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纤维素是植物细胞壁的主要多糖类组成成分[1]。根据细胞壁的组成成分和形成方式,可将植物细胞壁分为初生细胞壁和次生细胞壁[1]。初生细胞壁的主要成分是纤维素、半纤维素和果胶,通常在细胞板形成后开始沉积。木本植物木质部细胞在初生细胞壁形成后,进一步分化形成次生细胞壁。次生细胞壁的主要成分是纤维素、半纤维素和木质素,构成了木材的主要成分[2]。纤维素是由D-葡萄糖通过β-1, 4糖苷键连接而成的多聚物,其分子结构非常简单,但合成机制却极其复杂[3]。高等植物中纤维素的合成是在位于细胞膜上的纤维素合酶复合体(Cellulose Synthesis Complex,CSC)完成的,其中纤维素合酶(Cellulose Synthase/CESA)是CSC的主要组成成分,是参与纤维素合成的关键酶[3]。植物的CESA基因最早从棉花(Gossypium hirsutum)纤维中被克隆得到[4],随后,其他植物中的CESA基因相继被报道。拟南芥(Arabidopsis thaliana)基因组包含10个CESA基因,水稻(Oryza sativa)中含有8个CESA基因[5-6]。CESA蛋白序列高度保守,含有8个跨膜结构域,2个在N端,6个在C端[3]。在第2和第3跨膜域之间存在一个胞质环区,该胞质区里含有一些与底物结合与催化相关的残基,这些残基构成了一个保守的D, D, D, Q/RXXRW(天冬氨酸,天冬氨酸,谷氨酰胺-X-X-精氨酸-色氨酸)结构[3]。
目前的研究结果认为,3种CESA蛋白形成一个有功能的CSC[7]。拟南芥中负责次生壁纤维素合成的CSC是由CESA4、CESA7和CESA8组成,负责初生壁纤维素合成的CSC由CESA1、CESA3和CESA6及其类似基因CESA2/CESA5/CESA9组成[5, 8-14]。双分子荧光互补和酵母双杂实验证实,CESA4可与自身互作,且CESA4、CESA7和CESA8之间可以相互作用[15],而免疫共沉淀、双分子荧光互补和酵母双杂实验证明,初生壁CESA1、CESA3和CESA6自身以及相互之间可以相互作用[11, 16]。随后,Pull-down实验证实所有次生壁纤维素合成相关CESA基因之间存在互作关系[13, 17]。
次生细胞壁是木材的主要来源,其中纤维素含量高达40~50%,因而,参与次生壁纤维素合成的CESA基因将在木材形成中发挥关键作用。通过同源序列比对,毛果杨(Populus trichocarpa)基因组中鉴定出17个CESA基因[18-20],其中,负责次生壁纤维素合成CESA基因有5种(PtCESA4、PtCESA7A、PtCESA7B、PtCESA8A和PtCESA8B),相对于拟南芥(AtCESA4、AtCESA7、和AtCESA8)多出2种CESA基因。由于3种CESA蛋白组成一个有功能的CSC,因此,研究杨树5个次生壁CESA基因的表达模式和作用方式,有助于揭示这5个次生壁CESA基因在木本植物纤维素合成中的工作机理。目前,杨树次生壁CESA基因的表达模式已有部分研究,例如,Naoki等通过启动子驱动的GUS转基因材料研究了次生壁CESA基因的在木质部、韧皮部、根冠等组织的表达模式[21]。另外,Song等曾利用PtCESA7A或PtCESA8B的抗体以分化的木质部材料开展共沉淀研究,对共沉淀获得的复合体进行质谱分析检测到了所有5个杨树次生壁CESA基因,表明杨树5个次生壁CESA基因之间存在一定的互作关系,且均参与木材形成过程中的纤维素合成[22]。然而,目前关于杨树5个次生壁CESA基因以何种形式组成有功能的CSC,是随机组合还是有所偏好,尚无相关的研究。本文通过对杨树5个次生壁CESA基因的表达模式与互作模式展开分析,试图为揭示杨树5个次生壁CESA基因的作用模式提供一些线索。
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杂种杨84K(P. alba × P. glandulosa)组培苗用于基因遗传转化。84K组培苗在温度为23~25℃、光照为16/8 h(白天/黑夜)、光照强度为50 μmol·m-2·s-1的条件下培养。克隆获得2 Kb的CESA4、CESA7B、CESA8A与CESA8B基因的启动子区,通过Gateway反应重组到pMDC164载体。所有表达载体通过电击转化法转化入根癌农杆菌菌株GV3101,随后采用叶盘法进行遗传转化[23]。