百日草离体叶肉细胞分化为管状分子概述

李 娜，白 兰，郭学民

(河北科技师范学院生命科技学院，河北 秦皇岛，066600)

百日草离体叶肉细胞分化为管状分子概述

李 娜，白 兰，郭学民*

(河北科技师范学院生命科技学院，河北 秦皇岛，066600)

概述了植物细胞分化的模式系统——管状分子分化实验系统的建立以及基于该系统的管状分子分化的生理学、细胞学、生物化学和分子生物学等方面的研究进展，并对今后的研究方向进行了展望。

百日草；次生细胞壁；细胞分化；管状分子

管状分子(Traeheary Elements，TEs)是维管植物木质部内导管和管胞的总称，皆为长柱状细胞，次生壁木质化，成熟后均缺乏原生质体，其功能是输导水分、矿质元素和机械支持作用。导管和管胞差别是，管胞无穿孔，管胞间壁仅有具缘纹孔，借以实现物质转运；而导管分子间的某些区域具有穿孔，多个导管分子通过末端的穿孔连接形成一个长的管道，即导管，与管胞相比，其输导能力大大增强。管状分子来源于形成层，由形成层细胞经过细胞扩增、次生壁沉积、胞内物质自溶、形成端壁穿孔等步骤而形成[1, 2]。

管状分子分化已被作为植物细胞分化的模式系统，在形态结构、生化和分子组成、发育及生理功能上都具有明显的特点，是植物解剖学、发育生物学和细胞生物学的研究热点之一。多年来，各国学者建立了整体实验系统和离体实验系统，对管状分子的形态解剖学、生理学以及分子机制进行了一定的研究。其中，百日草(ZinniaelegansJacq.)叶肉细胞离体培养系统是目前分化效果最好的实验系统，人们利用该系统开创性地研究了离体条件下管状分子分化的基本过程，并对管状分子分化的细胞学、生理学、生物化学和分子生物学进行了卓有成效的研究。笔者拟对30多年来人们利用百日草叶肉细胞离体培养系统的研究成果进行概述，为进一步阐明管状分子分化机制提供基础。

1 离体实验系统的建立

因为管状分子形态随着分化进程而发生显著变化，其中包括环纹、螺纹和网纹次生细胞壁(SCW)的形成以及自溶作用。所以，管状分子分化被认为是植物细胞分化的模式系统。然而，大多数早期关于分化的研究，采用的是多细胞系统，这样的系统包含几种作为起始材料的细胞类型，为追踪单个细胞分化过程带来了困难。Kohlenbach和Schmidt[3]发现，以机械法分离的单个百日草叶肉细胞可直接分化为管状分子，这促使Fukuda和Komamine[4]在此基础上，建立了一个有效的、高频分化的实验系统。该系统已被全世界许多实验室广泛应用，有时根据特定情况仅仅对一些细节进行了修改[5～10]。

用液体介质浸渍百日草叶片，研磨后，以小网眼筛过滤，液体介质反复漂洗悬浮液，分离得到单个叶肉细胞。要注意：(1)材料要适当。取幼苗第一对叶，而非成年植物最嫩的叶片。(2)条件要适当。旋转培养转速为10 r/min，0.3～0.4 mol/L山梨醇调节渗透压，生长调节剂为0.1 mg/L a-萘乙酸(NAA)和1 mg/L 6-苄基腺嘌呤(6-BA)，起始细胞浓度为(0.4～3.8)×108细胞/L。这样，几乎30%分离叶肉细胞，在适当的离体培养条件下，可半同步地分化为管状分子。

2 细胞学、生理学和生物化学方面的研究

百日草实验系统的建立促进了管状分子分化诸多方面的研究(图1)。初步的细胞学和生理学研究表明，细胞分裂不是管状分子分化的前提条件，而一些DNA合成在分化中发挥重要作用[11～21]；以肌动蛋白依赖的微管重组，限定了次生细胞壁的特有格局[6，22～27]；分化过程是动态的，细胞变化表现在次生细胞壁沉积前细胞器数量的增加，次生细胞壁沉积开始不久次生细胞壁木质化启动[28～31]，原生质体逐渐自溶，初生壁非木质化部分的局部水解，分化过程终止[28，32～34]。另外，百日草系统已清晰地证明，管状分子分化受植物激素诸如生长素、细胞分裂素[4]、油菜素内酯[35，36]、赤霉素[37]、一氧化氮[38]、乙烯[39]、信号肽(例如CLE肽[40]、木质素[41]、植物磺肽素[42])的调控；另外，大量生化和免疫学研究，揭示了细胞壁成分诸如纤维素、木聚糖、木质素和其他次生壁特有分子的变化[29，30，43～50]，以及自溶过程中的各种事件，诸如蛋白质和核酸的降解[51，52]等。

