Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (1): 86-94.doi: 10.3864/j.issn.0578-1752.2021.01.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Expression Analysis of Light Harvesting Chlorophyll a/b Protein Gene CcLhca-J9 in Conyza canadensis

LI ZuRen1(),LUO DingFeng1,BAI HaoDong1,XU JingJing1,HAN JinCai1,XU Qiang2,WANG RuoZhong2,BAI LianYang1,2()   

  1. 1Hunan Provincial Key Laboratory for Biology and Control of Weeds, Hunan Academy of Agricultural Sciences, Changsha 410125
    2Hunan Provincial Key Laboratory for Phytohormones, Hunan Agricultural University, Changsha 410128
  • Received:2020-04-12 Accepted:2020-06-28 Online:2021-01-01 Published:2021-01-13
  • Contact: LianYang BAI E-mail:lizuren88214@sina.com;bailianyang2005@aliyun.com

RichHTML

63

PDF

596

Abstract:

【Background】Conyza canadensis, a pernicious weed, is harmful and lack of the effective management strategy. The caprylic acid can efficiently inhibit the photosynthesis of C. canadensis, which is a potential bio-herbicidal compound. Light harvesting chlorophyll a/b-binding protein (LHC) is the key complex protein in PSI, and plays an important role in photosynthesis.【Objective】The objective of this study is to explore the LHC gene in response to caprylic acid, and to provide a theoretical basis for the development of bio-herbicide.【Method】The open reading frame (ORF) of CcLhca-J9 was cloned from C. canadensis leaf by homologous PCR and RACE. The nucleotide sequence was analyzed by DNAMAN. The phylogenetic tree was constructed with the high similarity nucleotide from NCBI by neighbor-joining method and MEGA software. The molecular weight, isoelectric point and protein structure were analyzed using SWISS-MODEL and ExPaSy websites. Using homologous modeling results as a model, the affinity of caprylic acid and CcLhca-J9 protein was analyzed using AutoDock 4.2 software. RT-qPCR was used to analyze the difference of CcLhca-J9 expression level of caprylic acid, water and ferulic acid treatments.【Result】The CcLhca-J9 ORF is 744 bp, encoding 247 amino acid residues with a molecular weight of 26.766 kD and theoretical pI of 6.43, which belongs to Chloroa b-bind family protein. The phylogenetic analysis showed that the CcLhca-J9 had the most closed relationship with the Lhca protein of Tanacetum cinerariifolium (GEW73959.1) and Artemisia annua (PWA35049.1), and was in the same branch of Asteraceae group, with consistency of more than 85%, indicating that the gene family was conserved. The second structure of CcLhca-J9 protein had alpha helix, random coil, extended strand, beta turn. The homologous three-dimensional model of CcLhca-J9 protein was compared by 4y28.1.O (2.80Á), the tertiary structure was a monomolecular object with six chlorophyll a ligands, which was a typical light harvesting chlorophyll a/b-binding protein. Molecular docking result showed that hydrogen and p-π bond interaction were produced between caprylic acid and CcLhca-J9 amino acid residues Gly68, Phe67, Phe69 and Arg197. The result of RT-qPCR showed that the expression level of CcLhca-J9 was significantly decreased after 0-8 h caprylic acid treatment. Compared with ferulic acid control and water treatment, caprylic acid treatment inhibited the expression of CcLhca-J9.【Conclusion】CcLhca-J9 has the typical function of light harvesting chlorophyll a/b-binding protein, which may be involved in the process of inhibiting the growth of C. canadensis leaves and is a herbicidal target with the potential to develop herbicides.

