甜菜夜蛾卵黄原蛋白受体基因的RNA干扰

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赵静^,¹^,², 陶蓉¹, 郝德君^,¹, 肖留斌^,², 谭永安²

1南京林业大学南方现代林业协同创新中心/林学院,南京210037

2江苏省农业科学院植物保护研究所,南京 210014

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

ZHAO Jing^,¹^,², TAO Rong¹, HAO DeJun^,¹, XIAO LiuBin^,², TAN YongAn²

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

通讯作者: 郝德君，E-mail：dejunhao@163.com。肖留斌，E-mail：xlbwll@sohu.com

第一联系人: 赵静,E-mail： jingzhao0126@126.com。
收稿日期:2018-08-13接受日期:2018-10-2网络出版日期:2019-01-01

基金资助:

国家重点研发计划.6111661
国家现代农业产业技术体系.CARS-18-16
江苏省农业科学院院基金计划.6111612
江苏省农业科学院院基金计划.6111613

Received:2018-08-13Accepted:2018-10-2Online:2019-01-01

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.

Keywords：beet armyworm (Spodoptera exigua);vitellogenin receptor gene (VgR);RNA interference;ovary development

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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2019.01.006
ZHAO Jing, TAO Rong, HAO DeJun, XIAO LiuBin, TAN YongAn. RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm (Spodoptera exigua)[J]. Scientia Acricultura Sinica, 2019, 52(1): 56-64 doi:10.3864/j.issn.0578-1752.2019.01.006

0 引言

【研究意义】卵黄原蛋白（vitellogenin,Vg）的合成与摄取是昆虫卵黄形成的关键因素^[1,2],其含量高低直接影响雌成虫产卵潜力。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,对昆虫卵巢的成熟起着至关重要的作用,是研究控制害虫的潜在靶标^[3,4]。甜菜夜蛾（Spodoptera exigua）作为一种世界性分布的多食性重要害虫,由于其繁殖能力强及较高的抗药性,亟待开拓减少化学农药使用的新型无公害防控手段^[5,6,7]。因此,开展VgR的研究,对于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具有重要意义。【前人研究进展】目前,关于昆虫VgR的研究非常活跃,32种昆虫的VgR序列已被克隆和测序,其中大部分是鳞翅目、双翅目和膜翅目昆虫,包括家蚕（Bombyx mori）、斜纹夜蛾（Spodoptera litura）、埃及伊蚊（Aedes aegypti）、红火蚁（Solenopsis invicta）等^{[8,9,10,11,12,13,14,15,16,17,18,19]}。昆虫VgR在体内合成具有发育时期和组织的特异性。大多数昆虫的VgR属于卵巢特异性表达蛋白,例如橘小实蝇（Bactrocera dorsalis）^[14]、美洲大蠊（Periplaneta americana）^[15]、棉铃虫（Helicoverpa armigera）^[16]等昆虫的VgR仅在卵巢中表达。对于一些社会性昆虫如西方蜜蜂（Apis mellifera）^[12]和红斑尼葬甲（Nicrophorus vespilloides）^[18]的研究发现,VgR在头部、中肠等其他组织也有表达。不同昆虫VgR的表达启动期也不同,大多数昆虫VgR的表达发生在蛹末期或初羽化成虫之后,例如斜纹夜蛾^[9]、甜菜夜蛾^[17]、马德拉蜚蠊（Leucophaea maderae）^[19]等,但在家蚕中,VgR在雌性家蚕的整个发育周期均有表达,幼虫期表达水平较低,随后逐渐升高,在成虫期达到最大值^[8]。 RNA干扰（RNA interference,RNAi）是由dsRNA或siRNA通过序列配对的方式特异性地与靶标基因mRNA序列相结合,形成沉默复合体,降解靶标基因的mRNA^[20]。目前,RNAi技术已广泛地应用于鳞翅目、半翅目、膜翅目、鞘翅目等多种昆虫的基因功能研究^{[21,22,23,24,25,26]}。通过对昆虫生长发育及繁殖关键基因的鉴定和功能分析,可为开发植物或微生物介导的RNAi 技术的防治新途径提供理论依据及新的潜在靶标基因资源。PITINO等^[27]将桃蚜（Myzus persicae）的两个与生长发育相关的基因（MpC002和Rack-1）分别导入烟草和拟南芥中,用转基因植物饲喂桃蚜,导致桃蚜体内的MPC002或Rack-1的表达量降低了60%,后代数量减少。然而,目前关于昆虫VgR的功能研究仅仅局限于少数昆虫,主要关注其对昆虫卵黄形成、卵巢发育以及生殖能力的影响。如在家蚕和褐飞虱（Nilaparvata lugens）中,通过干扰VgR的表达,呈现卵巢发育受阻、不能产卵或产卵量下降等表型^[8,26]。【本研究切入点】笔者实验室前期研究已克隆甜菜夜蛾VgR并明确其时空表达特性^[17],但甜菜夜蛾VgR的功能尚不明确。【拟解决的关键问题】利用体外合成的甜菜夜蛾卵黄原蛋白受体保守序列dsRNA注射甜菜夜蛾,通过荧光定量PCR（RT-qPCR）分析注射后甜菜夜蛾VgR的表达情况,研究其对甜菜夜蛾的卵巢发育和产卵量的影响,明确VgR在卵黄合成中的作用,以期为利用RNAi技术防治甜菜夜蛾提供理论依据。

1 材料与方法

试验于2018年在江苏省农业科学院植物保护研究所完成。

1.1 材料

供试甜菜夜蛾虫源来自江苏省农业科学院植物保护研究所经济作物虫害与生防研究室。甜菜夜蛾饲养条件为温度（26.5±1）℃,相对湿度（65±5）%,光周期L﹕D=14 h﹕10 h,成虫期补充10%蜂蜜水。

1.2 甜菜夜蛾总RNA的提取和cDNA的合成

通过液氮研磨甜菜夜蛾雌成虫的腹部组织,按照试剂盒说明书用Trizol reagent（Invitrogen）提取总RNA,用50 μL的无RNA酶水溶解,-70℃保存备用。以总RNA为模板,通过M-MLV酶（Promega）反转录获得cDNA,于-20℃保存备用。