简略描述如下:将叶盘与转化了目的构建的农杆菌(OD600为0.4~0.8)共同孵育10 min,之后在分化培养基(MS+0.5 g·L-1 6-苄氨基腺嘌呤(6-Benzylaminopurine,6-BA)+ 0.05 mg·L-1萘乙酸(1-Naphthaleneacetic acid,NAA))上暗培养3天;暗培养后在添加3 mg·L-1潮霉素与200 mg·L-1特美汀的分化培养基上继续培养,获得生长良好的不定芽;将不定芽转移至添加3 mg·L-1潮霉素与200 mg·L-1特美汀的生根培养基(MS+0.05 mg·L-1 IAA + 0.02 mg·L-1 NAA)中继续培养,直到获得不定根生长良好的转基因植株。转基因植株获得后,通过克隆片段特异引物加载体特异引物进行PCR筛选验证转基因阳性植株。每个基因的转基因阳性植株至少拿到10个独立株系。转基因植株培养4 w后进行GUS染色。荧光素酶互补报告实验中所用烟草材料为本氏烟草(Nicotiana benthamiana),在温度为23~25℃、光照为16/8 h(白天/黑夜)的培养室中培养。播种后生长一个月左右可以用于荧光素酶互补报告实验。
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从TAIR (http://www.arabidopsis.org/)和Phytozome (http://www.phytozome.net/poplar.php)网站获取拟南芥和杨树的CESA基因家族的序列信息。采用MEGA 5.0软件中基于遗传距离的邻近结合法(Neighbor Joining,NJ)构建进化树。根据拟南芥AtCESA锌指结构和跨膜结构域信息(http://www.uniprot.org/),手工绘制杨树CESA锌指结构和跨膜结构域位置图。依据杨树次生壁CESA基因的基因号(PtCESA4:Potri.002G257900;PtCESA7A:Potri.006G181900;PtCESA7B:Potri.018G103900;PtCESA8A:Potri.011G069600;PtCESA8B:Potri.004G059600)在已报道的杨树幼嫩与成熟的茎、叶和根组织的RNA-seq数据[23]与剥皮再生过程再生组织表达量的基因芯片数据[24]中进行检索,获得基因的表达数据。杨树幼嫩与成熟的茎、叶和根组织的RNA-seq实验方法详见Liu等的方法[23],简略而言,使用RNeasy Plant Mini试剂盒和RNase-free DNase Ⅰ试剂盒(Qiagen)提取杨树幼嫩与成熟的茎、叶和根组织的总RNA,交由公司完成测序。剥皮再生过程再生组织表达量的基因芯片数据的实验方法详见Tang等的方法[24],简略而言,取生长时间一致的毛白杨在同一时间进行茎干的环剥,剥皮后第0、7、10、12、16、18和21 d用无菌刀片刮取树干表面再生组织,利用Affymetrix系统平台进行基因芯片的杂交、洗染和扫描。
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分别用0.1 mol·L-1NAA,0.1 μmol·L-1 6-BA,10 μmol·L-1赤霉素(Gibberellin,GA3),1 nmol·L-1油菜素内酯(epibrassinolide,BR)和1 μmol·L-1乙烯(ethylene),对组培培养4 w的84K植株进行处理。处理方法为,选取生长状态一致的84K组培苗,去除培养基,将根部置于上述浓度的激素溶液中,在处理0 h、3 h、6 h时取样进行次生壁CESA的表达量分析。取样部位为组培苗的茎。所有的试剂购于Sigma-Aldrich。在qRT-PCR实验中,取激素处理后的84K茎段,使用RNeasy Plant Mini试剂盒和RNase-free DNase Ⅰ试剂盒(Qiagen)提取总RNA。每个样品取2 μg RNA使用SuperScript Ⅲ first-strand synthesis system(Invitrogen)合成cDNA第一链。定量PCR的引物使用Primer 3软件设计,按照SYBR Premix Ex TaqTM Ⅱ试剂盒(TaKaRa)使用说明书准备反应混合液,实时定量PCR反应在7500 Fast Real-Time PCR System(Applied Biosystems)或Roche 480实时荧光定量PCR仪(Roche)上进行。PtACTIN作为内参,一次qRT-PCR实验包含四次技术重复。所有实时定量实验至少重复三次。实验所用qRT-PCR引物序列见表 1。
表 1 qRT-PCR引物序列
Table 1. Primers for qRT-PCR.
