RNA干扰对家蚕丝腺蜕皮激素相关基因表达的影响

昆虫变态发育过程中,蜕皮激素通过一系列的激素相关转录因子进行信号的转导和放大,从而完成对生长变态发育的调控,其中蜕皮激素受体(EcR)及转录因子BR-C和E74A可能作为早期因子发挥作用.为了研究这3个早期转录因子在鳞翅目昆虫中的功能,本研究采用体外合成dsRNA的方法,将合成的dsRNA分别注射熟蚕期的家蚕Bomby...     

飞蝗载脂蛋白受体基因LmLPR的表达及功能分析

【目的】载脂蛋白受体(Lipophorin receptor,LPR)在昆虫体内脂类物质的转运中发挥着重要的作用。基于飞蝗转录组数据库获得飞蝗载脂蛋白受体基因LmLPR,分析其基因特性和表达特征,并通过RNA干扰(RNA interference,RNAi)技术对其生物学功能进行分析,探究LmLPR在飞蝗脂类物质转运中的作用。【方法】基于课题组飞蝗转录组数据库,获得载脂蛋白受体基因,结合飞蝗基因组数据库,获得其全长开放阅读框(ORF)序列,克隆验证后对其编码蛋白序列特性进行分析;利用GENEDOC软件将该蛋白氨基酸序列与其他物种LPR氨基酸序列进行多序列比对,并使用MEGA5.1软件中的Neighbor-Joining方法,将该序列与其它物种的同源序列进行聚类分析;通过RT-qPCR方法对其在5龄第2天飞蝗不同组织部位和不同发育天数表皮中的表达特性进行分析;通过RNAi技术及伊红染色方法对其生物学功能进行分析。【结果】通过克隆和序列分析获得两种飞蝗LPR基因,即长型(LmLPR-L)和短型(LmLPR-S),序列特征分析发现LmLPR-L和LmLPR-S均有1个信号肽,4个表皮生长因子结构域,5个低密度脂蛋白受体YWTD结构域,以及1个跨膜域。不同于LmLPR-L,LmLPR-S在N端比LmLPR-L多一个低密度脂蛋白受体结构域(LmLPR-L含有6个,LmLPR-S含有7个),而在C端缺失38个氨基酸序列。系统进化树分析显示LmLPR-L和LmLPR-S与蜚蠊目德国小蠊BlattellagermanicaBgLPR-L和BgLPR-S聚为一支。RT-qPCR结果显示LmLPR-S在卵巢中高表达,在其他组织部位的表达量较低,而LmLPR-L在翅芽和卵巢中均高表达,在其他组织部位的表达量较低;时期表达显示LmLPR-S和LmLPR-L在蜕皮后表达量均呈现先升高后降低的趋势,分别在第2天和第3天的表达量最高。在4龄期通过RNAi抑制LmLPR的表达后,发现飞蝗能够正常蜕皮,伊红染色结果显示表皮通透性没有显著改变。【结论】飞蝗载脂蛋白受体基因LmLPR主要在翅芽和卵巢中高表达,其下调表达后不影响若虫的正常生长发育以及表皮的通透性。研究结果为深入探究载脂蛋白受体在昆虫表皮脂类物质转运过程中的作用机制提供了基础。     

飞蝗羧酸酯酶基因LmCarE25的体外重组表达

羧酸酯酶(Carboxylesterase,CarE)是一类在生物中广泛分布的多功能家族酶系,在昆虫抗药性形成机制中发挥重要作用。为探讨飞蝗Locusta migratoria(Meyen)羧酸酯酶基因LmCarE25的生物学功能,本文采用荧光实时定量PCR技术进行研究,发现其在飞蝗成虫各组织部位均有表达,其中胃盲囊、翅和肌肉中表达量较低,中肠、马氏管和脂肪体表达量较高。本文尝试在大肠杆菌和昆虫细胞体外重组表达该酶,构建重组表达载体pET32a-LmCarE25,SDS-PAGE结果显示,LmCarE25可在大肠杆菌BL21(DE3)和JM109中表达,蛋白分子量约为60 ku,但为包涵体;将LmCarE25基因插入真核表达载体pFastBacHTA中,借助Bac-to-Bac体系获得重组Bacmid,以sf9细胞系作为宿主细胞表达目的蛋白,Western-Blot检测结果显示,LmCarE25获得可溶性表达。上述结果为进一步深入研究飞蝗羧酸酯酶家族功能提供了基础资料。     