1.3 VgR基因片段的引物设计与扩增

根据笔者实验室前期已发表的甜菜夜蛾VgR的全长序列（GenBank登录号：KT899978）^[18],利用Primer5.0 软件在VgR基因功能区设计引物VgR-F和VgR-R。以甜菜夜蛾cDNA为模板扩增VgR特异性片段。绿色荧光蛋白基因（GFP）片段通过特异性引物（表1）从笔者实验室保存的GFP质粒上扩增。PCR反应程序：94℃预变性3 min,然后按照94℃ 45 s,60℃ 30 s,72℃ 20 s,进行35次循环反应,最后于72℃延伸10 min。通过琼脂糖凝胶电泳分离VgR与GFP的PCR产物,并用胶回收试剂盒（Axygen）纯化回收目的DNA片段。目的片段与pMD-19T载体（TaKaRa）连接,经PCR筛选阳性克隆后,通过AxyPrep质粒小量制备试剂盒（Axygen）提取质粒送上海生工生物工程有限公司测序。利用DNAMAN软件分析该基因序列的准确性,测序正确的质粒为pMD-19T-VgR与pMD-19T-GFP。

Table 1
表1
表1本研究所用引物
Table 1Primers 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

下划线表示T7RNA聚合酶启动子
T7RNA polymerase promoter is underlined

新窗口打开|下载CSV

1.4 VgR及GFP-dsRNA的合成与注射

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（VgR-TF与VgR-R、VgR-F与VgR-TR; GFP-TF与GFP-R、GFP-F与GFP-TR;表1）分别进行两个基因PCR产物正义链与反义链的扩增。用1%的琼脂糖凝胶进行电泳分离回收,用T7 Ribo MAX^TM Express RNAi System（Promega）合成试剂盒合成VgR双链RNA（VgR-dsRNA）和GFP双链RNA（GFP-dsRNA）,用无RNA酶水配制成2 μg·μL^-1终浓度的试剂备用。

选取甜菜夜蛾化蛹第2天、大小均一的雌蛹用于注射。应用10 μL微量进样器将3 μL VgR-dsRNA（2 μg·μL^-1）从腹部靠近尾端1/3处注射。在化蛹的第6天再次注射3 μL相同浓度的VgR-dsRNA。注射完毕后,针头停留约30 s后缓慢拔出,以免体液过多流出。试验设注射GFP-dsRNA和不注射两个对照组。每个处理80头雌蛹,重复3次,共计240头。

1.5 甜菜夜蛾VgR RNAi的效应检测

1.5.1 甜菜夜蛾VgR表达量的RT-qPCR检测注射dsRNA后,取甜菜夜蛾刚羽化、羽化24 h、羽化48 h雌成虫的卵巢组织,对所搜集组织样品进行总RNA提取,cDNA合成。以注射GFP-dsRNA和不注射为对照,每个样本设置3个生物学重复,每个重复4头虫体。以SYBR^?Premix Ex Taq^TM（TaKaRa）为染料,采用LightCycler? 480 实时PCR系统进行基因扩增以及数据分析。VgR及β-actin的RT-qPCR引物见表1。RT-qPCR总反应体系：SYBR Premix Ex Taq（2×）12.5 μL,forward primer（10 μmol·L^-1）0.5 μL,reverse primer（10 μmol·L^-1）0.5 μL,cDNA 2.0 μL,ddH₂O 9.5 μL,总体积25 μL。反应条件：95℃ 30 s;95℃ 5 s,60℃ 20 s,72℃ 10 s,40个循环;95℃ 15 s;60℃ 1 min;95℃ 15 s。最终结果的计算采用2^-ΔΔCt法（Ct表示循环数）。

1.5.2 RNAi后甜菜夜蛾卵巢发育进度的观察以及羽化率、产卵量的统计通过统计对照组（不注射、注射GFP-dsRNA）和注射VgR-dsRNA处理组羽化蛹皮的数量来计算甜菜夜蛾的羽化率。解剖对照组（不注射、注射GFP-dsRNA）和注射VgR-dsRNA处理组甜菜夜蛾刚羽化、羽化24 h、羽化48 h雌成虫（每个处理30头）的腹部,观察卵巢发育进度。测定不同处理组羽化24 h雌成虫（每个处理20头）的卵巢管长度及成熟卵粒直径大小。选择同日羽化的成虫20对,分别饲养,逐日统计其单雌产卵量。

1.6 数据统计与分析

VgR相对表达量、产卵量、羽化率等数据使用SPSS软件进行统计学分析,采用单因素方差分析并用Duncan’s新复极差法进行差异显著性检测,显著性检验水平P<0.05。

2 结果

2.1 目的片段的扩增

利用甜菜夜蛾卵巢的总RNA和GFP质粒为模板,分别扩增甜菜夜蛾VgR和GFP,获得的PCR产物电泳结果见图1,分别得到大小为327和417 bp的单一带,与预期片段大小一致。

图1

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图1VgR 和GFP 的PCR 扩增

M: Trans2K DNA marker; 1: VgR; 2: GFP
Fig. 1PCR amplification of VgR and GFP

2.2 dsRNA的合成

以pMD-19T-VgR与pMD-19T-GFP质粒作为DNA模板,通过PCR扩增,在目的序列的两端加上T7启动子。在T7RNA聚合酶的作用下,转录合成dsRNA,经过一系列洗涤、过柱,得到纯化的VgR-dsRNA和GFP-dsRNA,大小约为327和417 bp（图2）,与预期片段长度大小一致。

图2

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图2VgR体外转录产物

1：VgR体外转录产物dsRNA dsRNA-production of VgR transcripted in vitro;2：GFP对照 Positive control of GFP
Fig. 2Production of VgR transcripted in vitro

2.3 VgR-dsRNA注射对甜菜夜蛾VgR表达量的影响

RT-qPCR检测结果表明,与对照组相比,注射VgR-dsRNA后的VgR表达水平显著下降（图3）。刚羽化的雌成虫,注射VgR-dsRNA组的VgR表达量相比注射GFP-dsRNA的对照组下降了79.35%;羽化24 h的雌成虫,注射VgR-dsRNA组的VgR表达量相比注射GFP-dsRNA的对照组下降了84.22%;羽化48 h的雌成虫,注射VgR-dsRNA组的VgR表达量相比注射GFP-dsRNA的对照组下降了67.68%。VgR表达量在不注射dsRNA（CK）和注射GFP-dsRNA的两个对照组之间无显著差异（图3）。说明注射VgR的dsRNA对该基因具有明显的沉默效应。

图3

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图3dsRNA处理后甜菜夜蛾VgR的表达水平

柱上标有不同小写字母表示差异显著（P<0.05）。图5同
Fig. 3The 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