Key words: Conyza canadensis, light harvesting protein, gene clone, molecular docking, caprylic acid

Table 1

The primers used for CcLhca-J9 cloning and RT-qPCR"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 引物用途 Use of primers
CcJ9-1 Forward: GAGYTGCGGYATCBCCGCCGT
Reverse: GTTGTGCCATGGRTCAGCCAA		 核心片段扩增 Fragment cloning
CcJ9-F2 Forward: CGGATTTGACCCACTTCGTCT 5′ RACE扩增 5′ cDNA end amplification
CcJ9-R2 Reverse: TATTGCCAGTATGGTGGGTAGGGT 5′ RACE扩增 5′ cDNA end amplification
CcJ9-R3 Reverse: GGCAGCCCATTCTTGTGCTTTTAC 5′ RACE扩增 5′ cDNA end amplification
CcJ9-F3 Forward: GGCAACTGGGTAAAAGCACAAGAAT 3′ RACE扩增 3′ cDNA end amplification
CcJ9-F4 Forward: AAGACCCAAAAACATTCGCAGAGTA 3′ RACE扩增 3′ cDNA end amplification
qR-J9 Forward: CATTCTTGTGCTTTTACCCAG
Reverse: GAGGTTTAAAGAGTCCGAGCT		 实时荧光定量PCR RT-qPCR
ACTIN Forward: CCGATCCAGACGCTGTATTT
Reverse: TGCTGATCGTATGAGCAAGG		 实时荧光定量PCR RT-qPCR

Fig. 1

Amplification of CcLhca-J9"

Fig. 2

CcLhca-J9 gene nucleotide and the encoded amino acid sequences"

Fig. 3

Multiple sequence alignment of CcLhca-J9 and homologous gene coding region"

Fig. 4

Phylogenetic tree of CcLhca-J9 protein with homologous proteins from other species (neighbor-joining method, 1 000 replicates)"

Fig. 4

Phylogenetic tree of CcLhca-J9 protein with homologous proteins from other species (neighbor-joining method, 1 000 replicates)"

Fig. 5

Secondary structure of CcLhca-J9"

Fig. 5

Secondary structure of CcLhca-J9"

Fig. 6

Homology model structure of CcLhca-J9 6x CLA (chlorophyll a) ligands were shown with different colors"

Fig. 6

Homology model structure of CcLhca-J9 6x CLA (chlorophyll a) ligands were shown with different colors"

Fig. 7

Molecular docking schema chart between caprylic acid and CcLha-J9"

Fig. 7

Molecular docking schema chart between caprylic acid and CcLha-J9"

Fig. 8

Relative expression level of CcLhca-J9 after caprylic acid treatment"

Fig. 8

Relative expression level of CcLhca-J9 after caprylic acid treatment"

