,1, 徐彩娣1,3Potential Functions of Nilaparvata lugens GSK-3 in Regulating Glycogen and Trehalose Metabolism
DING YanJuan1, LIU YongKang1, LUO YuJia1, DENG YingMei1, XU HongXing2, TANG Bin,1, XU CaiDi1,3通讯作者:
收稿日期:2018-11-15接受日期:2018-12-29网络出版日期:2019-04-01
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Received:2018-11-15Accepted:2018-12-29Online:2019-04-01
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丁艳娟,E-mail：
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丁艳娟, 刘永康, 罗雨嘉, 邓颖梅, 徐红星, 唐斌, 徐彩娣. 褐飞虱GSK-3调控糖原与海藻糖代谢的潜在功能[J]. 中国农业科学, 2019, 52(7): 1237-1246 doi:10.3864/j.issn.0578-1752.2019.07.011
DING YanJuan, LIU YongKang, LUO YuJia, DENG YingMei, XU HongXing, TANG Bin, XU CaiDi.
0 引言
【研究意义】我国是水稻（Oryza sativa）种植大国,自1991年起,稻谷播种面积稳定在3 000万公顷左右,产量近年来基本维持在2.1亿吨。作为重要的粮食作物,水稻产量的稳定对我国民生具有非常重要的意义。但水稻在其生产以及储存过程经常遭到各种害虫的威胁。据报道,目前水稻害虫总数已经超过800种[1]。其中,褐飞虱（Nilaparvata lugens）是危害最为严重的一种水稻害虫。褐飞虱是单食性害虫,具有繁殖速度快、内禀增长率较高、生命周期很短、适应性强等特点[2,3,4,5]。另外,褐飞虱具有迁飞性,每年都会从东南亚迁飞到我国危害水稻生产,造成较广的危害范围,防治难度大[6]。因此,亟需寻找一种环保、高效的褐飞虱防治方法。【前人研究进展】海藻糖（trehalose）是一种普遍存在于低等植物、藻类、细菌、真菌、酵母、昆虫及其他无脊椎动物中的非还原性双糖[7,8],也是昆虫血淋巴中的重要能量物质,为昆虫进行一系列生命活动提供能源,并调控昆虫的生长、发育、蜕皮变态等过程,因此被称为昆虫的“血糖”[9]。昆虫体内的糖原以及海藻糖代谢直接影响到昆虫的一系列生命活动,在这个过程中,糖原合成酶激酶3（glycogen synthase kinase 3,简称GSK-3或GSK3）起到了至关重要的调控作用[10,11]。相关研究表明,在动物体内,糖原合成酶的主要功能便是在糖原合成的最后一步中催化尿苷二磷酸葡萄糖上的葡萄糖基C1在糖原的非还原性末端C4形成α-1,4-糖苷链,使原来的糖原分子增加一个葡萄糖单位。GSK-3可以通过使糖原合成酶磷酸化从而使糖原合成酶失活,抑制糖原合成最后一步的进行,并且还可以阻碍胰岛素信号通路的传导从而抑制糖原合成[12,13,14,15]。而糖原和海藻糖之间的转化密不可分,因此GSK-3的表达对糖原和海藻糖的合成代谢具有重要的影响。GSK-3通过使糖原合成酶磷酸化以及影响胰岛素信号通路从而抑制糖原的合成,而糖原与其他的糖类物质之间存在紧密的联系,它们可以通过酶的催化作用从而互转化。当昆虫体内GSK-3表达失常,其体内糖代谢过程便会失衡,从而昆虫的发育、蜕皮等一系列生命活动都会出现异常[10,16-20]。目前对GSK-3的调控作用也有一定的研究进展,比如Akt1可以通过调节GSK-3β活性来保证黑腹果蝇（Drosophila melanogaster）的雌性生殖系干细胞（germline stem cell）的维持[21]。【本研究切入点】前期研究表明GSK3β在各种信号传导途径中发挥着重要的作用,例如对果蝇的WNK神经起正调节作用[22]。但关于GSK-3与褐飞虱糖类物质代谢关系的研究鲜见报道,GSK-3在褐飞虱胰岛素信号通路中的作用也有待研究。【拟解决的关键问题】以重要的农业害虫褐飞虱的GSK-3为对象,研究其分子特性、时期表达特征,并探究褐飞虱体内GSK-3调控昆虫主要糖类物质代谢的功能,为将来通过调控昆虫“血糖”平衡或能量供应来控制害虫提供理论依据。1 材料与方法
试验于2017—2018年在杭州师范大学完成。1.1 供试昆虫及材料
褐飞虱采自中国水稻研究所,在本实验室饲养。水稻品种全部采用感虫水稻TN1（Taichung Native 1）。水稻种植步骤：首先将水稻种子浸入约70℃的温水中浸泡10 min左右,打破休眠;再将种子浸泡到自来水中放至于30℃人工气候培养箱泡种24 h;随后倒去泡种的水,自来水冲洗数次后用湿纱布包裹种子,置于30℃人工气候培养箱催芽24—48 h,种子发芽后播种于塑料盆;适当施肥促进小苗生长,待长至10 cm左右后,转移至大田插秧;待水稻生长至分蘖中期后将其移至养虫笼,每隔2—3 d更换一次水稻。褐飞虱饲养条件：温度（26±1）℃,光周期16 h/8 h,相对湿度为70%。从4龄若虫至发育为成虫后3 d的褐飞虱虫体,每12 h取材,用于发育表达模式研究。用于后期注射试验的材料均取5龄若虫后的褐飞虱。1.2 主要试剂
Trizol试剂盒（购自Lifetech Scientific Corporation）;pMD18-T、AMV反转录试剂盒、6×Loading buffer、DNA Marker DL2000及实时荧光定量PCR试剂（购自大连TaKaRa公司）;PCR引物(上海英潍捷基有限公司合成);T7 RiboMax Express RNAiSystem（Promega,Madison,USA）;氯仿、异丙醇、EDTA等常规试剂（国药集团化学试剂有限公司）;实时荧光定量PCR96孔透明板和光学粘性封膜等耗材（Bio-RAD,USA）。1.3 试验方法
1.3.1 GSK-3 cDNA序列分析 根据褐飞虱转录组中获得的GSK-3 cDNA编码序列,克隆测序后,利用NCBI中的ORF Finder（Open Reading Frame Finder）（1.3.2 总RNA抽提及cDNA合成 利用Trizol试剂盒并按照说明书提取褐飞虱虫体的总RNA。提取后用1%的琼脂糖凝胶电泳检测总RNA的质量,然后利用NanoDrop 2000分光光度计测定RNA的浓度及纯度。使用Prime Script?RT Reagent Kit with gDNA Eraser试剂盒配置体系并合成cDNA第一链。
1.3.3 dsRNA的合成 根据GSK-3 dsRNA特异性片段设计并合成特异性引物,进行PCR扩增,扩增产物再进行T克隆,随后用带T7启动子的引物进行交叉PCR反应,相关引物序列见表1。根据T7 RiboMAXTM Express RNAi System试剂盒的说明进行dsGSK-3的合成,随后采用NanoDropTM 2000分光光度计测定dsGSK-3的浓度,采用同样的方法合成GFP的dsRNA作为对照组[11]。
Table 1
表1
表1dsRNA合成及荧光定量PCR引物
Table 1
| 引物名称Primer name | 引物序列Primer sequence (5′-3′) | 引物名称Primer name | 引物序列Primer sequence (5′-3′) | |
|---|---|---|---|---|
| GSK-3 | F: GGAAAGTTGAATCAAAGTGCTCG R: AGGCTTTTGCCAGGGATG | qNlIlp1 | F: AACGATGCTGACTTGCAGATT R: CGTACACGCGGAATAAATCA | |
| dsGSK-3 | F: T7-CTGCGACAGCGGCGAAATG R: T7-CGGTGACAGATGCCCAGCGAGT | qNlIlp2 | F: TTCTCAGCCGCTCTAGCAAT R: CAGACGAAGGATCAGGGAAG | |
| dsGFP | F: T7-AAGGGCGAGGAGCTGTTCACCG R: T7-CAGCAGGACCATGTGATCGCGC | qNlIlp3 | F: ATACTGCGGCCAATAGCAAG R: TCTCAATCCCCAAAATCAGC | |
| qNlTRE1-1 | F: GCCATTGTGGACAGGGTG R: CGGTATGAACGAATAGAGCC | qNlIlp4 | F: TCCCGGACAGTTCTCACTTT R: TTGTATTCTCCGGAGGCAAG | |
