Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (15): 3108-3119.doi: 10.3864/j.issn.0578-1752.2020.15.011

• PLANT PROTECTION • Previous Articles     Next Articles

Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera

ZHANG DaoWei1(),KANG Kui1,YU YaYa2,KUANG FuPing1,PAN BiYing3,CHEN Jing2,TANG Bin1,3()   

  1. 1College of Biology and Agriculture, Zunyi Normal University/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, Zunyi 563006, Guizhou
    2School of Basic Medical Science, Zunyi Medical University, Zunyi 563006, Guizhou
    3College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
  • Received:2020-01-16 Accepted:2020-03-13 Online:2020-08-01 Published:2020-08-06
  • Contact: Bin TANG E-mail:zhangdw1000@163.com;tbzm611@163.com

RichHTML

9

PDF

170

Abstract:

【Objective】Phenoloxidase (PO) is an immune protein and plays an important regulatory role in insects. It exists as the form of inactive prophenoloxidase (PPO). In this study, based on the three PPO sequences of Sogatella furcifera by transcriptome sequencing, the biological functions of SfPPOs were further explored by analyzing the developmental and tissue expression patterns of different SfPPOs, suppressing the expression of SfPPOs and inducing by pathogenic bacteria.【Method】Taking three SfPPO sequences as research objects, the protein structure and homology with other insects were analyzed by bioinformatics method. In addition, dsRNA injected with green fluorescent protein (GFP) was used as a control group, and the inhibitory effect of target gene was detected by quantitative real-time PCR (qRT-PCR) after injecting with dsSfPPO. In order to investigate the role of SfPPO in the growth and immune response of S. furcifera, qRT-PCR was used to detect the expression of SfPPO at different developmental stages and in different tissues, as well as the induced expression of three SfPPOs after injecting with Escherichia coli and Bacillus subtilis.【Result】The open reading frames (ORF) of SfPPO1, SfPPO2-1, and SfPPO2-2 are 2 079, 999, and 2 070 bp in length, respectively, which encoding 692, 332, and 689 amino acids, respectively. The predicted protein molecular weights are 79.84, 37.67, and 79.53 kD, and the isoelectric points are 6.43, 9.56, and 6.20, respectively. Bioinformatics analysis showed that the three PPO proteins of S. furcifera had high homology and were closely related to Nilaparvata lugens. SfPPO1, SfPPO2-1 and SfPPO2-2 were all highly expressed on the 3rd day of adult, and the developmental expression patterns of SfPPO2-1 and SfPPO2-2 were similar. The tissue expression results showed that SfPPO1 and SfPPO2-1 were highly expressed in the epidermis and fat body, while SfPPO2-2 was highly expressed in the wing and fat body, and the expressions of all the three genes were relatively low in head, foot, midgut and Malpighian tubule. Compared with the dsGFP group, injecting with the dsRNA of target gene could significantly silence the expression of target gene, and when the SfPPO2-1 expression was silenced, SfPPO1 appeared to be significantly overexpressed, indicating that there might be compensatory function among different PPOs. The expression levels of SfPPO2-1 and SfPPO2-2 were significantly increased after 12 h of induction with E. coli, while the expression of SfPPO1 was significantly increased after 24 h of induction. In addition, the expression levels of SfPPO1, SfPPO2-1 and SfPPO2-2 were all significantly increased after 24 h of induction with B. subtilis.【Conclusion】The expression of SfPPO1, SfPPO2-1 and SfPPO2-2 has tissue and development specificity, and there are differences in immune response under the induction of different pathogenic bacteria. The results are helpful to explore the potential molecular mechanism of phenoloxidase in insect development and immunity.

Key words: Sogatella furcifera, prophenoloxidase (PPO), RNA interference (RNAi), quantitative real-time PCR (qRT-PCR), inducible expression, immune response

Table 1

Primers used for qRT-PCR and primers of dsPPOs and dsGFP"

