Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (1): 56-64.doi: 10.3864/j.issn.0578-1752.2019.01.006

• PLANT PROTECTION • Previous Articles     Next Articles

RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm (Spodoptera exigua)

ZHAO Jing1,2(),TAO Rong1,HAO DeJun1(),XIAO LiuBin2(),TAN YongAn2   

  1. 1Co-Innovation Center for the Sustainable Forestry in Southern China/College of Forestry, Nanjing Forestry University, Nanjing 210037
    2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2018-08-13 Accepted:2018-10-02 Online:2019-01-01 Published:2019-01-12
  • Contact: DeJun HAO,LiuBin XIAO E-mail:jingzhao0126@126.com;dejunhao@163.com;xlbwll@sohu.com

RichHTML

13

PDF

379

Abstract:

【Objective】Vitellogenin receptor (VgR) is the main receptor that mediates the endocytosis of insect vitellin. The objective of this study is to clarify the function of VgR of beet armyworm (Spodoptera exigua) through RNA interference (RNAi) method, and to provide a basis for further understanding the molecular mechanism of reproductive physiology and developing effective new methods for prevention and control.【Method】The fragment of VgR was amplified from the cDNA of female adults abdomen tissues of S. exigua by PCR which included the ligand-binding domain region. The green fluorescent protein gene (GFP) fragment was amplified from the GFP plasmid stored in the laboratory by specific primers. The fragment of VgR and GFP was then inserted into the pMD-19T for sequencing. The nucleic acid sequence was analyzed by DNAMAN software. The correct plasmid confirmed by sequencing acted as the DNA template. PCR amplification was performed using primers with T7 promoter. VgR and GFP dsRNA were synthesized with T7 RiboMAX TM Express RNAi System synthesis kit. The abdomens of S. exigua female pupae on 2nd and 6th day were injected with 3 μL double RNA by 10 μL microsyringe (2 μg·μL -1). RT-qPCR was used to detect the changes of VgR expression in 0-, 24-, 48-hour-old female adults. Meanwhile, eclosion rate and eggs per female were evaluated in control groups (blank control, GFP-dsRNA injection) and treatment group (VgR-dsRNA injection). 【Result】 The VgR and GFP gene fragments obtained by amplification were 327 and 417 bp, respectively. The VgR expression level of 0-, 24-, 48-hour-old female adults in the VgR-dsRNA group decreased by 79.35%, 84.22% and 67.68% compared with the GFP-dsRNA group, respectively. Through anatomical observation of the ovary of 0-, 24-, 48-hour-old female adults, it was found that compared with the GFP-dsRNA group, the ovary development of the VgR-dsRNA group was significantly delayed. Compared with the GFP-dsRNA group, the length of the ovary tube in the VgR-dsRNA group decreased by 23.92% for the 24-hour-old female adults. The GFP-dsRNA group has more mature eggs in the ovary with larger average diameter of (0.46±0.05) mm while the number of mature eggs in the VgR-dsRNA group was small with an average diameter of (0.23±0.02) mm. There was no significant difference of eclosion rate between GFP-dsRNA group and VgR-dsRNA group. In the VgR-dsRNA group, the average number of eggs per female was 170, while in the control groups (blank group, GFP-dsRNA group), the average number of eggs per female was 451 and 420, respectively. There was a significant difference in the amount of oviposition between control groups and treatment group. 【Conclusion】 The function of VgR was studied by dsRNA injection in vitro, which could significantly reduce the expression of VgR. VgR plays an irreplaceable role in the reproduction of S. exigua, which directly affects the ovary development and spawning capacity, and can be used as a potential target for controlling S. exigua.

Key words: beet armyworm (Spodoptera exigua), vitellogenin receptor gene (VgR), RNA interference, ovary development

Table 1

Primers used in this study"

引物名称Primer name 引物序列 Primer sequence (5′ to 3′) 产物长度Product length (bp)
dsRNA VgR-F CACAATCAAGACCGATACCA 327
VgR-R GTCCAGTCACTCCAGAACACT
VgR-TF TAATACGACTCACTATAGGG CACAATCAAGACCGATACCA
VgR-TR TAATACGACTCACTATAGGG GTCCAGTCACTCCAGAACACT
GFP-F CACAAGTTCAGCGTGTCCG 417
GFP-R CACCTTGATGCCGTTC
GFP-TF TAATACGACTCACTATAGGG CACAAGTTCAGCGTGTCCG
GFP-TR TAATACGACTCACTATAGGG CACCTTGATGCCGTTC
RT-qPCR VgR-QF GAAGGGAGGGAAGTGTCCTGAG 104
VgR-QR TGATGGTGAAAGAAACGCTGTG
β-actin-F CCAGCCTTCCTTCTTGGGTAT 93
β-actin -R AGGTCCTTACGGATGTCAACG

Fig. 1

PCR amplification of VgR and GFP"

Fig. 2

Production of VgR transcripted in vitro"

Fig. 3

The relative expression level of VgR in S. exigua after dsRNA injection Different lowercases on the columns indicate significantly different (P<0.05). The same as Fig. 5"

Fig. 4

The ovary development of S. exigua after dsRNA injection"

Fig. 5

Eclosion rate and fecundity after VgR-dsRNA injection"

[1] 戈林泉, 吴进才 . 昆虫卵黄蛋白及其激素调控的研究进展. 昆虫知识, 2010,47(2):236-246.
GE L Q, WU J C . Research progress in insect vitellin and its hormone regulation. Chinese Bulletin of Entomology, 2010,47(2):236-246. (in Chinese)
[2] ENGELMANN F . Insect vitellogenin: Identification, biosynthesis and role in vitellogenesis//TREHERNE J E, BERRIDGE M J, WIGGLESWORTH V B. Advanced in Insect Physiology, 1979,14:49-108.
doi: 10.1016/S0065-2806(08)60051-X
[3] 王加伟, 彭露, 邹明民, 杨一帆, 汪蕾, 尤民生 . 昆虫卵黄原蛋白受体(VgRs)及其主要功能综述. 环境昆虫学报, 2016,38(4):831-842.
doi: 10.3969/j.issn.1674-0858.2016.04.25
WANG J W, PENG L, ZOU M M, YANG Y F, WANG L, YOU M S . A review of insect vitellogenin receptors (VgRs) and their fundamental functions. Journal of Environmental Entomology, 2016,38(4):831-842. (in Chinese)
doi: 10.3969/j.issn.1674-0858.2016.04.25
[4] TUFAIL M, TAKEDA M . Insect vitellogenin/lipophorin receptors: Molecular structures, role in oogenesis, and regulatory mechanisms. Journal of Insect Physiology, 2009,55(2):87-103.
doi: 10.1016/j.jinsphys.2009.01.009
[5] 戴瀚洋, 孙洋, 柏立新, 赵静, 肖留斌, 谭永安 . 亚致死浓度甲维盐胁迫对甜菜夜蛾幼虫解毒酶系活力及其相关基因表达量的影响. 棉花学报, 2015,27(2):149-158.
doi: 10.11963/issn.1002-7807.201502008
DAI H Y, SUN Y, BAI L X, ZHAO J, XIAO L B, TAN Y A . Activities of detoxification enzymes and expressions of related genes in Spodoptera exigua larvae treated with sublethal concentrations of emamectin benzoate. Cotton Science, 2015,27(2):149-158. (in Chinese)
doi: 10.11963/issn.1002-7807.201502008
[6] 司升云, 周利琳, 王少丽, 江幸福, 许再福, 慕卫, 王冬升, 王小平, 陈浩涛, 杨亦桦, 吉训聪 . 甜菜夜蛾防控技术研究与示范——公益性行业(农业)科研专项 “甜菜夜蛾防控技术研究与示范” 研究进展. 应用昆虫学报, 2012,49(6):1432-1438.
SI S Y, ZHOU L L, WANG S L, JIANG X F, XU Z F, MU W, WANG D S, WANG X P, CHEN H T, YANG Y H, JI X C . Progress in research on prevention and control of beet armyworm Spodoptera exigua in China. Chinese Journal of Applied Entomology, 2012,49(6):1432-1438. (in Chinese)
[7] 文礼章, 张友军 . 我国甜菜夜蛾大尺度暴发频度与广域温度和广域降雨量关系的预测模型. 昆虫学报, 2010,53(12):1367-1381.
WEN L Z, ZHANG Y J . Modelling of the relationship between the frequency of large-scale outbreak of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) and the wide-area temperature and rainfall trends in China. Acta Entomologica Sinica, 2010,53(12):1367-1381. (in Chinese)
[8] LIN Y, MENG Y, WANG Y X, LUO J, KATSUMA S, YANG C W, BANNO Y, KUSAKABE T, SHIMADA T, XIA Q Y . Vitellogenin receptor mutation leads to the oogenesis mutant phenotype “scanty vitellin” of the silkworm, Bombyx mori. Journal of Biological Chemistry, 2013,288(19):13345-13355.
doi: 10.1074/jbc.M113.462556 pmid: 3650373
[9] SHU Y H, WANG J W, LU K, ZHOU J L, ZHOU Q, ZHANG G R . The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis. Insect Molecular Biology, 2011,20(1):61-73.
doi: 10.1111/j.1365-2583.2010.01054.x pmid: 20955241
[10] CHO K H, RAIKHEL A S . Organization and developmental expression of the mosquito vitellogenin receptor gene. Insect Molecular Biology, 2001,10(5):465-474.