| qNlTRE1-2 | F: GATCGCACGGATGTTTA R: AATGGCGTTCAAGTCAA | qNlTPS1 | F: AAGACTGAGGCGAATGGT R: AAGGTGGAAATGGAATGTG | |
| qNlTRE2 | F: TCACGGTTGTCCAAGTCT R: TGTTTCGTTTCGGCTGT | qNlTPS2 | F: AGAGTGGACCGCAACAACA R: TCAACGCCGAGAATGACTT | |
| qNlInR1 | F: GAGTGCAACCCGGAGTATGT R: TCTTGACGGCACACTTCTTG | qNlGP | F: GCTGCCTATGGCTATGGTATTC R: TCTGAGTGTTGACCCACTTCTTG | |
| qNlInR2 | F: CTCTTGCCGAACAGCCTTAC R: GGGTCGTTTAGTGGGTCTGA | qNlGS | F: GCTCCAAAGCCTATGTTTCTACTG R: TGGTAACCCCTGTCCCTCA | |
| T7 | GGATCCTAATACGACTCACTATAGG | qNl18S | F: CGCTACTACCGATTGAA R: GGAAACCTTGTTACGACTT |
新窗口打开|下载CSV
1.3.4 褐飞虱的显微注射 用于显微注射的材料分为dsGSK-3以及作为对照的dsGFP。在注射前向标准毛细管注射dsRNA确定显微注射器每次泵出dsRNA的体积,然后通过调整氮气压来调整泵出dsRNA的体积,使其泵出体积符合注射所需量。将用CO2麻醉后的5龄褐飞虱虫体腹部朝上放置于有琼脂胶板的一次性培养皿中,注射部位为第一对足中间偏下较软部位。
取5龄第1天的褐飞虱用于显微注射,注射量均为200 ng/头,每个处理组注射100头褐飞虱。注射后48 h取材,用于海藻糖、葡萄糖、糖原含量和海藻糖酶活性测定,以及相关基因表达量测定。
1.3.5 GSK-3发育表达模式及RNAi后海藻糖通路、胰岛素信号通路相关基因表达量测定 从4龄若虫至发育为成虫后3 d的褐飞虱虫体,每12 h取材,用于发育表达模式研究;取注射后48 h褐飞虱用于检测海藻糖通路及胰岛素信号通路关键基因表达量。采取平行取样,即每个处理组取3管,每管5头褐飞虱,最后每个样品得到3管平行cDNA,放置于-80℃冰箱保存。试验时,再进行重复点样,即每管cDNA做3个定量,3管平行cDNA 共得到9个数据,每个样品为平均值±标准误,保证数据的可靠性。qRT-PCR反应体系（20 μL）：10 μL SYBR Premix Ex Taq;1 μL上游/下游引物;1 μL cDNA;7 μL灭菌超纯水。定量引物见表1。反应程序：95℃预变性10 s,95℃解链5 s,59℃退火并延伸30 s,40个循环。
1.3.6 海藻糖、葡萄糖、总糖原含量以及海藻糖酶活性测定 取每个处理及对照组材料,加入100 μL PBS,研磨,再加100 μL PBS,超声破碎至无块状组织,破碎后加入800 μL PBS,4℃,1 000×g离心20 min。取350 μL上清,4℃,20 800×g超离心60 min。超离心后的上清用于葡萄糖、可溶性海藻糖酶（TRE1）和蛋白质（Pr1）的测定,沉淀用PBS悬浮后用于葡萄糖、膜结合海藻糖酶（TRE2）和蛋白质（Pr2）的测定,具体步骤按照试剂盒说明操作。
1.3.7 数据分析 取3个重复孔的平均CT值用于计算,即最后得到的数据为平均值±标准误。再带入2-ΔΔCT公式计算,对照组为褐飞虱注射dsGFP组的CT值。2-ΔΔCT计算公式：
2-ΔΔCT= 2-[(CT实验组-CT实验18S)- (CT对照组-CT对照18S)]
应用Excel软件绘制图表,并使用STATISTICA 8.0和SigmaPlot 10.0进行统计分析,采用One-Way ANOVA法进行差异显著性检验（P<0.05为差异显著,用*表示;P<0.01为差异极显著,用**表示）。
2 结果
2.1 GSK-3的序列结构特征
根据褐飞虱转录组数据及克隆验证后,发现褐飞虱GSK-3 cDNA开放阅读框全长1 914 bp,编码637个氨基酸,预测蛋白分子量为69.25 kD,等电点为9.15,为偏碱性蛋白,无信号肽结构（图1）。GSK-3蛋白含有20种氨基酸,其中丝氨酸（Ser）、甘氨酸（Gly）、亮氨酸（Leu）和天冬酰胺（Asn）等含量较丰富,所占比例分别为11.1%、8.0%、7.7%和7.5%。酸性氨基酸（带负电荷）为46个,碱性氨基酸（带正电荷）为78个。图1
新窗口打开|下载原图ZIP|生成PPT图1褐飞虱GSK-3的核苷酸和氨基酸序列
Fig. 1Nucleotide and amino acid sequence of N. lugens GSK-3
2.2 褐飞虱GSK-3发育表达模式
褐飞虱GSK-3在不同发育阶段的表达量不同,在4龄初期和末期、5龄48 h和成虫36 h表达量相对较高;在4龄24 h、5龄的初期及末期表达量相对较低（图2）。图2
新窗口打开|下载原图ZIP|生成PPT图2褐飞虱GSK-3发育表达情况
GSK-3在褐飞虱4龄（第1—4天）、 5龄（第1—7天）和成虫早期（第1—4天）不同发育天数的表达
柱上不同小写字母表示差异显著（P<0.05）
Fig. 2Relative expression level of GSK-3 in different developmental stages of N. lugens
The relative expression of GSK-3 in different days of 4th instar nymphs (day 1 to day 4), 5th instar nymphs (day 1 to day 7), and early stages of adult (day 1 to day 4)
Different lowercases on the bars indicate significant difference (P<0.05)
2.3 RNAi后GSK-3的表达
体外合成GSK-3的dsRNA注射到褐飞虱体内后,与注射dsGFP组相比较注射后48 h其mRNA表达水平极显著下降（图3）,该结果表明RNAi有效抑制了GSK-3的表达。图3
新窗口打开|下载原图ZIP|生成PPT图3RNAi后GSK-3的表达情况
Fig. 3Relative expression level of N. lugens GSK-3 after RNA interference
2.4 GSK-3 RNAi后对糖原及葡萄糖含量的影响
与注射dsGFP组相比较,GSK-3的dsRNA 注射后48 h时糖原含量极显著下降（P<0.01,图4-A）,葡萄糖含量下降,但无显著差异（P>0.05,图4-B）。图4
新窗口打开|下载原图ZIP|生成PPT图4GSK-3 RNAi后对糖原及葡萄糖含量的影响
Fig. 4Effect of GSK-3 RNA interference on glycogen and glucose contents of N. lugens
2.5 GSK-3 RNAi后对褐飞虱海藻糖代谢的影响
RNAi抑制GSK-3表达后48 h,海藻糖含量及海藻糖酶活性检测结果显示,与注射dsGFP的对照组相比,抑制后48 h褐飞虱海藻糖含量显著上升（P<0.05,图5-A）,可溶性和膜结合型两类海藻糖酶活性均显著下降（P<0.05,图5-B、5-C）。图5
新窗口打开|下载原图ZIP|生成PPT图5GSK-3 RNAi后对褐飞虱海藻糖代谢的影响
Fig. 5Effect of GSK-3 RNA interference on trehalase metabolism of N. lugens
2.6 GSK-3 RNAi后褐飞虱海藻糖代谢通路及胰岛素信号通路中相关基因的表达变化
采用RNAi抑制褐飞虱GSK-3的表达后,与注射dsGFP相比较,qRT-PCR检测结果显示褐飞虱胰岛素信号通路及海藻糖代谢通路相关基因中的TRE、InR、Ilp、TPS、GP、GS表达量均显著或极显著下降（图6）。图6
新窗口打开|下载原图ZIP|生成PPT图6GSK-3 RNAi后褐飞虱海藻糖代谢通路及胰岛素信号通路相关基因的相对表达水平
Fig. 6Relative expression level of regulated genes of trehalose metabolism pathway and insulin signaling pathway after GSK-3 RNA interference
3 讨论