引物名称
Primer name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)		 产物长度
Length (bp)
qSf18s CGCTACTACCGATTGAA GGAAACCTTGTTACGACTT 165
qSfPPO1 GACTCTGCCTCCCTACACC AGCGAGCGAACACGTTGC 176
dsSfPPO1 TGGGCATCAACCTACATCA TGCTAACAGCCTCGTACAAAC 360
dsSfPPO1-T7 T7-TGGGCATCAACCTACATCA T7-TGCTAACAGCCTCGTACAAAC 410
qSfPPO2-1 AGAACTCACCTCGCAACCA CTGAAGATGCTGCACCCTAG 116
dsSfPPO2-1 AGCATCTTCAGCATCAGCCTTTC T7-GGCATTCCCTCGGCGTTT 361
dsSfPPO2-1-T7 T7-AGCATCTTCAGCATCAGCCTTTC GGCATTCCCTCGGCGTTT 411
qSfPPO2-2 TGCTCGCATCACTCATCT ACAAATCTGTCCACCTCAA 177
dsSfPPO2-2 GGCATCAACCTACACCAT CCCTGCGTATTTATCACC 383
dsSfPPO2-2-T7 T7-GGCATCAACCTACACCAT T7-CCCTGCGTATTTATCACC 433
dsSfGFP AAGGGCGAGGAGCTGTTCACCG CAGCAGGACCATGTGATCGCGC
dsSfGFP-T7 T7-AAGGGCGAGGAGCTGTTCACCG T7-CAGCAGGACCATGTGATCGCGC

Fig. 1

Nucleotide and amino acid sequences of SfPPO Initiation and termination codons are indicated in bold and underlined. The nucleotide sequences reported in this study have been submitted to GenBank (Accession numbers: MN895440, MN895441, and MN895442)。A: SfPPO1; B: SfPPO2-1; C: SfPPO2-2"

Fig. 2

Phylogenetic tree of SfPPOs and PPO proteins from other insect species based on the amino acid sequence (neighbor-joining method)"

Fig. 3

Relative expression of SfPPOs at different developmental stages of S. furcifera Different lowercases on the bars indicate significant differences (P<0.05)。 The same as Fig. 4"

Fig. 4

Relative expression of SfPPOs in different tissues of S. furcifera"

Fig. 5

Relative expression of SfPPOs after injection for 48 h and 72 h"

Fig. 6

Response of SfPPOs after bacteria induction"

