Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn derivatization-HPLC method.

A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle' UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.

Clone and Function Verification of the OPR gene in Brassica napus Related to Linoleic Acid Synthesis.

BACKGROUND: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids (FA), and each fatty acid was regulated by different genes. Furthermore, different FA affect each other, which needs continuous and in-depth research to obtain more clear results in Brassica napus. RESULTS: In this paper, broad-scale miRNA expression profiles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were verified using a quantitative real-time PCR (RT-qPCR) analysis, we found that the target gene of bna-miR156b > c > g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35 s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T1 and T2 generation, the content of linoleic acid (LA) increased significantly in OE but deceased in OPRi. CONCLUSIONS: This is the first study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid profiles in oilseed for breeding programs.

Characterization of the promoter and 5'-UTR intron of oleic acid desaturase (FAD2) gene in Brassica napus.

In the present study, we characterized the transcriptional regulatory region (KF038144) controlling the expression of a constitutive FAD2 in Brassica napus. There are multiple FAD2 gene copies in B. napus genome. The FAD2 gene characterized and analyzed in the study is located on chromosome A5 and was designated as BnFAD2A5-1. BnFAD2A5-1 harbors an intron (1,192 bp) within its 5'-untranslated region (5'-UTR). This intron demonstrated promoter activity. Deletion analysis of the BnFAD2A5-1 promoter and intron through the ß-glucuronidase (GUS) reporter system revealed that the -220 to -1 bp is the minimum promoter region, while -220 to -110 bp and +34 to +285 bp are two important regions conferring high-levels of transcription. BnFAD2 transcripts were induced by light, low temperature, and abscisic acid (ABA). These observations demonstrated that not only the promoter but also the intron are involved in controlling the expression of the BnFAD2A5-1 gene. The intron-mediated regulation is an essential aspect of the gene expression regulation.