昆虫中GSK-3主要作为糖原合成的一个重要限速酶,其序列相对比较保守[23]。本研究通过对GSK-3序列的结构特征进行分析,发现GSK-3为偏碱性蛋白,无信号肽结构,序列高度保守。前期研究发现褐飞虱海藻糖代谢途径中的TPS和TRE表达被抑制后,糖原的合成也同时受到影响,多表现为糖原含量下降[24],并且能够直接影响昆虫几丁质合成与蜕皮[25]。并且当糖原合成酶（GS）和糖原磷酸化酶（GP）表达受到抑制后,能够介导海藻糖代谢调控几丁质合成途径（待发表）。相关研究结果显示,家蚕幼虫在取食期GSK-3α mRNA表达量相对较高,在蜕皮期表达量较低,在幼虫游走期表达量最低[23]。而且,褐飞虱蜕皮激素响应相关基因与其龄期表达模式有密切关系,对其蜕皮过程至关重要[26]。本研究中,褐飞虱GSK-3在5龄初期和末期表达量相对较低。褐飞虱不仅是一种重要的农业害虫,随着其基因组测序的完成[27],发现其非常适合作为基因功能研究的靶标昆虫[4-5,15]。RNA干扰是基因功能研究的有效工具,主要是通过显微注射dsRNA或siRNA抑制基因的表达[4],该技术已经广泛用于探索和研究昆虫的相关基因功能,当昆虫的关键发育基因被抑制后,其影响通常会表现在不同组织的表型上[5,28-31]。本试验中,褐飞虱GSK-3 dsRNA注射后48 h,该基因的表达与注射dsGFP相比较,表达量极显著下降,表明RNA干扰效果明显。在前期研究中发现GSK-3参与胰岛素、Wnt/β-连环蛋白、Hedgehog以及Notch等信号传导通路,在调控细胞的分化、代谢、凋亡以及基因表达等方面都起着重要作用[32]。GSK-3主要通过胰岛素受体底物-1（IRS-1）的Ser332位点实现对胰岛素信号通路的阻碍,从而抑制糖原合成[33]。本研究发现,GSK-3被抑制后48 h糖原和葡萄糖含量极显著降低或下降,表明GSK-3能够调控糖原和葡萄糖的合成。GSK-3还能通过糖原合成酶磷酸化以及影响胰岛素信号通路从而抑制糖原的合成,而糖原与其他的糖类物质之间存在紧密的联系,它们可以通过酶的催化作用从而相互转化[10]。这一点从本研究中也得到了验证,当GSK-3表达被抑制后,糖原和葡萄糖含量降低,转化为海藻糖而导致含量显著上升,作为其生理活动的能量来源。上述研究结果表明,GSK-3在多种细胞信号通路中都起到关键性的调节作用[28]。
GSK-3广泛存在于动物体内,主要在动物脑组织中表达,负责信号的传递[11,34-36]。在家蚕幼虫中注射家蚕素II时,其体内海藻糖浓度会降低,且海藻糖酶的活性得到提高;同时还能降低糖原含量和提高糖原酸化酶的活性[37],但是当家蚕成虫注射家蚕素时,其血淋巴中的海藻糖或脂质水平却没有受到影响[38]。本研究发现,当GSK-3表达抑制后48 h,TRE1-1及TRE2的表达极显著下降,同时,2种海藻糖酶活性也显著下降,海藻糖含量上升,表现出一致性。另外,2个TPS的表达也极显著下降,同时GSK-3被抑制后48 h能够极显著降低或降低糖原、葡萄糖含量,这与前期相关的研究结果一致[39],说明GSK-3可以通过调控TRE活性变化和TRE表达来影响3种糖类物质含量。相关研究报道胰岛素进入昆虫体内后,先激活Ilp和InR,再激活磷脂酰肌醇激酶（PI3K）,活化的PI3K催化磷脂酰肌醇的磷酸化,接着引起效应器Akt的磷酸化,并丧失磷酸化糖原合成激酶（GSK-3）活性,抑制其对糖原合成酶（GS）的磷酸化作用,促使GS变为有活性的分子,从而促进糖原的合成[15]。从本试验来看,GSK-3表达抑制后,降低了GS及GP的表达量,从而影响糖原及其他糖类物质的合成,即GSK-3表达抑制后能够下调胰岛素通路中的相关基因,从而调控海藻糖等糖类物质代谢。而GSK-3抑制后,葡萄糖含量变化不显著,推测是因为褐飞虱体内还存在其他糖代谢途径,例如胰岛素信号通路的其他基因对褐飞虱糖代谢的调控等。对于胰岛素途径中的InR和Ilp,当GSK-3的表达被抑制后,2个InR和4个Ilp的表达同样被抑制,间接表明InR能够调控GSK-3的表达,其具体机制有待于进一步研究。
4 结论
GSK-3 RNA干扰后可以有效抑制褐飞虱体内靶标基因的表达;GSK-3能够降低海藻糖和糖原代谢相关基因的表达及海藻糖酶活性,从而降低糖原和葡萄糖含量,提高海藻糖含量,并调控昆虫海藻糖平衡。（责任编辑 岳梅）
参考文献 原文顺序
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被引期刊影响因子
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DOI:10.3969/j.issn.1001-411X.2005.02.013URL [本文引用: 1]
The chromatographic peak distinction of 13 secondary compounds from two types of rice varieties were investigated by using high-performance liquid chromatography(HPLC). The result of multiple regression analysis was showed that peak 1, peak 2, peak 8 and peak 12 were major secondary compounds to affect the variety resistance to brown planthopper (BPH, Nilaparvata lugens) biotype II, whereas peak 3, peak 4, peak 5, peak 9, peak 11 and peak 12 were major secondary compounds to Bangladesh biotype of BPH. Two regression equations among the area values of the chromatographic peaks and the BPH-resistant level of rice variety to different biotypes were obtained. It was demonstrated that the resistant activity of rice varieties to different BPH biotypes was closely associated with qualitative and quantitative combinations of the secondary compounds. These secondary compounds were suggested to be used as the marks to evaluated and identified the rice variety resistance to different biotypes of BPH.
DOI:10.3969/j.issn.1001-411X.2005.02.013URL [本文引用: 1]
The chromatographic peak distinction of 13 secondary compounds from two types of rice varieties were investigated by using high-performance liquid chromatography(HPLC). The result of multiple regression analysis was showed that peak 1, peak 2, peak 8 and peak 12 were major secondary compounds to affect the variety resistance to brown planthopper (BPH, Nilaparvata lugens) biotype II, whereas peak 3, peak 4, peak 5, peak 9, peak 11 and peak 12 were major secondary compounds to Bangladesh biotype of BPH. Two regression equations among the area values of the chromatographic peaks and the BPH-resistant level of rice variety to different biotypes were obtained. It was demonstrated that the resistant activity of rice varieties to different BPH biotypes was closely associated with qualitative and quantitative combinations of the secondary compounds. These secondary compounds were suggested to be used as the marks to evaluated and identified the rice variety resistance to different biotypes of BPH.