2.4 VgR-dsRNA注射对甜菜夜蛾卵巢发育进度的影响

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组（图4-a、4-b、4-c）与注射GFP-dsRNA组（图4-e、4-f、4-g）相比,甜菜夜蛾的卵巢发育进度显著推迟。测定羽化24 h的甜菜夜蛾的卵巢管长度和卵粒大小发现,注射VgR-dsRNA组卵巢管明显缩短,成熟卵粒数量和卵粒大小明显小于注射GFP-dsRNA对照组。注射VgR-dsRNA组的甜菜夜蛾（羽化24 h）卵巢管长度为（24.01±1.52）mm,比注射GFP-dsRNA组的卵巢管长度（31.56±2.03）mm下降了23.92%（图4-b、4-f）;同时,注射GFP-dsRNA组的卵巢成熟卵粒较多,平均直径为（0.46±0.05）mm（图4-h）,而注射VgR-dsRNA组成熟卵粒较少,卵粒平均直径为（0.23±0.02）mm,大部分卵粒卵黄沉积较少,处于空瘪状态（图4-d）。对于羽化48 h的甜菜夜蛾卵巢,注射VgR-dsRNA组的干扰效率下降了67.68%,VgR表达量有所上升（图3）,此时卵母细胞中的卵黄沉积加快,卵黄含量增多,但是成熟卵粒数量还是明显少于GFP-dsRNA对照组（图4-c、4-g）。在解剖过程中,发现注射GFP-dsRNA组与空白对照组的卵巢发育进度并无显著差异。

图4

新窗口打开|下载原图ZIP|生成PPT
图4注射dsRNA后甜菜夜蛾卵巢发育情况

a—c：注射VgR-dsRNA后羽化不同时间段的雌成虫卵巢（0、24、48 h）The ovary of 0-, 24-, 48-hour-old adults after VgR-dsRNA injection;e—g：注射 GFP-dsRNA后羽化不同时间段的雌成虫卵巢（0、24、48 h）The ovariole of 0-, 24-, 48-hour-old adults after GFP-dsRNA injection;i—k：羽化不同时间段的雌成虫卵巢（0、24、48 h）The ovary of 0-, 24-, 48-hour-old adults;d：注射VgR-dsRNA后羽化24 h的雌成虫卵巢管 The ovariole of 24-hour-old adults after VgR-dsRNA injection;h：注射GFP-dsRNA后羽化24 h的雌成虫卵巢管 The ovariole of 24-hour-old adults after GFP-dsRNA injection;l：羽化24 h的雌成虫卵巢管 The ovariole of 24-hour-old female adults
Fig. 4The ovary development of S. exigua after dsRNA injection

2.5 VgR-dsRNA注射对甜菜夜蛾羽化率及产卵量的影响

以无注射dsRNA和注射GFP-dsRNA的甜菜夜蛾为对照,测定注射VgR-dsRNA后成虫的羽化率及单雌产卵量,结果见图5。可以看出,注射GFP-dsRNA和VgR-dsRNA的甜菜夜蛾的羽化率无显著差异,但较对照组羽化率略降低。注射VgR-dsRNA的甜菜夜蛾单雌平均产卵量只有170粒,而空白对照和注射GFP-dsRNA的单雌平均产卵量可达451和420粒。

图5

新窗口打开|下载原图ZIP|生成PPT
图5注射VgR-dsRNA后甜菜夜蛾的羽化率与产卵量

Fig. 5Eclosion rate and fecundity after VgR-dsRNA injection

3 讨论

昆虫细胞内存在将dsRNA切割成siRNA的Dicer酶,长链的dsRNA即可在昆虫中引起RNAi效应^[28]。目前将体外合成的dsRNA导入昆虫活体的方法有注射法和喂食法。与喂食法相比,注射法的优点在于便于控制剂量,产生效应快,但其局限性体现在操作难度较大。迄今为止,通过注射方法实现虫体内的RNAi已经在多种鳞翅目昆虫中得以实施,如烟草天蛾（Manduca sexta）^[29]、家蚕^[8]、甜菜夜蛾^[21]、棉铃虫^[16,30]以及斜纹夜蛾^[9]等。在前期研究中笔者克隆了甜菜夜蛾VgR的全长并明确了其表达规律,发现甜菜夜蛾VgR在卵巢特异性表达,其合成和启动期在蛹末期,表达高峰在成虫羽化48 h,这与甜菜夜蛾卵巢发育进度紧密相关^[17]。因此,选择在甜菜夜蛾VgR启动期前期（化蛹第2天）在腹部注射VgR-dsRNA,为了加强RNAi的效果,在蛹末期又注射了相同剂量的dsRNA,VgR表达量在甜菜夜蛾羽化24 h时降低了84%。在注射过程中,由于技术不熟练所造成的机械损伤对昆虫存活率和羽化率会有一定的影响^[21]。本研究中,注射VgR-dsRNA和GFP-dsRNA的试虫羽化率无显著差异,但较未注射的空白对照组羽化率略降低,可能是机械损伤导致。

昆虫有强大的生殖力,能在极短的时间内产生含有大量卵黄的成熟卵。卵黄原蛋白是卵黄的主要成分,它的存在或含量高低直接影响昆虫的生殖。目前,许多研究者为了探求合理的控制害虫生殖的方法,采用RNAi技术沉默介导Vg胞吞作用的卵黄原蛋白受体VgR。对于昆虫VgR进行RNAi后,重点研究其对昆虫卵黄沉积、卵巢发育进度以及生殖能力的影响。LU等^[26]通过向褐飞虱注射VgR-dsRNA,VgR表达量下调,卵巢表面的受体缺乏不能正常摄取Vg进入卵巢,血淋巴中Vg含量增多,最终导致褐飞虱卵巢发育停滞,不能产卵;SHANG等^[31]通过对蚜虫（Toxoptera citricidus）注射VgR-dsRNA,发现蚜虫的生殖周期有所变化,生殖前期变长,生殖期变短,后代数量下降。同时,向同一种昆虫注射不同的VgR-dsRNA,其沉默效果也可能不同;LIN等^[8]分别在家蚕蛹第1、4、7天注射两种VgR-dsRNA,发现VgR表达量下降程度不同,VgR mRNA敲除率高的家蚕不能产卵,而VgR mRNA敲除率低的家蚕最终卵粒变小变白;SHU等^[9]在斜纹夜蛾的蛹期注射两种dsRNA,表型基本类似,与对照相比,血淋巴中Vg含量增多,卵巢中的Vg和VgR含量下降,产卵量显著下降。与前人研究结果相似,本研究通过用RNAi方法降低VgR表达量后,甜菜夜蛾与生殖相关的卵巢发育受到了影响,卵母细胞中沉积的卵黄含量显著减少,卵巢管变短,直接反映生殖力的产卵量显著下降。这些结果表明VgR mRNA表达是昆虫Vg摄取和卵发育的基础。