棉铃虫章鱼胺受体2基因的分子鉴定

【目的】为了探讨章鱼胺受体2基因(Octopamine receptor 2,OA2B2)在棉铃虫Helicoverpa armigera(Hübner)体内表达谱及激素对其调控作用,为今后研究Ha OA2B2受体基因在转录调控中的作用,开发新型杀虫剂提供一定的理论依据。【方法】本实验采用QRT-PCR技术、激素处理、RNAi介导的蜕皮激素相关核受体沉默等方法对Ha OA2B2受体基因在棉铃虫不同发育阶段及组织中的表达谱与激素调控进行探究。【结果】生物信息学分析表明,Ha OA2B2受体基因编码框长度1 191 bp,编码396个氨基酸。Ha OA2B2受体基因氨基酸序列与家蚕Bombyx mori一致性高达89%。Ha OA2B2受体基因在棉铃虫各时期均有表达,尤其在5龄第3天转录水平达到最高;Ha OA2B2受体基因在棉铃虫幼虫头部表达量最高。蜕皮激素可促进Ha OA2B2受体基因的表达。【结论】分析了该基因的序列特征和表达谱,蜕皮激素对其表达量的影响,为今后研究Ha OA2B2受体基因在转录调控中的作用以及开发新型杀虫剂提供一定的理论依据。     

飞蝗发育相关基因Omb的克隆、原核表达及时空表达分析

【目的】本研究克隆飞蝗Locusta migratoria发育相关的optomotor-blind(Omb)基因,并对其进行序列和表达分析,旨在更好地了解Omb基因在飞蝗翅发育中的作用及为进一步蝗灾的治理和防治提供新的理论依据。【方法】利用RT-RCR扩增飞蝗Omb基因cDNA序列,采用生物信息学软件分析该基因的核苷酸和氨基酸序列,利用MEGA 6. 0构建昆虫纲分子系统进化树;构建重组表达载体pET-30a/LmOmb,转化到大肠杆菌Escherichia coli BL21(DE3)中,SDS-PAGE及Western blot鉴定重组表达蛋白;基于qPCR技术分析Omb基因在飞蝗不同发育时期及成虫不同组织中的表达谱。【结果】克隆获得飞蝗Omb基因部分cDNA序列,命名为LmOmb(GenBank登录号:MG867658),其长792 bp,编码264个氨基酸,在第37-219位氨基酸之间存在一个T-box superfamily保守结构域。同源序列比对分析表明Lm Omb与褐飞虱Nilaparvata lugens Nl Omb氨基酸序列一致性为93%。在IPTG诱导下目标蛋白以6×His标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR结果显示LmOmb基因在飞蝗不同发育时期均有表达,其中胚胎期的表达量最高,进入若虫期后表达量下降,且各龄若虫之间表达量相对平稳。Lm Omb基因在雌性和雄性成虫的胸部、足、腹部和翅中都有表达,且雌性和雄性之间表达量明显不同。【结论】LmOmb基因可能参与了飞蝗的胚胎发育。研究结果为进一步研究飞蝗LmOmb的功能提供了依据。     

飞蝗雌成虫脂肪体G蛋白偶联受体鉴定及其在卵发育中的功能

【目的】本研究旨在鉴定飞蝗Locusta migratoria雌成虫脂肪体中表达的G蛋白偶联受体(G protein-coupled receptor, GPCR)基因,揭示其在飞蝗卵发育和卵巢生长中的功能。【方法】基于前期获得的第1个促性腺周期内飞蝗雌成虫脂肪体转录组数据,鉴定和聚类分析GPCR基因;qRT-PCR检测GPCR基因在卵黄发生期飞蝗雌成虫脑、胸腹神经节、脂肪体、卵巢和中肠的组织特异性表达模式;通过RNAi实验分析GPCR基因在卵发育和卵巢生长中的功能。【结果】在飞蝗雌成虫脂肪体转录组中新鉴定到了29个GPCR基因。qRT-PCR分析显示,PTH/PTHrPR1和MSR分别在飞蝗雌成虫脂肪体和胸腹神经节中显著高表达,Mthl4,Mthl6和GPR119L在中肠中显著高表达,Octβ1R,CrzR,CCAPR,LGR2,mGluR和GABABR在脑中的表达水平显著高于在其他组织中的。RNAi筛选发现敲降5个GPCR基因CCAPR,PTH/PTHrPR1,ADGRL3,Mthl15和DHR的表达显著抑制飞蝗初级卵母细胞发育和卵巢生长。【结论】本研究结果有助于揭示昆虫高繁殖力的...     