DOI:10.1111/j.1570-7458.2008.00785.xURL [本文引用: 1]
The fine structure of the salivary sheaths in plant tissues can provide important information on homopteran probing and ingestion behaviors. Salivary sheaths secreted by the brown planthopper (BPH), Nilaparvata lugens (St氓l) (Homoptera: Delphacidae), and their tissue pathway were investigated using light, scanning electron, and transmission electron microscopy. About half of the salivary flanges on the surface of the food substrate were connected with internal salivary sheaths. Only 43% of the salivary sheaths showed side branches. Many sculpture-like protuberances and small cavities had been formed on the outer surface of the salivary sheath, but the sheath lumen circumferences were sealed. Brown planthoppers showed a preference for probing and leaving salivary sheaths in the susceptible rice variety TN1 rather than in the resistant variety B5 during the first 2 days of the experiments. The salivary sheaths in rice tissues reached the inner tissue layer of the leaf sheaths and stems, but were mostly observed to end in the first and second layer of the leaf sheaths. Brown planthoppers also preferred to probe into the thick segment of the outer leaf sheath. After ingestion by the insect, the cytoplasm in both phloem and companion cells degraded and the main organelles were lost. Numerous small vesicles were found in most of the phloem cells, but cell walls remained intact. Large numbers of symbiont-like structures were observed inside the salivary sheath lumen. These results indicated that BPH has complicated feeding behaviors, which warrants further investigation.
[本文引用: 3]
[本文引用: 3]
DOI:10.1371/journal.pone.0022137URLPMID:3136512 [本文引用: 1]
The brown planthopper (BPH)Nilaparvata lugens(Stal) is a serious pest of rice in Asia. Development of novel control strategies can be facilitated by comparison of BPH feeding behaviour on varieties exhibiting natural genetic variation, and then elucidation of the underlying mechanisms of resistance. BPH feeding behaviour was compared on 12 rice varieties over a 12 h period using the electrical penetration graph (EPG) and honeydew clocks. Seven feeding behaviours (waveforms) were identified and could be classified into two phases. The first phase involved patterns of sieve element location including non penetration (NP), pathway (N1+N2+N3), xylem (N5)[21]and two new feeding waveforms, derailed stylet mechanics (N6) and cell penetration (N7). The second feeding phase consisted of salivation into the sieve element (N4-a) and sieve element sap ingestion (N4-b). Production of honeydew drops correlated with N4-b waveform patterns providing independent confirmation of this feeding behaviour. Overall variation in feeding behaviour was highly correlated with previously published field resistance or susceptibility of the different rice varieties: BPH produced lower numbers of honeydew drops and had a shorter period of phloem feeding on resistant rice varieties, but there was no significant difference in the time to the first salivation (N4-b). These qualitative differences in behaviour suggest that resistance is caused by differences in sustained phloem ingestion, not by phloem location. Cluster analysis of the feeding and honeydew data split the 12 rice varieties into three groups: susceptible, moderately resistant and highly resistant. The screening methods that we have described uncover novel aspects of the resistance mechanism (or mechanisms) of rice to BPH and will in combination with molecular approaches allow identification and development of new control strategies.
