂）: the full-length cDNA sequence is obtained on the first time and it is 1712 bp. It contains a 1152 bp complete open reading frame. The coding regions on both sides of the wing are respectively provided with 5 ’-UTR277 bp and 3’-UTR 261 bp（excluding the poly A tail）. And the full-length c DNA sequence encods 383 amino acids in total.（2） Paeonia ostii fatty acid desaturase gene（PoFAD₂）encoding protein: the molecular formula of the gene encoding protein is C2070H3033N527O540S14, the relative molecular weight is 44389.9Da and isoelectric point of PI is 7.10; the instability index（Instability index） 40.12（>40）, it is unstable protein; total average hydrophobicity index（Grand average of hyd-ropathicity,GRAVY） is-0.122, it is hydrophilic without signal peptide, which belongs to the integral membrane protein and endoplasmic reticulum enzyme; the protein has 4 transmembrane regions, which locate at 55⁷7, 178²00, 223²45, ²72 250. And the fourth transmembrane regions is stronger than others. The protein includes a fatty acid desaturase shared by conserved functional domains, which is namely 12- fatty acid desaturase domain. The domain contains 3 highly conserved histidine clusters, which can enriching divalent iron ions and is the catalytic activation center of PoFAD₂ enzyme. In the secondary structure of this gene,alpha helix（alpha helix）, random coil（random coil） and beta-sheet accounted 36.55 %,31.85 % and 21.93 %; and beta angle（beta-turn）occupy 9.66 %, which scattered distributed in the whole protein. The homology and phylogenetic relationships with the peony is the highest recently. It indicates that the evolution of a fatty acid desaturase enzyme FAD₂ gene maintains a more conservative structure and function.（3）Expression analysis of Paeonia ostii fatty acid desaturase enzyme gene PoFAD₂: the expressions are different in different organs and tissues and the expression is the highest in the ovule. So we can deduce the expression of PoFAD₂ is seed specific expression. In seed development process, the trends of expression are consistent in three varieties, which is the trend of up- down- up and the peak of expression is 75 d.（4）The content determination of fatty acid in the seeds development processs of Paeonia ostii:The trends of oleic acid, linoleic acid and linolenic acid are consist, which show increasing first and then decreasing trend, and have the peak in 75 days. It is same with the trend of oleic acid desaturase enzyme expression in Paeonia ostii seeds development process.