全 文 ：植物学报 Chinese Bulletin of Botany 2013, 48 (4): 389–397, www.chinbullbotany.com
doi: 10.3724/SP.J.1259.2013.00389
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收稿日期: 2012-07-27; 接受日期: 2012-12-27
基金项目: 山东省自然科学基金(No.ZR2011CL002)和临沂大学科研计划(No.HX09104)
* 通讯作者。 E-mail: liulinlyu163@163.com
拟南芥胼胝质合酶基因GSL8参与细胞壁形成和根端静止
中心建立与维持
刘林1*, 全先庆1, 赵小梅1, 黄力华1, 冯尚彩2, 黄坤艳1, 周晓燕1, 粟文婷1
1临沂大学生命科学学院, 临沂 276005; 2临沂大学现代中药研究所, 临沂 276005
摘要 用T-DNA插入和RNA干扰技术敲除拟南芥(Arabidopsis thaliana)胼胝质合酶基因GSL8, 在光学和透射电子显微镜
下观察野生型和突变体种子的细胞壁和胚根结构, 比较未敲除和敲除该基因幼苗细胞壁及根端分生组织结构。结果表明,
敲除该基因可导致细胞壁发育不良, 壁上出现大小不等的缺口, 缺口处没有质膜将相邻细胞分隔。用T-DNA插入法抑制该
基因表达, 发现在种子发育阶段胚根不能形成正常的静止中心。用小RNA干扰技术抑制该基因表达, 结果显示根端失去静
止中心。综合以上结果, 认为该基因不仅参与细胞壁发育, 也参与根端静止中心的建立与维持。
关键词 拟南芥, 胼胝质合酶基因, 细胞壁, 静止中心, 根
刘林, 全先庆, 赵小梅, 黄力华, 冯尚彩, 黄坤艳, 周晓燕, 粟文婷 (2013). 拟南芥胼胝质合酶基因GSL8参与细胞壁形成
和根端静止中心建立与维持. 植物学报 48, 389–397.
胼胝质是植物细胞合成的β-1,3-键连接的葡聚
糖, 在植物生长和发育过程中具有重要作用。胼胝质
参与减数分裂(Stone and Clarke, 1992; Samuels et
al., 1995; Lucas and Lee, 2004)、有丝分裂和细胞壁
发 育 (Kakimoto and Shibaoka, 1988, 1992;
Samuels et al., 1995; Samuels and Staehelin,
1996; Rensing et al., 2002)。
拟南芥(Arabidopsis thaliana)有12个胼胝质合酶
基因, 亦称为类葡聚糖合酶基因(glucan synthase-
like genes, GSL genes), 命名为GSL1─12 (Richm-
ond and Somerville, 2000; Hong et al., 2001; Enns
et al., 2005)。自AGRIKOLA(Arabidopsis Genome
RNAi Knock-out Line Analysis)计划实施以来, 这些
基因已有半数得到研究, 研究显示它们参与多种生物
学过程。GSL2参与小孢子发生(Dong et al., 2005)和
花粉管生长(Nishikawa et al., 2005); GSL1和GSL5
协同参与小孢子发育和受精作用(Enns et al., 2005);
GSL5参与病原物入侵诱发的防御反应(Jacobs et al.,
2003); GSL7参与韧皮部运输的调控(Barratt et al.,
2011); GSL10参与雄配子体第1次有丝分裂, 即小孢
子的不均等分裂, 并与生殖细胞进入营养细胞的过程
有关(Töller et al., 2008; Huang et al., 2009)。
GSL8参与雄配子体发育过程中小孢子的有丝分
裂(Töller et al., 2008), 也参与气孔发育(Chen et al.,
2009)。根的生长习性与根端分生组织中的静止中心
关系密切, T-DNA插入突变体的根基本不生长(Chen
et al., 2009), 这是否意味着根端分生组织缺少静止
中心, 目前尚无报道。因此, 本实验用T-DNA插入法
和小RNA干扰技术抑制GSL8基因表达, 以期揭示该
基因在拟南芥生长发育过程中的作用, 旨在阐明其与
根端分生组织静止中心的关系。
1 材料与方法
1.1 实验材料和植株培养
选取哥伦比亚野生型拟南芥 (Arabidopsis thaliana
L.), T-DNA插入突变体gsl8-4(SALK_109342)源自
Nottingham Arabidopsis Stock Centre, 双元发夹
RNA载体源自CATMA。
取野生型种子和T-DNA插入突变体gsl8-4杂合型
种子, 用20%漂白液(含0.1%Triton X-100)消毒5分
钟, 蒸馏水冲洗后在4°C低温下处理3天。将种子播种
·研究报告·
