Cloning And Characterization Of Stearoyl-fatty Acid Desaturase From Descurainia Sophia

In this thesis we used RT-PCR and RACE method to clone 6 cDNA sequences of Descurainia sophia, designated Dsacpd9, Dsald9, Dsd12CHL, Dsd12ER, Dsw3CHL, Dsw3ER, putatively encoding aâ–³9 Stearoyl-ACP-fatty acid desaturase, aâ–³9 Stearoyl fatty acid desaturase, a chloroplastâ–³12 fatty acid desaturase, a ERâ–³12 fatty acid desaturase, a chloroplastω3 fatty acid desaturase and a ERω3 fatty acid desaturase. At the same time we also cloned the partial sequence of 183 rRNA of Descurainia Sophia which provide us the control gene. Sequence analysis indicated that Dsacpd9 had an open reading frame of 1203 bp encoding 400 amino acids of 45.3kDa; Dsald9 had an open reading frame of 1134 bp encoding 377 amino acids of 43.2kDa; Dsd12CHL had an open reading frame of 1344 bp encoding 447 amino acids of 51.1kDa;Dsd12ER had an open reading frame of 1152 bp encoding 383amino acids of 44.1kDa; Dsw3CHL had an open reading frame of 1356 bp encoding 451 amino acids of 51.6kDa; Dsw3ER had an open reading frame of 1161 bp encoding 386 amino acids of 44.0kDa. Bioinformatics analysis on these desaturase genes and their deduced amino acid sequences implied that all the enzymes except nsacpd9, involved in this work, were membrane-bound enzymes. According to the subcellular localization analysis, Dsacpd9 was a soluble desaturase locating chloroplast; Dsald9 was a chloroplast desaturase, and had three transmembrane regions; nsdl2CHL was a chloroplast desaturase, and had two transmembrane regions; Dsd12ER was a desaturase in ER, and had six transmembrane regions; Dsw3CHL was a chloroplast desaturase, and had three transmembrane regions; Dsw3ER was a desaturase in ER, and had three transmembrane regions.Temporal expression analysis suggested that Dsacpd9, Dsd12CHL and Dsw3ER expressed in all the tissues of Descurainia Sophia; and other three desaturase gene presented specific expression in different tissues: Dsald9 only expressed in stems, leaves, petals; nsd12ER only expressed in roots, stems, petals and developing seeds; Dsw3CHL only expressed in roots, stems, leaves and petals.Heterologous expression in S. cerevisiae strain INVSc1 of Stearoyl-fatty acid desaturase demonstrated that Dsd12CHL and Dsd12ER exhibitedâ–³¹²-fatty acid desaturase activity by the appearance of linoleic acid; Dsw3CHL and Dsw3ER exhibitedω3-fatty acid desaturase activity by the appearance of linolenic acid; Dsacpd9 and Dsald9 exhibitedâ–³⁹-fatty acid desaturase activity by the increase of the ratio of C18:1/C18:0 and the analysis of bioinformatics soft. Therefore, our study about the cloning and characterization of Stearoyl-fatty acid desaturase from Descurainia Sophia can provide the basis in theory for the genetic improvement and the application of gene engineering. At the same time we also provide the better resource of gene for other transgenic plants.