,1,2, 潘碧莹1, 邱玲玉1, 黄镇2, 周泰2, 叶林2, 唐斌1, 王世贵,1The Structure Characteristics and Biological Functions on Regulating Trehalose Metabolism of Two NlTret1s in Nilaparvata lugens
YU WeiDong,1,2, PAN BiYing1, QIU LingYu1, HUANG Zhen2, ZHOU Tai2, YE Lin2, TANG Bin1, WANG ShiGui,1通讯作者:
责任编辑: 岳梅
收稿日期:2020-07-18接受日期:2020-09-3网络出版日期:2020-12-01
| 基金资助: |
Received:2020-07-18Accepted:2020-09-3Online:2020-12-01
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於卫东, 潘碧莹, 邱玲玉, 黄镇, 周泰, 叶林, 唐斌, 王世贵. 两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能[J]. 中国农业科学, 2020, 53(23): 4802-4812 doi:10.3864/j.issn.0578-1752.2020.23.007
YU WeiDong, PAN BiYing, QIU LingYu, HUANG Zhen, ZHOU Tai, YE Lin, TANG Bin, WANG ShiGui.
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0 引言
【研究意义】我国水稻（Oryza sativa）年种植面积为3 000万公顷,总产量约达2亿吨以上[1]。作为一种被广泛种植和食用的粮食作物,水稻生产的质量与产量关系着国家粮食安全。然而其生产和储存过程中常常受到各种害虫的威胁,其中褐飞虱（Nilaparvata lugens）是对水稻生产危害最为严重的害虫[2,3,4]。20世纪70年代后,水稻种植密度提高、土壤肥力增加及品种交替频繁等因素使褐飞虱的威胁日益加剧[5]。目前,生产中主要依靠化学杀虫剂来防治褐飞虱,但化学杀虫剂的过度使用对环境和天敌均造成一定影响,并诱发抗药性[6,7,8,9,10]。因此,环保且高效的新型绿色防控方法的开发迫在眉睫。【前人研究进展】海藻糖被称为“昆虫的血糖”,在昆虫的生长发育和抗逆等方面具有重要作用[11,12,13,14,15]。昆虫体内海藻糖合成途径主要为TPS-TPP途径,海藻糖的合成依赖于海藻糖合成酶（trehalose-6-phosphate synthase,TPS）的催化作用,而海藻糖分解代谢需要海藻糖酶（trehalase,TRE）的帮助,其中海藻糖酶又可分为两种,可溶型（TRE1）和膜结合型（TRE2）[16]。目前对昆虫海藻糖代谢的研究主要集中于海藻糖的合成或分解途径[15,17],对于昆虫血糖-海藻糖转运过程的研究却非常少。众所周知不管是海藻糖还是葡萄糖,它们都不能像水或者脂肪一样直接穿过细胞膜,而需要在特殊的物质——糖转运蛋白（sugar transporter,ST）的帮助下才能顺利地进出细胞,并发挥具体的功能。糖转运蛋白属于MFS（major facilitator superfamily）超家族,是动物中最丰富的小分子转运体[18],葡萄糖转运蛋白和海藻糖转运蛋白是其中的两种。在医学研究中,葡萄糖转运蛋白被报道与I型糖尿病有关,也被视为癌症的治疗靶点[19]。在植物中,糖转运蛋白也被证实与果实的成熟等有关[20]。在昆虫中,海藻糖转运蛋白1（trehalose transporter1,Tret1）被发现可响应细胞内和细胞外梯度转运海藻糖[21]。此外,有研究表明褐飞虱不能直接利用所吸食的水稻中的蔗糖,但糖转运蛋白可以介导蔗糖进入飞虱体内[22,23]。目前,在褐飞虱中已经鉴定出18个推定的糖转运蛋白,其中7个在肠道中高度表达[24]。进一步的研究显示,NlST1和NlST16是葡萄糖转运蛋白而NIST6是果糖转运蛋白[25]。【本研究切入点】褐飞虱其他糖转运蛋白尤其是海藻糖转运蛋白的功能并未有更深入的研究。此外,褐飞虱虽然是一种农业害虫,但是也非常适合作为基因功能研究的靶标昆虫[26,27]。【拟解决的关键问题】通过对两个NlTret1的结构以及在调控海藻糖代谢中的潜在功能进行探究,评估NlTret1作为新型杀虫剂靶点的可能性,为开发绿色农药提供理论依据。1 材料与方法
试验于2018年7月至2019年7月在杭州师范大学完成。1.1 NlTret1及蛋白序列分析
首先通过美国国家生物技术信息中心（NCBI）网站搜索获得两条NlTret1基因序列,然后通过NCBI网站上的BLAST-N和BLAST-X工具,将NlTret1的cDNA序列与GenBank中存在的其他海藻糖转运蛋白的基因序列进行比较。使用多序列比对网站（1.2 供试昆虫
供试褐飞虱为杭州师范大学动物适应与进化重点实验室饲养种群,其初始虫源来自中国水稻研究所杭州种群。饲养用水稻均为感虫品系TN1（Taichung Native 1）。褐飞虱饲养条件：温度（25±1）℃,光周期16L:8D,相对湿度（70±5）%。待TN1水稻上的褐飞虱若虫长至5龄后,用于后期RNAi显微注射试验。1.3 褐飞虱总RNA的抽提及第一链cDNA的合成
褐飞虱总RNA抽提采用Trizol法,抽提过程中使用的EP管等均需为RNase-free。提取得到的总RNA用1%的琼脂糖凝胶电泳先检测质量,然后用NanoDrop 2000分光光度计测定提取RNA的浓度及纯度。使用PrimeScript?RT reagent Kit With gDNA Eraser试剂盒配置体系,进行第一链cDNA的合成,合成的cDNA保存于-20℃冰箱。1.4 dsRNA的合成
用Primer 5软件设计两个NlTret1的dsRNA特异性片段并使用合成的特异性引物进行PCR扩增,产物进行T克隆,具体引物序列见表1。随后用带T7启动子的引物进行交叉PCR反应,根据T7 RiboMAXTM Express RNAi System试剂盒的说明进行dsRNA合成,待dsRNA合成后,采用NanoDropTM 2000测定合成的dsRNA浓度,同时以绿色荧光蛋白基因（GFP）作为对照组,采用同样的方法合成GFP的dsRNA。1.5 褐飞虱的显微注射
用于显微注射的材料为5龄第1天的褐飞虱。首先打开显微注射仪,调节氮气压力,并在莱卡EZ4解剖镜下通过注射标准毛细管确定每次注射的dsRNA的体积（0.1 μL）。将CO2麻醉后的褐飞虱虫体腹面朝上放置于提前制备好的琼脂糖胶台的凹槽中,在莱卡EZ4解剖镜下于褐飞虱第二和第三对足间的腹基节连接处分别注射dsNlTret1-like X1、dsNlTret1-2 X1和dsGFP,注射量为50 ng/头。最后将注射好的褐飞虱转移至装有新鲜水稻的玻璃管中,分别在48 h后收取依然存活的褐飞虱用于后续试验。每个处理取6个平行样,其中3个平行样用于基因表达情况检测,每个平行样10头褐飞虱;另外3个平行样用于糖含量和酶活性测定,每个平行样20头褐飞虱。1.6 RNAi后褐飞虱体内海藻糖代谢通路相关基因表达量测定