doi: 10.1046/j.0962-1075.2001.00285.x pmid: 11881811
[11] CHEN M E, LEWIS D K, KEELEY L L , PIETRANTONIO P V. cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Insect Molecular Biology, 2004,13(2):195-204.
doi: 10.1111/j.0962-1075.2004.00477.x pmid: 15056367
[12] GUIDUGLI-LAZZARINI K R, DO NASCIMENTO A M, TANAKA E D, PIULACHS M D, HARTFELDER K, BITONDI M G, SIMOES Z L . Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers. Journal of Insect Physiology, 2008,54(7):1138-1147.
doi: 10.1016/j.jinsphys.2008.04.021 pmid: 18606165
[13] LIU Q N, ZHU B J, LIU C L, WEI G Q, WANG Z G . Characterization of vitellogenin receptor (VgR) from the Chinese oak silkworm, Antheraea pernyi. Bulletin of Insectology, 2011,64(2):167-174.
[14] LIN W J, CHIEN C Y, TSAI C L, CHEN M E . A nonovary-specific vitellogenin receptor from the oriental fruit fly, Bactrocera dorsalis (Hendel). Archives of Insect Biochemistry and Physiology, 2015,90(4):169-180.
doi: 10.1002/arch.21252 pmid: 26280361
[15] TUFAIL M, TAKEDA M . Molecular cloning, characterization and regulation of the cockroach vitellogenin receptor during oogenesis. Insect Molecular Biology, 2005,14(4):389-401.
doi: 10.1111/j.1365-2583.2005.00570.x pmid: 16033432
[16] ZHANG W N, MA L, XIAO H J, XIE B T, SMAGGHE G, GUO Y Y, LIANG G M . Molecular characterization and function analysis of the vitellogenin receptor from the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae). PLoS ONE, 2016,11(5):e0155785.
doi: 10.1371/journal.pone.0155785 pmid: 27192057
[17] ZHAO J, SUN Y, XIAO L B, TAN Y A, JIANG Y P, BAI L X . Vitellogenin and vitellogenin receptor gene expression profiles in Spodoptera exigua are related to host plant suitability. Pest Management Science, 2018,74(4):950-958.
doi: 10.1002/ps.4794
[18] ROY-ZOKAN E M, CUNNINGHAM C B, HEBB L E, MCKINNEY E C, MOORE A J . Vitellogenin and vitellogenin receptor gene expression is associated with male and female parenting in a subsocial insect. Proceedings of the Royal Society B Biological Sciences, 2015,282(1809):20150787.
doi: 10.1098/rspb.2015.0787 pmid: 4590458
[19] TUFAIL M, TAKEDA M . Molecular cloning and developmental expression pattern of the vitellogenin receptor from the cockroach, Leucophaea maderae. Insect Biochemistry and Molecular Biology, 2007,37(3):235-245.
doi: 10.1016/j.ibmb.2006.11.007 pmid: 17296498
[20] 杨中侠, 文礼章, 吴青君, 王少丽, 徐宝云, 张友军 . RNAi技术在昆虫功能基因研究中的应用进展. 昆虫学报, 2008,51(10):1077-1082.
doi: 10.3321/j.issn:0454-6296.2008.10.012
YANG Z X, WEN L Z, WU Q J, WANG S L, XU B Y, ZHANG Y J . Application of RNA interference in studying gene functions in insects. Acta Entomologica Sinica, 2008,51(10):1077-1082. (in Chinese)
doi: 10.3321/j.issn:0454-6296.2008.10.012
[21] 周耀振, 修伟明, 董双林 . 应用RNA干扰技术对甜菜夜蛾信息素结合蛋白功能的研究. 南京农业大学学报, 2009,32(3):58-62.
doi: 10.3321/j.issn:1000-2030.2009.03.011
ZHOU Y Z, XIU W M, DONG S L . Functional study of pheromone binding protein by using RNAi in male beet armyworm, Spodoptera exigua Hiibner. Journal of Nanjing Agricultural University, 2009,32(3):58-62. (in Chinese)
doi: 10.3321/j.issn:1000-2030.2009.03.011
[22] TURNER C T, DAVY M W, MACDIARMID R M, PLUMMER K M, BIRCH N P, NEWCOMB R D . RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. Insect Molecular Biology, 2006,15(3):383-391.
doi: 10.1111/j.1365-2583.2006.00656.x pmid: 16756557