图1 百日草叶肉细胞管状分子分化模型

在生长素、细胞分裂素、植物磺肽素、木质素、油菜素内酯、乙烯、赤霉素作用下，离体叶肉细胞可被诱导分化为管状分子，但是CLE肽却抑制分化。

3 相关基因的鉴定与描述

人们也用分子生物学方法，进一步分析了百日草实验系统中管状分子分化的分子机制(图1)。运用同源克隆法(例如，核酸酶ZEN1[53]，肉桂醇脱氢酶和过氧物酶[54]，b-微管蛋白[55]，油菜素内酯合成酶[56]和Rho/Rac small GTPases[57])，差示筛选法(例如，分化标记，管状分子分化相关的(TED)2-4(Tracheary Element Differentiation-related(TED)2-4)[58～60]，果胶裂解酶[61])，消减杂交法(例如，核糖核酸酶[62]及分化标记、蛋白酶和木质素合成酶[10，63～65])、全面的转录组分析微阵列[66～68]和cDNA-AFLP[69]，分别鉴定了许多与编码管状分子特定事件相关蛋白质的cDNA，和限定管状分子分化特定阶段的标记蛋白。因为百日草转化方法尚不稳定，所以其分离基因的功能分析受到很大制约(例如，果胶裂解酶ZePel[61]，TED4[70]，过氧物酶ZPO-C[54])。然而，最近，通过基因枪法和电穿孔转染法，瞬间将基因或双链RNAs导入百日草细胞，成功地描述了其他基因的功能[57，71～73]，这可能为管状分子分化相关基因功能的分析提供了有益的线索。

4 其他植物的研究

在利用百日草实验系统，研究管状分子分化调控的基础上，建立了拟南芥人工培养细胞的管状分子分化系统，该系统在油菜素内酯调控下，有30%～50%继代细胞分化为管状分子[74，75]。后续的基因芯片分析表明，多数拟南芥基因在管状分子分化期间特异表达[74]，这些基因包括编码植物特定NAC-domain转录因子VND1-7的基因家族，借以最终揭示长久以来寻找的管状分子分化的转录开关[74]。在某些植物和人工培养细胞中，VND基因被作为管状分子异常分化的有效诱导物[76～79]。

5 待解决的问题和未来研究展望

管状分子分化百日草叶肉细胞离体实验系统的研究在草本植物中广泛展开。该系统为诱导和促进管状分子分化的研究建立了有效的平台，并获得了一些关键基因和调节因子，初步揭示了管状分子分化的机制，为在单细胞水平上认识细胞分化和转分化途径、分化过程影响因素提供了可能，但复杂、精确的分子机制的构建仍需进一步研究。(1)管状分子分化具有复杂的时空特异性，调控网络综合而庞大，虽然生长素和细胞分裂素是管状分子分化的基本调节激素，已有学者对2者进行了大量研究并取得一定的成果，但是其他激素诸如赤霉素、乙烯、脱落酸、油菜素内酯等作用效果研究资料极少，所以，植物激素作用机制的研究尚未明确。(2)管状分子分化是植物细胞凋亡的典型例子，成熟的管状分子丧失了细胞核和细胞内容物，成为死的、中空的管状细胞。目前对管状分子细胞凋亡的信号转导途径知之甚少，需要进一步探索。(3)以草本植物百日草，甚至拟南芥为材料得到的管状分子分化的初步机制，是否适合木本植物，尚需验证。(4)虽然百日草系统较为成熟，管状分子分化率和同步性较高，但随着研究的深入，仍需进一步改进和优化游离单细胞的离体培养方法，摸索分化的最佳条件，提高分化率和同步性。(5)未来需要综合利用分子生物学、细胞生物学、生物信息学和系统生物学的研究方法，从多角度、多层次、多学科开展研究工作，结合模式植物拟南芥实验系统和实验植物分子遗传学，在不久的将来有可能全面阐明管状分子分化的机制。

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(责任编辑：朱宝昌)

Progress on Differentiation of Isolated Mesophyll Cells into Tracheary Elements in Common Zinnia (ZinniaelegansJacq.)

LI Na, BAI Lan, GUO Xuemin

(College of Life Science & Technology, Hebei Normal University of Science & Technology, Qinhuangdao Hebei, 066600, China)

Differentiation of tracheary elements (TEs) has been regarded as a model system for cytodifferentiation in plants. Fukuda and Komamine established an efficient experimental system for TE differentiation from isolated single mesophyll cells ofZinniaelegans, which provided an excellent platform for the study of TEs differentiation at single cell level. The establishment of the system and the progress on physiology, cytology, biochemistry and molecular biology of the system-based TEs differentiation were summarized in this paper. At last, the future research direction of TEs differentiation was prospected.

Zinniaelegans; secondary cell wall; cytodifferentiation; tracheary element

10.3969/J.ISSN.1672-7983.2016.04.010

河北科技师范学院博士后启动基金项目(项目编号：2013YB021)。

2016-11-23;修改稿收到日期: 2016-12-21

S681.9

A

1672-7983(2016)04-0061-07

李娜(1991-)，女，硕士研究生。主要研究方向：植物发育细胞生物学。

*通讯作者，男，博士，教授。主要研究方向：植物结构生理学。E-mail: xueminguo@126.com。