将显微注射48 h后不同组别的褐飞虱分装于3个平行管中,抽提总RNA后反转录得到3管平行的cDNA,参照SYBR? Premix Ex TaqTM试剂盒的方法进行qRT-PCR检测。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灭菌水。选用褐飞虱18S核糖体核糖核苷酸基因（18S ribosomal RNA,18S）作为内参基因[28],具体引物序列见表1。qRT-PCR程序：95℃预变性10 s,95℃解链5 s,59℃退火并延伸30 s,39个循环。每个处理3个生物学重复,每个生物学重复含3个技术重复。检测基因的相对拷贝数用2-ΔΔCT方法进行分析[29]。1.7 总糖原、海藻糖、葡萄糖含量以及海藻糖酶活性测定
取试验组以及对照组材料,加入200 μL PBS,用研磨棒充分研磨后放入超声破碎仪进行超声破碎,破碎后再加入800 μL PBS,4℃,1 000×g离心20 min。取350 μL上清,4℃,20 800×g超离心1 h,剩余上清用于总蛋白、总糖原和海藻糖含量的测定。超离心后的上清用于测定葡萄糖、蛋白质含量以及可溶型海藻糖酶活性,沉淀悬浮于300 μL的PBS后用于测定葡萄糖、蛋白质含量和膜结合型海藻糖酶活性。具体步骤参照ZHANG等描述的方法[30]。1.8 数据分析
应用Excel软件整理、分析数据,通过SigmaPlot 10.0和SPSS软件进行显著性分析和作图,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）。Table 1
表1
表1实时荧光定量PCR和dsNlTret1、dsGFP的引物序列
Table 1
| 引物名称 Primer name | 正向引物 Forward primer (5′-3′) | 反向引物 Reverse primer (5′-3′) | 产物长度 Length (bp) |
|---|---|---|---|
| dsNlTret1-like X1 | GCAAACAACAGCGAGCAA | CCAAGAGGCACCCATCC | 472 |
| dsNlTret1-like X1-T7 | T7-GCAAACAACAGCGAGCAA | T7-CCAAGAGGCACCCATCC | 552 |
| dsNlTret1-2 X1 | CTTCGTTCACCAGCACCT | CAAATGGCACTTATTATCGTC | 547 |
| dsNlTret1-2 X1-T7 | T7-CTTCGTTCACCAGCACCT | T7-CAAATGGCACTTATTATCGTC | 597 |
| dsGFP | AAGGGCGAGGAGCTGTTCACCG | CAGCAGGACCATGTGATCGCGC | 657 |
| dsGFP-T7 | T7-AAGGGCGAGGAGCTGTTCACCG | T7-CAGCAGGACCATGTGATCGCGC | 707 |
| qNl18S | CGCTACTACCGATTGAA | GGAAACCTTGTTACGACTT | 165 |
| qNlTret1-like X1 | GTGGGAATCGTGAACATGGG | ATGGTCATGAGTGTGCTGGA | 101 |
| qNlTret1-2 X1 | TATGTGTCGGCTGGGTTCTT | CTCTGAGCCAGCACAATGAC | 190 |
| qNlTPS1 | AAGACTGAGGCGAATGGT | AAGGTGGAAATGGAATGTG | 154 |
| qNlTPS2 | AGAGTGGACCGCAACAACA | TCAACGCCGAGAATGACTT | 161 |
| qNlTRE1-1 | GCCATTGTGGACAGGGTG | CGGTATGAACGAATAGAGCC | 132 |
| qNlTRE1-2 | GATCGCACGGATGTTTA | AATGGCGTTCAAGTCAA | 178 |
| qNlTRE2 | TCACGGTTGTCCAAGTCT | TGTTTCGTTTCGGCTGT | 197 |
新窗口打开|下载CSV
2 结果
2.1 NlTret1蛋白序列分析
两条NlTret1（NlTret1-like X1和NlTret1-2 X1）的开放阅读框长度分别为1 920和1 578 bp,分别编码639和525个氨基酸,预测蛋白分子量分别为69.29和58.71 kD,等电点分别为8.32和8.36（图1-A）。蛋白质疏水性预测显示NlTret1-like X1和NlTret1-2 X1的GRAVY（grand average of hydropathicity）值分别为0.313和0.354,表明这两个转运蛋白主要由疏水性氨基酸组成。二级和三级结构显示这两个NlTret1的结构均较为简单,主要为螺旋和卷曲（图1-A、1-C）。SMART分析以及三级结构预测发现NlTret1-like X1和NlTret1-2 X1分别包含12个和10个跨膜结构域（图1-B、1-C）,表明它们均为跨膜蛋白。此外,保守结构域分析结果显示两者均属于MFS超家族（图1-B）。图1
新窗口打开|下载原图ZIP|生成PPT图1NlTret1序列及其结构分析
Fig. 1Amino acid sequences and structural analysis of NlTret1s
GenBank登录号为XP_022183984.1（NlTret1-like X1）和XP_022195528.1（NlTret1-2 X1）Initiation and termination GenBank accession numbers: XP_022183984.1 (NlTret1-like X1) and XP_022195528.1 (NlTret1-2 X1)。A：NlTret1二级结构预测Prediction of the secondary structure of NlTret1s;B：NlTret1结构域分析（蓝色方块表示跨膜结构,粉色方块表示低复杂区域）Analysis of domain of NlTret1s (Blue blocks represent transmembrane region and pink blocks represent low complexity region);C：NlTret1三级结构预测Prediction of the tertiary structure of NlTret1s
2.2 NlTret1系统进化树
进化分析结果显示,NlTret1-like X1和NlTret1-2 X1分别与其他昆虫的Tret1-like X1和Tret1 X1聚在一支,其中NlTret1-like X1与黄蔗蚜（Sipha flava）、高粱蚜（Melanaphis sacchari）、棉蚜（Aphis gossypii）、玉米缢管蚜（Rhopalosiphum maidis）、桃蚜（Myzus persicae）及豌豆蚜（Acyrthosiphon pisum）的Tret1聚在一支;NlTret1-2 X1则与臭虫（Cimex lectularius）和茶翅蝽（Halyomorpha halys）聚在一支,表明褐飞虱的这两个Tret1与其他昆虫的Tret1具有较高的同源性,并且与同为半翅目的上述昆虫亲缘关系较近（图2）。图2
