Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs)
Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs)
复制标题
两种非生物胁迫响应性菊芋（Helianthus tuberosus）果聚糖 1-外切水解酶（1-FEH）的克隆和功能表征
DOI：
10.1007/s11103-014-0262-1
复制
发表时间：
2015-01-01
影响因子：
5.1
通讯作者：
Zhao, Haiyan
中科院分区：
文献类型：
--
作者：
Xu, Huanhuan;Liang, Mingxiang;Zhao, Haiyan
先前有报道称菊芋（Helianthus tuberosus）中存在两种果聚糖水解酶，其中一种天然的果聚糖 - β - 果糖苷酶（1 - FEH）通过SDS - PAGE纯化至均一，但未克隆到相应的cDNA。在此，我们从菊芋中克隆了两个全长的1 - FEH cDNA序列，分别命名为Ht1 - FEH I和Ht1 - FEH II，它们与菊苣的1 - FEH I和1 - FEH II具有高度的同源性。在毕赤酵母X - 33中对相应重组蛋白的功能鉴定表明，两种Ht1 - FEHs对β(2,1) - 连接的果聚糖都具有较高的水解酶活性，但对β(2,6) - 连接的果聚糖和蔗糖活性较低或无活性。与其他植物的FEHs一样，重组Ht1 - FEHs的活性受到蔗糖的强烈抑制。实时定量PCR分析表明，Ht1 - FEH I转录本在菊芋发育中的叶片和茎中大量积累，而Ht1 - FEH II的表达水平在块茎萌发过程中在块茎中升高，这意味着两种Ht1 - FEHs发挥不同的作用。在NaCl处理的植株茎中，Ht1 - FEH I和II转录本的水平均显著升高。NaCl处理也诱导了块茎中两种Ht1 - FEHs的转录，而PEG处理则略微抑制了块茎中Ht1 - FEH II的表达。通过HPLC对糖代谢酶活性和碳水化合物浓度的分析表明，在NaCl和PEG处理下，1 - FEHs的酶活性升高，但果糖含量降低。鉴于FEH水解果聚糖产生果糖，我们讨论了在遭受非生物胁迫的菊芋中1 - FEH活性与果聚糖动态之间不一致的可能解释。
Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-beta-fructosidase (1-FEH) was purified to homogeneity by SDS-PAGE, but no corresponding cDNA was cloned. Here, we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke, named Ht1-FEH I and Ht1-FEH II, which showed high levels of identity with chicory 1-FEH I and 1-FEH II. Functional characterization of the corresponding recombinant proteins in Pichia pastoris X-33 demonstrated that both Ht1-FEHs had high levels of hydrolase activity towards beta(2,1)-linked fructans, but low or no activity towards beta(2,6)-linked levan and sucrose. Like other plant FEHs, the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose. Real-time quantitative PCR analysis showed that Ht1-FEH I transcripts accumulated to high levels in the developing leaves and stems of artichoke, whereas the expression levels of Ht1-FEH II increased in tubers during tuber sprouting, which implies that the two Ht1-FEHs play different roles. The levels of both Ht1-FEH I and II transcript were significantly increased in the stems of NaCl-treated plants. NaCl treatment also induced transcription of both Ht1-FEHs in the tubers, while PEG treatments slightly inhibited the expression of Ht1-FEH II in tubers. Analysis of sugar-metabolizing enzyme activities and carbohydrate concentration via HPLC showed that the enzyme activities of 1-FEHs were increased but the fructose content was decreased under NaCl and PEG treatments. Given that FEH hydrolyzes fructan to yield Fru, we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress.