Citrus fruit bitter flavors:: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus
Citrus fruit bitter flavors:: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus
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DOI：
10.1111/j.1365-313x.2004.02193.x
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发表时间：
2004-10-01
期刊：
影响因子：
7.2
通讯作者：
Eyal, Y
中科院分区：
文献类型：
--
作者：
Frydman, A;Weisshaus, O;Eyal, Y
柑橘属植物积累了大量的黄烷酮，这些黄烷酮会影响水果的风味，并已被证明有益于人类健康。苦味物种，如葡萄柚和柚子，会积累苦味黄烷酮-7-O-新橙皮甙，部分原因是其特有的味道。无苦味的物种，如柑橘和橙子，只积累无味的黄烷酮-7-O-芸香苷。柑橘黄烷酮糖苷生物合成的关键风味决定步骤是由鼠李糖基转移酶催化的； 1,2 鼠李糖基转移酶 (1,2RhaT) 催化苦味新橙皮苷的生物合成，而 1,6 鼠李糖基转移酶 (1,6RhaT) 催化无味的芸香苷的生物合成。我们报告了柚子中编码柑橘 1,2RhaT 的基因 Cm1,2RhaT 的分离和功能表征。使用转基因植物细胞培养物对底物进行生物转化，对 Cm1,2RhaT 重组酶进行功能分析。表达重组 Cm1,2RhaT 的转基因 BY2 烟草细胞将黄烷酮和黄酮（但不是黄酮醇）生物转化为 7-O-新橙皮苷。免疫印迹分析证实，1,2RhaT 蛋白仅在苦柑橘品种中表达，而 1,6RhaT 酶（其活性先前在非苦柑橘品种中已有记录）不存在交叉反应。 Cm1,2RhaT 在 RNA 水平上的表达在幼果和叶片中显着，但在相应的成熟组织中表达较低，因此与先前建立的黄烷酮-新橙皮苷积累的发育模式很好地相关。类黄酮糖基转移酶基因家族的系统发育分析将 Cm1,2RhaT 与唯一其他具有功能特征的类黄酮-葡萄糖苷鼠李糖基转移酶基因放在一个单独的基因簇上，表明专门从事类黄酮苷糖基部分糖基化的鼠李糖基转移酶具有共同的进化起源。
Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.