[1] 李杨汉. 中国杂草志. 北京: 中国农业出版社, 1998.
LI Y H. Weed of China. Beijing: China Agriculture Press, 1998. (in Chinese)
[2] METZGER B A, SOLTANI N, RAEDER A J, HOOKER D C, ROBINSON D E, SIKKEMA P T. Multiple herbicide-resistant horseweed (Conyza canadensis) dose response to tolpyralate and tolpyralate plus atrazine and comparison to industry standard herbicides in corn. Weed Technology, 2019,33(2):366-373.
doi: 10.1017/wet.2019.20
[3] SONG X L, WU J J, ZHANG H J, QIANG S. Occurrence of glyphosate-resistant horseweed (Conyza canadensis) population in China. Agricultural Sciences in China, 2011,10(7):1049-1055.
doi: 10.1016/S1671-2927(11)60093-X
[4] 朱文达, 曹坳程, 喻大昭, 魏守辉. 小飞蓬对油菜产量性状的影响及其经济域值. 华中农业大学学报, 2008,27(2):217-222.
ZHU W D, CAO A C, YU D Z, WEI S H. Influence of Conyza canadensis on the yield character of oil seed rape and its economic threshold. Journal of Huazhong Agricultural University, 2008,27(2):217-222. (in Chinese)
[5] LI Z R, AMIST N, BAI L Y. Allelopathy in sustainable weeds management. Allelopathy Journal, 2019,48(2):109-138.
[6] DUKE S O. Why have no new herbicide modes of action appeared in recent years?. Pesticide Management Science, 2012,68(4):505-512.
doi: 10.1002/ps.2333
[7] DUKE S O, DAYAN F E. Discovery of new herbicide modes of action with natural phytotoxins//Discovery and Synthesis of Crop Protection Products. ACS Symposium Series, 2015,1204:79-92.
[8] WANG P, GRIMM B. Comparative analysis of light-harvesting antennae and state transition in chlorine and cpSRP mutants. Plant Physiology, 2016,172(3):1519-1531.
doi: 10.1104/pp.16.01009 pmid: 27663408
[9] BAI W J, LU C, WANG X. Recent advances in the total synthesis of tetramic acid-containing natural products. Journal of Chemistry, 2016,2016:8510278.
[10] DOMINGOS L, DESRUS A, MÊME S, MÊME W. L-Phosphinothricin modulation of inwardly rectifying K+ channels increased excitability in striatal medium-sized spiny neurons. Archives of Toxicology, 2016,90(7):1719-1727.
doi: 10.1007/s00204-016-1721-z pmid: 27136897
[11] SALIMON J, SALIH N, YOUSIF E. Industrial development and applications of plant oils and their biobased oleochemicals. Arabian Journal of Chemistry, 2012,5(2):135-145.
doi: 10.1016/j.arabjc.2010.08.007
[12] 李祖任, 黄勤勤, 彭琼, 周勇, 周小毛, 柏连阳. 植物源羊脂酸的除草活性及其响应机制. 植物保护学报, 2018,45(5):1161-1167.
LI Z R, HUANG Q Q, PENG Q, ZHOU Y, ZHOU X M, BAI L Y. Herbicidal activity and response mechanism of botanical caprylic acid. Journal of Plant Protection, 2018,45(5):1161-1167. (in Chinese)
[13] LI Z R, SHEN W H, ZHOU X M, ZHAN Q C, PENG Q, WANG L F, WU L M, YANG H N, BAI L Y. Changes in Conyza canadensis (L.) cronquist leaf anatomy under caprylic acid stress. Pakistan Journal of Botany, 2019,51(4):1223-1229.
[14] LI Z R, KUANG W, LIU Y B, PENG D, BAI L Y. Proteomic analysis of horseweed (Conyza canadensis) subjected to caprylic acid stress. Proteomics, 2019,19(9):e1800294.
doi: 10.1002/pmic.201800294 pmid: 30865362
[15] KROL M, SPANGFORT M D, HUNER N P, OQUIST G, GUSTAFSSON P, JANSSON S. Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant. Plant Physiology, 1995,107:873-883.
