黄兴^,1, 习金根¹, 陈涛², 覃旭², 谭施北¹, 陈河龙³, 易克贤^,1,*1中国热带农业科学院环境与植物保护研究所/农业农村部热带作物有害生物综合治理重点实验室/海南省热带农业有害生物监测与控制重点实验室, 海南海口 571101
²广西壮族自治区亚热带作物研究所, 广西南宁 530001
³中国热带农业科学院热带生物技术研究所, 海南海口571101

Identification and expression of PAL genes in sisal

HUANG Xing^,1, XI Jin-Gen¹, CHEN Tao², QIN Xu², TAN Shi-Bei¹, CHEN He-Long³, YI Ke-Xian^,1,*1Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Integrated Pest Management on Tropical Crops, Ministry of Agriculture and Rural Affairs/Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Haikou 571101, Hainan, China
²Guangxi Subtropical Crops Research Institute, Nanning 530001, Guangxi, China
³Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China

通讯作者: *易克贤, E-mail: yikexian@126.com

收稿日期:2020-05-30接受日期:2020-09-13网络出版日期:2021-06-12

基金资助:

国家重点研发计划项目.2018YFD0201100 国家现代农业产业技术体系建设专项.CARS-16 海南省自然科学基金项目.319QN275 海南省自然科学基金项目.320RC698 广西重点研发计划项目.桂科AB18221105 “一带一路”热带项目.BARTP-08

Received:2020-05-30Accepted:2020-09-13Online:2021-06-12

Fund supported:

The National Key Research and Development Program of China.2018YFD0201100 The China Agriculture Research System.CARS-16 The Hainan Provincial Natural Science Foundation of China.319QN275 The Hainan Provincial Natural Science Foundation of China.320RC698 The Guangxi Key Research and Development Program.桂科AB18221105 The Belt and Road Tropical Project.BARTP-08

作者简介 About authors
E-mail: huangxing@catas.cn









摘要
剑麻是热带地区重要的纤维作物, 但其分子生物学研究基础薄弱, 纤维发育机制尚未明确。苯丙氨酸裂解酶(phenylalanine ammonia-lyase, PAL)是纤维重要组分木质素生物合成的起始酶, 近年来转录组测序技术快速发展, 使开展剑麻PAL基因相关研究更为便利。本文根据已报道转录组数据成功鉴定出2个含完整编码序列的剑麻PAL基因, 其在剑麻叶片发育过程中的表达模式与前人报道的PAL在纤维发育过程中的活性变化规律一致, 表明其与木质素生物合成密切相关。遗传进化分析结果显示, 剑麻和番麻PAL基因进化关系更近, 选择压力分析结果显示, 剑麻和番麻PAL基因序列选择压力一致且高于太匮龙舌兰PAL基因, 这一现象可能由剑麻和番麻纤维性状的趋同进化引起。此外, 剑麻PAL基因在铜铅胁迫后差异表达不显著, 其可能在重金属胁迫后受到转录后调控。值得一提的是, 在烟草疫霉侵染后, 剑麻PAL基因表达水平上调倍数较高, 其可能同时参与苯丙烷类代谢途径中抗病相关次生代谢产物的合成和细胞壁介导的免疫机制。因此开展剑麻PAL基因功能解析可加深对剑麻纤维发育机制和抗病机制的理解, 对培育高产、优质、多抗剑麻新品种具有重要意义。
关键词： 剑麻;苯丙氨酸裂解酶基因;遗传进化;选择压力;表达模式;逆境胁迫

Abstract
Sisal is an important fiber crop in tropical areas, but its research foundation of molecular biology is relatively weak, and the mechanism of fiber development still remains unclear. Phenylalanine ammonia-lyase (PAL) is the first enzyme of lignin bio-synthesis, which is an important component of fiber. According to published transcriptome data, two sisal PAL genes with complete coding sequences were successfully identified. Their expression patterns during sisal leaf development were consistent with previously reported PAL activity changes during fiber development, indicating that PAL was closely related to lignin bio-synthesis. Phylogenetic analysis showed that sisal PALs were closely related with Agave americana. Selection pressure analysis showed similar selection pressure of PALs in sisal and A. americana, which were higher than those in A. tequilana. This might be caused by the convergent evolution of fiber-related traits in sisal and A. americana. In addition, sisal PALs were not significantly expressed under neither copper nor lead stress, which might be caused by post-transcriptional regulation under heavy metal stresses. It was worth noting that the expression of sisal PALs was highly up-regulated after Phytophthora nicotianae Breda inoculation. Sisal PALs might participate in the bio-synthesis of disease resistance-related secondary metabolites in phenylpropanoid pathway, as well as plant cell-wall mediated immunity. Therefore, functional characterization of sisal PALs could improve the understanding of mechanisms in fiber development and disease resistance, which is of great importance for breeding new sisal varieties with high yield, high quality and multiple resistance.
Keywords：sisal;PAL gene;phylogenetic analysis;selection pressure;expression pattern;adverse stress