引物名Primer 引物序列Sequence PtCesA4rtF GAGTTGGAGAAATCATCA PtCesA4rtR GAGAGTTAGTTCCTTCAG PtCesA7ArtF CTTCCATGTGCACCTTTGAAGCCA PtCesA7ArtR TCAGGAGCTCGAGGTTCTATGCTA PtCesA7BrtF AACCACTGCAACCATCTCA PtCesA7BrtR ATGTTCCATGACAGCTCAGG PtCesA8ArtF GTATTCTGGGGCTAAACCTTCG PtCesA8ArtR TCTCGCAGTTCATGTAACTCAAC PtCesA8BrtF AAGGACCAAGCAAACATT PtCesA8BrtR AAGTGGATGTAACGGTAAG -
荧光素酶互补报告体系所用载体由中科院遗传所周奕华老师课题组惠赠。按照文献中操作步骤进行荧光素酶互补报告实验[25],简言之,将克隆得到的次生壁PtCESA蛋白CDS序列通过酶切连接的方法分别构建到包含荧光素酶蛋白N端序列的NLuc载体或C端序列的Cluc载体,构建好的载体通过农杆菌GV3101介导转化本氏烟草叶片,培养3 d后,在叶片上涂布荧光素,利用IndiGO软件检测荧光信号。
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GUS的组织化学染色按照如下步骤进行[23]:培养4 w的转基因组培苗,在预冷的90%丙酮中固定约2 h。固定结束后,在冰上将组织材料使用GUS染色缓冲液(50 mmol·L-1 sodium phosphate (pH7.0),2 mmol·L-1 potassium ferrocyanide,2 mmol·L-1 potassium ferricyanide,10 mmol·L-1 EDTA,0.2% Triton X-100 (v/v))洗涤3次,再转移至GUS染色液(染色缓冲液添加20% (v/v) methanol和1 mM X-Gluc),于37℃温浴12 h后,使用70%乙醇脱色。对CESA4、CESA7B、CESA8A与CESA8B基因启动子驱动的GUS转基因材料,每一个转基因材料至少选择5个株系进行GUS染色,每个转基因材料的染色至少重复三次,以保证结果的可靠性。
杨树次生壁纤维素合酶的表达与互作模式分析
Interaction and Expression of Secondary Wall CESAs in Populus
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摘要:
目的 纤维素的合成在木材形成过程中具有重要的作用,纤维素合酶(Cellulose Synthase,CESA)是参与纤维素合成的关键酶。由于3种CESA形成一个有功能的纤维素合酶复合体,而杨树中包含CESA4、CESA7A、CESA7B、CESA8A和CESA8B5种次生壁CESA,本文从这5种CESA的表达模式与互作分析入手,探讨了其在次生壁纤维素合成中的工作模式。 方法 利用RNA-seq与基因芯片数据进行基因表达分析,揭示5种次生壁CESA在根、茎、叶组织的表达模式与次生维管再生过程中的表达变化。利用启动子驱动的GUS转基因材料GUS染色分析与实时定量PCR揭示5种次生壁CESA在各个组织的表达模式与在激素处理下的响应模式。利用荧光素酶互补实验揭示CESA7A、CESA7B、CESA8A与CESA8B之间的互作模式。 结果 基因表达分析表明,杨树5种次生壁CESA基因在杨树成熟茎中高表达,尤其在次生维管组织发育的后期高表达,表明其主要参与木材次生壁纤维素的合成。对启动子驱动的GUS转基因材料观察表明,CESA4、CESA7B、CESA8A和CESA8B的GUS信号在杨树茎和叶中较强,但这些次生壁CESA的表达模式存在一定的差异,这种差异在叶脉中表现地尤为明显。此外,在赤霉素GA3和细胞分裂素6-BA处理下,杨树次生壁CESA的表达量显著上调;在生长素NAA、油菜素内酯BR和乙烯处理下,杨树次生壁CESA的表达量下调,但不同的CESA对激素响应的表达量变化幅度存在差异。荧光素酶互补实验表明,杨树次生壁CESA7B和CESA8B、CESA7B和CESA8A、CESA7A与CESA8B之间存在互作,说明它们之间可以形成复合体。 结论 这些数据显示杨树5种次生壁CESA基因在不同组织与不同激素处理下的表达变化存在一定的差异,而它们之间均能互作,提示杨树5个次生壁CESA基因虽然具有同等的能力形成功能性的纤维素合酶复合体,但在不同组织、不同激素作用下可能有不同的组合方式,进而会导致木材成分的差异。 Abstract:Objective Cellulose synthesis is important for the wood formation of woody plant.CESAsplay important roles in the cellulose synthesis.Since that three kind of CESAs constitute a functional cellulose synthesis complex and five secondary wall CESAs existed in the poplar genome, we examined the expression pattern and interaction of these CESAs in Populus toreveal the work model of them in secondary wall cellulose synthesis. Method RNA-seq and microarray data analysis were used to