[23] CIUDAD L, PIULACHS M D, BELLES X . Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant. The FEBS Journal, 2006,273(2):325-335.
doi: 10.1111/j.1742-4658.2005.05066.x pmid: 16403020
[24] ARAUJO R N, SANTOS A, PINTO F S, GONTIJO N F, LEHANE M J, PEREIRA M H . RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochemistry and Molecular Biology, 2006,36(9):683-693.
doi: 10.1016/j.ibmb.2006.05.012 pmid: 16935217
[25] BAUM J A, BOGAERT T, CLINTON W, HECK G R, FELDMANN P, ILAGAN O, JOHNSON S, PLAETINCK G, MUNYIKWA T, PLEAU M, VAUGHN T, ROBERTS J . Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 2007,25(11):1322-1326.
doi: 10.1038/nbt1359
[26] LU K, SHU Y H, ZHOU J L, ZHANG X Y, ZHANG X Y, CHEN M X, YAO Q, ZHOU Q, ZHANG W Q . Molecular characterization and RNA interference analysis of vitellogenin receptor from Nilaparvata lugens (Stål). Journal of Insect Physiology, 2015,73:20-29.
doi: 10.1016/j.jinsphys.2015.01.007 pmid: 25617689
[27] PITINO M, COLEMAN A D, MAFFEI M E, RIDOUT C J, HOGENHOUT S A . Silencing of aphid genes by dsRNA feeding from plants. PLoS ONE, 2011,6(10):e25709.
doi: 10.1371/journal.pone.0025709 pmid: 3187792
[28] KENNERDELL J R, CARTHEW R W . Heritable gene silencing in Drosophila using double-stranded RNA. Nature Biotechnology, 2000,18(8):896-898.
doi: 10.1038/78531 pmid: 10932163
[29] LEVIN D M, BREUER L N, ZHUANG S, ANDERSON S A, NARDI J B, KANOST M R . A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 2005,35(5):369-380.
doi: 10.1016/j.ibmb.2005.01.003 pmid: 15804572
[30] SIVAKUMAR S, RAJAGOPAL R, VENKATESH G R, SRIVASTAVA A, BHATNAGAR R K . Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac. Journal of Biological Chemistry, 2007,282(10):7312-7319.
doi: 10.1074/jbc.M607442200
[31] SHANG F, NIU J Z, DING B Y, ZHANG Q, YE C, ZHANG W, SMAGGHE G, WANG J J . Vitellogenin and its receptor play essential roles in the development and reproduction of the brown citrus aphid, Aphis (Toxoptera) citricidus. Insect Molecular Biology, 2018,27(2):221-233.
doi: 10.1111/imb.12366 pmid: 29226991
[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] ZHANG DaoWei,KANG Kui,YU YaYa,KUANG FuPing,PAN BiYing,CHEN Jing,TANG Bin. Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera [J]. Scientia Agricultura Sinica, 2020, 53(15): 3108-3119.
[8] 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.
[9] 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.
[10] 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.
[11] 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.
[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] 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.
[14] Yue CHEN,TianXingZi WANG,Shuo YANG,Tong ZHANG,JinJiao MA,GaoWei YAN,YuQing LIU,Yan ZHOU,JiaNan SHI,JinPing LAN,Jian WEI,ShiJuan DOU,LiJuan LIU,Ming YANG,LiYun LI,GuoZhen LIU. Expression Profiling and Functional Characterization of Rice Transcription Factor OsWRKY68 [J]. Scientia Agricultura Sinica, 2019, 52(12): 2021-2032.
[15] HE JingLan,ZHANG Ming,LIU RuiYing,WAN GuiJun,PAN WeiDong,CHEN FaJun. Effects of the Interference of Key Magnetic Response Genes on the Longevity of Brown Planthopper (Nilaparvata lugens) Under Near-Zero Magnetic Field [J]. Scientia Agricultura Sinica, 2019, 52(1): 45-55.
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