DOI:10.1038/s41598-017-01754-9URLPMID:5431645 [本文引用: 1]
Trehalose is a non-reducing disaccharide that serves as the main sugar component of haemolymph in insects. Trehalose hydrolysis enzyme, called trehalase, is highly conserved from bacteria to humans. However, our understanding of the physiological role of trehalase remains incomplete. Here, we analyze the phenotypes of severalTrehalase(Treh) loss-of-function alleles in a comparative manner inDrosophila. The previously reported mutant phenotype ofTrehaffecting neuroepithelial stem cell maintenance and differentiation in the optic lobe is caused by second-site alleles in addition toTreh. We further report that the survival rate ofTrehnull mutants is significantly influenced by dietary conditions.Trehmutant larvae are lethal not only on a low-sugar diet but also under low-protein diet conditions. A reduction in adaptation ability under poor food conditions inTrehmutants is mainly caused by the overaccumulation of trehalose rather than the loss ofTreh, because the additional loss ofTps1mitigates the lethal effect ofTrehmutants. These results demonstrate that proper trehalose metabolism plays a critical role in adaptation under various environmental conditions.
[本文引用: 1]
[本文引用: 1]
DOI:10.3969/j.issn.0452-8255.2008.05.035 [本文引用: 1]
海藻糖（trehalose）是由2个葡萄糖分子通过α,α-1,1糖苷键连接的一种非还原性双糖。海藻糖作为昆虫的血糖,对于生物的能量代谢和抗逆等方面具有重要的作用。文章从昆虫海藻糖的发现、海藻糖的化学性质、昆虫中海藻糖的生理作用、代谢途径等方面进行综述,并对昆虫中海藻糖的进一步研究作了展望。
DOI:10.3969/j.issn.0452-8255.2008.05.035 [本文引用: 1]
海藻糖（trehalose）是由2个葡萄糖分子通过α,α-1,1糖苷键连接的一种非还原性双糖。海藻糖作为昆虫的血糖,对于生物的能量代谢和抗逆等方面具有重要的作用。文章从昆虫海藻糖的发现、海藻糖的化学性质、昆虫中海藻糖的生理作用、代谢途径等方面进行综述,并对昆虫中海藻糖的进一步研究作了展望。
DOI:10.1124/jpet.102.047381URLPMID:12626660 [本文引用: 3]
Abstract Glycogen synthase kinase-3 (GSK-3) was shown to be a key factor in attenuation of the cellular action of insulin. We speculated that inhibition of GSK-3 might have a potential therapeutic value in treatment of insulin resistance and type 2 diabetes. Here, we present a novel class of specific phosphorylated peptides inhibitors of GSK-3, which in sharp contrast to other protein kinase inhibitors that are ATP analogs, are substrate-competitive. We show that the GSK-3 peptide inhibitor activated glycogen synthase activity 2.5-fold in human embryonic kidney 293 cells, and increased glucose uptake in primary mouse adipocytes in the absence or presence of insulin compared with cells treated with two respective peptide controls. In addition, an i.p. administration of GSK-3 peptide inhibitor to normal or insulin-resistant obese C57BL/6J mice, improved their performance on glucose tolerance tests compared with control-treated animals. We present here a novel rational strategy for developing specific GSK-3 inhibitors and point toward GSK-3 as a promising therapeutic target in insulin resistance and type-2 diabetes.
DOI:10.1016/j.ejmech.2015.10.018URLPMID:26562543 [本文引用: 3]
Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis invivo as well as invitro . Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition.
[本文引用: 1]
DOI:10.1016/j.abb.2016.03.020URLPMID:27036853 [本文引用: 1]
61Glycogen synthase incorporates β-phosphate of UDP-glucose into glycogen.6132P-labeling of glycogen using [β-32P]UDP-glucose as substrate monitors the reaction.61The32P associated with glycogen is not due to the non-covalent binding of [β-32P]UDP.
DOI:10.7666/d.y1557094URL [本文引用: 1]
目的通过研究Akt/GSK- 3β信号通路与高血压所致心肌肥厚的关系及厄贝沙坦的干预作用,探讨ARB逆转心肌肥厚的新机制。方法选择32例高血压所致心肌肥厚患者作为观察组,予厄 贝沙坦治疗,测定治疗前后的左室重量(LVM)及左室重量指数(LVMI),采用ELISA方法测定Akt及GSK-3β治疗前后的表达并进行比较。选择 30例条件匹配的健康人作为对照组。结果1.观察组与对照组(药物干预治疗前)Akt及GSK-3β的磷酸化水平测定结果提示:Akt观察组明显升高(观 察组vs对照组:3.38±1.08vs0.95±0.14,P0.05);GSK-3β明显降低(观察组0.29±0.14vs对照组 0.04±0.05,P0.05),提示高血压左室肥厚患者Akt/GSK-3β表达存在显著差异;2.观察组经厄贝沙坦治疗前后Akt及GSK-3β的 磷酸化水平有显著性差异:Akt表达降低(治疗前3.38±1.08vs治疗后2.56±0.87),GSK-3β表达增高(治疗前 0.30±0.14vs治疗后0.64±0.28),均P0.05;3.线性回归显示Akt及GSK-3β表达与LVM相关,且前者成正相关,后者呈负相 关。结论1.Akt/GSK-3β信号通路与高血压所致心肌肥厚具相关性,Akt表达呈正相关,GSK-3β表达呈负相关;2.厄贝沙坦治疗后该信号通路 表达改变具有显著性差异,提示厄贝沙坦可能通过作用于Akt/GSK-3β信号通路逆转左室肥厚。
DOI:10.7666/d.y1557094URL [本文引用: 1]
目的通过研究Akt/GSK- 3β信号通路与高血压所致心肌肥厚的关系及厄贝沙坦的干预作用,探讨ARB逆转心肌肥厚的新机制。方法选择32例高血压所致心肌肥厚患者作为观察组,予厄 贝沙坦治疗,测定治疗前后的左室重量(LVM)及左室重量指数(LVMI),采用ELISA方法测定Akt及GSK-3β治疗前后的表达并进行比较。选择 30例条件匹配的健康人作为对照组。结果1.观察组与对照组(药物干预治疗前)Akt及GSK-3β的磷酸化水平测定结果提示:Akt观察组明显升高(观 察组vs对照组:3.38±1.08vs0.95±0.14,P0.05);GSK-3β明显降低(观察组0.29±0.14vs对照组 0.04±0.05,P0.05),提示高血压左室肥厚患者Akt/GSK-3β表达存在显著差异;2.观察组经厄贝沙坦治疗前后Akt及GSK-3β的 磷酸化水平有显著性差异:Akt表达降低(治疗前3.38±1.08vs治疗后2.56±0.87),GSK-3β表达增高(治疗前 0.30±0.14vs治疗后0.64±0.28),均P0.05;3.线性回归显示Akt及GSK-3β表达与LVM相关,且前者成正相关,后者呈负相 关。结论1.Akt/GSK-3β信号通路与高血压所致心肌肥厚具相关性,Akt表达呈正相关,GSK-3β表达呈负相关;2.厄贝沙坦治疗后该信号通路 表达改变具有显著性差异,提示厄贝沙坦可能通过作用于Akt/GSK-3β信号通路逆转左室肥厚。
[D].