390 植物学报 48(4) 2013
于MS培养基(pH5.8)上, 环境温度为25°C, 光周期为
16小时光照/8小时黑暗, 10天后移栽到土壤中。
将带有pTA7002-GSL8-RNAi的种子以同样方法
进行消毒和低温处理, 播种在添加50 mg·L–1潮霉素
和20 µg·L–1地塞米松的MS培养基上。另取一部分种
子播种在只添加50 mg·L–1潮霉素的MS培养基上(对
照)。环境温度为25°C, 光周期为16小时光照/8小时黑
暗。
1.2 地塞米松诱导的GSL8基因RNA干扰载体构
建与转化
以 dsGSL8-SalI-d1(5-GGGCGTCGACCGCAAGAC-
CCTTCCTCTATAT-3) 和 dsGSL8-SpeI-r1(5-CCC-
GACTAGTCGCATATCTCATTAAAGCAGGA-3) 为
引物 , 对GSL8特异性发夹RNA载体质粒 (CATMA
2a35110)进行PCR扩增。扩增产物用SalI和SpeI切割
并克隆到含地塞米松诱导性启动子UAS的双元转化
载体pTA7002上, 得到该基因的干扰RNA载体pTA-
7002-GSL8-RNAi。将干扰RNA载体pTA7002-GSL8-
RNAi导入根癌农杆菌GV3101中, 然后用花蕾浸泡法
将pTA7002-GSL8-RNAi载体转化入哥伦比亚野生型
拟南芥。
收集浸泡过的花蕾中产生的种子, 经消毒和低温
处理3天后播种到添加50 mg·L–1潮霉素的MS培养基
上。10天后, 不含pTA7002-GSL8-RNAi的幼苗死亡,
正常生长的幼苗为转化成功的植株。将转化成功的植
株移栽到土壤基质中培养, 所产种子带有pTA7002-
GSL8-RNAi。将收获的种子播种到含有地塞米松的培
养基上, pTA7002-GSL8-RNAi上的启动子UAS受地
塞米松诱导而启动表达。
1.3 RNA提取和RT-PCR检测
从4种材料中提取总RNA, 用以检测GSL8基因的
mRNA水平。4种材料包括哥伦比亚野生型8日龄幼
苗、纯合型突变体gsl8-4未脱水种子、生长在添加50
mg·L–1潮霉素和20 µg·L–1地塞米松的MS培养基上的
8日龄幼苗及生长在添加50 mg·L–1潮霉素MS培养基
上作为对照的8日龄幼苗。用Trizol试剂从植物组织中
提取总RNA, 除去混杂DNA。以dNTP为原料, 以寡
dT链为引物, 用反转录酶(M-MLV RTase)对1 µg纯
化的RNA进行反转录。PCR扩增程序如下: 96°C3分
钟; 94°C30秒, 55°C45秒, 72°C30秒; 30个循环。
1.4 透射电子显微观察
为比较野生型与敲除GSL8基因的种子胚根中的静止
中心, 从T-DNA插入突变体植株中选取尚未脱水的
纯合型gsl8种子, 取其胚根; 从野生型尚未脱水的种
子取胚根。为比较幼苗根端静止中心, 取生长在添加
50 mg·L–1潮霉素和20 µg·L–1地塞米松的MS培养基
上的8日龄幼苗根尖; 从生长在只添加50 mg·L–1潮霉
素的MS培养基上取8日龄幼苗(对照)根尖。所取材料
先用2%戊二醛(50 mmol·L–1磷酸缓冲液配制, pH6.8)
于 4°C下初固定 4小时 , 再用 1%四氧化锇 (50
mmol·L–1磷酸缓冲液配制, pH6.8)于4°C下后固定4小
时 , 然后用浓度梯度10%的上行系列乙醇脱水 ,
Epon812树脂包埋, 再进行超薄切片, 切片厚度为70
nm。切片用醋酸双氧铀和柠檬酸铅双重染色, 在透射
电子显微镜下观察并拍照。
2 结果与讨论
2.1 敲除GSL8基因引起细胞壁变化
哥伦比亚野生型幼苗具有正常的表型, 根生长旺盛
(图1A), 从这些幼苗中检测到GSL8基因的表达(图
1F)。T-DNA插入突变体gsl8幼苗表型不正常, 根只有
少量生长(图1B), 这些幼苗中没有GSL8基因的表达
(图1F)。生长在只添加50 mg·L–1潮霉素的MS培养基
上含pTA7002-GSL8-RNAi基因的幼苗也具有正常的
表型 , 根生长旺盛 (图1C), 在这些幼苗中检测到
GSL8的mRNA(图1G)。生长在添加50 mg·L–1潮霉素
和20 µg·L–1地塞米松的MS培养基上的幼苗形态不正
常, 根的生长非常有限(图1D), 在这些幼苗中基本检
测不到GSL8的mRNA(图1G)。在哥伦比亚野生型种
子中也检测到GSL8基因的表达, 而在纯合突变体种
子中检测不到GSL8基因的表达(图1E)。
用2种方法敲除GSL8基因后, 胚及幼苗各部分
都出现大量不完整的细胞壁。切片观察结果显示, 细
胞壁上存在大小不等的缺口, 缺口处没有质膜, 相邻
细胞之间的原生质体完全相通, 失去隔离(图2A, B)。
在野生型及含pTA7002-GSL8-RNAi而未表达的幼苗
中不存在细胞壁不完整的情况(图2C)。细胞壁缺口与
胞间连丝有本质区别, 胞间连丝是由特化内质网和质
刘林等: 拟南芥胼胝质合酶基因 GSL8参与细胞壁形成和根端静止中心建立与维持 391