[1] 徐安隆, 陈曙, 贺文爱, 张木清, 邱永福. 水稻抗褐飞虱与白背飞虱研究进展. 南方农业学报, 2015,46(9):1628-1635.
XU A L, CHEN S, HE W A, ZHANG M Q, QIU Y F. Progress on rice resistance to brown planthopper and white-backed planthopper. Journal of Southern Agriculture, 2015,46(9):1628-1635. (in Chinese)
[2] KANOST M R, JIANG H, YU X Q. Innate immune responses of a lepidopteran insect,Manduca sexta. Immunological Reviews, 2004,198(1):97-105.
[3] GILLESPIE J P, KANOST M R, TRENCZEK T. Biological mediators of insect immunity. Annual Review of Entomology, 1997,42:611-643.
doi: 10.1146/annurev.ento.42.1.611 pmid: 9017902
[4] STRAND M R. The insect cellular immune response. Insect Science, 2008,15(1):1-14.
[5] ASHIDA M, BREY P. Recent advances in research on insect prophenoloxidase cascade//BREY P, HULTMARK D. Molecular Mechanisms of Immune Responses in Insects. London: Chapman and Hall, 1997: 135-172.
[6] CERENIUS L, LEE B L, SÖDERHÄLL K. The proPO-system: pros and cons for its role in invertebrate immunity. Trends in Immunology, 2008,29(6):263-271.
doi: 10.1016/j.it.2008.02.009 pmid: 18457993
[7] KANOST M R, GORMAN M J. Phenoloxidases in insect immunity//BECKAGE N E. Insect Immunology. Elsevier Inc., 2008: 69-96.
[8] LEMAITRE B, HOFFMANN J. The host defense of Drosophila melanogaster. Annual Review of Immunology, 2007,25:697-743.
pmid: 17201680
[9] LAI S C, CHEN C C, HOU R F. Immunolocalization of prophenoloxidase in the process of wound healing in the mosquito Armigeres subalbatus (Diptera: Culicidae). Journal of Medical Entomology, 2002,39(2):266-274.
pmid: 11931025
[10] NAM H J, JANG I H, YOU H, LEE K A, LEE W J. Genetic evidence of a redox-dependent systemic wound response via Hayan protease-phenoloxidase system in Drosophila. The EMBO Journal, 2012,31(5):1253-1265.
pmid: 22227521
[11] 陈晓宇, 时敏, 陈学新. 昆虫酚氧化酶原激活酶研究. 应用昆虫学报, 2015,52(2):281-288.
CHEN X Y, SHI M, CHEN X X. The prophenoloxidase activating proteinase in insects. Chinese Journal of Applied Entomology, 2015,52(2):281-288. (in Chinese)
[12] 吕鸿声. 昆虫免疫学原理. 上海: 上海科学技术出版社, 2008.
LÜ H S. Principles of Insect Immunology. Shanghai: Shanghai Scientific and Technical Publishers, 2008. (in Chinese)
[13] 张道伟, 陈静. 酚氧化酶参与德国小蠊对大肠杆菌的免疫应答. 昆虫学报, 2014,57(10):1123-1132.
ZHANG D W, CHEN J. Phenoloxidase is involved in regulating immune response to Escherichia coli in Blattella germanica (Blattodea: Blattellidae). Acta Entomologica Sinica, 2014,57(10):1123-1132. (in Chinese)
[14] 陈静, 张道伟, 钱正敏. 褐飞虱两个酚氧化酶原基因的特性及细菌诱导后的表达分析. 中国生物防治学报, 2018,34(1):44-51.
CHEN J, ZHANG D W, QIAN Z M. Characteristics and bacterial-induced expression analysis of two prophenoloxidase genes in the brown planthopper, Nilaparvata lugens (Hemiptera Delphacidae). Chinese Journal of Biological Control, 2018,34(1):44-51. (in Chinese)
[15] MAQBOOL M, MOBASHIR M, HODA N. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease. European Journal of Medicinal Chemistry, 2016,107:63-81.
pmid: 26562543
[16] ZHAO L N, YANG M M, SHEN Q D, LIU X J, SHI Z K, WANG S G, TANG B. Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference. Scientific Reports, 2016,6:27841.
pmid: 27328657
[17] CHEN Q W, JIN S, ZHANG L, SHEN Q D, WEI P, WEI C M, WANG S G, TANG B. Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum (Coleoptera: Tenebrionidae) revealed by RNA interference. Bulletin of Entomological Research, 2018,108(3):388-399.
doi: 10.1017/S000748531700089X pmid: 28920565
[18] LU A, ZHANG Q, ZHANG J, YANG B, WU K, XIE W, LUAN Y X, LING E. Insect prophenoloxidase: The view beyond immunity. Frontiers in Physiology, 2014,5:252.
doi: 10.3389/fphys.2014.00252 pmid: 25071597
[19] WERTHEIM B, KRAAIJEVELD A R, SCHUSTER E, BLANC E, HOPKINS M, PLETCHER S D, STRAND M R, PARTRIDGE L, GODFRAY H C. Genome-wide gene expression in response to parasitoid attack in Drosophila. Genome Biology, 2005,6(11):R94.
pmid: 16277749