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本文引用格式
黄兴, 习金根, 陈涛, 覃旭, 谭施北, 陈河龙, 易克贤. 剑麻苯丙氨酸裂解酶基因的鉴定及表达分析[J]. 作物学报, 2021, 47(6): 1082-1089. doi:10.3724/SP.J.1006.2021.04116
HUANG Xing, XI Jin-Gen, CHEN Tao, QIN Xu, TAN Shi-Bei, CHEN He-Long, YI Ke-Xian. Identification and expression of PAL genes in sisal[J]. Acta Agronomica Sinica, 2021, 47(6): 1082-1089. doi:10.3724/SP.J.1006.2021.04116


剑麻是我国热带、亚热带地区栽培的重要硬质纤维作物, 其纤维具有质地坚韧、强度高、耐摩擦、耐酸碱腐蚀、不易打滑等特性, 广泛应用于渔业、航海、工矿、汽车、造纸、纤维复合材料等特种行业^[1]。国内剑麻主栽品种为H11648, 其主要收获部位叶片生物量大但含水率较高, 纤维含量仅占约5%, 其纤维性状存在较大改良提升空间^[2]。目前剑麻研究基础相对薄弱, 特别是分子生物学研究水平较低。剑麻纤维相关研究多集中于田间农艺性状, 其分子机制研究相对较少^[3]。近年来高通量测序技术的快速发展, 为剑麻分子生物学研究带来了新的发展机遇。Huang等^[4]报道了剑麻叶片转录组测序及组装, 并通过同源序列克隆方法获得5个剑麻纤维素合成酶基因, 为解析剑麻纤维合成机制提供了重要基础。Deng等^[5]在已公布的剑麻转录组序列中鉴定出24个SAUR家族基因, 其中6个在剑麻叶片发育过程中差异表达。此外, Huang等^[6]通过转录组比较有效揭示了剑麻种间进化差异, 并通过选择压力分析筛选出6个纤维性状相关候选基因。相比之下, 剑麻木质素合成代谢相关研究尚未见报道。作为纤维的重要组成部分, 木质素合成代谢机制较为复杂, 其生物合成途径大致分为两步: 先是木质素单体的合成, 而后由木质素单体聚合成具有生物活性的木质素^[7]。木质素单体合成由十几种酶参与调控, 苯丙氨酸裂解酶(phenylalanine ammonia-lyase, PAL)位于木质素生物合成途径的起步位置, 在木质素生物合成途径中起关键调控作用^[8]。拟南芥基因组中包含4个PAL基因, 相关研究报道已证实其在木质素、黄酮类、酚类化合物等次生代谢产物合成过程中的重要作用^[9,10,11]。水稻中也报道了PAL基因的全基因组鉴定, 同时发现其在水稻应答铬胁迫过程中起重要调控作用^[12]。随着完成基因组测序的植物种类逐年增加, 除拟南芥、水稻外的其他植物也相继报道了PAL基因家族的全基因组分析, 例如毛果杨、葡萄、西瓜等^[13,14,15]。剑麻尚未开展基因组测序, 但转录组测序数据的公布为PAL基因鉴定工作提供了可用的生物信息^[4]。本文将在已公布的剑麻及其近缘物种转录组中通过同源序列比对鉴定PAL家族基因, 并开展遗传进化分析、选择压力分析及表达分析, 阐明剑麻PAL基因的遗传进化规律及其在叶片发育、重金属胁迫及病原侵染等过程中的表达模式, 以期为进一步解析剑麻PAL基因的生物学功能及作用机制提供参考。

1 材料与方法

1.1 材料种植与处理

剑麻主栽品种H11648种植于中国热带农业科学院环境与植物保护研究所(19.99°N, 110.33°E)。2年生的盆栽剑麻用于不同叶片发育时期取样, 取心叶、未展开叶和完全展开叶(与中轴夹角大于45°) 3个发育时期^[5]。选取1年生盆栽剑麻进行逆境胁迫处理, 重金属盐处理采用硫酸铜和硝酸铅溶液浇灌, 清水浇灌作为对照, 根据前人报道, 分别设1 g L^-1 (CuSO₄·5H₂O)和1.3 g L^{-1 [16,17]} 2个重金属盐浓度。每个处理浇灌500 mL重金属溶液, 处理后约2周叶片因重金属毒害发生卷曲时进行取样^[5]。病原侵染处理根据前人报道采用刺伤接种法接种剑麻斑马纹病病原烟草疫霉, 对照处理仅刺伤不接种病原, 接种5 d后取样^[18]。每个发育时期、胁迫处理及对照均在不同植株取样3次作为生物学重复, 取样后立即置于液氮速冻, 通过植物总RNA提取试剂盒(天根生化科技(北京)有限公司)提取RNA并保存于-80°C超低温冰箱备用。