沙葱萤叶甲对蜕皮激素响应的转录组分析

为建立沙葱萤叶甲Gauleruca daurica成虫对蜕皮激素(20-hydroxyecdysone, 20E)响应的转录组数据库,挖掘对20E响应的基因以及代谢和信号通路,并在转录组水平探讨20E调控生殖滞育的分子机制,本研究采用Illumina HiSeqTM4000高通量测序平台对20E及二甲基亚砜(dimethyl sulfoxide, DMSO)处理后的沙葱萤叶甲成虫进行了转录组测序,共获得80 313个unigene;与阴性对照DMSO相比,共获得201个差异表达基因,其中106个上调、95个下调。GO和KEGG富集分析表明,多数差异表达功能基因富集于各种代谢通路,其中核黄素代谢(riboflavin metabolism)、溶酶体(lysosome)和泛酸与乙酰辅酶A合成(pantothenate and CoA biosynthesis)通路显著富集(q<0.05)。结果表明,20E可能通过影响多种代谢通路调控沙葱萤叶甲的生殖滞育。     

蜕皮激素及其对蟹虾养殖业的影响

一、蜕皮激素概况昆虫和甲壳动物蜕皮激素的研究是本世纪四十年代开始的。当时日本学者福田等人曾对家蚕进行结扎、断头及器官移植等试验,发现蚕的前胸腺的分泌活动与蜕皮及变态有着内在的密切联系。1954-1956年,德国卡尔森等从蚕蛹中分离到微量天然蜕皮激素(50mg/1000kg),仅使用0.0075μg就可以使结扎的丽蝇腹部化蛹。1965年,由美国霍夫迈斯特等鉴定了α-蜕皮激素的化学结构,证明它是一种广泛存在于昆虫及甲壳类动物体内的甾族化合物(即类固醇)。后     

向日葵AGO和DCL基因家族的鉴定和表达分析

为了解向日葵(Helianthus annuus)的Argonaute (AGO)和Dicer-like (DCL)的功能,利用拟南芥(Arabidopsis thaliana)的AGO和DCL基因序列在向日葵基因组数据库中进行同源比对,对向日葵AGO和DCL家族成员进行生物信息学分析。结果表明,从向日葵中鉴定到15个Ha AGOs和5个HaDCLs家族成员;这2类基因在染色体上的分布均不均匀。系统发育分析表明,与拟南芥相似,HaAGO家族成员可分为3个分支,HaDCL可分为4个分支;所有的HaAGO都具有保守的N domain、DUF1785、PAZ和PIWI结构域,Ha DCL家族成员都含有PAZ和RIBOc结构域。表达分析表明,Ha DCL3a和Ha DCL3b在茎和花序中高度表达;亚细胞定位表明Ha AGO多定位于细胞核。这表明向日葵中可能存在典型的RNAi干扰机制,并可能参与了协调向日葵的生长发育过程。     

麦红吸浆虫蜕皮激素受体（EcR）基因的克隆与表达分析

为了研究蜕皮激素受体（EcR）在麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育活动中的作用, 利用RT PCR和RACE技术克隆得到了麦红吸浆虫蜕皮激素受体基因cDNA全长, 并通过Real-time quantitative PCR研究了其表达情况。该cDNA全长序列被命名为SmEcR （GenBank登录号： KC491135）, 其开放阅读框长1 386 bp, 编码461个氨基酸残基。其蛋白预测分子量52.90 kD, 理论等电点6.24。该蛋白与其他已报道的昆虫EcR蛋白具有很高的同源性, 其中与迟眼蕈蚊Bradysia coprophila中相应蛋白的氨基酸序列一致性高达92%。SmEcR在麦红吸浆虫不同滞育时期和不同虫态中均有表达, 且在不同滞育时期、 不同虫态中的表达量差异很大。在滞育不同时期以11月表达量最高, 12月表达量最低; 在不同虫态以麦穗幼虫中的表达量较低, 而成虫中的表达水平很高。本研究为进一步明确SmEcR在麦红吸浆虫滞育调控中的作用奠定了基础。     