[20] HERNÁNDEZ-LÓPEZ J, GOLLAS-GALVÁN T, GÓMEZ-JIMÉNEZ S, PORTILLO-CLARK G, VARGAS-ALBORES F. In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes. Fish and Shellfish Immunology, 2003,14(2):105-114.
pmid: 12526875
[21] 张小玉, 汪贞, 黄泽夫, 王国秀, 刘绪生. 棉铃虫不同虫态及虫龄血淋巴中酚氧化酶活力的比较. 应用昆虫学报, 2006,43(3):330-332.
ZHANG X Y, WANG Z, HUANG Z F, WANG G X, LIU X S. Dynamics of phenoloxidase activity in haemolymph of Helicoverpa armigera at different stages and instars. Chinese Journal of Applied Entomology, 2006,43(3):330-332. (in Chinese)
[22] 冯从经, 杜予州, 陆自强, 符文俊. 亚洲玉米螟幼虫血清中酚氧化酶原的性质. 昆虫学报, 2005,48(5):649-654.
FENG C J, DU Y Z, LU Z Q, FU W J. Characterization of prophenoloxidase from the hemolymph of Ostrinia furnacalis (Guenée). Acta Entomologica Sinica, 2005,48(5):649-654. (in Chinese)
[23] 张永亮, 朱勇. 家蚕和野桑蚕酚氧化酶原基因的组织表达差异比较. 蚕业科学, 2009,35(3):630-633.
ZHANG Y L, ZHU Y. A comparison on tissue-specific expressions of prophenoloxidase gene in Bombyx mori and Bombyx mandarina. Science of Sericulture, 2009,35(3):630-633. (in Chinese)
[24] LOU Y H, PAN P L, YE Y X, CHENG C, XU H J, ZHANG C X. Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper. Insect Molecular Biology, 2018,27(3):393-403.
pmid: 29465791
[25] 韩艳华, 刘梦姚, 张洪志, 王曼姿, 高飞, 王孟卿, 刘晨曦, 陈红印, 张礼生. RNA干扰技术在昆虫滞育机制研究中的应用. 环境昆虫学报, 2018,40(6):1248-1254.
HAN Y H, LIU M Y, ZHANG H Z, WANG M Z, GAO F, WANG M Q, LIU C X, CHEN H Y, ZHANG L S. Application of RNA interference on researches of diapause mechanism in insects. Journal of Environmental Entomology, 2018,40(6):1248-1254. (in Chinese)
[26] XI Y, PAN P L, ZHANG C X. The β-N-acetylhexosaminidase gene family in the brown planthopper, Nilaparvata lugens. Insect Molecular Biology, 2015,24(6):601-610.
pmid: 26304035
[27] FIRE A, XU S, MONTGOMERY M K, KOSTAS S A, DRIVER S E, MELLO C C. Potent and specific genetic interference by double- stranded RNA in Caenorhabditis elegans. Nature, 1998,391(6669):806-811.
doi: 10.1038/35888 pmid: 9486653
[28] ZHU Q S, ARAKANE Y, BEEMAN R W, KRAMER K J, MUTHUKRISHNAN S. Functional specialization among insect chitinase family genes revealed by RNA interference. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(18):6650-6655.
pmid: 18436642
[29] FU K Y, LI Q, ZHOU L T, MENG Q W, LÜ F G, GUO W C, LI G Q. Knockdown of juvenile hormone acid methyl transferase severely affects the performance of Leptinotarsa decemlineata (Say) larvae and adults. Pest Management Science, 2016,72(6):1231-1241.
doi: 10.1002/ps.4103 pmid: 26299648
[30] CHEN J, TANG B, CHEN H, YAO Q, HUANG X F, CHEN J, ZHANG D W, ZHANG W Q. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference. PLoS ONE, 2010,5(4):e10133.
doi: 10.1371/journal.pone.0010133 pmid: 20405036
[31] 徐亚玲, 李文楚. 昆虫酚氧化酶作用机制的研究进展. 安徽农业科学, 2010,38(27):14844-14846.
XU Y L, LI W C. Research progress in activation mechanisms of phenoloxidse in insects. Journal of Anhui Agricultural Sciences, 2010,38(27):14844-14846. (in Chinese)
[32] 刘守柱, 薛超彬, 罗万春. 黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化. 昆虫学报, 2009,52(9):941-945.
LIU S Z, XUE C B, LUO W C. Phenoloxidase activity during cuticle sclerotization in larvae of Tenebrio molitor (Coleoptera: Tenebrionidae). Acta Entomologica Sinica, 2009,52(9):941-945. (in Chinese)
[33] 王荫长. 昆虫生物化学. 北京: 中国农业出版社, 2001: 245-270.
WANG Y C. Insect Biochemistry Beijing: China Agriculture Press, 2001: 245-270. (in Chinese)
[34] LI Y, WANG Y, JIANG H, DENG J. Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes. Proceedings of the National Academy of Sciences of the United States of America, 2009,106(40):17002-17006.
doi: 10.1073/pnas.0906095106 pmid: 19805072
[35] 李国荣, 张士璀, 李红岩, 王昌留. 酚氧化酶研究概况Ⅰ: 特性、功能、分布和在胚胎发育中的变化. 海洋科学, 2003,27(4):4-8.
LI G R, ZHANG S C, LI H Y, WANG C L. Advance on the study of phenoloxidase Ⅰ: Properties, functions, distribution and change during embryonic stage. Marine Sciences, 2003,27(4):4-8. (in Chinese)