甜菜夜蛾是一种重要的世界性害虫,具有繁殖能力强、易产生抗药性等特点^[5,6]。VgR在昆虫的生殖中起着不可替代的作用,是卵黄发生的基础,直接影响昆虫卵母细胞的生理与形态发育、卵子的数量^[3]。本研究通过体外注射dsRNA的方法研究VgR的功能,表明通过RNAi可以显著减少甜菜夜蛾的产卵量,起到调控甜菜夜蛾生殖暴发的作用,但是如何将VgR-dsRNA应用于甜菜夜蛾的防治（dsRNA工程微生物或转基因作物）还需要进一步深入研究。

4 结论

通过合成甜菜夜蛾VgR功能区序列的dsRNA,体外注射后,能够显著降低VgR表达,同时甜菜夜蛾的卵巢发育受到了影响,卵母细胞中沉积的卵黄含量显著减少,卵巢管变短,直接反映生殖力的产卵量显著下降。VgR是甜菜夜蛾Vg摄取和卵发育的基础,可作为控制甜菜夜蛾危害的潜在靶标。

参考文献原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[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.

URL [本文引用: 1]

卵黄蛋白的结构及其合成、摄取过程与激素的调控机理是目前昆虫生理学的研究热点之一.近几年,随着分子克隆技术、基因工程手段和生物信息学的发展,对卵黄蛋白基因的研究将为寻找害虫生物防治提供新途径.本文对昆虫卵黄蛋白及其激素调控进行了综述.为防治害虫再猖獗的发生和促进大量繁殖益虫提供重要的理论依据.

GE L

, WU J

. Research progress in insect vitellin and its hormone regulation
Chinese Bulletin of Entomology, 2010,47(2):236-246. (in Chinese)

URL [本文引用: 1]

卵黄蛋白的结构及其合成、摄取过程与激素的调控机理是目前昆虫生理学的研究热点之一.近几年,随着分子克隆技术、基因工程手段和生物信息学的发展,对卵黄蛋白基因的研究将为寻找害虫生物防治提供新途径.本文对昆虫卵黄蛋白及其激素调控进行了综述.为防治害虫再猖獗的发生和促进大量繁殖益虫提供重要的理论依据.

[2]

ENGELMANN

. 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 URL [本文引用: 1]

[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 URL [本文引用: 2]

卵黄原蛋白受体（VgRs）属于低密度脂蛋白受体家族成员,具有该家族典型的保守结构域,包括配体结合域,表皮生长因子前体同源域,跨膜域,O-联糖功能域,以及胞质尾域。昆虫VgRs通常具有卵巢特异性,是卵黄原蛋白Vg的专一性胞吞作用受体,可介导Vg进入昆虫卵母细胞,而后沉淀积累形成昆虫生殖必须的卵黄蛋白YP。VgRs介导的胞吞作用是一个动态循环过程,它是卵黄发生的基础,对昆虫卵母细胞发育起着至关重要的作用。近年来的研究表明,VgRs不仅与卵巢激活、卵黄发生与卵子形成密切相关,而且在昆虫信息交流、社会分化、行为构建以及免疫调控等中也起到了至关重要的作用,已成为潜在的害虫控制新靶标。本文首次对昆虫VgRs基因的序列信息,分子结构,系统进化,表达模式以及调控功能等方面进行了综述,旨在为了解VgRs基因的研究进展及前景提供参考,对进一步改进害虫生态控制的策略和措施也具有指导意义。

WANG J

, PENG

, ZOU M

, YANG Y

, WANG

, YOU M

. 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 URL [本文引用: 2]

TUFAIL

, TAKEDA

. 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 URL [本文引用: 1]

[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 URL [本文引用: 2]

为明晰几种重要解毒酶在甜菜夜蛾针对甲维盐抗性形成过程中的可能作用,本文研究分析了甲维盐处理后甜菜夜蛾幼虫体内主要解毒酶活力及其对应基因表达量的变化趋势.结果表明,在甲维盐(剂量为:LC5、LC20、 LC50)胁迫72 h后,甜菜夜蛾体内多功能氧化酶(MFO)比活力及SeCYP450表达量均较对照极显著上升(P＜0.01),且二者均随着处理浓度的增大而先上升后下降.同时,谷胱甘肽S-转移酶(GST)的比活力也呈现出相同的变化趋势.而SeGSTs与SeGSTs1在胁迫后的表达趋势与GST比活力的并不一致,SeGSTs表达量随着处理浓度的上升而极显著升高(P＜0.01),而SeGSTs1则随着处理浓度的上升而下降.在3个亚致死浓度甲维盐处理下, 酯酶(EST)比活力均被显著抑制(P＜0.05),同样的趋势也反映在SeCarE表达量的显著变化上(P＜0.05).此外,MFO、EST的比活力变化趋势与其对应基因SeCYP450、SeCarE表达谱之间存在极显著相关性(P＜0.01);而GST活力只与SeGSTs表达量具有极显著相关性 (P＜0.01),与SeGSTs1表达量之间则不存在显著相关性.因此,MFO、GST及其相关基因SeCYP450、SeGSTs、SeGSTs1有可能参与到甜菜夜蛾对甲维盐抗药性的演化过程中.

DAI H

, SUN

, BAI L

, ZHAO

, XIAO L

, TAN Y

. 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 URL [本文引用: 2]

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.

