Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii
Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii
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DOI：
10.1021/jf052711s
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发表时间：
2006-04-19
影响因子：
6.1
通讯作者：
Wang, AY
中科院分区：
文献类型：
--
作者：
Hsieh, CW;Liu, LK;Wang, AY
从绿色绿竹（Bambusaoldhamii）竹笋中克隆了3个编码酸性转化酶的Bo β果cDNA。根据其产物的氨基酸序列和系统发育分析，确定Bo β fruct1和Bo β fruct2编码细胞壁转化酶，而Bo β fruct3编码液泡转化酶。在毕赤酵母中产生由Bo β fruct2和Bo β fruct3编码的重组蛋白，并使用硫酸铵分级分离和固定化金属亲和层析纯化至接近均一。分离的酶的最适pH值，PL值和底物特异性与植物细胞壁或液泡转化酶的最适pH值，PL值和底物特异性是一致的。Bo β fruct1和Bo β fruct2在芽基部区域的生长依赖性表达强调了它们在蔗糖卸载和为芽生长提供底物中的作用。它的高蔗糖亲和力表明，Bo β fruct2编码的酶是重要的维持源和库器官之间的蔗糖梯度，而Bo β fruct3的主要表达的区域中的活跃的细胞分化和扩展表明在细胞增殖调节和细胞扩大的功能。
Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3 encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pl values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.