1.2 剑麻PAL基因鉴定与分析

以4个拟南芥和9个水稻PAL基因为检索序列^[12], 采用TBlastx方法^[19]检索剑麻(Agave H11648)^[4]转录组数据库。检索获得的基因由ORF-FINDER分析其编码序列, 仅保留含有完整编码序列的PAL基因用于后续分析^[20]。通过ProtParam预测剑麻PAL蛋白长度、分子量和理论等电点^[21], 并通过CELLO进行亚细胞定位预测^[22]。以含有完整编码序列的剑麻PAL基因为检索序列, 检索其近缘物种沙漠龙舌兰(Agave deserti)、太匮龙舌兰(Agave tequilana)和番麻(Agave americana)转录组数据库^[23,24], 以获得剑麻PAL基因在3个近缘物种中的同源序列。选取拟南芥(4个)、水稻(9个)、玉米(17个)、短柄草(8个)、高粱(8个)、棉花(8个)、亚麻(4个)、芦笋(4个)及4个龙舌兰属物种PAL序列进行遗传进化分析, 采用ClustalX 2.0软件进行多重序列比对并构建Neighbor- Joining进化树, Bootstrap分析采用1000次重复^[25]。

1.3 剑麻PAL基因选择压力分析

以龙舌兰属野生种沙漠龙舌兰为参照, 将剑麻、太匮龙舌兰和番麻的PAL基因序列分别与沙漠龙舌兰PAL基因序列进行种间选择压力分析^[6]。通过DnaSP软件(5.0版本)分析2个基因序列间的同义突变频率(synonymous substitution, Ks)、非同义突变频率(non-synonymous substitution, Ka)及两者间的比值(Ka/Ks)^[26]。此外, 进一步通过滑窗法分析序列间的Ka/Ks值, 窗口长度为30个碱基(bp), 步移长度为6 bp ^[6]。

1.4 剑麻PAL基因表达分析

由已报道的剑麻、沙漠龙舌兰、太匮龙舌兰和番麻转录组中获取^[4,23-24]转录组表达数据(RPKM)。将已提取的剑麻RNA样品通过GoScript Reverse Transcription System (Promega, 美国)反转录为cDNA进行实时定量PCR分析, 仪器采用QuantStudio 6实时荧光定量PCR系统(Thermo Fisher, 美国)。扩增体系为20 μL, 其中包括0.5 μL正向引物、0.5 μL反向引物、1 μL cDNA模板、10 μL TransStart Tip Green qPCR Supermix (北京全式金生物技术有限公司)、0.4 μL Passive Reference Dye (北京全式金生物技术有限公司)和7.6 μL双蒸水。反应程序为94℃ 30 s; 94℃ 3 min, 94℃ 10 s, 58℃ 30 s, 共40个循环; 溶解曲线循环。以剑麻PP2A基因(protein phosphatase 2A)为内参基因, 每个样品重复3次作为技术重复, 结果用2^-ΔΔCt法进行相对定量分析^[27]。采用Primer 3软件^[28]设计引物, 其序列见表1。

Table 1
表1
表1实时定量PCR引物
Table 1Primers of qRT-PCR in the study

基因 Gene	正向引物 Forward primer (5°-3°)	反向引物 Reverse primer (5°-3°)	产物长度 Product size (bp)
AhPAL1	AGCAGTGATTGGGTGATGGA	GAGGAGGGTGTTGATTCGGA	216
AhPAL2	GCGATTGGGAAGCTCATGTT	GAGATGAGGCCCAGTGAGTT	239
AhPP2A	CCTCCTCCTCCTTCGGTTTG	GCCATGAATGTCACCGCAGA	235

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2 结果与分析

2.1 剑麻PAL基因的鉴定

同源序列检索共获得2个含有完整编码序列的剑麻PAL基因, 将其命名为AhPAL1、AhPAL2, 并上传至GenBank数据库(表2)。其序列长度分别为2112 bp和2118 bp, 分别编码703个和705个氨基酸。其蛋白分子量均超过76 kD, 理论等电点分别为5.85和5.93, 亚细胞定位预测均位于细胞质。

Table 2
表2
表2剑麻PAL基因及其蛋白理化性质、亚细胞定位预测
Table 2PAL genes and its protein physicochemical properties and subcellular localization in sisal