[本文引用: 3]
[D].
[本文引用: 3]
DOI:10.11924/j.issn.1000-6850.casb15120111URL [本文引用: 1]
为了更好地指导海藻糖基因工程菌株的构建,进一步提高海藻糖的产量,综述了国内外近30年,尤其是最近10年海藻糖合成途径及分子生物学方面的研究,表明不同的生物中共存在5条海藻糖合成途径,虽然各途径中的酶与基因已基本分离到,工程菌株的构建也渐次展开,但大部分工程菌局限于用来研究途径中单一酶的理化性质,对海藻糖产量提高方面的研究还存在不足。随着现代生物技术尤其是DNA重组技术、宏基因组学与合成生物学的迅速发展,构建工程菌进行异源高效表达将成为今后海藻糖研究的一个热点。
DOI:10.11924/j.issn.1000-6850.casb15120111URL [本文引用: 1]
为了更好地指导海藻糖基因工程菌株的构建,进一步提高海藻糖的产量,综述了国内外近30年,尤其是最近10年海藻糖合成途径及分子生物学方面的研究,表明不同的生物中共存在5条海藻糖合成途径,虽然各途径中的酶与基因已基本分离到,工程菌株的构建也渐次展开,但大部分工程菌局限于用来研究途径中单一酶的理化性质,对海藻糖产量提高方面的研究还存在不足。随着现代生物技术尤其是DNA重组技术、宏基因组学与合成生物学的迅速发展,构建工程菌进行异源高效表达将成为今后海藻糖研究的一个热点。
DOI:10.1186/1471-2148-6-109URL
DOI:10.16380/j.kcxb.2015.10.002URLMagsci
【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 <em>Blattella germanica </em>TPS基因，研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列，利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因，分别命名为 <em>BgTPS</em>1 (GenBank登录号：KR050213) 和 <em>BgTPS</em>2 (GenBank登录号：KR050214)。其中，<em>BgTPS</em>1基因cDNA序列全长2 987 bp，开放阅读框 (ORF) 2 502 bp，编码833个氨基酸；<em>BgTPS</em>2基因cDNA序列全长3 212 bp，开放阅读框2 469 bp，编码822个氨基酸。<em>BgTPS</em>1和<em>BgTPS</em>2基因都主要在5龄幼虫脂肪体中表达，且<em>BgTPS</em>2基因的表达量为<em>BgTPS</em>1基因表达量的3.9倍。在两种不同极端温度诱导下，<em>BgTPS</em>1和<em>BgTPS</em>2基因mRNA均上调表达。其中，<em>BgTPS2 </em>的表达量始终显著高于 <em>BgTPS</em>1。在0℃时，<em>BgTPS</em>1和<em>BgTPS</em>2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达，且<em>BgTPS</em>2基因的表达量显著高于<em>BgTPS</em>1基因；低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。
DOI:10.16380/j.kcxb.2015.10.002URLMagsci
【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 <em>Blattella germanica </em>TPS基因，研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列，利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因，分别命名为 <em>BgTPS</em>1 (GenBank登录号：KR050213) 和 <em>BgTPS</em>2 (GenBank登录号：KR050214)。其中，<em>BgTPS</em>1基因cDNA序列全长2 987 bp，开放阅读框 (ORF) 2 502 bp，编码833个氨基酸；<em>BgTPS</em>2基因cDNA序列全长3 212 bp，开放阅读框2 469 bp，编码822个氨基酸。<em>BgTPS</em>1和<em>BgTPS</em>2基因都主要在5龄幼虫脂肪体中表达，且<em>BgTPS</em>2基因的表达量为<em>BgTPS</em>1基因表达量的3.9倍。在两种不同极端温度诱导下，<em>BgTPS</em>1和<em>BgTPS</em>2基因mRNA均上调表达。其中，<em>BgTPS2 </em>的表达量始终显著高于 <em>BgTPS</em>1。在0℃时，<em>BgTPS</em>1和<em>BgTPS</em>2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达，且<em>BgTPS</em>2基因的表达量显著高于<em>BgTPS</em>1基因；低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。
DOI:10.1111/j.1742-4658.2010.07811.xURLPMID:3037560
Larvae of an anhydrobiotic insect, Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTps/) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTps did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis.
DOI:10.1093/glycob/cwu125URLPMID:25429048 [本文引用: 1]
Trehalose, a non-reducing disaccharide, is widespread throughout the biological world. It is the major blood sugar in insects playing a crucial role as an instant source of energy and in dealing with abiotic stresses. The hydrolysis of trehalose is under the enzymatic control of trehalase. The enzyme trehalase is gaining interest in insect physiology as it regulates energy metabolism and glucose generation via trehalose catabolism. The two forms of insect trehalase namely, Tre-1 and Tre-2, are important in energy supply, growth, metamorphosis, stress recovery, chitin synthesis and insect flight. Insect trehalase has not been reviewed in depth and the information available is quite scattered. The present mini review discusses our recent understanding of the regulation, mechanism and biochemical characterization of insect trehalase with respect to its physiological role in vital life functions. We also highlight the molecular and biochemical properties of insect trehalase that makes it amenable to competitive inhibition by most glycosidase inhibitors. Due to its crucial role in carbon metabolism in insects, application of inhibitors against trehalose can form a promising area towards formulating strategies for insect pest control.