新窗口打开|下载原图ZIP|生成PPT图2基于氨基酸序列构建的NlTret1与其他昆虫Tret1的系统发育树(邻接法)
Fig. 2Phylogenetic tree of NlTret1s and Tret1 proteins from other insect species based on the amino acid sequence (neighbor- joining method)
Tret1蛋白来源物种及其GenBank登录号 Source species of Tret1 proteins and their GenBank accession numbers。豌豆蚜Acyrthosiphon pisum：ApTret1 (XP_001943832.1)、ApTret1 X1 (XP_003247868.1);桃蚜Myzus persicae：MpTret1-like (XP_022175298.1)、MpTret1-like X1 (XP_022168826.1);高粱蚜Melanaphis sacchari：MsTret1-like (XP_025205275.1)、MsTret1-like X1 (XP_025193454.1);玉米缢管蚜Rhopalosiphum maidis：RmTret1-like (XP_026817841.1)、RmTret1-like X1 (XP_026807124.1);棉蚜Aphis gossypii：AgTret1-like (XP_027847026.1)、AgTret1-like X1 (XP_027852240.1);黑豆蚜Aphis craccivora：AcTret1-like (KAF0773727.1);黄蔗蚜Sipha flava：SfTret1-like (XP_025415984.1)、SfTret1-like X1 (XP_025419752.1);褐飞虱Nilaparvata lugens：NlTret1-2 X1 (XP_022195528.1)、NlTret1-like X1 (XP_022183984.1);臭虫Cimex lectularius：ClTret1-like (XP_014254493.1);茶翅蝽Halyomorpha halys：HhTret1-like (XP_014285740.1);湿木白蚁Zootermopsis nevadensis：ZnTret1-like (XP_021920751.1);第二隐白蚁Cryptotermes secundus：CsTret1 X1 (XP_023724429.1);埃及伊蚊Aedes aegypti：AaTret1 (XP_001654366.1);猫虱Ctenocephalides felis：CfTret1-like (XP_026477563.1);菜粉蝶Pieris rapae：PrTret1-like (XP_022131116.1);麦茎蜂Cephus cinctus：CcTret1 X1 (XP_015587164.1);瘿蜂Belonocnema treatae：BtTret1-like (XP_033208929.1);汗蜂Dufourea novaeangliae：DnTret1 (KZC12919.1);彩带蜂Nomia melanderi：NmTret1-like X1 (XP_031847112.1);银额果蝇Drosophila albomicans：DaTret1 X1 (XP_034103480.1);铜绿蝇Lucilia cuprina：LcTret1 X1 (XP_023292603.1);德国小蠊Blattella germanica：BgTret1 (PSN42188.1);柑橘木虱Diaphorina citri：DcTret1-like (XP_026682851.1)
2.3 RNAi后褐飞虱体内两个NlTret1的表达情况
与注射dsGFP组相比,干扰NlTret1-like X1或NlTret1-2 X1 48 h后褐飞虱体内靶标基因的表达量均极显著下降（P<0.01）（图3）,dsNlTret1-like X1或dsNlTret1-2 X1的干扰效率分别为66.38%和89.45%,表明靶标基因的干扰有效。图3
新窗口打开|下载原图ZIP|生成PPT图3注射48 h后NlTret1-like X1和NlTret1-2 X1的表达量
Fig. 3Relative expression level of NlTret1-like X1 and NlTret1-2 X1 after injection for 48 h in N. lugens
2.4 RNAi后褐飞虱体内海藻糖代谢通路相关基因的表达情况
与注射dsGFP组相比,干扰NlTret1-like X1 48 h后褐飞虱体内TPS1、TPS2、TRE1-1、TRE1-2和TRE2的表达量均极显著下降;而在干扰NlTret1-2 X1 48 h后褐飞虱体内TPS1、TPS2和TRE1-1也极显著低表达,但TRE1-2和TRE2的相对表达量极显著上升（P<0.01）（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4RNAi后褐飞虱海藻糖代谢通路相关基因的表达量
Fig. 4Relative expression level of regulated genes of trehalose metabolic pathway in N. lugens after RNAi
2.5 RNAi后对褐飞虱体内糖原、海藻糖和葡萄糖含量的影响
与注射dsGFP组相比,干扰NlTret1-like X1 48 h体内海藻糖含量极显著增加（P<0.01）,而其他糖类物质含量变化均不显著。干扰NlTret1-2 X1 48 h后,褐飞虱体内的海藻糖、葡萄糖和糖原含量均无显著变化（图5）。图5
新窗口打开|下载原图ZIP|生成PPT图5RNAi 48 h后褐飞虱体内海藻糖、葡萄糖和糖原的含量
N.S.代表差异不显著。下同
Fig. 5Contents of trehalose, glucose and glycogen in N. lugens after RNAi
N.S. indicates no significant difference. The same as below
2.6 RNAi后对褐飞虱体内海藻糖酶活性的影响
与注射dsGFP组相比,褐飞虱体内可溶性海藻糖酶和膜结合型海藻糖酶活性在干扰NlTret1-like X1 48 h后均极显著下降（P<0.01）,而在干扰NlTret1-2 X1后未出现显著变化（图6）。图6
新窗口打开|下载原图ZIP|生成PPT图6RNAi后褐飞虱体内的海藻糖酶活性
Fig. 6Enzyme activity of trehalase in N. lugens after RNAi
3 讨论