基因 Gene	碱基长度 Gene length (bp)	蛋白长度 Protein length (aa)	蛋白分子量 Molecular weight (kD)	理论等电点 pI	亚细胞定位 Subcellular localization
AhPAL1	2112	703	76,474.38	5.85	细胞质(2.462) Cytoplasmic (2.462)
AhPAL2	2118	705	76,267.12	5.93	细胞质(2.245) Cytoplasmic (2.245)

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2.2 剑麻PAL基因系统进化分析

以拟南芥、水稻PAL基因为参照, 选取纤维作物(棉花、亚麻)、禾本科作物(玉米、短柄草、高粱)、天门冬目作物芦笋、剑麻PAL基因及其近缘物种PAL基因(表3)进行遗传进化分析, 结果如图1所示。70个蛋白序列被分成2个分支, 其中分支I中的亚分支a均为天门冬目作物序列, 亚分支b均为拟南芥、棉花、亚麻序列, 分支II则均为禾本科作物序列。此外, 剑麻和番麻序列均聚类在相同分支, 沙漠龙舌兰和太匮龙舌兰序列也均聚类在同一分支。

Table 3
表3
表3龙舌兰属PAL基因序列号
Table 3Accessions of PALs in Agave

基因 Gene	物种 Species	GenBank序列号 GenBank accession
AhPAL1	Agave H11648	MT536163
AhPAL2	Agave H11648	MT536164
AmPAL1	Agave americana	GBHM01016452.1
AmPAL2	Agave americana	GBHM01016955.1
AdPAL1	Agave deserti	GAHT01019079.1
AdPAL2	Agave deserti	GAHT01004501.1
AqPAL1	Agave tequilana	GAHU01002518.1
AqPAL2	Agave tequilana	GAHU01004755.1

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图1

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图1拟南芥、水稻及龙舌兰属PAL基因遗传进化分析

At: 拟南芥; Ah: 剑麻; Am: 番麻; Ad: 沙漠龙舌兰; Aq: 太匮龙舌兰; Ao: 芦笋; Lus: 亚麻; Gorai: 棉花; Os: 水稻; GRMZM: 玉米; Bradi: 短柄草; Sobic: 高粱。利用ClustalX 2.0软件进行氨基酸序列的多重比对并构建Neighbor-Joining进化树(Bootstrap分析采用1000次重复)。
Fig. 1Phylogenetic analysis of PALs in Arabidopsis, rice, and Agave species

At: Arabidopsis thaliana; Ah: Agave H11648; Am: Agave americana; Ad: Agave deserti; Aq: Agave tequilana; Ao: Asparagus officinalis; Lus: Linum usitatissimum; Gorai: Gossypium raimondii; Os: Oryza sativa; GRMZM: Zea mays; Bradi: Brachypodium sylvaticum; Sobic: Sorghum bicolor. Multiple alignment of amino acid sequences was carried out and Neighbor-Joining evolutionary tree was constructed by ClustalX 2.0 software (Bootstrap values were tested for 1000 trails).

2.3 剑麻PAL基因选择压力分析

以龙舌兰属野生种沙漠龙舌兰为参照, 将其余龙舌兰属物种PAL基因序列分别与沙漠龙舌兰PAL基因序列进行种间选择压力分析(表4)。Ka/Ks值大于1表明相应基因受到正向选择, 小于等于1表明其受到纯化选择^[6]。龙舌兰属PAL基因种间Ka/Ks值均小于1, 表明PAL基因在龙舌兰属物种进化过程中受纯化选择作用。进一步通过滑窗法分析龙舌兰属PAL基因种间选择压力发现, 剑麻和番麻PAL基因Ka/Ks变化趋势较为接近, 其首尾区段选择压力高于中部区段(图2)。AhPAL2和AmPAL2的部分区段Ka/Ks值大于1, 表明该区域受正选择效应。此外, 太匮龙舌兰PAL基因Ka/Ks值变化趋势与剑麻/番麻差异较大, AqPAL1受选择压力较小, AqPAL1仅前端受选择压力较大, 但两者Ka/Ks值均未超过1。

Table 4
表4
表4龙舌兰属PAL基因选择压力分析
Table 4Selection pressure analysis of PALs in Agave

基因 Gene	物种/物种 Specie/specie	同义突变频率 Ks	非同义突变频率 Ka	非同义突变频率/同义突变频率 Ka/Ks
PAL1	Ad/Ah	0.1176	0.0098	0.083333
	Ad/Am	0.2956	0.0155	0.052436
	Ad/Aq	0.0179	0.0019	0.106145
PAL2	Ad/Ah	0.3324	0.0271	0.081528
	Ad/Am	0.3347	0.0280	0.083657
	Ad/Aq	0.0872	0.0129	0.147936

物种缩写同图1。
Abbreviations of species are the same as those given in Fig. 1. Ks: synonymous substitution; Ka: non-synonymous substitution.