[36] 杨伟克, 刘增虎, 钟健, 董占鹏. 家蚕眠期血淋巴酚氧化酶活性变化及其基因表达分析. 南方农业学报, 2019,50(2):391-396.
YANG W K, LIU Z H, ZHONG J, DONG Z P. Variation of activity of phenoloxidase and its gene expression in hemolymph of Bombyx mori larvae during molting stage. Journal of Southern Agriculture, 2019,50(2):391-396. (in Chinese)
[1] GUAN RuoBing,LI HaiChao,MIAO XueXia. Commercialization Status and Existing Problems of RNA Biopesticides [J]. Scientia Agricultura Sinica, 2022, 55(15): 2949-2960.
[2] YIN Fei,LI ZhenYu,SAMINA Shabbir,LIN QingSheng. Expression and Function Analysis of Cytochrome P450 Genes in Plutella xylostella with Different Chlorantraniliprole Resistance [J]. Scientia Agricultura Sinica, 2022, 55(13): 2562-2571.
[3] WU Wei,XU HuiLi,WANG ZhengLiang,YU XiaoPing. Cloning and Function Analysis of a Serine Protease Inhibitor Gene Nlserpin2 in Nilaparvata lugens [J]. Scientia Agricultura Sinica, 2022, 55(12): 2338-2346.
[4] CHEN ErHu,MENG HongJie,CHEN Yan,TANG PeiAn. Cuticle Protein Genes TcCP14.6 and TcLCPA3A are Involved in Phosphine Resistance of Tribolium castaneum [J]. Scientia Agricultura Sinica, 2022, 55(11): 2150-2160.
[5] Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids [J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.
[6] GE XinZhu,SHI YuXing,WANG ShaSha,LIU ZhiHui,CAI WenJie,ZHOU Min,WANG ShiGui,TANG Bin. Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Its Regulation of Trehalose Metabolism [J]. Scientia Agricultura Sinica, 2021, 54(23): 5021-5031.
[7] LIU XiaoJian,GUO Jun,ZHANG XueYao,MA EnBo,ZHANG JianZhen. Molecular Characteristics and Function Analysis of Nuclear Receptor Gene LmE75 in Locusta migratoria [J]. Scientia Agricultura Sinica, 2020, 53(11): 2219-2231.
[8] YAO LiXiao,FAN HaiFang,ZHANG QingWen,HE YongRui,XU LanZhen,LEI TianGang,PENG AiHong,LI Qiang,ZOU XiuPing,CHEN ShanChun. Function of Citrus Bacterial Canker Resistance-Related Transcription Factor CitMYB20 [J]. Scientia Agricultura Sinica, 2020, 53(10): 1997-2008.
[9] MA Wen,LIU Jiao,ZHANG XueYao,SHEN GuoHua,QIN XueMei,ZHANG JianQin. Enzymatic Characteristics and Metabolic Analysis to Malathion and p,p’-DDT of LmGSTS2 from Locusta migratoria [J]. Scientia Agricultura Sinica, 2019, 52(8): 1389-1399.
[10] DING YanJuan,LIU YongKang,LUO YuJia,DENG YingMei,XU HongXing,TANG Bin,XU CaiDi. Potential Functions of Nilaparvata lugens GSK-3 in Regulating Glycogen and Trehalose Metabolism [J]. Scientia Agricultura Sinica, 2019, 52(7): 1237-1246.
[11] TANG Bin,SHEN QiDa,ZENG BoPing,XIAO ZhongJiu,QIU LingYu,PAN BiYing,LI Kun,ZHANG DaoWei. Characteristics, Developmental Expression and RNAi Effect Analysis of a Novel Trehalose-6-Phosphate Synthase Gene in Nilaparvata lugens [J]. Scientia Agricultura Sinica, 2019, 52(3): 466-477.
[12] JunBo PENG,XingHong LI,Wei ZHANG,Ying ZHOU,JinBao HUANG,JiYe YAN. Pathogenicity and Gene Expression Pattern of the Exocrine Protein LtGH61A of Grape Canker Fungus [J]. Scientia Agricultura Sinica, 2019, 52(24): 4518-4526.
[13] LIU FanQi,WAN GuiJun,ZENG LuYing,LI ChunXu,PAN WeiDong,CHEN FaJun. Selection of Stable Internal Reference Genes for Transcript Expression Analyses in Laodelphax striatellus Under Near-Zero Magnetic Field [J]. Scientia Agricultura Sinica, 2019, 52(19): 3346-3356.
[14] ZHANG DaoWei,YU YaYa,PAN BiYing,KANG Kui,ZENG BoPing,CHEN Jing,TANG Bin. Regulation Function of Trehalose-6-phosphate Synthase Genes on Chitin Synthesis in Sogatella furcifera [J]. Scientia Agricultura Sinica, 2019, 52(19): 3357-3366.
[15] FU ZhongMin,CHEN HuaZhi,LIU SiYa,ZHU ZhiWei,FAN XiaoXue,FAN YuanChan,WAN JieQi,ZHANG Lu,XIONG CuiLing,XU GuoJun,CHEN DaFu,GUO Rui. Immune Responses of Apis mellifera ligustia to Nosema ceranae Stress [J]. Scientia Agricultura Sinica, 2019, 52(17): 3069-3082.
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