Molecular characterization of soybean squalene synthases (GmSQSs) and genetic engineering for enhanced phytosterols in Arabidopsis seeds
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Squalene synthase (SQS), which converts fanesyl diphosphate (FPP) to squalene, represents a crucial branch point of the isoprenoid pathway and is considered a key enzyme in diverting carbon flux towards the synthesis of sterol end products. The present work focuses on identification and characterization of the soybean squalene synthase (GmSQSs) family and genetic engineering to enhance sterol content in plant seeds. Two putative GmSQSs encoding soybean squalene synthase were found and named GmSQS1 and GmSQS2, with GmSQS1 having been cloned earlier (Hata et al., 1997). GmSQS2 is a novel gene found in this study with one open reading frame of 413 amino acids. To survey the gene expression of GmSQS1 and GmSQS2 in soybean plants, qRT-PCR analysis was performed. GmSQS1 and GmSQS2 express ubiquitously in soybean plants. GmSQS1 expresses highly in root, while GmSQS2 is more abundant in leaves. This thesis also demonstrates that GmSQS1 has function in regulating carbon flux from upstream to downstream of the sterol pathway to form end products: sitosterol, stigmasterol, and campesterol. GmSQS1 was overexpressed in Arabidopsis using constitutive and seed specific promoters. Total end product sterols increased up to 50% compared to wild type (WT). In addition, sitosterol and campesterol increased up to 50% and stigmasterol is up to 5-fold.
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