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图2

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图2龙舌兰属PAL基因选择压力分析

物种缩写同图1。使用DnaSP进行滑窗法分析, 窗口长度为30 bp, 步移长度为6 bp。
Fig. 2Selection pressure analysis of PALs in Agave

Abbreviations of species are the same as those given in Fig. 1. Sliding window analysis was conducted by DnaSP with a window size of 30 bp and a step size of six bp.

2.4 剑麻PAL基因表达分析

通过已公布的转录组数据获取龙舌兰属PAL基因表达信息发现, PAL1仅在太匮龙舌兰中表达水平较高, PAL2在剑麻和番麻中的表达水平均低于其余两物种, 表明PAL基因在龙舌兰属不同物种中存在表达模式差异(图3)。

图3

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图3龙舌兰属PAL基因转录组表达分析

物种缩写同图1。
Fig. 3Transcriptome expression of PALs in Agave

Abbreviations of species are the same as those given in Fig. 1.


qRT-PCR检测结果显示, 剑麻PAL基因在3个时期表达趋势均为先上调之后下调(图4)。其中AhPAL1仅在未展开叶中显著上调, 而AhPAL2仅在展开叶中显著下调。逆境胁迫表达结果显示, 2个基因在铜铅胁迫下差异表达均不显著, 仅AhPAL1在铜胁迫后轻微下调、在铅胁迫后轻微上调。此外, 2个基因在烟草疫霉侵染后均极显著上调。

图4

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图4剑麻PAL基因在叶片发育和逆境胁迫下的相对表达量

L0: 心叶; L1: 未展开叶; L2: 完全展开叶; CK: 空白对照; CU: 铜胁迫处理; PB: 铅胁迫处理; PN: 烟草疫霉侵染。*, **分别表示在0.05和0.01水平差异显著。
Fig. 4Relative expression of AhPALs at different leaf developmental stages and under abotic/biotic stresses in sisal

L0: shoot; L1: unexpended leaf; L2: expended leaf; CK: control; CU: copper stress; PB: lead stress; PN: Phytophthora nicotianae Breda inoculation. * and ** indicate significant differences at the 0.05 and 0.01 probability levels, respectively.

3 讨论

PAL是苯丙烷类代谢途径的起始酶, 其在植物生长发育及环境适应性等方面起重要调控作用^[29]。前人研究已证实PAL与木质素含量直接相关, 因此克隆PAL基因并解析其在剑麻纤维发育过程中的功能, 对改良剑麻纤维相关性状具重要意义^[7]。剑麻基因组尚未公布, 其基因组较大、杂合度较高使基因组组装难度较大, 近年来转录组测序技术快速发展,为剑麻基因研究提供了快速、高通量且价格低廉的技术手段^[4]。本文成功从已发表的剑麻叶片转录组中鉴定出2个含完整编码序列的PAL基因, 表明上述2个基因主要表达部位之一为叶片。根据已开展PAL基因全基因组分析的植物推测, 剑麻极可能含多个PAL基因^[30]。除AhPAL1和AhPAL2外其他剑麻PAL基因可能因在叶片中表达水平较低而未能通过转录组测序组装出完整编码序列。剑麻主要收获部位为叶片, 因此其叶片中的木质素合成途径可能主要由AhPAL1和AhPAL2参与调控。苎麻相关研究发现, 纤维发育至成熟过程中木质素含量逐渐增加, 而PAL活性则先上升后下降^[31]。剑麻叶片收获以完全展开叶片为主(与中轴夹角>45°), 叶片完全展开时纤维趋于成熟, 因此本文选取3个叶片发育时期与剑麻纤维发育的前、中、后3个时期相对应。在此过程中, 剑麻PAL基因的表达模式(图4)与苎麻PAL活性变化趋势一致, 表明剑麻PAL基因与木质素合成密切相关。

遗传进化分析结果显示, 双子叶作物、禾本科作物和天门冬目作物PAL基因分别聚类在不同分支中, 表明PAL基因具有一定程度的物种特异性进化, 前人报道也证实了在胡桃科和禾本科植物中存在物种特异的PAL基因家族扩张^[30]。此外, 剑麻和番麻PAL基因聚类在相同分支, 同时沙漠龙舌兰和太匮龙舌兰PAL基因也聚类在相同分支(图1)。但由4种龙舌兰科植物叶绿体序列构建的进化树则显示剑麻和太匮龙舌兰亲缘关系较近, 而沙漠龙舌兰与其余三者亲缘关系均较远^[6]。上述结果间的差异可能由剑麻和番麻纤维性状趋同进化所导致^[32]。番麻叶片生物量与剑麻相当, 也可用于纤维加工, 但其叶缘尖刺较多, 收获不便, 因而以纤维生产为用途的规模种植较少^[33]。本文进一步分析龙舌兰属PAL基因的选择压力发现, 其均未受到正向选择(表4), 仅剑麻和番麻PAL基因部分区段受到正向选择, 太匮龙舌兰PAL基因区段受到的选择压力均较小(图2)。此外, 转录组数据显示, PAL基因在剑麻和番麻叶片中的表达水平与其余两者存在差异(图3), 同时纤维素合成酶基因也存在相近表达模式^[4], 表明剑麻和番麻纤维发育调控机制与其余两者存在较大差异。现有数据未能完全解释其调控机制存在差异的原因, 仍需开展更深入的基因功能及其上下游调控机制的研究。