pmid: 7748263
[16] 石兰馨, 张晓平, 梁厚果. 捕光叶绿素a/b结合蛋白和cab基因. 植物生理学通讯, 1995,31(6):470-476.
SHI L X, ZHANG X P, LIANG H G. Light-harvesting chlorophyll a/b-binding protein and CAB gene. Plant Physiology Communications, 1995,31(6):470-476. (in Chinese)
[17] HEY D, ROTHBART M, HERBST J, WANG P, MULLER J, WITTMANN D, GRUHL K, GRIMM B. LIL3, a light-harvesting complex protein, links terpenoid and tetrapyrrole biosynthesis in Arabidopsis thaliana. Plant Physiology, 2017,174(2):1037-1050.
doi: 10.1104/pp.17.00505 pmid: 28432258
[18] 阳江华, 张希财, 邹智. 橡胶树捕光叶绿素a/b结合蛋白基因CAB2的克隆与分析. 西南林业大学学报 (自然科学版), 2019,39(1):88-94.
YANG J H, ZHANG X C, ZOU Z. Molecular cloning and analysis of HbCAB2, a chlorophyll a/b-binding protein-encoding gene from Hevea brasiliensis. Journal of Southwest Forestry University (Natural Science Edition), 2019,39(1):88-94. (in Chinese)
[19] 袁定阳, 余东, 谭炎宁, 孙志忠, 韶也, 孙学武, 段美娟. RT-PCR克隆籼稻叶绿素a/b结合蛋白基因全长cDNA及序列的in silico分析. 基因组学与应用生物学, 2012,31(2):173-177.
YUAN D Y, YU D, TAN Y N, SUN Z Z, SHAO Y, SUN X W, DUAN M J. RT-PCR Cloning and in silico analysis of a full-length cDNA gene encoding the light-harvesting chlorophyll a/b-binding protein in rice (Oryza sativa L. subsp. indica). Genomics and Applied Biology, 2012,31(2):173-177. (in Chinese)
[20] SAGA Y, MIYAGI K. Characterization of 3-acetyl chlorophyll a and 3-acetyl protochlorophyll a accommodated in the b800 binding sites of photosynthetic light-harvesting complex 2 in the purple photosynthetic bacterium Rhodoblastus acidophilus. Photochemistry and Photobiology, 2018,94(4):698-704.
doi: 10.1111/php.12919 pmid: 29569330
[21] 蒋梦婷, 朱宁, 龚洪泳, 侯应军, 余心怡, 渠慎春. ‘南通小方柿’赤霉素不敏感基因DkGAI2的克隆与功能分析. 中国农业科学, 2019,52(19):3417-3429.
doi: 10.3864/j.issn.0578-1752.2019.19.012
JIANG M T, ZHU N, GONG H Y, HOU Y J, YU X Y, QU S C. Cloning and function analysis of gibberellin insensitive DkGAI2 gene in Nantongxiaofangshi (Diospyros kaki Linn. cv. nantongxiaofangshi). Scientia Agricultura Sinica, 2019,52(19):3417-3429. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2019.19.012
[22] BIENERT S, WATERHOUSE A, DE BEER T A P, TAURIELLO G, STUDER G, BORDOLI L, SCHWEDE T. The SWISS-MODEL repository—new features and functionality. Nucleic Acids Research, 2017,45(Database issue):D313-D319.
doi: 10.1093/nar/gkw1132
[23] SUBRAMANIVAN V, SEKAR R, PRAVEENKUMAR A, SELVAM R. Molecular modeling studies of repandusinic acid as potent small molecule for hepatitis B virus through molecular docking and ADME analysis. Quantitative Biology, 2019,7(4):302-312.
doi: 10.1007/s40484-019-0179-4
[24] 宋维源, 侯钰, 赵剑宇, 刘小凤, 张小兰. 黄瓜CsRPL1/2的克隆及其功能分析. 中国农业科学, 2020,53(1):148-159.
doi: 10.3864/j.issn.0578-1752.2020.01.014
SONG W Y, HOU Y, ZHAO J Y, LIU X F, ZHANG X L. Cloning and functional analysis of CsRPL1/2 in cucumber. Scientia Agricultura Sinica, 2020,53(1):148-159. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2020.01.014
[25] 翟玉山, 邓宇晴, 董萌, 徐倩, 程光远, 彭磊, 林彦铨, 徐景升. 甘蔗捕光叶绿素a/b结合蛋白基因ScLhca3的克隆及表达. 作物学报, 2016,42(9):1332-1341.
doi: 10.3724/SP.J.1006.2016.01332