examine the expression profile of the five secondary wall CESAsin root, stem and leaf, and in the regeneration of secondary vascular bundle, respectively. Promotor driving GUS expression assay and qRT-PCR were carried out to reveal the tissue expression pattern and hormone response of the five secondary wall CESAs. Firefly luciferase complementation assay was used to check the interaction between CESA7A, CESA7B, CESA8A and CESA8B. Result Expression analysis showed that all the five secondary wall CESAsexhibited high expression in the mature stem and preferred to express in the later stage of stem development, suggesting that they involved in the secondary wall synthesis during wood formation. Promotor driving GUS expression assay of CESA4, CESA7B, CESA8A and CESA8B demonstrated high expression of these secondary wall CESAs in stem and leaf, and some differences, however, were existed in the detail expression pattern, especially in leaf veins. In addition, the expression of secondary wall CESAswere upregulated during GA3 and 6-BA treatment, while down-regulated during NAA, BR and ethylene treatment. The expression variation of these CESAs in response to different hormones were different. Firefly luciferase complementation assayshowed that CESA7A and CESA7B could interact with CESA8A and CESA8B, indicating that they parallelly determine the final secondary wall cellulose synthesis complex. Conclusion The five secondary wall CESAsshowed different expression from one another in different tissue and under different hormone treatment. However, these CESAs could interact with each other.Thesedata suggest that poplar secondary wall CESAs could form the cellulose synthesis complex in equal possibility, but the complex might be varied in different tissues and in response to different hormones, which might have an effect on the wood chemical composition. -
Key words:
- CESA
- / Secondary cell wall
- / Expression analysis
- / Interaction
- / Hormone
- / Populus
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表 1 qRT-PCR引物序列
Table 1. Primers for qRT-PCR.
引物名Primer 引物序列Sequence PtCesA4rtF GAGTTGGAGAAATCATCA PtCesA4rtR GAGAGTTAGTTCCTTCAG PtCesA7ArtF CTTCCATGTGCACCTTTGAAGCCA PtCesA7ArtR TCAGGAGCTCGAGGTTCTATGCTA PtCesA7BrtF AACCACTGCAACCATCTCA PtCesA7BrtR ATGTTCCATGACAGCTCAGG PtCesA8ArtF GTATTCTGGGGCTAAACCTTCG PtCesA8ArtR TCTCGCAGTTCATGTAACTCAAC PtCesA8BrtF AAGGACCAAGCAAACATT PtCesA8BrtR AAGTGGATGTAACGGTAAG -
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