Zheng Wenqing, Zhang Yuan, Zhang Qian, Wu Ruihua, Wang Xinwei, Feng Shengnian, Chen Shaoliang, Lu Cunfu, Du Liang
Beijing Advanced Innovation Center of Tree Breeding by Molecular Design, Beijing Forestry University, Beijing, China.
College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China.
Front Plant Sci. 2020 May 19;11:600. doi: 10.3389/fpls.2020.00600. eCollection 2020.
Plant hexokinases (HXKs) are a class of multifunctional proteins that not only act as the enzymes required for hexose phosphorylation but also serve as sugar sensors that repress the expression of some photosynthetic genes when internal glucose level increases and regulators of cell metabolism and some sugar-related signaling pathways independent on their catalytic actives. The HXKs have been studied in many plants; however, limited information is available on HXKs of moso bamboo (). In this study, we identified and characterized 12 hexokinase genes in moso bamboo. Phylogenetic analysis revealed that the moso bamboo hexokinases (PeHXKs) were classifiable into five subfamilies which represented the three types of hexokinases in plants. Gene structure and conserved motif analysis showed that the PeHXK genes contained diverse numbers of introns and exons and that the encoded proteins showed similar motif organization within each subfamily. Multiple sequence alignment revealed that the PeHXK proteins contained conserved domains, such as phosphate 1 (P1), phosphate 2 (P2), adenosine, and a sugar-binding domain. Evolutionary divergence analysis indicated that the PeHXK, OsHXK, and BdHXK families underwent negative selection and experienced a large-scale duplication event approximately 19-319 million years ago. Expression analysis of the PeHXK genes in the leaf, stem, root, and rhizome of moso bamboo seedlings indicated that the PeHXKs perform pivotal functions in the development of moso bamboo. A protein subcellular localization assay showed that PeHXK5a, PeHXK8, and PeHXK3b were predominantly localized in mitochondria, and PeHXK8 protein was also detected in the nucleus. The HXK activity of the PeHXK5a, PeHXK8, and PeHXK3b was verified by a functional complementation assay using the HXK-deficient triple-mutant yeast strain YSH7.4-3C (, , and ), and the results showed that the three PeHXKs had the plant HXK-specific enzyme traits. The present findings would provide a foundation for further functional analysis of the PeHXK gene family.
植物己糖激酶（HXKs）是一类多功能蛋白，它们不仅作为己糖磷酸化所需的酶，还作为糖传感器，当内部葡萄糖水平升高时抑制某些光合基因的表达，并且是细胞代谢和一些与糖相关的信号通路的调节剂，与它们的催化活性无关。己糖激酶已在许多植物中得到研究；然而，关于毛竹的己糖激酶的信息有限。在本研究中，我们鉴定并表征了毛竹中的12个己糖激酶基因。系统发育分析表明，毛竹己糖激酶（PeHXKs）可分为五个亚家族，代表了植物中的三种己糖激酶类型。基因结构和保守基序分析表明，PeHXK基因所含内含子和外显子数量不同，且每个亚家族中编码的蛋白质具有相似的基序组织。多序列比对显示，PeHXK蛋白包含保守结构域，如磷酸1（P1）、磷酸2（P2）、腺苷和一个糖结合结构域。进化分歧分析表明，PeHXK、OsHXK和BdHXK家族经历了负选择，并在大约1.9亿至3.19亿年前经历了一次大规模复制事件。对毛竹幼苗叶、茎、根和根茎中PeHXK基因的表达分析表明，PeHXKs在毛竹发育中发挥关键作用。蛋白质亚细胞定位分析表明，PeHXK5a、PeHXK8和PeHXK3b主要定位于线粒体，并且在细胞核中也检测到PeHXK8蛋白。通过使用HXK缺陷型三突变酵母菌株YSH7.4-3C（，，和）的功能互补试验验证了PeHXK5a、PeHXK8和PeHXK3b的HXK活性，结果表明这三个PeHXKs具有植物HXK特异性酶特性。本研究结果将为进一步对PeHXK基因家族进行功能分析提供基础。
