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作物学报 ›› 2021, Vol. 47 ›› Issue (6): 1082-1089.doi: 10.3724/SP.J.1006.2021.04116

• 专题：主要麻类作物基因组学与遗传改良 • 上一篇    下一篇

剑麻苯丙氨酸裂解酶基因的鉴定及表达分析

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. 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
  • 收稿日期:2020-05-30 接受日期:2020-09-13 出版日期:2021-06-12 网络出版日期:2020-10-10
  • 通讯作者: 易克贤
  • 作者简介:E-mail: huangxing@catas.cn
  • 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)

Identification and expression of PAL genes in sisal

HUANG Xing1(), XI Jin-Gen1, CHEN Tao2, QIN Xu2, TAN Shi-Bei1, CHEN He-Long3, YI Ke-Xian1,*()   

  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
  • Received:2020-05-30 Accepted:2020-09-13 Published:2021-06-12 Published online:2020-10-10
  • Contact: YI Ke-Xian
  • Supported by:
    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)

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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.

Key words: sisal, PAL gene, phylogenetic analysis, selection pressure, expression pattern, adverse stress

表1

实时定量PCR引物"

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

表2

剑麻PAL基因及其蛋白理化性质、亚细胞定位预测"

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)

表3

龙舌兰属PAL基因序列号"

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

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)。"

表4

龙舌兰属PAL基因选择压力分析"

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

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。"

图3

龙舌兰属PAL基因转录组表达分析 物种缩写同图1。"

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。"

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