URL [本文引用: 2]

针对当前我国蔬菜生产中甜菜夜蛾Spodoptera exigua(Hübner)高抗药性、猖獗为害、防控困难的现状,项目组对甜菜夜蛾的发生规律、越冬与迁飞、抗药性、生物防治与综合防控等进行了系统的研究。明确了甜菜夜蛾在我国各地的发生规律及发生动态,从南到北发生时间呈楔形,而发生量总体东高西低,从南到北呈中部高南北低的马鞍形。阐述了甜菜夜蛾"无卵子发生-飞行拮抗"的迁飞特性,提出了接力棒式季节性南北往返迁飞模式,初步阐明了甜菜夜蛾在我国东部地区的迁飞路线与迁飞时间。提出并认证了甜菜夜蛾在我国的越冬区域。明确了全国各主要发生区甜菜夜蛾对10种杀虫剂的抗药性水平,探明了甜菜夜蛾对茚虫威、甲氨基阿维菌素苯甲酸盐的抗性风险、交互抗性、种群适合度、抗性遗传规律和抗性机理,提出了抗药性治理措施。分别在海南、湖南、湖北、上海、天津等地进行了甜菜夜蛾田间药效试验,筛选出环境友好型高效杀虫剂7种。掌握了马尼拉陡胸茧蜂、夜蛾黑卵蜂和淡足侧沟茧蜂人工大量繁殖技术,改进了甜菜夜蛾核型多角体病毒生产工艺,解决了寄生蜂和病毒规模化生产及田间释放关键技术,达到规模化生产的要求。改进优化了甜菜夜蛾性引诱剂配方、研发出新型诱捕装置,并投入生产应用。集成出4套农业生产轻简化实用技术,华南、华中、华北、华东地区根据当地发生特点制定了4套防控方案。在全国甜菜夜蛾主要发生区建立试验基地16个,核心示范基地29个,年示范面积总计约3800hm2,取得显著的经济、社会和生态效益。

SI S

, ZHOU L

, WANG S

, JIANG X

, XU Z

, MU

, WANG D

, WANG X

, CHEN H

, YANG Y

, JI X

. 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)

URL [本文引用: 2]

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.

URL Magsci [本文引用: 1]

甜菜夜蛾Spodoptera exigua (Hübner)是我国多种农作物上的重要害虫, 在我国许多地区频繁暴发成灾。为探索甜菜夜蛾种群动态规律并建立种群数量发生趋势预测模型, 作者应用时间序列分析和逐步回归分析方法研究了我国广域（较大范围）温度和广域降雨量变化趋势对我国广域甜菜夜蛾年暴发频度的影响规律。结果表明: 甜菜夜蛾发生的长期趋势和年间波动状况均与广域温度和广域降雨量具有复杂的影响关系。在1979-2008年间, 我国甜菜夜蛾暴发频度呈现出波浪式上升趋势, 其暴发指数平均年递增率为0.076, 而我国广域温度（以27个省市级气象台数据统计为例）在1990-2008年间的平均年递升率为<?xml:namespace prefix = st1 /><st1:chmetcnv UnitName="℃" SourceValue=".039" HasSpace="False" Negative="False" NumberType="1" TCSC="0" w:st="on">0.039℃</st1:chmetcnv>, 即我国甜菜夜蛾暴发频度上升趋势与我国广域温度升高趋势同向而行。作者从52个因素（当年和上年1-12月各月及全年日均温和月均降雨量）中筛选出了具有显著回归影响（P<0.05或0.01）的10个因素进入回归模型, 初步找出了能够预测广域甜菜夜蛾暴发趋势指数的温度与降雨量或其组合因素, 并使其模型达到99%以上的历史符合率和预测准确度。作者认为, 广域温、 雨因素与广域甜菜夜蛾暴发趋势指数的这种密切相关性, 不是偶然的巧合, 而是必然的环境（温度和降雨量）作用于生物（甜菜夜蛾）的因果关系。

WEN L

, ZHANG Y

. 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)

URL Magsci [本文引用: 1]

LIN

, MENG

, WANG Y

, LUO

, KATSUMA

, YANG C

, BANNO

, KUSAKABE

, SHIMADA

, XIA Q

. 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 URLPMID:3650373 [本文引用: 5]

In insects, the vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. The oogenesis mutant scanty vitellin (vit) of Bombyx mori (Bm) lacks vitellin and 30-kDa proteins, but B. mori egg-specific protein and BmVg are normal. The vit eggs are white and smaller compared with the pale yellow eggs of the wild type and are embryonic lethal. This study found that a mutation in the B. mori VgR gene (BmVgR) is responsible for the vit phenotype. We cloned the cDNA sequences encoding WT and vit BmVgR. The functional domains of BmVgR are similar to those of other low-density lipoprotein receptors. When compared with the wild type, a 235-bp genomic sequence in vit BmVgR is substituted for a 7-bp sequence. This mutation has resulted in a 50-amino acid deletion in the third Class B region of the first epidermal growth factor (EGF1) domain. BmVgR is expressed specifically in oocytes, and the transcriptional level is changed dramatically and consistently with maturation of oocytes during the previtellogenic periods. Linkage analysis confirmed that BmVgR is mutated in the vit mutant. The coimmunoprecipitation assay confirmed that mutated BmVgR is able to bind BmVg but that BmVg cannot be dissociated under acidic conditions. The WT phenotype determined by RNA interference was similar to that of the vit phenotype for nutritional deficiency, such as BmVg and 30-kDa proteins. These results showed that BmVgR has an important role in transporting proteins for egg formation and embryonic development in B. mori.

[9]

SHU Y

, WANG J

, LU

, ZHOU J

, ZHOU

, ZHANG G

. The ?rst 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 URLPMID:20955241 [本文引用: 4]

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) superfamily, and is an important carrier for the uptake of vitellogenin (Vg) into developing oocytes of all oviparous species. The first full-length message for a VgR from a Lepidopteran insect was cloned and sequenced from the ovary of Spodoptera litura Fabricius (GenBank accession no. GU983858). The coding region consisted of 5370 bp flanked by a 49 bp 5′-untranslated region (UTR) and a 177 bp 3′-UTR, which encoded a 1798-residue protein with a predicted molecular weight (MW) of 201.69 kDa. S. litura VgR (SlVgR)comprised two ligand binding sites with four LDLR class A repeats in the first domain and seven in the second domain, an epidermal growth factor-like domain containing an LDLR class B repeat and a YWXD motif, a transmembrane domain and a cytoplasmic domain. A phylogenetic relationship placed SlVgR as a separate group from the other insects. SlVgR messenger RNA (mRNA) was specifically expressed in the ovarian tissues. The developmental expression patterns showed that VgR mRNA was first transcribed in 6th day female pupae and the maximum level of VgR mRNA appeared in 36-h-old adults. Immunoblot analysis detected an ovary-specific VgR protein with a MW of 65200 kDa, whose development profiles were consistent with VgR mRNA expression patterns. RNA inteference (RNAi) specifically disrupted the VgR gene by injection of 3 or 5 08g VgR double-stranded RNA per insect in 4th or 6th day pupae. RNAi of SlVgR led to a phenotype characterized by high Vg accumulation in the haemolymph, low Vg deposition in the ovary and the failure of insect spawning. These results mean that VgR is critical for binding Vg and transporting it into the oocytes of the insect ovary, thus playing an important role in insect reproduction.