DOI:10.1016/j.ydbio.2018.04.028URLPMID:29729259 [本文引用: 1]
Abstract Tissue-specific stem cells are tied to the nutritional and physiological environment of adult organisms. Adipocytes have key endocrine and nutrient-sensing roles and have emerged as major players in relaying dietary information to regulate other organs. For example, previous studies in Drosophila melanogaster revealed that amino acid sensing as well as diet-dependent metabolic pathways function in adipocytes to influence the maintenance of female germline stem cells (GSCs). How nutrient-sensing pathways acting within adipocytes influence adult stem cell lineages, however, is just beginning to be elucidated. Here, we report that insulin/insulin-like growth factor signaling in adipocytes promotes GSC maintenance, early germline cyst survival, and vitellogenesis. Further, adipocytes use distinct mechanisms downstream of insulin receptor activation to control these aspects of oogenesis, all of which are independent of FOXO. We find that GSC maintenance is modulated by Akt1 through GSK-3, early germline cyst survival is downstream of adipocyte Akt1 but independent of GSK-3, and vitellogenesis is regulated through an Akt1-independent pathway in adipocytes. These results indicate that, in addition to employing different types of nutrient sensing, adipocytes can use distinct axes of a single nutrient-sensing pathway to regulate multiple stages of the GSC lineage in the ovary.
DOI:10.1371/journal.pone.0193204URL [本文引用: 1]
The with no lysine (WNK) protein kinase family is conserved among many species. Some mutations in human WNK gene are associated with pseudohypoaldosteronism type II, a form of hypertension, and hereditary sensory and autonomic neuropathy type 2A. In kidney, WNK regulates the activity of STE20/SPS1-related, proline alanine-rich kinase and/or oxidative-stress responsive 1, which in turn regulate ion co-transporters. The misregulation of this pathway is involved in the pathogenesis of pseudohypoaldosteronism type II. In the neural system, WNK is involved in the specification of the cholinergic neuron, but the pathogenesis of hereditary sensory and autonomic neuropathy type 2A is still unknown. To better understand the WNK pathway, we isolated WNK-associated genes using Drosophila. We identified Glycogen synthase kinase 308 (GSK308)/Shaggy (Sgg) as a candidate gene that was shown to interact with the WNK signaling pathway in both Drosophila and mammalian cells. Furthermore, GSK308 was involved in neural specification downstream of WNK. These results suggest that GSK308/Sgg functions as a positive effector in the WNK signaling pathway.
URL [本文引用: 2]
【Objective】The amino acid sequence of Bombyx mori glycogen synthase kinase 3α (BmGSK3α) was analyzed in this study.The mRNA transcription level of BmGSK3α was further stuied in different developed stages and after molting hormone and starvation treatments.【Method】Temporal expression profiles of BmGSK3α,effects of hormone and starvation treatment on BmGSK3α transcript were investigated by using Real-time PCR methods.【Result】The sequence analysis of BmGSK3α found that BmGSK3α gene consists of 2 exons and one intron of 1 411 bp in size.The predicted molecular weight and pI of BmGSK3α are 33.78 ku and 7.61 respectively.Amino acid sequence alignments indicated that BmGSK3α shared high identity with other reported GSK proteins (from 79% to 91%).The expression level of BmGSK3α increased in larva feeding stage and decreased in molting stage.BmGSK3α transcript had no significant difference after 6 h treatment of 20E and increased after 12 h of 20E treatment then decreased gradually.Further starvation treatment found that starvation had limited influence on BmGSK3α expression.【Conclusion】BmGSK3α is conserved in evolution process of insects.The expression level of BmGSK3α is regulated by 20E and starvation has limited influence on BmGSK3α expression.
URL [本文引用: 2]
【Objective】The amino acid sequence of Bombyx mori glycogen synthase kinase 3α (BmGSK3α) was analyzed in this study.The mRNA transcription level of BmGSK3α was further stuied in different developed stages and after molting hormone and starvation treatments.【Method】Temporal expression profiles of BmGSK3α,effects of hormone and starvation treatment on BmGSK3α transcript were investigated by using Real-time PCR methods.【Result】The sequence analysis of BmGSK3α found that BmGSK3α gene consists of 2 exons and one intron of 1 411 bp in size.The predicted molecular weight and pI of BmGSK3α are 33.78 ku and 7.61 respectively.Amino acid sequence alignments indicated that BmGSK3α shared high identity with other reported GSK proteins (from 79% to 91%).The expression level of BmGSK3α increased in larva feeding stage and decreased in molting stage.BmGSK3α transcript had no significant difference after 6 h treatment of 20E and increased after 12 h of 20E treatment then decreased gradually.Further starvation treatment found that starvation had limited influence on BmGSK3α expression.【Conclusion】BmGSK3α is conserved in evolution process of insects.The expression level of BmGSK3α is regulated by 20E and starvation has limited influence on BmGSK3α expression.
[本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2017.06.006URL [本文引用: 1]