不同昆虫的Tret具有相似的氨基酸序列,且与葡萄糖转运蛋白超家族相似,因此有研究表明Tret1可能是葡萄糖转运蛋白超家族的新成员[31]。此外,也有研究证实,嗜眠摇蚊（Polypedilum vanderplanki）Tret1不仅可以转运海藻糖,还可以转运葡萄糖类似物[21]。在葡萄糖转运蛋白（glucose transporter,Glut）超家族的几乎所有成员（包括Tret1）中都可以看到序列相似性,但它们的生化特性（例如底物选择性和动力学）差异很大,如Glut1的底物是葡萄糖、半乳糖、甘露糖和葡萄糖胺,而Glut5和H+-肌醇协同转运蛋白（H+-myo-inositol cotransporter,HMIT）的特定底物分别是果糖和肌醇[32]。说明除了保守区域中的氨基酸残基外,其他氨基酸残基也可能对每个转运蛋白的特异性产生影响。本研究通过生物信息学方法分析了褐飞虱的两个Tret1序列,发现它们的二级结构主要是螺旋及卷曲,且均属于MFS超家族。但其二级结构以及保守结构域外的氨基酸残基均存在差异,推测它们的海藻糖转运功能可能存在差异。此外,这两个NlTret1同其他糖转运蛋白相似,拥有较多个跨膜区域,其中NlTret1-like X1有12个跨膜区域,NlTret1-2 X1有10个跨膜区域（图1）。SAIER研究表明,MFS超家族成员蛋白的二级结构预测显示其大多都具有12次α-螺旋跨膜结构域,而其他一些具有14或24次α-螺旋的可能是进化过程中以12次跨膜α-螺旋为基础产生的[33]。NlTret1-2 X1只具有10个跨膜区域,可能是由于所得到的片段并不是全长。海藻糖是昆虫中的主要血淋巴糖,而海藻糖转运蛋白则负责海藻糖的运输并调节海藻糖在不同组织中的分布,在昆虫的营养稳态和胁迫耐受性中起着重要作用[11-15,24]。此外,自在秀丽隐杆线虫（Caenorhabditis elegans）的研究中首次发现RNAi现象之后,RNAi技术已成为昆虫基因功能研究、基因表达调控、害虫控制、新型农药开发等方面的有力工具[34,35,36]。本研究在验证基因干扰有效的前提下（图3）,检测了与海藻糖代谢相关的一些基因表达情况以及部分生化指标,发现干扰NlTret1-like X1后,褐飞虱体内所有的TPS和TRE均呈极显著低表达（图4）,但TPS的表达量高于TRE的表达量,且可溶性海藻糖酶及膜结合型海藻糖酶的活性均极显著下降（图6）,因此在干扰NlTret1-like X1后,褐飞虱体内的海藻糖被积累（图5）。干扰NlTret1-2 X1后TPS的表达量均显著下降且TRE1-2和TRE2均极显著高表达（图4）,但海藻糖酶活性无明显变化（图6）,推测可能是褐飞虱体内的糖原和葡萄糖在向海藻糖转化,导致褐飞虱体内的海藻糖含量无显著变化（图5）。在大猿叶虫（Colaphellus bowringi）中发现了两个Tret1,其中Tret1a在脂肪体中高表达,且RNAi后会导致脂肪体中海藻糖含量升高;Tret1b则在卵巢中高表达,然而在干扰其表达后,卵巢内的海藻糖含量却呈极显著下降趋势[37]。表明不同的Tret1可能在不同的组织或不同的生理过程中起作用。但由于大猿叶虫的研究主要针对脂肪体和卵巢组织进行,与本研究选取整个虫体存在差异,笔者推测褐飞虱的两个Tret1可能也在不同组织间发挥着不同功能,NlTret1-like X1可能在特异性转运海藻糖参与能量供应中起到更为显著的作用。
4 结论
褐飞虱的两个Tret1结构均较为简单,主要为螺旋和卷曲;此外,它们均属于MFS超家族,具有较多的跨膜结构域;褐飞虱的Tret1与其他昆虫的Tret1具有较高的同源性,且与其他半翅目昆虫如黄蔗蚜等的亲缘关系较近;注射dsNlTret1-like X1和dsNlTret1-2 X1均能有效抑制本基因的表达;dsNlTret1-like X1和dsNlTret1-2 X1注射到褐飞虱体内,能够打破体内海藻糖代谢的平衡。推测褐飞虱的这两个Tret1可能在不同组织间发挥着不同的功能,且NlTret1- like X1在特异性转运海藻糖参与能量供应中起到更为显著的作用。（责任编辑 岳梅）
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DOI:10.1093/mp/sst030URL [本文引用: 1]
The brown planthopper (BPH) is the most notorious pest of rice (Oryza sativa). Studies of rice-BPH interaction have contributed to development of new rice varieties, offering an effective means for long-lasting control of BPH. Here, we review the status of knowledge of the molecular basis of rice-BPH interaction, from the perspective of immunity. The BPH has complicated feeding behaviors on rice, which are mainly related to host resistance. Now, 24 resistance genes have been detected in rice, indicating gene-for-gene relationships with biotypes of the BPH. However, only one BPH resistance gene (Bph14) was identified and characterized using map-based cloning. Bph14 encodes an immune receptor of NB-LRR family, providing a means for studying the molecular mechanisms of rice resistance to BPH. Plant hormones (e. g. salicylic acid and jasmonate/ethylene), Ca2+, mitogen-activated protein kinases (MAPKs), and OsRac1 play important roles in the immune response of rice to BPH. Signal transduction leads to modifying expression of defense-related genes and defense mechanisms against BPH, including sieve tube sealing, production of secondary metabolites, and induction of proteinase inhibitor. A model for the molecular interactions between rice and the BPH is proposed, although many details remain to be investigated that are valuable for molecular design of BPH-resistant rice varieties.
DOI:10.1038/srep38581URLPMID:27924841 [本文引用: 1]
Viruses may induce changes in plant hosts and vectors to enhance their transmission. The white-backed planthopper (WBPH) and brown planthopper (BPH) are vectors of Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV), respectively, which cause serious rice diseases. We herein describe the effects of SRBSDV and RRSV infections on host-selection behaviour of vector and non-vector planthoppers at different disease stages. The Y-tube olfactometer choice and free-choice tests indicated that SRBSDV and RRSV infections altered the attractiveness of rice plants to vector and non-vector planthoppers. The attractiveness was mainly mediated by rice volatiles, and varied with disease progression. The attractiveness of the SRBSDV- or RRSV-infected rice plants to the virus-free WBPHs or BPHs initially decreased, then increased, and finally decreased again. For the viruliferous WBPHs and BPHs, SRBSDV or RRSV infection increased the attractiveness of plants more for the non-vector than for the vector planthoppers. Furthermore, we observed that the attractiveness of infected plants to planthoppers was positively correlated with the virus titres. The titre effects were greater for virus-free than for viruliferous planthoppers. Down-regulated defence genes OsAOS1, OsICS, and OsACS2 and up-regulated volatile-biosynthesis genes OsLIS, OsCAS, and OsHPL3 expression in infected plants may influence their attractiveness.
DOI:10.1038/s41598-018-26881-9URLPMID:29855556 [本文引用: 1]
To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.
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DOI:10.1303/aez.2000.177URL [本文引用: 1]
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DOI:10.1002/arch.20043URLPMID:15756698 [本文引用: 1]
The first neonicotinoid insecticide introduced to the market was imidacloprid in 1991 followed by several others belonging to the same chemical class and with the same mode of action. The development of neonicotinoid insecticides has provided growers with invaluable new tools for managing some of the world's most destructive crop pests, primarily those of the order Hemiptera (aphids, whiteflies, and planthoppers) and Coleoptera (beetles), including species with a long history of resistance to earlier-used products. To date, neonicotinoids have proved relatively resilient to the development of resistance, especially when considering aphids such as Myzus persicae and Phorodon humuli. Although the susceptibility of M. persicae may vary up to 20-fold between populations, this does not appear to compromise the field performance of neonicotinoids. Stronger resistance has been confirmed in some populations of the whitefly, Bemisia tabaci, and the Colorado potato beetle, Leptinotarsa decemlineata. Resistance in B- and Q-type B. tabaci appears to be linked to enhanced oxidative detoxification of neonicotinoids due to overexpression of monooxygenases. No evidence for target-site resistance has been found in whiteflies, whereas the possibility of target-site resistance in L. decemlineata is being investigated further. Strategies to combat neonicotinoid resistance must take account of the cross-resistance characteristics of these mechanisms, the ecology of target pests on different host plants, and the implications of increasing diversification of the neonicotinoid market due to a continuing introduction of new molecules.
DOI:10.1016/j.cropro.2007.08.004URL [本文引用: 1]
DOI:10.1016/j.aspen.2011.09.004URL [本文引用: 1]
The brown planthopper (BPH), Nilaparvata lugens (Stal), which periodically erupted in tropical Asian rice before the 1960s, became a major threat after farmers adopted green revolution technologies in the 1960s. Management and policy changes in the 1980s and 1990s emphasized non-insecticidal tactics to avert BPH outbreaks. However, insecticides have resurfaced as the primary means for controlling rice insect pests and tropical Asian countries have recently experienced planthopper outbreaks in record numbers. Our review of factors that have contributed to the outbreaks points to insecticides as the most tangible outbreak factor primarily because of their harmful effects on natural enemies. BPH resistance to insecticides and especially imidacloprid has increased the probability of outbreaks as farmers have applied increasing quantities of insecticide in an attempt to combat resistant populations. Similarly, heavy use of nitrogen fertilizer, especially on hybrid rice, has increased the potential for outbreaks. Other factors triggering outbreaks are less documented, but we discuss the possibility that the high outbreak synchrony in geographically separated populations of BPH may suggest a "Moran effect" such as climate that promotes an environment favoring above-average increases in BPH populations. Also, we hypothesize that BPH functions as a metapopulation and, as such, periodic outbreaks could be a natural phenomenon requiring resupply of planthoppers into vacant areas to ensure genetic linkage among subpopulations. We conclude with a series of recommendations for research and policy changes aimed at better understanding the cause of BPH outbreaks and for developing sustainable management practices to prevent future outbreaks. (C) Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2011. Published by Elsevier B.V.
