Regulation of sugar composition in fruit by metabolic engineering

水果的重要品质是味道，在大多数水果中，消费者对味道的偏好主要是甜味。己糖的甜度不同，果糖的甜度大约是葡萄糖的两倍。由于许多水果积累的己糖异构体、果糖和葡萄糖的含量大致相同，因此通过代谢工程提高果糖的比例有可能提高水果的甜味。在这项工作中，描述了两个番茄果糖激酶基因Frk1和Frk2的表达模式，以及在转基因番茄植株中抑制其表达的策略。利用Frk1和Frk2探针进行原位杂交，发现这两个基因在番茄果实中的表达存在差异。Frk2 mRNA在番茄幼果中特异定位于淀粉合成和种子发育，而Frk1 mRNA则广泛分布。northern blot和原位杂交结果表明，Frk1在植物细胞的果糖代谢中起主要作用，而Frk2特异性定位于淀粉合成细胞和发育中的种子相关细胞等糖的输入和利用细胞。利用Frk1 cDNA和E8启动子构建反义基因，将其克隆到pBl121二元载体上。种植了大约50个变形植株，并从中收获了果实。虽然内源性Frk1 mRNA水平在几个t转化子中下降，但在转化子中未检测到果糖激酶活性和果糖含量的任何变化。因此，利用Frk1和Frk2与35S启动子进行转化工作。结果，在一些带有Frk2反义基因的转化子中观察到果糖激酶活性降低。活性的变化可能影响水果中的糖成分。

The important quality of fruit is flavor and in most fruit, consumer flavor preferences are dominated by sweetness. There is a difference in sweetness of hexose sugars, with fructose being approximately twice as sweet as glucose. Because many fruit accumulate approximately equal levels of the hexose isomers, fructose and glucose, metabolic engineering to enhance the proportion of fructose has the potential to enhance fruit sweetness. In this work, the expression patterns of two tomato fructokinase genes, Frk1 and Frk2, are described as well as a strategy to suppress their expression in transgenic tomato plants.In situ hybridization using Frk1 and Frk2 probes showed differential expression of these two genes in tomato fruit. Frk2 mRNA is localized specifically for starch synthesis and seed development in young tomato fruit whereas Frk1 mRNA is widely distributed. The results from northern blot and in situ hybridization suggest that Frk1 plays a primary role in fructosemetabolism in plant cells while Frk2 is localized specifically in cells that import and utilize moresugar such as starch synthesizing cells and developing seed-related cells. An antisense gene was constructed using Frk1 cDNA and E8 promoter that are cloned to pBl121 binary vector. Approximately 50 transformants were grown and fruit were harvested from them. Although the levels of endogenous Frk1 mRNA were decreased in several t ransformants, any changes in fruct okinase activity and fructose content were not detected in the transformants. Therefore transformation works using Frk1 and Frk2 with 35S promoter were carried out. As a result, decreases in fructokinase activity were observed in some transformants with Frk2 antisense genes. The changes in activity possibly affect sugar composition in fruit.