图1 GSL8基因敲除突变体与野生型拟南芥幼苗形态和GSL8基因的表达
(A) 野生型幼苗; (B) T-DNA插入突变体gsl8幼苗; (C) pTA7002-GSL8-RNAi基因未表达的幼苗; (D) pTA7002-GSL8-RNAi基因表达
的幼苗; (E) 野生型种子中检测到GSL8基因的表达, 纯合突变体种子中检测不到GSL8的表达; (F) 野生型幼苗中检测到GSL8基因
的表达, 纯合突变体幼苗中检测不到GSL8基因的表达; (G) 在仅添加潮霉素的MS培养基上生长的幼苗中检测到GSL8的表达, 同时
添加潮霉素和地塞米松的MS培养基上生长的幼苗中基本检测不到GSL8的表达

Figure 1 Seedlings of GSL8 knockout mutants and detection of GSL8 expression in Arabidopsis
(A) A wild-type (WT) seedling; (B) A T-DNA mutant seedling; (C) A seedling without expression of pTA7002-GSL8-RNAi; (D) A
seedling with expression of pTA7002-GSL8-RNAi; (E) The expression level of GSL8 is detectable in WT seeds, while not de-
tectable in homozygous mutant seeds; (F) The expression level of GSL8 is detectable in WT seedlings, while not detectable in
homozygous mutant seedlings; (G) The expression level of GSL8 is detectable in the control seedlings grown on MS medium
with addition of hygromycin, while not detectable in the seedlings grown on MS medium with addition of hygromycin and dexa-
methasone