DOI:10.1007/BF01919312URLPMID:8706810 [本文引用: 2]
Trehalose is a non-reducing disaccharide comprising two glucose molecules. It is present in high concentration as the main haemolymph (blood) sugar in insects. The synthesis of trehalose in the fat body (an organ analogous in function to a combination of liver and adipose tissue in vertebrates) is stimulated by neuropeptides (hypertrehalosaemic hormones), released from the corpora cardiaca, a neurohaemal organ associated with the brain. The peptides cause a decrease in the content of fructose 2,6-biphosphate in fat body cells. Fructose 2,6-biphosphate, acting synergistically with AMP, is a potent activator of the glycolytic enzyme 6-phosphofructokinase-1 and a strong inhibitor of the gluconeogenic enzyme fructose 1,6-biphosphatase. This indicates that fructose 2,6-biphosphate is a key metabolic signal in the regulation of trehalose synthesis in insects. Trehalose is hydrolysed by trehalase (E.C. 3.2.1.28). The activity of this enzyme is regulated in flight muscle, but the mechanism by which this is achieved is unknown. Trehalase from locust flight muscle is a glycoprotein bound to membranes of the microsomal fraction. The enzyme can be activated by detergents in vitro and by short flight intervals in vivo, which indicates that changes in the membrane environment modulate trehalase activity under physiological conditions.
DOI:10.1093/glycob/cwg047URLPMID:12626396 [本文引用: 1]
Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.
DOI:10.1111/j.1744-7909.2008.00736.xURL [本文引用: 1]
Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In-silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.
DOI:10.1016/j.jinsphys.2010.02.009URLPMID:20193689 [本文引用: 1]
Trehalose is an important disaccharide and a key regulation factor for the development of many organisms, including plants, bacteria, fungi and insects. In order to study the trehalose synthesis pathway, a cDNA for a trehalose-6-phosphate synthase from Spodoptera exigua (SeTPS) was cloned which contained an open reading frame of 2481 nucleotides encoding a protein of 826 amino acids with a predicted molecular weight of 92.65kDa. The SeTPS genome has 12 exons and 11 introns. Northern blot and RT-PCR analyses showed that SeTPS mRNA was expressed in the fat body and in the ovary. Competitive RT-PCR revealed that SeTPS mRNA was expressed in the fat body at different developmental stages and was present at a high level in day 1 S. exigua pupae. The concentrations of trehalose and glucose in the hemolymph were determined by HPLC and showed that they varied at different developmental stages and were negatively correlated to each other. The survival rates of the insects injected with dsRNA corresponding to SeTPS gene reached 53.95%, 49.06%, 34.86% and 33.24% for 36, 48, 60 and 204h post-injection respectively which were significantly lower than those of the insects in three control groups. These findings provide new data on the tissue distribution, expression patterns and potential function of the trehalose-6-phosphate synthase gene.
DOI:10.1093/glycob/cwu125URLPMID:25429048 [本文引用: 3]
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:10.1186/1471-2148-6-109URLPMID:17178000 [本文引用: 1]
BACKGROUND: The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes. RESULTS: In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein. CONCLUSION: We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.
DOI:10.3389/fphys.2018.00030URLPMID:29445344 [本文引用: 1]
The non-reducing disaccharide trehalose is widely distributed among various organisms. It plays a crucial role as an instant source of energy, being the major blood sugar in insects. In addition, it helps countering abiotic stresses. Trehalose synthesis in insects and other invertebrates is thought to occur via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. In many insects, the TPP gene has not been identified, whereas multiple TPS genes that encode proteins harboring TPS/OtsA and TPP/OtsB conserved domains have been found and cloned in the same species. The function of the TPS gene in insects and other invertebrates has not been reviewed in depth, and the available information is quite fragmented. The present review discusses the current understanding of the trehalose synthesis pathway, TPS genetic architecture, biochemistry, physiological function, and potential sensitivity to insecticides. We note the variability in the number of TPS genes in different invertebrate species, consider whether trehalose synthesis may rely only on the TPS gene, and discuss the results of in vitro TPS overexpression experiment. Tissue expression profile and developmental characteristics of the TPS gene indicate that it is important in energy production, growth and development, metamorphosis, stress recovery, chitin synthesis, insect flight, and other biological processes. We highlight the molecular and biochemical properties of insect TPS that make it a suitable target of potential pest control inhibitors. The application of trehalose synthesis inhibitors is a promising direction in insect pest control because vertebrates do not synthesize trehalose; therefore, TPS inhibitors would be relatively safe for humans and higher animals, making them ideal insecticidal agents without off-target effects.
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DOI:10.1073/pnas.0702538104URLPMID:17606922 [本文引用: 2]
Trehalose is potentially a useful cryo- or anhydroprotectant molecule for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. In this study, we isolated and characterized the functions of a facilitated trehalose transporter [trehalose transporter 1 (TRET1)] from an anhydrobiotic insect, Polypedilum vanderplanki. Tret1 cDNA encodes a 504-aa protein with 12 predicted transmembrane structures. Tret1 expression was induced by either desiccation or salinity stress. Expression was predominant in the fat body and occurred concomitantly with the accumulation of trehalose, indicating that TRET1 is involved in transporting trehalose synthesized in the fat body into the hemolymph. Functional expression of TRET1 in Xenopus oocytes showed that transport activity was stereochemically specific for trehalose and independent of extracellular pH (between 4.0 and 9.0) and electrochemical membrane potential. These results indicate that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. The extraordinarily high values for apparent Km (>or=100 mM) and Vmax (>or=500 pmol/min per oocyte) for trehalose both indicate that TRET1 is a high-capacity transporter of trehalose. Furthermore, TRET1 was found to function in mammalian cells, suggesting that it confers trehalose permeability on cells, including those of vertebrates as well as insects. These characteristic features imply that TRET1 in combination with trehalose has high potential for basic and practical applications in vivo.
DOI:10.1016/j.ibmb.2015.07.015URLPMID:26226652 [本文引用: 1]
Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.