[10]

CHO K

, RAIKHEL A

. 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 URLPMID:11881811 [本文引用: 1]

Vitellogenin is a precursor of the major yolk protein, vitellin. It is internalized by developing oocytes via receptor-mediated endocytosis. Previously, we characterized the vitellogenin receptor (VgR) from oocytes of the mosquito Aedes aegypti [ Sappington, T.W., Kokoza, V.A., Cho, W.L. and Raikhel, A.S. (1996 ) Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor. Proc Natl Acad Sci USA 93: 8934 8939]. The VgR receptor has a unique structure with two putative ligand-binding domains. In order to understand the regulation of this important molecule, we characterized the VgR gene structure and its expression during vitellogenesis in the mosquito A. aegypti . We report here that the VgR gene was separated by five introns that have an average length of 60 bp, except for the second intron which was more than 20 kb long. Most introns were located within the coding regions of the first protein domain. We isolated two allelic variations of the VgR gene, VgR1 and VgR2 , the nucleotide sequences of which differing only in their 5'-flanking regions. Considering their frequency in the mosquito genome, VgR2 appeared to be a major allele. The expression of VgR mRNA was studied by the Northern blot analysis and in situ hybridization. The level of the VgR transcript started to rise in the ovary one day post-eclosion. It continued its dramatic rise during the vitellogenic period, reaching its peak at 24 h PBM. The VgR transcript was present exclusively in ovaries where it was seen in oocytes and nurse cells of primary follicles and germ-line cells of the germarium.

[11]

CHEN M

, LEWIS D

, KEELEY 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 URLPMID:15056367 [本文引用: 1]

We describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA from the imported fire ant, Solenopsis invicta , an invasive pest. Using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, fragments encompassing the entire coding region of a putative VgR were cloned and sequenced. The complete 5764 bp cDNA encodes a 1782 residue protein with a predicted molecular mass of 201.3 kDa (= Si VgR). Northern blot analysis demonstrated that the 7.4 kb Si VgR transcript was present only in ovaries of reproductive females (virgin alates and queens). The temporal profile of transcriptional expression showed that Si VgR mRNA increased with age in virgin alate females and that this was up-regulated by methoprene, a juvenile hormone (JH) analogue. This suggests that the Si VgR gene is JH regulated.

[12]

GUIDUGLI-LAZZARINI K

, DO NASCIMENTO A

, TANAKA E

, PIULACHS M

, HARTFELDER

, BITONDI M

, SIMOES Z

. 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 URLPMID:18606165 [本文引用: 2]

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

[13]

LIU Q

, ZHU B

, LIU C

, WEI G

, WANG Z

. Characterization of vitellogenin receptor (VgR) from the Chinese oak silkworm, Antheraea pernyi
Bulletin of Insectology, 2011,64(2):167-174.

[本文引用: 1]

[14]

LIN W

, CHIEN C

, TSAI C

, CHEN M

. 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 URLPMID:26280361 [本文引用: 2]

ABSTRACT The yolk protein precursor, vitellogenin (Vg), is absorbed into growing oocytes via receptor-mediated endocytosis for embryonic development. In this study, a Vg receptor (VgR) cDNA of the oriental fruit fly ( Bactrocera dorsalis Hendel) was cloned via RT-PCR and RACE (GenBank accession no. KR535603) and its expression analyzed. The BdVgR cDNA has a length of 6,585 bp encoding 1,923 amino acids. It has a conserved motif arrangement with other insect VgRs, and showed high identity to the B. cucurbitae VgR (91.4%). The expression of BdVgR mRNA and proteins was shown in both ovary and fat body. This is the first report on a nonovary-specific VgR from a nonsocial insect. In ovary, the expression of BdVgR mRNA and proteins was inconsistent, with the transcription, but not protein, level high on D0. In fat body, the expression levels of BdVgR mRNA and proteins were high on days 5 and 6. The function of BdVgR in the fat body is not clear. However, it may be involved in reuptake of yolk proteins from the hemolymph as an amino acid reservoir or as autocrine regulation of yolk protein expression.

[15]

TUFAIL

, TAKEDA

. 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 URLPMID:16033432 [本文引用: 2]

The vitellogenin receptor (VgR) belongs to the low density lipoprotein receptor (LDLR) superfamily, and mediates the uptake of vitellogenin (Vg) into developing oocytes of all oviparous species. We cloned and characterized a VgR from previtellogenic ovaries of the cockroach, Periplaneta americana ( Pa ). This is the first report on a VgR from a hemimetabolous insect. The cDNA, comprising 5722 bp, encoded a 1790-residue mature protein with a predicted molecular mass of 200.5 kDa. We next characterized the ovarian expression pattern, developmental regulation and cellular distribution of the VgR mRNA and protein. Northern blot analysis confirmed that a 7.2 kb transcript was specifically expressed in ovarian tissues at high levels throughout ovarian development, especially in previtellogenic ovaries and in ovaries before adult emergence. RNA in situ hybridization and immunocytochemistry localized the VgR mRNA and protein to germ-line derived cells, the oocytes, and revealed that VgR gene transcription and translation begin very early during oocyte differentiation in the germarium. Immunoblot analysis detected an ovary-specific VgR protein of 210 kDa that was present in previtellogenic ovaries on the day of female emergence. The VgR protein signal strengthened every day and was intense after initiation of vitellogenesis and onset of Vg uptake. The immunoblotting of vitellins demonstrated that Vg uptake occurred on day 5, one day after Vg first appeared in the haemolymph, indicating that the receptor-endocytotic machinery starts functioning soon after the ligand becomes available.

[16]

ZHANG W

, MA

, XIAO H

, XIE B

, SMAGGHE

, GUO Y

, LIANG G

. 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 URLPMID:27192057 [本文引用: 3]

Developing oocytes accumulate plentiful yolk protein during oogenesis through receptor-mediated endocytosis. The vitellogenin receptor (VgR), belonging to the low-density lipoprotein receptor (LDLR) family, regulates the absorption of yolk protein. In this work, the full-length vitellogenin receptor (HaVgR) in the cotton bollwormHelicoverpa armigerawas identified, encoding a 1817 residue protein. Sequence alignment revealed that the sequence ofHaVgR contained all of the conservative structural motifs of LDLR family members, and phylogenetic analysis indicated thatHaVgR had a high identity among Lepidoptera and was distinct from that of other insects. Consistent with other insects,HaVgRwas specifically expressed in ovarian tissue. The developmental expression pattern showed thatHaVgRwas first transcribed in the newly metamorphosed female adults, reached a peak in 2-day-old adults and then declined. Western blot analysis also revealed an ovarian-specific and developing expression pattern, which was consistent with theHaVgRmRNA transcription. Moreover, RNAi-mediatedHaVgRknockdown strongly reduced the VgR expression in both the mRNA and protein levels, which inhibited the yolk protein deposition in the ovaries, led to the dramatic accumulation of vitellogenin and the up-regulation ofHaVgexpression in hemolymph, and eventually resulted in a declined fecundity. Together, all of these findings demonstrate thatHaVgRis a specific receptor in uptake and transportation of yolk protein for the maturation of oocytes and that it plays a critical role in female reproduction.