前人研究表明, 铜铅胁迫处理后PAL活性显著提高, 但其基因表达水平未显著变化, 表明PAL基因在重金属胁迫下存在转录后调控^[34,35]。与此类似, 剑麻PAL基因在铜铅胁迫后表达量变化不显著(图4), 表明剑麻中也存在转录后调控。最新研究发现, 水稻PAL基因与抗病性密切相关, 水稻基因组包含9个PAL基因, 其中OsPAL4参与介导了对多种真菌病害的广谱抗性^[36]。剑麻PAL基因在烟草疫霉侵染后显著上调, 表明其参与了剑麻抗病响应机制(图4), 但剑麻PAL基因如何通过调控苯丙烷类代谢途径参与抗病机制仍需开展更深入的研究。重金属胁迫通常未对细胞壁造成影响, 相比之下, 病原入侵时会破坏植物细胞壁结构, 植物因细胞壁结构完整性受到破坏而启动天然防御机制并修复受损的细胞壁结构, 即细胞壁介导的免疫机制(plant cell wall-mediated immunity)^[37]。因此, 剑麻PAL基因在病原入侵时上调倍数远超纤维发育中期, 其在调控木质素生物合成以修复细胞壁的同时也参与了多种抗病相关次生代谢产物的合成, 例如水杨酸等^[38]。因此开展剑麻PAL基因功能解析不仅可加深对剑麻纤维发育机制的理解, 也可提升对剑麻抗病机制的认知, 对培育高产、优质、多抗剑麻新品种具有重要意义。

4 结论

本文从剑麻转录组中鉴定出2个PAL基因成员, 与番麻亲缘关系较近。剑麻PAL基因进化过程中未受到正向选择, 仅部分区段受到了较强的进化选择压力。PAL基因在龙舌兰属不同物种叶片中存在表达模式差异, 同时剑麻PAL基因广泛参与调控纤维发育和逆境胁迫应答。PAL基因的鉴定有助于解析剑麻纤维发育及抗病机制, 在剑麻高产、优质、多抗剑麻新品种选育中具有重要应用价值。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] Li Y, Mai Y W, Ye L. Sisal fibre and its composites: a review of recent developments
Comp Sci Technol, 2000,60:2037-2055.
[本文引用: 1]

[2] 许能琨, 余让水, 孙光明. 氮磷钾钙镁肥不同用量对剑麻产量质量和矿质组分的影响
热带作物学报, 1994,15(1):39-45.
[本文引用: 1]

Xu N K, Yu R S, Sun G M. Effects of different levels of NPKCaMg fertilizers on yield, fibre quality and nutrient content of Agave H.11648
Chin J Trop Crops, 1994,15(1):39-45 (in Chinese with English abstract).
[本文引用: 1]

[3] 黄兴, 陈涛, 习金根, 贺春萍, 吴伟怀, 梁艳琼, 郑金龙, 李锐, 易克贤. 剑麻单叶农艺性状与鲜叶产量的相关性研究
中国麻业科学, 2018,40(2):70-74.
[本文引用: 1]

Huang X, Chen T, Xi J G, He C P, Wu W H, Liang Y Q, Zheng J L, Li R, Yi K X. The Correlation between single leaf traits and fresh yield of sisal
Plant Fiber Sci China, 2018,40(2):70-74 (in Chinese with English abstract).
[本文引用: 1]

[4] Huang X, Xiao M, Xi J, He C, Zheng J, Chen H, Gao J, Zhang S, Wu W, Liang Y, Xie L, Yi K. De novo transcriptome assembly of Agave H11648 by Illumina sequencing and identification of cellulose synthase genes in Agave species
Genes, 2019,10:103.
[本文引用: 6]

[5] Deng G, Huang X, Xie L, Tan S, Gbokie T J, Bao Y, Xie Z, Yi K. Identification and expression of SAUR genes in the CAM plant agave
Genes, 2019,10:555.
[本文引用: 3]

[6] Huang X, Wang B, Xi J, Zhang Y, He C, Zheng J, Gao J, Chen H, Zhang S, Wu W, Liang Y, Yi K. Transcriptome comparison reveals distinct selection patterns in domesticated and wild Agave species, the important CAM plants
Int J Genomics, 2018,2018:5716518.
[本文引用: 5]