Protein phosphorylation in the larval epidermis of Locusta migratoria: Role of cyclic AMP and 20-hydroxyecdysone

Cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinase (PK) activities were quantified in the wing-pad epidermis of Locusta migratoria during the fifth larval instar. A low level of cyclic nucleotide-independent PK activity was detected in this tissue, whereas no PK-C (Ca²⁺- and phospholipid-dependent enzyme) activity was found. The main cyclic nucleotide-PK activity was cAMP-PK and large changes were observed during the intermoult. Concomitant increases in cAMP-PK activity and cAMP-dependent phosphorylation of several endogenous polypeptide substrates occurred during the last part of the intermoult cycle. The most marked ³²P-incorporation was observed in a 43 kDa polypeptide. These changes could be correlated with the peak of 20-OH-ecdysone in both hemolymph and integument.In vitro incubation of wing-pads with 20-OH-ecdysone (48 h) did not change cAMP-PK activity, but there were major changes in the pattern of endogenous phosphoproteins in particular the appearance of 43 and 45 kDa phosphoproteins. Two-dimensional analysis revealed several groups of phosphorylated spots. The results indicate a close relationship between certain cAMP-dependent phosphorylated polypeptides and the action of 20-OH-ecdysone.     

Effects of juvenile hormone analogs and 20-hydroxyecdysone on diapause termination in eggs of Locusta migratoria and Oxya yezoensis

To understand the hormonal control of embryonic diapause, juvenile hormone analogs (JHAs), methoprene and hydroprene, and 20-hydroxyecdysone (20E) were applied onto diapause eggs of Locusta migratoria and Oxya yezoensis. These insects enter diapause at the mid-stage of embryogenesis prior to blastokinesis. Topical application of JHAs significantly facilitated diapause termination in both species but JHA-treated embryos underwent abnormal morphogenesis, pigmentation and sclerotization without dorsal closure. The Locusta eggs immersed in the 20E solution for 24h terminated diapause in a dose-dependent manner. We also investigated phosphorylation of extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinase (MAPK), during diapause-terminating process of Locusta migratoria and found that ERK was activated either by cold exposure or JHA treatment. The possible involvement of the hormones and ERK in embryonic diapause and the possibility of ecdysteroids synthesis by prothoracic glands of diapause embryo were proposed.     

Age-related changes in the response to adipokinetic hormone in Locusta migratoria

ABSTRACT. In fifth instar nymphs of Locusta there is only a feeble adipokinetic response to extracts of corpora cardiaca. In fledglings, this poor response persists for a few days but then increases dramatically to reach a plateau by day 8. The response declines to almost zero as the locusts age beyond 35 days of adult life. This pattern of change in response is similar in both males and females but there are some differences in magnitude depending upon whether the response is measured as changes in haemolymph total lipid (vanillin-positive material) or total diglyceride (gas liquid chromatography). The poor response to adipokinetic hormone in nymphs and newly fledged locusts is not a result of shortage of stored lipid in the fat body and cannot be improved by injection of extra hormone.     

Cuticle protein in Locusta migratoria

• 1.1. The total protein content and the soluble proteins of the cuticle of male fifth instar and newly moulted adult locusts were analysed.
• 2.2. Protein was deposited in cuticle in a stepwise manner whereas chitin deposition was continuous.
• 3.3. Resorption of cuticular protein preceded resorption of chitin following apolysis.
• 4.4. Immunological and electrophoretic analyses indicated that certain cuticular proteins were also present in the haemolymph.
• 5.5. Certain water soluble proteins appeared to be characteristic of the newly deposited cuticle and were presumably bound to the cuticular matrix as development continued.
• 6.6. Despite an increase in total soluble protein following apolysis, no evidence of new proteins was detected. It was deduced that the initial step in protein resorption from the old cuticle is the release of the protein from its cuticular bonds.