[17]

ZHAO

, SUN

, XIAO L

, TAN Y

, JIANG Y

, BAI L

. 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 URL [本文引用: 4]

Abstract BACKGROUND The beet armyworm Spodoptera exigua , a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and the host plant is a basic and important component of early forecasting of beet armyworm outbreaks. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. RESULTS In this study, key life-history parameters of S. exigua reared on distinct hosts were investigated; the host plants could be ranked as lettuce > shallot > tomato > celery in their order of suitability. Full-length S. exigua vitellogenin receptor ( SeVgR ) cDNA was cloned, and sex-, stage- and tissue-specific expression characteristics were assessed. Spodoptera exigua vitellogenin ( SeVg ) and SeVgR expression levels were markedly modulated by host nutrients ( P < 0.05). SeVg and SeVgR expression levels were significantly higher in S. exigua reared on lettuce, the most preferred and most nutritive host, than in those reared on tomato and celery. Interestingly, significant linear regression correlations were found between SeVg and SeVgR expression levels and key S. exigua life-history parameters, especially life span, pupa weight, and female fecundity ( P < 0.01). CONCLUSION Host plant type and suitability could affect the expression pattern of SeVg and SeVgR , which influenced S. exigua fecundity. Vg and VgR have the potential to be used as molecular markers of S. exigua fecundity and for forecasting outbreaks of S. exigua on different hosts. 2017 Society of Chemical Industry

[18]

ROY-ZOKAN E

, CUNNINGHAM C

, HEBB L

, MCKINNEY E

, MOORE A

. 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 URLPMID:4590458 [本文引用: 3]

Complex social behaviour in Hymenoptera has been hypothesized to evolve by co-opting reproductive pathways (the ovarian ground plan hypothesis, OGPH) and gene networks (the reproductive ground plan hypothesis, RGPH). In support of these hypotheses, in eusocial Hymenoptera where there is reproductive division of labour, the yolk precursor protein vitellogenin (Vg) influences the expression of worker social behaviour. We suggest that co-opting genes involved in reproduction may occur more generally than just in the evolution of eusociality; i.e. underlie earlier stages of social evolution such as the evolution of parental care, given that reproduction and parental care rarely overlap. We therefore examinedvitellogenin(vg) gene expression associated with parental care in the subsocial beetleNicrophorus vespilloides. We found a significant reduction in the expression ofvgand its receptor,vgr, in head tissue during active parental care, and confirmed that the receptor is expressed in the brains of both sexes. Ours is the first study to show thatvgris expressed in the brain of a non-eusocial insect. Given the association between behaviour and gene expression in both sexes, and the presence of vitellogenin receptors in the brain, we suggest that Vg was co-opted early in the evolution of sociality to have a regulatory function. This extends the association of Vg in parenting to subsocial species and outside of the Hymenoptera, and supports the hypothesis that the OGPH is general and that heterochrony in gene expression is important in the evolution of social behaviour and precedes subsequent evolutionary specialization of social roles.

[19]

TUFAIL

, TAKEDA

. 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 URLPMID:17296498 [本文引用: 2]

A cDNA encoding the vitellogenin receptor (VgR) of the cockroach, Leucophaea maderae ( Lm) was cloned and sequenced. The coding region consisted of 5454 bp flanked by a 67 bp 5′-untranslated region (UTR) and a 168 bp 3′-UTR, which encoded a 1792-residues mature protein with a predicted molecular mass of 199.84 kDa. The deduced amino acid sequence of the LmVgR cDNA revealed two ligand-binding domains with 5 repeats in the first domain and 8 in the second domain similar to those of other insect VgRs. Northern hybridization analysis revealed the presence of a 657.3 kb transcript that was specifically expressed only in the ovarian tissues. The developmental expression profile demonstrated that LmVgR mRNA exists throughout the ovarian development, and that the transcriptional level is especially high in the previtellogenic periods. The Immunoblot analysis detected an ovary-specific VgR protein with a M r of 65215 kDa under reducing conditions. The VgR protein continues to increase until day 3 of adult emergence, declines thereafter slightly until day 6, then rises again and reaches to its maximum on day 9 during the vitellogenic periods. Compared to other insect VgRs, the LmVgR primary protein structure shows an overall amino acid identity between 33% and 65% to their corresponding gene products. The similarity was high when compared with other cockroach VgRs described from Periplaneta americana (55%) and Blattella germanica (65%). A proposed phylogenetic (neighbor-joining) relationship suggests that LmVgR and B. germanica VgR have closer ancestry than from P. americana VgR. The cytoplasmic tail of LmVgR contains a leucine-isoleucine internalization signal similar to other insect counterparts unlike the NPXY motif of other LDLR family members. The cockroaches VgRs harbor, in addition, a putative NPTF motif for internalization. The sequence reported in this paper has been submitted to GenBank (accession number: AB255883).

[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 URL Magsci [本文引用: 1]

RNA干扰（RNA interference，RNAi）是指外源或内源的双链RNA（dsRNA）特异性地引起基因表达沉默的现象，它作为一种有效的工具用来产生转录后沉默，从而抑制特定基因的表达，成为基因功能研究的一种新方法，除了在模式昆虫如果蝇Drosophila中广泛应用之外，也在非模式昆虫中得到成功应用。近年来，RNAi技术在导入方法和基因功能分析方面都取得了飞速发展，且与转基因技术相结合成功应用于害虫防治领域。本文综述了RNAi技术在导入方法、昆虫功能基因组功能分析及害虫防治等领域新近的研究成果，并展望了该技术的应用前景。

YANG Z

, WEN L

, WU Q

, WANG S

, XU B

, ZHANG Y

. 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 URL Magsci [本文引用: 1]

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 URL [本文引用: 3]

采用RNA干扰技术,通过腹腔注射法将甜菜夜蛾信息素结合蛋白1(SexigPBP1)双链RNA导入雄蛾体内,48 h后定量PCR检测表明,SexigPBP1的mRNA表达量被敲减90%以上;同时,触角电位(EAG)测定结果表明,注射处理48 h后雄蛾对雌蛾性信息素主组分Z9,E12-14∶Ac的EAG反应降低了约60%,证明SexigPBP1在雄蛾对该组分的感受中起重要作用。

ZHOU Y

, XIU W

, DONG S

. 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 URL [本文引用: 3]

TURNER C

, DAVY M

, MACDIARMID R

, PLUMMER K

, BIRCH N

, NEWCOMB R

. 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 URLPMID:16756557 [本文引用: 1]

RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene ( EposCXE1 ) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene ( EposPBP1 ) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

[23]

CIUDAD

, PIULACHS M

, BELLES

. 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 URLPMID:16403020 [本文引用: 1]

During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)-mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica , as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre-adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster , which have the yl ( VgR ) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.