[7] 李潞滨, 刘蕾, 何聪芬, 董银卯, 彭镇华. 木质素生物合成关键酶基因的研究进展
分子植物育种, 2007,5(增刊1):45-51.
[本文引用: 2]

Li L B, Liu L, He C F, Dong Y M, Peng Z H. Research progresses on the genes encoding the key enzymes in biosynthetic pathway of lignin
Mol Plant Breed, 2007,5(S1):45-51 (in Chinese with English abstract).
[本文引用: 2]

[8] 石海燕, 张玉星. 木质素生物合成途径中关键酶基因的分子特征
中国农学通报, 2011,27(5):288-291.
[本文引用: 1]

Shi H Y, Zhang Y X. Molecular characterization of key enzyme genes related to the pathway of lignin biosynthesis
Chin Agric Sci Bull, 2011,27(5):288-291 (in Chinese with English abstract).
[本文引用: 1]

[9] Wanner L A, Li G, Ware D, Somssich I E, Davis K R. The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana
Plant Mol Biol, 1995,27:327-338.
[本文引用: 1]

[10] Olsen K M, Lea U S, Slimestad R, Verheul M, Lillo C. Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis.
J Plant Physiol, 2008,165:1491-1499.
[本文引用: 1]

[11] Raes J, Rohde A, Christensen J H, Van Y D P, Boerjan W. Genome-wide characterization of the lignification toolbox in Arabidopsis
Plant Physiol, 2003,133:1051-1071.
[本文引用: 1]

[12] Yu X Z, Fan W J, Lin Y J, Zhang F F, Gupta D K. Differential expression of the PAL gene family in rice seedlings exposed to chromium by microarray analysis
Ecotoxicology, 2018,27:325-335.
[本文引用: 2]

[13] Jaillon O, Aury J M, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla
Nature, 2007,449:463.
[本文引用: 1]

[14] Shi R, Sun Y H, Li Q, Heber S, Sederoff R, Chiang V L. Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes
Plant Cell Physiol, 2010,51:144-163.
[本文引用: 1]

[15] Wada K C, Mizuuchi K, Koshio A, Kaneko K, Mitsui T, Takeno K. Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis
J Plant Physiol, 2014,171:895-902.
[本文引用: 1]

[16] 李福燕, 张黎明, 李许明, 郭彬, 陈柳燕, 漆智平. 剑麻对铜的耐性与累积效应研究初探
中国农学通报, 2006,22(12):417-420.
[本文引用: 1]

Li F Y, Zhang L M, Li X M, Guo B, Chen L Y, Qi Z P. Sisal tolerance of cupreous and its accumulation preliminary explore
Chin Agric Sci Bull, 2006,22(12):417-420 (in Chinese with English abstract).
[本文引用: 1]

[17] 陈柳燕, 张黎明, 李福燕, 郭彬, 李许明, 廖香俊, 漆智平. 剑麻对重金属铅的吸收特性与累积规律初探
农业环境科学学报, 2007,26:1879-1883.
[本文引用: 1]

Chen L Y, Zhang L M, Li F Y, Guo B, Li X M, Liao X J, Qi Z P. A primary research on sisal’s uptake property and the accumulation rule to Pb ions
J Agro-Environ Sci, 2007,26:1879-1883 (in Chinese with English abstract).
[本文引用: 1]

[18] 汪平, 高建明, 杨峰, 郑金龙, 刘巧莲, 陈河龙, 易克贤. 烟草疫霉侵染前后剑麻叶片转录组学研究
热带作物学报, 2014,35:576-582.
[本文引用: 1]

Wang P, Gao J M, Yang F, Zheng J L, Liu Q L, Chen H L, Yi K X. Transcriptome of sisal leaf pretreated with Phytophthora nicotianae Breda
Chin J Trop Crops, 2014,35:576-582 (in Chinese with English abstract).
[本文引用: 1]

[19] Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool
J Mol Biol, 1990,215:403-410.
[本文引用: 1]

[20] Rombel I T, Sykes K F, Rayner S, Johnston S A. ORF-FINDER: a vector for high-throughput gene identification
Gene, 2002,282:33-41.
[本文引用: 1]

[21] Wilkins M R, Gasteiger E, Bairoch A, Sanchez J C, Williams K L, Appel R D, Hochstrasser D F. Protein identification and analysis tools in the ExPASy server
Methods Mol Biol, 1999,112:531-552.
[本文引用: 1]

[22] Yu C, Chen Y, Lu C, Hwang J. Prediction of protein subcellular localization
Proteins, 2006,64:643-651.
[本文引用: 1]

