Effects Of DGAT1 Silencing On Triacylglycerol Biosynthesis In Transgenic Tobacco

Diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) drives the final and only committed acylation step in the synthesis of triacylglycerol (TAG). TAGs are quantitatively the most important storage form of energy for eukaryotic cells. In plants, TAG mainly accumulates in seeds, pollens and fruits of many species. However, DGAT1 transcripts are also detected in most tissues of plant, including roots, leaves, stems, petals, flowers, anthers, developing siliques, young seedlings and germinating seeds. To date, many studies of DGAT1 gene have been focused on determining its effect on seed oil accumulation and the fatty acid composition of TAG in seeds. In this study, the intron-contained construct expressing hpRNA of NtDGAT1 was created to express double strand RNA in transgenic tobacco. According to the principle of RNA interference (RNAi), the hpRNA construct could silence Nicotiana tabacum endogenous DGAT1 gene effectively. The transgenic tobacco expressing silence phenotype will provide the foundation to better understand the function of DGAT1 gene. We not only analyzed the effects of DGAT1 silencing on seed oil accumulation, but also thoroughly studied the TAG content and DGAT1 transcript level in different organs of the transgenic line designated Sil7. In addition, as lipids, proteins, and sugars are the major storage substances in tobacco seeds, the metabolism relations among lipids, proteins, and sugars were prelimimarily illuminated by the measurement of the contents of these three substances in transgenic tobacco seeds. The results are as followed: The 679-bp fragment corresponding to nt 615-1293 of the gene encoding DGAT1 in Nicotiana tabacum was used to construct the vector expressing hpRNA. The expression vector was transferred into wild type tobacco (Wisconsin 38). Northern-blot analysis showed that, compared with that of wild type, there were only very low DGAT1 mRNA levels in flowers and developing seeds of transgenic tobacco. This result demonstrates that hpRNA constructs can target specifically against and effectively silence the DGAT1 gene. In addition, we obtained a high efficiency of transgenics (67%, 8 of 12 transgenic lines expressing silence phenotype of reduced oil content). It showed that such a method is very useful in the research of functional gene expression by specific degradation of the target gene and it will be a powerful tool for gene engineering in plants. To investigate the effect of DGAT1 expression silence on different organs in transgenic tobacco, we analyzed the triacylglycerol content and fatty acid composition of TAGs in different organs of Sil7. RT-PCR analysis was also adopted to compare the DGAT1 transcription levels in different organs of wild-type tobacco with that of Sil7. The results showed that the lower DGAT transcript level in transgenic tobacco had a positive correlation with the reduction of TAG content in different organs. Therefore, there may present a specific corresponding between DGAT1 expression level and TAG content in plants. In addition, the still presence of TAG in different organs of Sil7 suggests that, either DGAT1 enzyme in transgenic tobacco is null and then other enzymes, such as DGAT2 and PDAT, participate in the biosynthesis of TAG, or the activity of the enzyme is only partially affected. We also analyzed the fatty acid composition of TAGs in different organs of Sil7. The result showed that, in contrast with that of wild type tobacco, the acyl compositions of TAGs in different organs, including leaves, stems, roots, and petals, were changed. The ratio of 18:3/18:2 was elevated in these organs, whereas no obvious change was observed in seeds. The analysis of triacylglycerol content in seeds of 8 transgenic lines showed that DGAT1 gene silence in transgenic tobacco induced the decrease of seed TAG content and the reduced average 1000-seed weight. At the same time, the protein and sugar contents in seeds increased. The reduced accumulation of oil but increased accumulation of protein and sugar in seeds of transgenic tobacco suggests that there be an adverse relation between the biosynthesis of triacylglycerol and the synthesis of protein and sugar.