Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)
Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)
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DOI：
10.1093/jxb/erl034
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发表时间：
2006-08-01
影响因子：
6.9
通讯作者：
Prud'homme, Marie-Pascale
中科院分区：
文献类型：
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作者：
Lasseur, Bertrand;Lothier, Jeremy;Prud'homme, Marie-Pascale
果聚糖是多年生黑麦草的主要贮藏物质。为了解释该物种中普遍存在的基于新酮糖的果聚糖合成，构建了L。以洋葱果聚糖：果聚糖6G-fructosyltransferase（6G-FFT）为探针，对多年生植物进行了筛选。分离到与液泡型果糖基转移酶和转化酶具有显著同源性的全长Lp6G-FFT克隆。通过在毕赤酵母中异源表达来测试cDNA的功能性。重组蛋白具有6G-FFT和果聚糖：果聚糖1-果糖基转移酶（1-FFT）活性，最大6G-FFT/1-FFT比值为2。研究了6G-FFT的活性与发育阶段、组织分布和碳水化合物状态表达的变化，并与蔗糖：蔗糖1-果糖基转移酶（1-SST）进行了比较。Lp6 G-FFT和Lp1-SST主要表达于伸长叶片基部和叶鞘中。这两个基因的表达下降沿着叶轴，在平行的空间发生的果聚糖和果糖基转移酶的活动。令人惊讶的是，Lp6G-FFT在光合活性组织中高度表达，其中检测到非常低的可提取的果糖基转移酶活性和果聚糖量，表明表达的转录后调节。Lp6G-FFT基因的表达增加，只有在延长叶片的叶片，鞘，延长叶基部的蔗糖含量增加类似。Lp6G-FFT基因表达的调节取决于根据其库-源状态的组织。
Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.