【作者】 张发云；

【导师】 许亦农；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 植物学， 2005， 博士

【摘要】 二脂酰甘油酰基转移酶(DGAT; EC 2.3.1.20)是催化三脂酰甘油(TAG)合成的最后也是最关键步骤的酶。TAG是真核细胞中最重要的能量存储形式。在植物中,TAG主要在种子、花粉和许多物种的果实中积累。然而,DGAT1基因的转录本也存在于植物的其它器官中,这些器官包括根、茎、叶、花瓣、花粉囊、未成熟的角果、幼苗以及正在发芽的种子等。迄今为止,许多针对DGAT1基因的研究都集中于DGAT1基因的表达在对种子油脂的积累以及对种子中TAG的脂肪酸组成所起的作用上。在本研究中,我们通过构建烟草DGAT1基因带有内含子的发卡RNA(hpRNA)结构,使之在转基因烟草植株中表达双链RNA(dsRNA),利用RNAi原理达到使烟草内源DGAT1基因沉默的目的。转基因沉默烟草植株的获得将会为更好地研究DGAT1基因的功能奠定基础。本实验不仅研究分析了DGAT1基因的抑制对烟草种子油脂积累的影响,还对表现出沉默性状的转基因植株Sil7的不同器官中TAG的含量以及DGAT1的转录水平等进行了研究分析。此外,通过对转基因烟草不同株系种子中的主要贮藏物质——油脂、蛋白质和糖的含量测定,初步揭示出在烟草种子中三者生物合成代谢之间存在的相关性。主要研究结果如下: 采用烟草DGAT1基因的第615～1293碱基之间679bp的片段构建了能表达发卡RNA(hpRNA)结构的表达载体,并转化烟草(Nicotiana tabacum)Wisconsin 38。Northern杂交分析发现,与野生型对照烟草(WT)相比,在沉默植株的花和发育状态种子中DGAT1基因的转录水平有很大降低,这表明该发卡结构能够高效率地引起烟草DGAT1基因的沉默。此外,在对Sil1至Sil12共12株转基因烟草进行油脂含量分析的结果表明,其中有8株表现出油脂降低的性状,转基因沉默效率达到67%。这表明:利用RNAi的方法可对目标基因进行特异降解来研究基因的功能,因此是一个在研究基因的表达功能上十分有效的方法,而且已成为植物基因工程的有力工具。为了研究DGAT1基因的沉默对转基因植株不同器官的影响,本实验分析了转基因植株Sil7的不同器官中TAG的含量和脂肪酸组成,并采用RT-PCR方法对野生型对照和转基因植株中DGAT1基因的转录水平进行了比较分析。研究发更多还原

【Abstract】 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.更多还原