欢迎访问作物学报，今天是
作物学报

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)

RichHTML

18

PDF

1126

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。"

[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.
[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.
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).
[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.
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).
[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.
[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.
[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.
[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.
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).
[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.
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).
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
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).
[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.
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).
[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.
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).
[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.
[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.
[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.
[22] Yu C, Chen Y, Lu C, Hwang J. Prediction of protein subcellular localization. Proteins, 2006,64:643-651.
[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.
[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.
[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.
[26] Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 2009,25:1451-1452.
[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.
[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.
[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.
[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.
[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.
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).
[32] Pichersky E, Lewinsohn E. Convergent evolution in plant specialized metabolism. Annu Rev Plant Biol, 2011,62:549-566.
[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.
[34] Kováčik 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.
[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.
pmid: 21503278
[36] Wang R, Wang G L, Ning Y. PALs: emerging key players in broad-spectrum disease resistance. Trends Plant Sci, 2019,24:785-787.
[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.
[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.
pmid: 24684436
[1] 孙淑凤, 许振南, 黄嘉鑫, 翁建峰, 李新海. 玉米MAPK家族全基因组鉴定及其对拟轮枝镰孢菌感染的响应[J]. 作物学报, 2026, 52(5): 1291-1308.
[2] 胡城祯, 高维东, 孔斌雪, 王建飞, 车卓, 杨德龙, 陈涛. 小麦TaAPC11基因家族鉴定及TaAPC11-5B参与干旱胁迫的生物学功能研究[J]. 作物学报, 2026, 52(1): 148-164.
[3] 闫知兰, 赵芹, 常甜达, 王一鸣, 王碧辉, 王鹏, 黄春国, 张会, 王利祥, 郝晓鹏, 赵波. 豆科作物AOX基因鉴定及其在普通菜豆响应非生物胁迫中的表达模式研究[J]. 作物学报, 2025, 51(7): 1769-1783.
[4] 沈傲, 刘敏, 倪迪安, 刘炜. 谷子m6A甲基转移酶基因SiMTA1的启动子序列特征和基因表达模式分析[J]. 作物学报, 2025, 51(7): 1969-1978.
[5] 徐林珊, 郜耿东, 王宇, 王家星, 杨吉招, 武亚瑞, 张宵寒, 常影, 李真, 谢雄泽, 龚德平, 王晶, 葛贤宏. 甘蓝型油菜漆酶基因家族成员表达模式及与茎秆抗折力的关联分析[J]. 作物学报, 2025, 51(1): 134-148.
[6] 宋倩娜, 宋慧洋, 李京昊, 段永红, 梅超, 冯瑞云. 马铃薯转录因子StFBH3对非生物逆境胁迫的响应分析[J]. 作物学报, 2025, 51(1): 247-259.
[7] 杨煜琛, 靳雅荣, 骆金婵, 祝鑫, 李葳航, 贾纪原, 王小珊, 黄德均, 黄琳凯. 珍珠粟WD40基因家族鉴定及表达特征分析[J]. 作物学报, 2024, 50(9): 2219-2236.
[8] 郭思语, 赵克勇, 代正罡, 邹华文, 吴忠义, 张春. 玉米N-乙酰转移酶ZmNAT1基因响应非生物胁迫的功能分析[J]. 作物学报, 2024, 50(8): 2001-2013.
[9] 艾蓉, 张春, 悦曼芳, 邹华文, 吴忠义. 玉米转录因子ZmEREB211对非生物逆境胁迫的应答[J]. 作物学报, 2023, 49(9): 2433-2445.
[10] 左春阳, 李亚玮, 李焱龙, 金双侠, 朱龙付, 张献龙, 闵玲. 陆地棉漆酶基因家族成员表达模式分析[J]. 作物学报, 2023, 49(9): 2344-2361.
[11] 马春敏, 李维希, 李芳军, 田晓莉, 李召虎. 陆地棉硝酸盐转运体NRT基因家族鉴定及表达分析[J]. 作物学报, 2023, 49(6): 1496-1517.
[12] 贾玉库, 高宏欢, 冯健超, 郝紫瑞, 王晨阳, 谢迎新, 郭天财, 马冬云. 小麦G2-like转录因子家族基因鉴定与表达模式分析[J]. 作物学报, 2023, 49(5): 1410-1425.
[13] 韩贝, 孙思敏, 孙伟男, 杨细燕, 张献龙. 植物体细胞胚胎发生的分子机制[J]. 作物学报, 2023, 49(2): 299-309.
[14] 杨佳宝, 张展, 周至铭, 吕新华, 孙黎. 向日葵HaLACS9基因的克隆与功能分析[J]. 作物学报, 2023, 49(2): 426-437.
[15] 陈吴钧, 刘江栋, 蒋凯旋, 王幼平, 蒋金金. 甘蓝型油菜BnKNOX基因家族的鉴定与分析[J]. 作物学报, 2023, 49(11): 2991-3006.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2