膜组成的直径约40 nm的复杂结构, 由质膜和特化内
质网构成。特化内质网构成胞间连丝的连丝小管, 连丝
小管两端通常与典型的内质网相连(图2D)。连丝小管与
质膜之间有电子密度稍低的空隙, 即胞质腔。显然, 细
胞质组分通过缺口发生细胞间转移不受缺口的调控;
通过胞间连丝转移时, 则要受到胞间连丝严格控制。
2.2 敲除GSL8基因引起根端静止中心变化
对比拟南芥野生型和T-DNA插入突变体gsl8种子胚
根, 结果显示根端差异显著(图3)。野生型种子胚根端
部细胞形状规则, 排列整齐, 层次分明, 在中柱的下
面可观察到典型的静止中心。静止中心细胞比周围细
胞大, 排列整齐, 形状基本一致, 呈扁平状, 外侧呈
斜面 , 因而上窄下宽 , 呈现梯形轮廓 (图3C, D)。
T-DNA插入突变体种子胚根端部细胞形状不规则 ,
排列紊乱, 没有明显的层次, 依据细胞形状和大小观
察不到静止中心; 在中柱的下面, 即相当于静止中心
的位置, 细胞形状不规则, 大小不一致, 排列不整齐
(图3A, B)。显然, 由于GSL8基因表达受抑制, 使胚胎
发育时期不能建立正常的静止中心。
对比用RNAi干扰技术敲除GSL8基因与未敲除
GSL8的幼苗根尖, 结果显示根端差异显著(图4)。生
长在含50 mg·L–1潮霉素而不含地塞米松MS培养基上
的幼苗, GSL8基因正常表达(图1G)。与野生型种子胚
根(图3C, D)类似, 这些幼苗根端细胞形状规则, 排列
较整齐, 有较明显的层次, 中柱下面有典型的静止中
心(图4A)。生长在含50 mg·L–1潮霉素和20 µg·L–1地
塞米松的MS培养基上的幼苗, GSL8基因基本不表达
(图1G)。对这些幼苗的根尖进行连续切片, 结果表明
这些幼苗根端细胞形状不规则, 排列不整齐, 缺少明
392 植物学报 48(4) 2013


图2 GSL8基因敲除后的拟南芥细胞壁
(A) 基因敲除后细胞壁上有缺口(箭头所示); (B) 放大显示图(A)中箭头所示细胞壁缺口; (C) 野生型根端分生组织细胞具完整的细
胞壁(箭头所示); (D) 放大显示图(C)中箭头所示位置的胞间连丝。CS: 胞质腔; DT: 连丝小管; ER: 内质网; PM: 质膜; CW: 细胞壁

Figure 2 Cell walls in GSL8 knockout mutants of Arabidopsis
(A) Root apical meristemic cells in knockout mutants, showing a wall gap (arrow); (B) Highlighting the gap that is arrowed in
figure A; (C) Root apical meristemic cells in wild-type, showing complete walls; (D) Highlighting a plasmodesma in the arrowed
region in Figure C. CS: Cytoplasmic sleeve; DT: Desmotubule; ER: Endoplasmic reticulum; PM: Plasma membrane; CW: Cell wall

显的层次, 中柱下面没有静止中心(图4B, C)。显然,
敲除GSL8基因引起了幼苗根端静止中心消失。
2.3 敲除GSL8基因引起根端分生组织细胞结构
异常
在野生型和未进行小RNA干扰幼苗的根端, 分生组
织细胞形状规则, 细胞核相对较大, 核仁电子密度较
高, 核质电子密度略低, 细胞质电子密度中等, 基本
不含液泡, 或者含极少的小液泡(图5A)。与此形成鲜
明的对比, 敲除GSL8基因的幼苗根端分生组织细胞
发生畸形, 形状不规则, 细胞核缩小, 细胞内出现大
量小液泡, 小液泡合并后形成大液泡, 液泡内可见大
刘林等: 拟南芥胼胝质合酶基因 GSL8参与细胞壁形成和根端静止中心建立与维持 393


图3 拟南芥T-DNA插入突变体与野生型种子胚根端部比较
(A), (B) T-DNA插入突变体种子胚根端部, 细胞排列紊乱, 没有典型的静止中心, 箭头示相当于野生型胚根静止中心位置上形状不
规则的细胞; (C), (D) 野生型种子胚根端部, 细胞排列整齐, 有典型的静止中心, 箭头示静止中心细胞