ZHAI Y S, DENG Y Q, DONG M, XU Q, CHENG G Y, PENG L, LIN Y Q, XU J S. Cloning and characterization of light harvesting chlorophyll a/b-binding protein coding gene (ScLhca3) in sugarcane. Acta Agronomica Sinica, 2016,42(9):1332-1341. (in Chinese)
doi: 10.3724/SP.J.1006.2016.01332
[26] WEI L, CAO Y, BAI L H, LIANG X, DENG T T, LI J, QIAO D R. Cloning and expression of a gene coding for the major light- harvesting chlorophyll a/b protein of photosystem II in the green alga Dunaliella salina. Journal of Applied Phycology, 2007,19:89-94.
doi: 10.1007/s10811-006-9116-9
[27] YANG D H, ANDERSSON B, ARO E M, OHAD I. The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation. Photosynthesis Research, 2001,68(2):163-174.
doi: 10.1023/A:1011849919438
[28] 李真, 刘明英, 韩小娇, 乔桂荣, 蒋晶, 邢世岩. 东南景天捕光叶绿素a/b结合蛋白基因salhcb2的分离及功能. 浙江农林大学学报, 2014,31(6):838-846.
doi: 10.11833/j.issn.2095-0756.2014.06.003
LI Z, LIU M Y, HAN X J, QIAO G R, JIANG J, XING S Y. Characterization of a light-harvesting chlorophyll a/b binding protein (LHCB) gene, salhcb2, in Sedum alfredii. Journal of Zhejiang A & F University, 2014,31(6):838-846. (in Chinese)
doi: 10.11833/j.issn.2095-0756.2014.06.003
[29] YANG D H, PAULSEN H, ANDERSSON B. The N-terminal domain of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for its acclimative proteolysis. FEBS Letters, 2000,466(2/3):385-388.
doi: 10.1016/S0014-5793(00)01107-8
[30] 刘小兰, 杨霞, 孙命, 刘晓红, 赵茹, 缪方明. 新型光系统Ⅱ抑制剂的设计、合成和生物活性测定. 化学学报, 2002,60(3):487-492.
LIU X L, YANG X, SUN M, LIU X H, ZHAO R, MIAO F M. Design, synthesis and biological activity determination of new type photosystemⅡinhibitors. Acta Chimica Sinica, 2002,60(3):487-492. (in Chinese)
[31] MICHEL H, EPP O, DEISENHOFER J. Pigment—protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis. The EMBO Journal, 1986,5(10):2445-2451.
pmid: 16453713
[32] NASIR M N, LEGRAND V, GATARD S, BOUQUILLON S, LAURENT P, NOTT K. Molecular analysis of the interfacial and membrane-interacting properties of D-xylose-based bolaforms. Planta, 2012,216(4):630-638.
doi: 10.1007/s00425-002-0886-2 pmid: 12569405
[33] CHEN S G, KANG Y, ZHANG M, WANG X X, STRASSER R J, ZHOU B, QIANG S. Differential sensitivity to the potential bioherbicide tenuazonic acid probed by the JIP-test based on fast chlorophyll fluorescence kinetics. Environmental and Experimental Botany, 2015,112:1-15.
doi: 10.1016/j.envexpbot.2014.11.009
[1] YAO Hong, SHI ShouRong, ZHAO RuQian. The Potential and Mechanism of Chlorogenic Acid to Alleviate Intestinal Inflammation in Chickens Based on Network Pharmacology and Molecular Docking [J]. Scientia Agricultura Sinica, 2025, 58(3): 600-616.
[2] WANG MengYun, DENG LiYuan, YU Yang, YANG YuHeng, FANG AnFei, TIAN BinNian, WANG Jing, BI ChaoWei. Effects of Amino Acid Substitutions at Different Sites of Succinate Dehydrogenase on the Sensitivity of Magnaporthe oryzae to Benzovindiflupyr [J]. Scientia Agricultura Sinica, 2025, 58(24): 5175-5189.
[3] LIU ZhuoLin, LIU HongYun. The Potential and Mechanisms of Apigenin to Relieve Heat Stress and Hypoxia in Dairy Cows Based on Network Pharmacology and Molecular Docking [J]. Scientia Agricultura Sinica, 2024, 57(5): 1010-1022.
