甜菜夜蛾卵黄原蛋白受体基因的RNA干扰

doi:10.3864/j.issn.0578-1752.2019.01.006

摘要/Abstract

摘要：

【目的】卵黄原蛋白受体（vitellogenin receptor,VgR）是介导昆虫卵黄原蛋白胞吞作用的主要受体,通过RNA干扰（RNAi）方法研究甜菜夜蛾（Spodoptera exigua）VgR的功能,为深入了解甜菜夜蛾生殖生理的分子机制及开发有效防治新方法提供依据。【方法】以甜菜夜蛾雌成虫腹部的cDNA为模板,通过PCR克隆得到甜菜夜蛾VgR基因片段,其包含配体结合域功能区。绿色荧光蛋白基因（GFP）片段通过特异性引物从笔者实验室保存的GFP质粒上扩增。将VgR和GFP 目的片段连入pMD-19T载体后进行测序,利用DNAMAN软件分析基因序列的准确性。以测序验证正确的质粒作为DNA模板,利用带T7启动子的引物进行PCR扩增。用T7 RiboMAX ^TM Express RNAi System合成试剂盒合成VgR-dsRNA和GFP-dsRNA。应用10 μL微量进样器在化蛹第2、6天的甜菜夜蛾雌蛹腹部注射3 μL双链RNA（2 μg·μL ^-1）。利用RT-qPCR技术检测甜菜夜蛾刚羽化、羽化24 h、羽化48 h雌成虫的VgR表达量变化,同时统计对照组（空白对照、注射GFP-dsRNA）和处理组（注射VgR-dsRNA）甜菜夜蛾的羽化率及单雌产卵量。【结果】扩增得到VgR和GFP基因片段,大小分别为327和417 bp。RT-qPCR 检测结果表明,与对照组相比,注射dsRNA后甜菜夜蛾的VgR表达水平显著下降。对于刚羽化、羽化24 h、羽化48 h的雌成虫,注射VgR-dsRNA处理组的VgR表达量相比注射GFP-dsRNA的对照组分别下降了79.35%、84.22%、67.68%。通过解剖观察刚羽化、羽化24 h、羽化48 h的甜菜夜蛾卵巢,发现注射VgR-dsRNA处理组与注射GFP-dsRNA对照组相比, 卵巢发育进度显著推迟。对于羽化24 h的甜菜夜蛾,与注射GFP-dsRNA组相比,注射VgR-dsRNA处理组卵巢管长度下降了23.92％;注射GFP-dsRNA组的卵巢成熟卵粒较多,平均直径为（0.46±0.05）mm,而注射VgR-dsRNA处理组成熟卵粒数量较少且相对较小,平均直径为（0.23±0.02）mm。注射GFP-dsRNA组和VgR-dsRNA组的羽化率无显著差异。注射VgR-dsRNA处理组的单雌平均产卵量只有170粒,而对照组（空白对照和注射GFP-dsRNA）单雌平均产卵量可达到451粒和420粒,处理组与对照组的产卵量存在显著差异。【结论】通过体外注射dsRNA的方法研究VgR的功能,能够显著降低VgR表达。VgR在甜菜夜蛾的生殖中起着不可替代的作用,直接影响甜菜夜蛾卵巢的发育与产卵量,可以作为控制甜菜夜蛾的潜在靶标。

关键词: 甜菜夜蛾, 卵黄原蛋白受体基因, RNA干扰, 卵巢发育

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

赵静, 陶蓉, 郝德君, 肖留斌, 谭永安. 甜菜夜蛾卵黄原蛋白受体基因的RNA干扰[J]. 中国农业科学, 2019, 52(1): 56-64.

ZHAO Jing, TAO Rong, HAO DeJun, XIAO LiuBin, TAN YongAn. RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm (Spodoptera exigua)[J]. Scientia Agricultura Sinica, 2019, 52(1): 56-64.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.01.006

https://www.chinaagrisci.com/CN/Y2019/V52/I1/56

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 31

[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

	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