Figure 3 Comparison of embryonic root apices in seeds of T-DNA mutant and wild-type Arabidopsis
(A), (B) The embryonic root apex of T-DNA mutant displays irregular arrangement of cells and absence of morphologically typical
quiescent center; Arrows indicate irregular shaped cells in the region that should be taken up by quiescent center cells in
wild-type; (C), (D) The embryonic root apex of wild-type reveals regular arrangement of cells and presence of quiescent center;
Arrows indicate quiescent center cells


量电子密度较高的絮状物质。伴随着细胞液泡化程度
加深, 细胞质电子密度增大, 并急剧减少。在液泡程
度非常高的细胞中, 细胞质非常少, 被大液泡挤压到
细胞边缘, 呈一薄层(图5B)。显然, 敲除GSL8基因引
起了分生组织细胞形态结构异常, 出现高度液泡化。
2.4 讨论
敲除拟南芥胼胝质合酶基因GSL8, 细胞壁上形成大
小不等的缺口, 这与以往报道的胞质分裂调控基因突
变体非常类似。Liu等 (1995)首先从豌豆 (Pisum

394 植物学报 48(4) 2013


图4 拟南芥RNAi幼苗与对照幼苗根端比较
(A) 对照幼苗根端有静止中心(箭头所示), 根冠细胞排列整齐; (B) 6日龄RNAi幼苗根端无静止中心, 根冠细胞排列不整齐; (C) 8日
龄RNAi幼苗根端结构严重紊乱

Figure 4 Comparison of root apices in RNAi and wild-type Arabidopsis seedlings
(A) The root apex of the wild-type seedling contains quiescent center (arrows) and its root cap cells are well organized; (B) The
root apex of the 6-day-old RNAi seedling has lost its quiescent center and its root cap cells are not arranged regularly; (C) The
root apex of the 8-day-old RNAi seedling displays a serious disorganization of cells




图5 敲除GSL8基因引起拟南芥根端分生组织细胞结构异常
(A) 对照幼苗根端分生组织细胞无明显液泡; (B) RNAi幼苗根端分生组织细胞液泡明显, 内含细胞质消化残迹(箭头所示)。V: 液泡

Figure 5 Structural abnormality of Arabidopsis root apical meristemic cells caused by knockout of GSL8
(A) Root apical meristemic cells in the wild-type seedling are free of prominent vacuoles; (B) Root apical meristemic cells in the
RNAi seedling possess remarkable vacuoles, where traces of digested cytoplasm can be seen (arrows). V: vacuole

sativum)中分离到一种胞质分裂缺陷型突变体, 命名
为cyd。该突变体胚发育过程中, 胞质分裂异常, 不能
形成完整的细胞板, 导致细胞壁不完整, 细胞壁上形
成缺口。随后, Lukowitz等(1996)发现拟南芥编码一
种突触融合蛋白家族基因, 该基因突变体knolle在胚
发育过程中也出现胞质分裂不正常, 细胞壁不完整。
刘林等: 拟南芥胼胝质合酶基因 GSL8参与细胞壁形成和根端静止中心建立与维持 395
拟南芥甘露糖-1-磷酸鸟嘌呤转移酶基因突变体cyt1
在胚发生过程中, 细胞壁发育严重缺陷, 细胞壁上出
现缺口, 不能形成正常胚(Nickle and Meinke, 1998;
Lukowitz et al., 2001)。编码一种KNOLLE结合蛋白
sec1的基因突变体keule, 其胚发育过程中同样表现
出胞质分裂不完全 , 细胞壁不完整(Assaad et al.,
2001)。拟南芥内-1,4-β-葡聚糖酶基因KORRIGAN
(KOR)突变体不能形成完整的细胞板, 导致细胞壁不
完整, 出现多核细胞, 致使幼苗形态异常(Zuo et al.,
2000)。显然, 拟南芥胼胝质合酶基因GSL8也参与了
细胞壁的发育。
敲除胼胝质合酶基因GSL8, 拟南芥幼苗表型不
正常, 初生根只有微量生长。尽管还缺少其它细胞学
和遗传学证据, 但从显微观察结果判断, 这些不正常
的幼苗根端缺少静止中心。静止中心是根端分生组织
中暂时脱离细胞周期的细胞, 绝大多数植物具有静止
中心(Wilcox, 1962; Peterson and Vermeer, 1980),
只有少数在恶劣环境中生长的植物才出现静止中心
消失的现象(Raju et al., 1964; Rodríguez-Rodríguez
et al., 2003)。静止中心在胚发育过程中(Clowes,
1978; Willemsen et al., 1998; Wenzel and Rost,
2001)或在种子萌发后建立(Clowes, 1958; Byrne,
1973; Webster and Langenauer, 1973; Bitonti et al.,
1992)。静止中心与根的生长密切相关, 具静止中心
的根有不断生长的能力; 静止中心一旦消失, 根就停
止生长(Varney and McCully, 1991; Skene et al.,
1998; Rodríguez-Rodríguez et al., 2003)。例如, 生
长在沙漠极端环境中的一些仙人掌科植物, 胚根缺少
静止中心, 结果种子萌发后胚根不生长, 或只发生有
限的几次细胞分裂, 表现有限生长习性(Dubrovsky,
1997; Dubrovsky et al., 1998)。环境胁迫(如严重干
旱 )首先引起静止中心的消失 , 然后根停止生长
Dubrovsky and Gómez-Lomelí, 2003)。静止中心是
保证根在不同环境条件下持续发育的故障排除系统
(Barlow, 1994), 是根不断生长的前提条件(Kerk and
Feldman, 1994, 1995; van den Berg et al., 1997;
Jürgens, 2001)。拟南芥根具无限生长的习性, 敲除
胼胝质合酶基因GSL8, 胚根不能建立静止中心, 幼
苗根端静止中心消失, 导致幼苗根只进行少量生长或
不生长。
综上所述, 本研究得出以下结论: 敲除拟南芥胼
胝质合酶基因GSL8, 造成细胞壁不完整, 胚发育时
期胚根不能建立正常的静止中心, 最终幼苗根端静止
中心消失。
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Arabidopsis Callose Synthase Gene GSL8 is Required for Cell
Wall Formation and Establishment and Maintenance of
Quiescent Center
Lin Liu1*, Xianqing Quan1, Xiaomei Zhao1, Lihua Huang1, Shangcai Feng2, Kunyan Huang1,
Xiaoyan Zhou1, Wenting Su1
1College of Life Sciences, Linyi University, Linyi 276005, China; 2Modern TCM Institute, Linyi University, Linyi 276005, China
Abstract Callose synthase gene GSL8 in Arabidopsis thaliana was knocked out by means of T-DNA insertion or RNA
interference. Cell walls and root apical organization in seeds and seedlings of the knockout lines and wild-type were
compared by using light and transmission electron microscopes. Gaps of various sizes were found to occur in cell walls in
all knockout lines, and there were no plasma membranes in the gap regions to separate the neighboring cells. No normal
quiescent center was established in the embryonic root apices during seed development in the T-DNA mutants. Fur-
thermore, the quiescent center was demonstrated to disappear from the root apices of seedlings as the gene GSL8 was
silenced by RNA interference. Based on these observations, it is suggested that the gene GSL8 was involved in the for-
mation of cell wall as well as the establishment and maintenance of quiescent center in the root apex.
Key words Arabidopsis thaliana, callose synthase gene, cell wall, quiescent center, root
Liu L, Quan XQ, Zhao XM, Huang LH, Feng SC, Huang KY, Zhou XY, Su WT (2013). Arabidopsis callose synthase
gene GSL8 is required for cell wall formation and establishment and maintenance of quiescent center. Chin Bull Bot 48,
389–397.
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* Author for correspondence. E-mail: liulinlyu163@163.com
(责任编辑: 白羽红)