[4] WANG ZhuangZhuang, DONG ShaoYun, ZHOU Qi, MIAO Han, LIU XiaoPing, XU KuiPeng, GU XingFang, ZHANG ShengPing. Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit [J]. Scientia Agricultura Sinica, 2023, 56(3): 508-518.
[5] NIAN HeFen, ZHANG YuXi, LI BoLiao, CHEN XiuLin, LUO Kun, LI GuangWei. Expression and Ligand Binding Characteristics of GfunOBP2 from Grapholita funebrana [J]. Scientia Agricultura Sinica, 2023, 56(12): 2302-2316.
[6] LI GuiXiang,LI XiuHuan,HAO XinChang,LI ZhiWen,LIU Feng,LIU XiLi. Sensitivity of Corynespora cassiicola to Three Common Fungicides and Its Resistance to Fluopyram from Shandong Province [J]. Scientia Agricultura Sinica, 2022, 55(7): 1359-1370.
[7] XiaoHe LIU,GuiSheng QIU,ZhaoGuo TONG,HuaiJiang ZHANG,WenTao YAN,Qiang YUE,LiNa SUN. Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii [J]. Scientia Agricultura Sinica, 2021, 54(5): 945-958.
[8] QIN JianHui,LI JinQiao,ZHAO Xu,LI KeBin,CAO YaZhong,YIN Jiao. Expression, Purification and Functional Analysis of Odorant Binding Protein 11 (OBP11) in Anomala corpulenta [J]. Scientia Agricultura Sinica, 2021, 54(14): 3017-3028.
[9] TAN Jing, SONG XinMi, FU XiaoBin, TANG MingZhu, WU Fan, HUA QiYun, LI HongLiang. Functional Mode and Immunocytochemical Localization of Chemosensory Protein 1 (CSP1) in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2017, 50(15): 3052-3062.
[10] LI HuiYuan, TIAN ChunYu, ZHENG YuYing, WU Tao. Cloning and Expression Analysis of Cadmium Tolerance Related Gene CsNAC019 in Cucumber [J]. Scientia Agricultura Sinica, 2017, 50(10): 1852-1861.
[11] BO Hui-wen, YU Mi-na, YU Jun-jie, YIN Xiao-le, DING Hui, WANG Ya-hui, LIU Yong-feng. Molecular Cloning Flanking Sequences of T-DNA Insertion from the Ustilaginoidea virens Mutant Strain B1241 [J]. Scientia Agricultura Sinica, 2016, 49(9): 1685-1695.
[12] XU Hai-feng, LIU Jing-xuan, WANG Yi-cheng, ZUO Wei-fang, QU Chang-zhi, WANG De-yun, ZHANG Jing, JIANG Sheng-hui, WANG Nan, CHEN Xue-sen. Isolation and Expression Analysis of a Vacuolar Glucose Transporter Gene MdVGT1 in Apple [J]. Scientia Agricultura Sinica, 2016, 49(23): 4584-4592.
[13] ZHANG Xiao, HU Xiao-dan, ZHONG Jian-feng, WU Ai-hua, XU Chong-xin, LIU Yuan, ZHANG Cun-zheng, XIE Ya-jing, LIU Xian-jin. Expression and Molecular Simulation of Alkaline Phosphatase Receptor of Plutella xyllostella [J]. Scientia Agricultura Sinica, 2016, 49(23): 4555-4565.
[14] SHEN Yu-hua, XU Zhen-jun, TANG Li-hong, YANG Xiao-po, HUANG Wen-jie, WU Xiao-bin, ZHANG Wen-bo. Cloning and Function Analysis of the MsNAC2 Gene with NAC Transcription Factor from Alfalfa [J]. Scientia Agricultura Sinica, 2015, 48(15): 2925-2938.
[15] SUN Li-na, ZHANG Huai-jiang, YAN Wen-tao, MA Chun-sen, QIU Gui-sheng. Molecular Cloning and Expression Profiling of a Ryanodine Receptor Gene in the Peach Fruit Moth (Carposina sasakii) [J]. Scientia Agricultura Sinica, 2015, 48(10): 1971-1981.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd