Regulation of sugar composition in fruit by metabolic engineering

水果的重要品质是风味，在大多数水果中，消费者的风味偏好主要是甜味。己糖的甜度不同，果糖的甜度大约是葡萄糖的两倍。由于许多水果积累了大致相等水平的己糖异构体、果糖和葡萄糖，因此提高果糖比例的代谢工程具有提高水果甜度的潜力。本研究以转基因番茄为材料，研究了番茄果糖激酶基因Frk1和Frk2在番茄果实中的表达模式以及抑制其表达的策略，并利用Frk1和Frk2探针进行原位杂交，结果表明这两个基因在番茄果实中的表达存在差异。Frk2 mRNA在番茄幼果中定位于淀粉合成和种子发育，而Frk1 mRNA广泛分布。北方杂交和原位杂交的结果表明，Frk 1在植物细胞果糖代谢中起主要作用，而Frk 2则特异定位于淀粉合成细胞和种子发育相关细胞等输入和利用糖较多的细胞中。将Frk1 cDNA和E8启动子克隆到pB1121双元载体上，构建了Frk1反义基因。生长约50个转化体并从它们收获果实。虽然在几个转化体中内源Frk1 mRNA的水平降低，但在转化体中未检测到果糖激酶活性和果糖含量的任何变化。因此，使用具有35S启动子的Frk1和Frk2进行转化工作。其结果是，果糖激酶活性的降低，观察到在一些转化与Frk2反义基因。活性的变化可能影响果实中糖的组成。

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.