DOI:10.1016/j.ibmb.2007.07.001URL [本文引用: 1]
Abstract
Phloem-sap feeding Hemipteran insects have access to a sucrose-rich diet but are dependent on sucrose hydrolysis and hexose transport for carbon nutrition. A cDNA library from Nilaparvata lugens (rice brown planthopper) was screened for clones encoding potential transmembrane transporters. A selected cDNA, NlHT1, encodes a 53 kDa polypeptide with sequence similarity to facilitative hexose transporters of eukaryotes and prokaryotes, including GLUT1, the human erythrocyte hexose transporter. NlHT1 was expressed as a recombinant protein in the methylotropic yeast Pichia pastoris, and was identified in a membrane fraction isolated from transformed yeast cells. Transport experiments using membrane vesicles containing NlHT1 showed that the protein is a saturable, sodium independent transporter, with a relatively low affinity for glucose (Km 3.0 mM), which can be inhibited by cytochalasin B. Competition experiments with fructose demonstrate NlHT1 is glucose specific. In situ localisation studies revealed that NlHT1 mRNA is expressed in N. lugens gut tissue, mainly in midgut regions, and that expression is absent in hindgut and Malpighian tubules. NlHT1 is therefore likely to play an important role in glucose transport from the gut, and in carbon nutrition in vivo. This is the first report of a facilitative glucose transporter from a phloem-feeding insect pest.DOI:10.1016/j.ibmb.2010.07.008URL [本文引用: 2]
Abstract
The brown planthopper (BPH), Nilaparvata lugens, attacks rice plants and feeds on their phloem sap, which contains large amounts of sugars. The main sugar component of phloem sap is sucrose, a disaccharide composed of glucose and fructose. Sugars appear to be incorporated into the planthopper body by sugar transporters in the midgut. A total of 93 expressed sequence tags (ESTs) for putative sugar transporters were obtained from a BPH EST database, and 18 putative sugar transporter genes (Nlst1–18) were identified. The most abundantly expressed of these genes was Nlst1. This gene has previously been identified in the BPH as the glucose transporter gene NlHT1, which belongs to the major facilitator superfamily. Nlst1, 4, 6, 9, 12, 16, and 18 were highly expressed in the midgut, and Nlst2, 7, 8, 10, 15, 17, and 18 were highly expressed during the embryonic stages. Functional analyses were performed using Xenopus oocytes expressing NlST1 or 6. This showed that NlST6 is a facilitative glucose/fructose transporter that mediates sugar uptake from rice phloem sap in the BPH midgut in a manner similar to NlST1.Graphical abstract
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DOI:10.1016/j.ibmb.2006.12.005URL [本文引用: 1]
Abstract
The hydrolysis of sucrose, the principal dietary source of carbon for aphids, is catalysed by a gut α-glucosidase/transglucosidase activity. An α-glucosidase, referred to as APS1, was identified in both a gut-specific cDNA library and a sucrase-enriched membrane preparation from guts of the pea aphid Acyrthosiphon pisum by a combination of genomic and proteomic techniques. APS1 contains a predicted signal peptide, and has a predicted molecular mass of 68 kDa (unprocessed) or 66.4 kDa (mature protein). It has amino acid sequence similarity to α-glucosidases (EC 3.2.1.20) of glycoside hydrolase family 13 in other insects. The predicted APS1 protein contains two domains: an N-terminal catalytic domain, and a C-terminal hydrophobic domain. In situ localisation and RT-PCR studies revealed that APS1 mRNA was expressed in the gut distal to the stomach, the same localisation as sucrase activity. When expressed heterologously in Xenopus embryos, APS1 was membrane-bound and had sucrase activity. It is concluded that APS1 is a dominant, and possibly sole, protein mediating sucrase activity in the aphid gut.DOI:10.1073/pnas.1809381115URLPMID:30061410 [本文引用: 1]
RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.
DOI:10.1111/imb.12133URLPMID:25224926 [本文引用: 1]
Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-beta-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.
DOI:10.1038/srep27841URLPMID:27328657 [本文引用: 1]
RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice.
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
DOI:10.3389/fphys.2017.00750URLPMID:29033849 [本文引用: 1]
The brown planthopper, Nilaparvata lugens is one of the most serious pests of rice, and there is so far no effective way to manage this pest. However, RNA interference not only can be used to study gene function, but also provide potential opportunities for novel pest management. The development of wing plays a key role in insect physiological activities and mainly involves chitin. Hence, the regulating role of trehalase (TRE) genes on wing bud formation has been studied by RNAi. In this paper, the activity levels of TRE and the contents of the two sugars trehalose and glucose were negatively correlated indicating the potential role of TRE in the molting process. In addition, NlTRE1-1 and NlTRE2 were expressed at higher levels in wing bud tissue than in other tissues, and abnormal molting and wing deformity or curling were noted 48 h after the insect was injected with any double-stranded TRE (dsTRE), even though different TREs have compensatory functions. The expression levels of NlCHS1b, NlCht1, NlCht2, NlCht6, NlCht7, NlCht8, NlCht10, NlIDGF, and NlENGase decreased significantly 48 h after the insect was injected with a mixture of three kinds of dsTREs. Similarly, the TRE inhibitor validamycin can inhibit NlCHS1 and NlCht gene expression. However, the wing deformity was the result of the NlIDGF, NlENGase, NlAP, and NlTSH genes being inhibited when a single dsTRE was injected. These results demonstrate that silencing of TRE gene expression can lead to wing deformities due to the down-regulation of the AP and TSH genes involved in wing development and that the TRE inhibitor validamycin can co-regulate chitin metabolism and the expression of wing development-related genes in wing bud tissue. The results provide a new approach for the prevention and management of N. lugens.