[24]

ARAUJO R

, SANTOS

, PINTO F

, GONTIJO N

, LEHANE M

, PEREIRA M

. 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 URLPMID:16935217 [本文引用: 1]

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 μg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75±14% and that feeding 1 μg/μL of NP2 dsRNA into second-instar nymphs (approx. 13 μg in total) reduced gene expression by 42±10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

[25]

BAUM J

, BOGAERT

, CLINTON

, HECK G

, FELDMANN

, ILAGAN

, JOHNSON

, PLAETINCK

, MUNYIKWA

, PLEAU

, VAUGHN

, ROBERTS

. Control of coleopteran insect pests through RNA interference
Nature Biotechnology, 2007,25(11):1322-1326.

DOI:10.1038/nbt1359 URL [本文引用: 1]

[26]

, SHU Y

, ZHOU J

, ZHANG X

, CHEN M

, YAO

, ZHOU

, ZHANG W

. Molecular characterization and RNA interference analysis of vitellogenin receptor from Nilaparvata lugens (St?l)
Journal of Insect Physiology, 2015,73:20-29.

DOI:10.1016/j.jinsphys.2015.01.007 URLPMID:25617689 [本文引用: 3]

Vitellogenin receptors (VgRs), members of the low-density lipoprotein receptor (LDLR) superfamily, are responsible for taking vitellogenin (Vg) into developing oocytes. Here the first full-length VgR cDNA from a hemipteran insect, the brown planthopper (Nilaparvata lugens), was cloned and sequenced. The complete mRNA sequence was 6174bp in length with an open reading frame (ORF) of 5796bp encoding 1931 amino acid residues. N. lugens VgR (NlVgR) contained two ligand-binding domains with five LDLR Class A cysteine-rich repeats in the first domain and eight in the second domain, which was similar to other insect VgRs. NlVgR was specifically expressed in the ovary, and the mRNA level started to increase after adult female emergence, with a peak on day 7 in the adult stage, and then declined. Western blot analysis of NlVgR protein revealed an ovary-specific expression pattern, which was consistent with NlVgR transcript detection. Injection with NlVgR double-stranded RNA (dsRNA) significantly disturbed NlVgR, which led to a decrease in NlVg protein content in the ovaries, an accumulation of NlVg protein in the hemolymph, the arrested development of ovaries, and the failure of insects to reproduce. Besides, NlVgR expression was significantly upregulated after the topical application of juvenile hormone (JH) III. These results suggest that VgR is critical for Vg uptaking of oocytes and it plays an important role in insect fecundity.

[27]

PITINO

, COLEMAN A

, MAFFEI M

, RIDOUT C

, HOGENHOUT S

. Silencing of aphid genes by dsRNA feeding from plants
PLoS ONE, 2011,6(10):e25709.

DOI:10.1371/journal.pone.0025709 URLPMID:3187792 [本文引用: 1]

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs. In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphidM. persicaewas selected because it has a broad plant host range that includes the model plantsNicotiana benthamianaandArabidopsis thalianafor which transgenic materials can relatively quickly be generated. We targetedM. persicae Rack1, which is predominantly expressed in the gut, andM. persicae C002(MpC002), which is predominantly expressed in the salivary glands. The aphids were fed onN. benthamianaleaf disks transiently producing dsRNA corresponding to these genes and onA. thalianaplants stably producing the dsRNAs.MpC002andRack-1expression were knocked down by up to 60% on transgenicN. benthamianaandA. thaliana. Moreover, silencedM. persicaeproduced less progeny consistent with these genes having essential functions. Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, theN. benthamianaleaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

[28]

KENNERDELL J

, CARTHEW R

. Heritable gene silencing in Drosophila using double-stranded RNA
Nature Biotechnology, 2000,18(8):896-898.

DOI:10.1038/78531 URLPMID:10932163 [本文引用: 1]

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

[29]

LEVIN D

, BREUER L

, ZHUANG

, ANDERSON S

, NARDI J

, KANOST M

. 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 URLPMID:15804572 [本文引用: 1]

http://linkinghub.elsevier.com/retrieve/pii/S0965174805000044

[30]

SIVAKUMAR

, RAJAGOPAL

, VENKATESH G

, SRIVASTAVA

, BHATNAGAR R

. 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 URL [本文引用: 1]

[31]

SHANG

, NIU J

, DING B

, ZHANG

, YE

, ZHANG

, SMAGGHE

, WANG 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 URLPMID:29226991 [本文引用: 1]

Abstract Vitellogenin (Vg) and its receptor (VgR) play a key role in the reproductive process and development of insects. Aphids are a group of high-fecundity insect species with pseudoplacental viviparity, but the roles of their Vg and VgR genes have not been investigated yet. The brown citrus aphid, Aphis ( Toxoptera ) citricidus , is a major insect pest of citrus and the main vector of Citrus tristeza closterovirus . In this study, we identified and characterized these two genes, designated as AcVg and AcVgR , from the brown citrus aphid. We found that AcVg has lost the DUF1943 domain that is present in other insect Vgs. Silencing of AcVg and AcVgR led to a delay in the nymph dult transition, a prolonged prereproductive period, and a shortened reproductive period, which in turn resulted in slower embryonic development and fewer new-born nymphs. Interestingly, silencing of AcVg decreased the transcript level of AcVgR , but silencing of AcVgR resulted in increased transcript levels of AcVg . In addition, silencing of Vg / VgR had similar phenotypes between alate and apterous morphs, suggesting that the functions of these two genes are the same in the two wing morphs of the aphid. Our results demonstrate that Vg and VgR are involved in various aspects of aphid development and reproduction. Further studies on the synthesis of Vg could help to elucidate the reproductive mechanism and provide information that will be useful for developing new pest control strategies.