[23] Gross S M, Martin J A, Simpson J, Abraham-Juarez M J, Wang Z, Visel A. De novo transcriptome assembly of drought tolerant CAM plants,Agave deserti and Agave tequilana
BMC Genomics, 2013,14:563.
[本文引用: 2]

[24] Abraham P E, Yin H, Borland A M, Weighill D, Lim S D, De Paoli H C, Engle N, Jones P C, Agh R, Weston D J, Wullschleger S D, Tschaplinski T, Jacobson D, Cushman J C, Hettich R L, Tuskan G A, Yang X. Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
Nat Plants, 2016,2:16178.
[本文引用: 2]

[25] Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G. Clustal W and Clustal X version 2.0
Bioinformatics, 2007,23:2947-2948.
[本文引用: 1]

[26] Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data
Bioinformatics, 2009,25:1451-1452.
[本文引用: 1]

[27] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method
Methods, 2001,25:402-408.
[本文引用: 1]

[28] Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth B C, Remm M, Rozen S G. Primer3—new capabilities and interfaces
Nucleic Acids Res, 2012,40:e115.
[本文引用: 1]

[29] Huang J, Gu M, Lai Z, Fan B, Shi K, Zhou Y H, Yu J Q, Chen Z. Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress
Plant Physiol, 2010,153:1526-1538.
[本文引用: 1]

[30] Yan F, Li H Z, Zhao P. Genome-wide identification and transcriptional expression of the PAL gene family in common walnut(Juglans regia L.)
Genes, 2019,10:46.
[本文引用: 2]

[31] 李建军, 郭清泉, 陈建荣. 苎麻木质素形成的相关酶类研究
中国麻业科学, 2009,31(2):119-124.
[本文引用: 1]

Li J J, Guo Q Q, Chen J R. Study on related enzymes of lignin synthesis in ramie
Plant Fiber Sci China, 2009,31(2):119-124 (in Chinese with English abstract).
[本文引用: 1]

[32] Pichersky E, Lewinsohn E. Convergent evolution in plant specialized metabolism
Annu Rev Plant Biol, 2011,62:549-566.
[本文引用: 1]

[33] Sghaier A, Chaabouni Y, Msahli S, Sakli F. Morphological and crystalline characterization of NaOH and NaOCl treated Agave americana L. fiber
Ind Crops Prod, 2012,36:257-266.
[本文引用: 1]

[34] Ková?ik J, Klejdus B, Hedbavny J, Zoń J. Copper uptake is differentially modulated by phenylalanine ammonia-lyase inhibition in diploid and tetraploid chamomile
J Agric Food Chem, 2010,58:10270-10276.
[本文引用: 1]

[35] Pawlak-Sprada S, Arasimowicz-Jelonek M, Podgórska M, Deckert J. Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part I. Effects of cadmium and lead on phenylalanine ammonia-lyase gene expression, enzyme activity and lignin content
Acta Biochim Pol, 2011,58:211-216.
URLPMID:21503278 [本文引用: 1]
Species-specific changes in expression of phenylalanine ammonia-lyase (PAL) and lignin content were detected in roots of soybean (Glycine max L.) and lupine (Lupinus luteus L.) seedlings treated with different concentrations of cadmium (Cd(2+), 0-25 mg/l) or lead (Pb(2+), 0-350 mg/l). The stimulatory effect of both metals was observed in mRNA coding for PAL in soybean. In the case of lupine, changes of PAL mRNA level were dependent on the metal used: Cd(2+) caused a decrease, whereas Pb(2+) an increase of PAL transcript level. The activity of PAL was enhanced in both plant species at higher metal concentrations (15-25 mg/l of Cd(2+) or 150-350 mg/l of Pb(2+)); however it was not directly correlated with PAL mRNA. This suggests a transcriptional and posttranscriptional control of PAL expression under heavy metals stress. In soybean, Cd(2+) or Pb(2+) treatment increased lignin content, while in lupine the effect was opposite. The decreased lignin accumulation in lupine roots in response to heavy metals, despite an increased PAL activity, suggests that the activated phenylpropanoid pathway was involved in the synthesis of secondary metabolites other than lignin.

[36] Wang R, Wang G L, Ning Y. PALs: emerging key players in broad-spectrum disease resistance
Trends Plant Sci, 2019,24:785-787.
[本文引用: 1]

[37] Bacete L, Mélida H, Miedes E, Molina A. Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses
Plant J, 2018,93:614-636.
[本文引用: 1]

[38] Duan L, Liu H B, Li X H, Xiao J H, Wang S P. Multiple phytohormones and phytoalexins are involved in disease resistance to Magnaporthe oryzae invaded from roots in rice
Physiol Plant, 2014,152:486-500.
URLPMID:24684436 [本文引用: 1]