DOI:10.1016/j.ibmb.2009.12.006URL [本文引用: 1]
Abstract
We recently cloned a trehalose transporter gene (Tret1) that contributes to anhydrobiosis induction in the sleeping chironomid Polypedilum vanderplanki Hinton. Because trehalose is the main haemolymph sugar in most insects, they might possess Tret1 orthologs involved in maintaining haemolymph trehalose levels. We cloned Tret1 orthologs from four species in three insect orders. The similarities of the amino acid sequence to TRET1 in P. vanderplanki were 58.5–80.4%. Phylogenetic analysis suggested the Tret1 sequences were conserved in insects. The Xenopus oocyte expression system showed apparent differences in the Km and Vmax values for trehalose transport activity among the six proteins encoded by the corresponding orthologs. The TRET1 orthologs of Anopheles gambiae (Km: 45.74±3.58mM) and Bombyx mori (71.58±6.45mM) showed low trehalose affinity, whereas those of Apis mellifera (9.42±2.37mM) and Drosophila melanogaster (10.94±7.70mM) showed high affinity. This difference in kinetics might be reflected in the haemolymph trehalose:glucose ratio of each species. Tret1 was expressed not only in the fat body but also in muscle and testis. These findings suggest that insect TRET1 is responsible for the release of trehalose from the fat body and the incorporation of trehalose into other tissues that require a carbon source, thereby regulating trehalose levels in the haemolymph.DOI:10.1007/s00424-003-1085-0URLPMID:12750891 [本文引用: 1]
The SLC2 family of glucose and polyol transporters comprises 13 members, the glucose transporters (GLUT) 1-12 and the H(+)- myo-inositol cotransporter (HMIT). These proteins all contain 12 transmembrane domains with both the amino and carboxy-terminal ends located on the cytoplasmic side of the plasma membrane and a N-linked oligosaccharide side-chain located either on the first or fifth extracellular loop. Based on sequence comparison, the GLUT isoforms can be grouped into three classes: class I comprises GLUT1-4; class II, GLUT6, 8, 10, and 12 and class III, GLUT5, 7, 9, 11 and HMIT. Despite their sequence similarity and the presence of class-specific signature sequences, these transporters carry various hexoses and HMIT is a H(+)/ myo-inositol co-transporter. Furthermore, the substrate transported by some isoforms has not yet been identified. Tissue- and cell-specific expression of the well-characterized GLUT isoforms underlies their specific role in the control of whole-body glucose homeostasis. Numerous studies with transgenic or knockout mice indeed support an important role for these transporters in the control of glucose utilization, glucose storage and glucose sensing. Much remains to be learned about the transport functions of the recently discovered isoforms (GLUT6-13 and HMIT) and their physiological role in the metabolism of glucose, myo-inositol and perhaps other substrates.
DOI:10.1016/s1369-5274(99)00016-8URLPMID:10508720 [本文引用: 1]
In the study of transmembrane transport, molecular phylogeny provides a reliable guide to protein structure, catalytic and noncatalytic transport mechanisms, mode of energy coupling and substrate specificity. It also allows prediction of the evolutionary history of a transporter family, leading to estimations of its age, source, and route of appearance. Phylogenetic analyses, therefore, provide a rational basis for the characterization and classification of transporters. A universal classification system has been described, based on both function and phylogeny, which has been designed to be applicable to all currently recognized and yet-to-be discovered transport proteins found in living organisms on Earth.
DOI:10.1002/ps.4337URLPMID:27299308 [本文引用: 1]
BACKGROUND: RNA interference (RNAi) technology can potentially serve as a suitable strategy to control the African sweet potato weevil Cylas puncticollis (SPW), which is a critical pest in sub-Saharan Africa. Important prerequisites are required to use RNAi in pest control, such as the presence of an efficient RNAi response and the identification of suitable target genes. RESULTS: Here we evaluated the toxicity of dsRNAs targeting essential genes by injection and oral feeding in SPW. In injection assays, 12 of 24 dsRNAs were as toxic as the one targeting Snf7, a gene used commercially against Diabrotica virgifera virgifera. Three dsRNAs with high insecticidal activity were then chosen for oral feeding experiments. The data confirmed that oral delivery can elicit a significant toxicity, albeit lower compared with injection. Subsequently, ex vivo assays revealed that dsRNA is affected by degradation in the SPW digestive system, possibly explaining the lower RNAi effect by oral ingestion. CONCLUSION: We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy. (c) 2016 Society of Chemical Industry.
DOI:10.1111/imb.12380URLPMID:29465791 [本文引用: 1]
In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference-based insect pest management.
DOI:10.1111/imb.12187URLPMID:26304035 [本文引用: 1]
beta-N-Acetylhexosaminidases (HEXs) are enzymes that can degrade the chitin oligosaccharides that are produced by the activity of chitinases on chitin in insects. Using bioinformatic methods based on genome and transcriptome databases, 11 beta-N-acetylhexosaminidase genes (NlHexs) in Nilaparvata lugens were identified and characterized. Phylogenetic analysis revealed a six-grouped tree topology. The O-Linked N-acetylglucosaminidase (O-GlcNAcase) group includes NlHex11, which harbours a catalytic domain that differs from that of the other 10 NlHexs. Observations of the expression of NlHexs during different developmental stages revealed that NlHex4 is expressed with periodicity during moulting. Although the tissue-specific expression patterns of most NlHexs were nonspecific, NlHex4 was found to be expressed mainly in the female reproductive system as well as in the integument. RNA interference (RNAi) demonstrated failure to shed the old cuticle only in the nymphs treated with double-stranded RNA (dsRNA) targeting NlHex4, and these nymphs eventually died; no observable morphological abnormalities were found in insects treated with dsRNAs targeting the other 10 NlHexs. Based on this study and our previous analyses, a '5 + 1 + 3' pattern of chitinolytic enzymes is proposed, in which five chitinases, one NlHEX and three chitin deacetylases are required for moulting in N. lugens. A better understanding of chitin metabolism in the hemimetabolous insect, N. lugens, would be achieved by considering three chitinolytic enzyme families: chitinase, chitin deacetylase and beta-N-acetylhexosaminidase.
DOI:10.1016/j.aspen.2020.05.011URL [本文引用: 1]