     

东北地区亚洲飞蝗染色体核型分析

采用常规压片法对吉林省亚洲飞蝗Locusta migratoria migratoria(L.)的染色体核型进行分析.研究结果表明:亚洲飞蝗性别决定机制为X0型,染色体数目为2n♂=23,染色体组式4L+4M+3S+X,全部为端部着丝粒染色体,NF=23.染色体中最长(L1)与最短(S11)染色体之比大于4:1,臂比大...     

西藏飞蝗的生物学特性

西藏飞蝗Locusta.migratoria tibetensis Chen在四川甘孜州1年发生1代,某些地方(乡城县)1年发生不完整的2代,即以卵越冬,翌年3月下旬开始孵化出土,4月中、下旬为孵化盛期,1~3龄始盛期为4月中旬~5月中旬,高峰期为5月下旬,7月上旬初始羽化,7月下旬~8月上旬为羽化盛期,8月上旬始见产卵,8月下旬~9月上旬为产卵盛期,第1代成虫较早产下的卵块在条件适宜的情况下可于当年9月上旬孵化出土,但孵化出的蝗蝻不能越冬。该虫卵、全蝻期及全世代的发育起点温度分别为14.2,16.1,14.6℃,有效积温为179.1日.度、360.0日.度、787.8日.度。在18,21,24,27和30℃等5种恒温条件下其平均世代历期214.4,133.3,79.2,66.3和50.7d。     

Thermal response and reversibility of diapause in the eggs of Locusta migratoria

Abstract. Eggs of a local population of Locusta migratoria L. (Orthoptera: Acrididae) collected near Hirosaki (40.5^oN) entered diapause when incubated at temperatures between 20 and 35^oC. For diapause development the optimum temperature was 10^oC. The lower thermal threshold for post-diapause development was 14.7^oC. After chilling at 10^oC for 20 days, the rate of hatching varied with incubation temperature, being 0, 61% and 81% at 20, 25 and 30^oC, respectively. After chilling for 40 days or more, however, almost all eggs hatched at 20–30^oC. Diapause with a reduced intensity seemed to be eliminated easily by a high temperature of 25 or 30^oC.
When eggs chilled at 10^oC for 20 days were kept at 20^oC for 7 days or more before incubation at 25^oC, almost all eggs maintained diapause. Most eggs chilled at 10^oC for three 10 day periods separated by 3 days of warming at 25^oC failed to terminate diapause. Daily alternations of 10^oC (18 h) and 25^oC (6h) decreased the diapause-terminating effect of chilling. These facts suggest that diapause intensity can be restored if eggs chilled insufficiently are exposed to a moderately high temperature. This reversible change in diapause intensity would play an important role in maintaining diapause before winter.     

Ecdysteroids During Embryogenesis in Locusta migratoria

SYNOPSIS. Ovaries of Locusta migratoria synthesize large amountsof ecdysteroids at the end of oöcyte maturation. The predominantecdysteroids in mature ovaries are conjugated 2-deoxyecdysone(100 µM) and conjugated ecdysone (50 µM) which outnumberthe corresponding free compounds by 50–100 fold. Thesevarious ecdysteroids persist during ovulation and are recoveredfrom newly-laid eggs. The conjugated maternal ecdysteroids aregradually metabolized as embryonic development proceeds; theyhave disappeared as such on day 6 after oviposition, that isafter blastokinesis and shortly after dorsal closure. Concomitantlyto this metabolism of the maternal conjugated ecdysteroids,other ecdysteroid conjugates appear in the eggs which have differentchromatographic behaviors and some of which are conjugates ofecdysone metabolites formed by the embryo. The data availableso far are compatible with the hypothesis that the maternalconjugates are hydrolysed to free 2-deoxyecdysone and ecdysoneby the embryo during early stages of development and subsequentlyconjugated to inactivation compounds. During the later stagesof embryonic development however, a de novo synthesis of ecdysoneis probable, the maternal conjugates having been metabolizedduring the earlier stages.     

Chiasma distribution in asynaptic Locusta migratoria

The formation of chiasmata in six full sib male partially asynaptic individuals of Locusta migratoria has been studied. The mean chiasma frequency per cell was 2.3 both at diplotene and metaphase I. Chiasmata tended to be distributed evenly among the bivalents. The frequency and distribution of the chiasmata in each type of bivalent (L, M, or S) depended on the level of asynapsis and on interference between the bivalents. Short bivalents were the most affected by interference, while M bivalents associated independently of L and S bivalent behaviour.