【目的】昆虫海藻糖酶能够调控几丁质代谢并控制蜕皮过程。本研究通过TRE表达被抑制后,检测褐飞虱（Nilaparvata lugens）蜕皮状况、几丁质含量及几丁质合成酶（chitin synthase,CHS）和几丁质酶（chitinase,Cht）基因表达情况,探究不同的海藻糖酶（trehalase,TRE）在褐飞虱表皮中对几丁质代谢的调控作用。【方法】采用RNAi技术,以实验室饲养种群褐飞虱为材料,通过向其体内注射双链RNA（dsRNA）分别抑制单个海藻糖酶基因或同时抑制多个海藻糖酶基因,注射48 h后通过Trizol法提取褐飞虱总RNA,反转录试剂盒合成第一链DNA后采用实时荧光定量PCR（qRT-PCR）技术检测该基因的表达情况,确定RNAi效果。氢氧化钾法测定48 h褐飞虱整体几丁质含量变化并对蜕皮困难虫体进行拍照;最后采用qRT-PCR检测褐飞虱CHS和Cht在mRNA水平上的相对表达量变化,分析TRE在调控几丁质代谢中的作用。【结果】与注射dsGFP相比较,其余各注射组褐飞虱整体几丁质含量显著下降,其中dsTRE1混合注射组与Validamycin注射组呈极显著下降,同时褐飞虱出现蜕皮困难等现象。qRT-PCR检测结果显示单个TRE的dsRNA注射后该基因的表达被抑制,但是部分TRE的表达有互补性上升。其中TRE1-2和TRE2在各注射组处理下表达均下降,dsTRE1s对TRE2的表达也有抑制效果,整体上dsTRE1混合注射组和海藻糖酶抑制剂Validamycin抑制效果明显;dsTRE注射组抑制CHS表达效果不明显,Validamycin能够显著降低CHS1和CHS1a在表皮中的表达,且2种dsTRE1注射后CHS1表达在上升,dsTRE1-2注射后表皮中的CHS1a的表达上升;Cht1和Cht8在dsTRE各注射组及Validamycin处理中表达下降或显著下降,dsTRE1-1注射后Cht2和Cht5表达显著上升;dsTRE1-2注射后Cht1、Cht6和Cht8表达下降,Cht2和Cht4表达显著上升;dsTRE2处理组中Cht1、Cht8和Cht10表达下降而Cht9表达显著上升;dsTRE1s注射后,Cht1和Cht5表达显著下降,而
DOI:10.3864/j.issn.0578-1752.2017.06.006URL [本文引用: 1]
【目的】昆虫海藻糖酶能够调控几丁质代谢并控制蜕皮过程。本研究通过TRE表达被抑制后,检测褐飞虱（Nilaparvata lugens）蜕皮状况、几丁质含量及几丁质合成酶（chitin synthase,CHS）和几丁质酶（chitinase,Cht）基因表达情况,探究不同的海藻糖酶（trehalase,TRE）在褐飞虱表皮中对几丁质代谢的调控作用。【方法】采用RNAi技术,以实验室饲养种群褐飞虱为材料,通过向其体内注射双链RNA（dsRNA）分别抑制单个海藻糖酶基因或同时抑制多个海藻糖酶基因,注射48 h后通过Trizol法提取褐飞虱总RNA,反转录试剂盒合成第一链DNA后采用实时荧光定量PCR（qRT-PCR）技术检测该基因的表达情况,确定RNAi效果。氢氧化钾法测定48 h褐飞虱整体几丁质含量变化并对蜕皮困难虫体进行拍照;最后采用qRT-PCR检测褐飞虱CHS和Cht在mRNA水平上的相对表达量变化,分析TRE在调控几丁质代谢中的作用。【结果】与注射dsGFP相比较,其余各注射组褐飞虱整体几丁质含量显著下降,其中dsTRE1混合注射组与Validamycin注射组呈极显著下降,同时褐飞虱出现蜕皮困难等现象。qRT-PCR检测结果显示单个TRE的dsRNA注射后该基因的表达被抑制,但是部分TRE的表达有互补性上升。其中TRE1-2和TRE2在各注射组处理下表达均下降,dsTRE1s对TRE2的表达也有抑制效果,整体上dsTRE1混合注射组和海藻糖酶抑制剂Validamycin抑制效果明显;dsTRE注射组抑制CHS表达效果不明显,Validamycin能够显著降低CHS1和CHS1a在表皮中的表达,且2种dsTRE1注射后CHS1表达在上升,dsTRE1-2注射后表皮中的CHS1a的表达上升;Cht1和Cht8在dsTRE各注射组及Validamycin处理中表达下降或显著下降,dsTRE1-1注射后Cht2和Cht5表达显著上升;dsTRE1-2注射后Cht1、Cht6和Cht8表达下降,Cht2和Cht4表达显著上升;dsTRE2处理组中Cht1、Cht8和Cht10表达下降而Cht9表达显著上升;dsTRE1s注射后,Cht1和Cht5表达显著下降,而
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DOI:10.1186/s13059-014-0521-0URLPMID:4269174 [本文引用: 1]
Abstract BACKGROUND: The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts. RESULTS: We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal's exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host. CONCLUSIONS: Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper.
DOI:10.1042/bj3590001URL [本文引用: 2]
DOI:10.1073/pnas.0800739105URLPMID:18436642
The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval-larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.
DOI:10.1002/ps.4103URLPMID:26299648 [本文引用: 1]
BACKGROUND Juvenile hormone (JH) plays a critical role in the regulation of metamorphosis in Leptinotarsa decemlineata , a notorious defoliator of potato. JH acid methyltransferase (JHAMT) is involved in one of the final steps of JH biosynthesis. RESULTS A putative JHAMT cDNA ( LdJHAMT ) was cloned. Two double-stranded RNAs (dsRNAs) (ds JHAMT1 and ds JHAMT2 ) against LdJHAMT were constructed and bacterially expressed. Experiments were conducted to investigate the effectiveness of RNAi in both second- and fourth-instar larvae. Dietary introduction of ds JHAMT1 and ds JHAMT2 successfully knocked down the target gene, lowered JH titre in the haemolymph and reduced the transcript of Kr眉ppel homologue 1 gene. Ingestion of ds JHAMT caused larval death and weight loss, shortened larval developmental period and impaired pupation. Moreover, the ds JHAMT -fed pupae exhibited lower adult emergence rates. The resulting adults weighed an average of 50 mg less than the control group, and the females did not deposit eggs. Application of pyriproxyfen to the ds JHAMT -fed insects rescued all the negative effects. CONCLUSIONS LdJHAMT expresses functional JHAMT enzyme. The RNAi targeting LdJHAMT could be used for control of L. decemlineata . 2015 Society of Chemical Industry
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DOI:10.1074/jbc.M410610200URLPMID:15574412 [本文引用: 1]
The ability of () to phosphorylate () is a potential inhibitory mechanism for resistance in . However, the serine site(s) phosphorylated by within had not been yet identified. Using an N-terminal deleted mutant and two fragments, -1 1-320 and -2 1-350, we localized site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the consensus motif (SXXXS) within -2 or , to alanine abolished their by . This suggested that Ser332 is a site and that Ser336 serves as the "priming" site typically required for action. Indeed, of prevented . Furthermore, the phosphorylated derived from the sequence was readily phosphorylated by , in contrast to the nonphosphorylated , which was not phosphorylated by the enzyme. When mutants S332A(), S336A(), or S332A/336A() were expressed in ovary overexpressing , their -induced tyrosine levels increased compared with that of wild-type (WT) . This effect was stronger in the double mutant S332A/336A() and led to enhanced -mediated activation of . Finally, immunoblot analysis with polyclonal directed against phosphorylated at Ser332 confirmed in cultured . Moreover, treatment with the inhibitor reduced Ser332 , whereas overexpression of enhanced this . In summary, our studies identify Ser332 as the target in , indicating its physiological relevance and demonstrating its novel inhibitory role in .
DOI:10.1016/0167-4838(84)90047-5URL [本文引用: 1]
DOI:10.1016/j.ejmech.2016.09.058URLPMID:27689729
61GSK-3 is an attractive pharmacological target for a number of disorders.61An overview of GSK-3 involvement in various signalling pathways is presented.61Detailed description of small molecule inhibitors of GSK-3 is provided.61The present review is helpful for the researchers planning to work on various aspects of GSK-3.
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DOI:10.1016/S0305-0491(97)00166-1URLPMID:9440228 [本文引用: 1]
Abstract The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.
DOI:10.1016/S0022-1910(99)00074-8URLPMID:12770287 [本文引用: 1]
Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.
DOI:10.1038/nature14286URLPMID:25799997 [本文引用: 1]
Abstract Wing polyphenism is an evolutionarily successful feature found in a wide range of insects. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.
