烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

中国野桑蚕性信息素合成激活肽(PBAN)基因的克隆及序列分析

根据家蚕(Bombyx mori)性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)基因DNA序列设计引物,扩增获得中国野桑蚕(Bombyx mandarina China)PBAN基因。分析表明,PBAN由33个氨基酸组成,在第14个氨基酸异亮氨酸和第15个氨基酸酪氨酸之间插入了698bp的内含子。根据PBAN及其基因cDNA、DNA序列分别构建分子进化树,结果显示3个水平比对结果构建的分子进化树有较好的一致性,推测PBAN基因可能适合于科、属之间的进化分析;并且PBAN基因内含子没有表现出特有的进化信息,推测PBAN基因内含子的进化与PBAN全长基因的进化在进化速率上并没有显著差别。相对于PBAN及α—SGNP、γ—SGNP,β—SGNP的进化速率相对较快,推测β—SGNP序列可能适合用于种间的进化分析。     

PBAN/pyrokinin神经肽类及其基因的研究进展

昆虫在其生长发育过程中,如胚胎发育、蜕皮变态、滞育、迁飞、代谢、生殖等都离不开神经肽的调控。信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)和Pyrokinin神经肽是C端具有五肽FXPRL(X=S,V,T,G等)(苯丙-X-脯-精-亮氨酸)序列的一类神经肽,在昆虫的生长发育中起重要的生理功能,如性信息素的合成、控制表皮色素、促进胚胎滞育和刺激内脏肌肉收缩等重要的生理功能。因此近几年对PBAN/pyrokinin神经肽的鉴定、加工、作用和降解方式的研究成为研究的热点,为研制高效、低毒、专一性强、无公害的杀虫剂提供了思路。介绍了PBAN/pyrokinin神经肽类及其基因的研究进展,并对PBAN/pyrokinin神经肽在害虫防治中的应用进行了展望。     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

昆虫血清素受体蛋白的生物信息学分析

【目的】昆虫血清素(5-羟色胺)受体已知有5个亚型。本文旨在系统分析昆虫5-羟色胺受体亚型蛋白的结构和进化关系。【方法】首先对文献报道已明确亚型7种昆虫的5-羟色胺受体(23个亚型序列)进行生物信息学分析,然后采用多序列比对和进化树构建的方法对NCBI数据库中推测可能为昆虫5-羟色胺受体蛋白序列进行分析。【结果】发现47个推测是昆虫5-羟色胺受体的蛋白序列中,有40个蛋白序列属于昆虫5-羟色胺受体,其余7个未能确认的昆虫5-羟色胺受体的蛋白序列都具有7个跨膜区域,属于G蛋白偶联受体家族,但不一定为5-羟色胺受体。【结论】本文对昆虫5-羟色胺受体蛋白的系统进化树分析,间接地证明了本文确认的昆虫5-羟色胺受体亚型注释信息的准确性,发现分类上同属一个目的昆虫5-HT受体序列的亲缘性较近。本研究为昆虫5-羟色胺受体的结构和功能分析提供基础。     

铃夜蛾属昆虫性信息素生物合成及内分泌调控

综述了铃夜蛾属Helicoverpa昆虫性信息素生物合成途径及内分泌因子的调控作用 ,包括信息素生物合成激活神经肽 (PBAN)和信息素生物合成抑制肽 (PSP)等的来源、结构和作用机制及一些种中保幼激素 (JH)和章鱼胺 (OA)对性信息素生物合成的作用 ,并展望了未来的研究方向。     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。     

实夜蛾属和铃夜蛾属昆虫性信息素通讯系统的研究进展

实夜蛾属Heliothis和铃夜蛾属Helicoverpa昆虫的性信息素通讯系统主要包括雌蛾的性信息素合成和雄蛾对性信息素接收两个方面,每方面都有分子、细胞、系统水平上进行协同作用的生物过程。性信息素生物合成激活肽（PBAN）与其受体作用,启动信号转导系统,从而激活合成性信息素的酶系统来合成性信息素,利用化学和生物测定的方法鉴定出具有诱蛾活性的性信息素腺体组分及行为功能;性信息素分子与性信息素结合蛋白（PBP）的复合体同受体相互作用,启动信号转导系统,诱导产生神经信号,从而引起一系列性行为反应。这些生物过程受到各种内部和外部因素的影响。     

二化螟APN1的原核表达及其与Cry2Aa蛋白的结合特性研究

【目的】Bt杀虫蛋白发挥杀虫活性的重要前提是Cry蛋白能够与昆虫中肠上皮细胞刷状缘膜囊(BBMVs)上的受体蛋白结合。在前期获得二化螟氨肽酶N1(Aminopeptidase N,APN1)基因全长序列的基础上,明确二化螟APN1多肽片段与Cry2Aa的结合能力。【方法】将二化螟APN1序列片段在大肠杆菌BL21(DE3)中表达,利用蛋白质单向电泳和ligand blotting技术分析二化螟APN1多肽片段与Cry2Aa的结合能力。【结果】重组载体可在表达菌株BL21(DE3)中表达一个约70 ku的蛋白,纯化后的多肽条带单一,纯度较好。Ligand blot分析结果显示,表达的二化螟APN1多肽片段可以与活化的Cry2Aa杀虫蛋白结合,且结合条带随着重组蛋白上样量的降低而减弱。【结论】APN1多肽片段可以与Cry2Aa结合,为阐明APN1基因的功能奠定基础,也为其他Bt蛋白的受体蛋白相关研究提供新的借鉴。     

杨柳田头菇B交配型基因信息素受体片段的克隆与分析

利用简并PCR及DNA步移法,从杨柳田头菇Agrocybe salicacola YAASM0711菌株中扩增得到了一个4 231 bp的核酸片段.经过比对及序列预测,所获得序列中含有杨柳田头菇交配型编码基因中的信息素受体部分,其序列长度为1194 bp,包含4个内含子,5个外显子的长度分别为217 bp,113 bp,67 bp,138bp,449 bp.拼接后的ORF全长984 bp,编码327个氨基酸残基.该序列与灰盖鬼伞Coprinus cinerea、双色蜡蘑Laccaria bicolor信息素受体氨基酸序列较为相似,含有7个跨膜区.信息素受体遗传进化分析显示,其与多个物种信息素受体聚集在一起,可能与真菌信息素受体的多种起源有关.     

Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer,Chilo suppressalis

The rice stem borer,Chilo suppressalis Walker is one of the most importantinsect pests on rice in Asia,north Africa and southern Europe.Transgenic Bt rice has beendeveloped in the laboratory with good resistance to this pest and other Lepidopteran insects,which will provide a possible alternative tool for this pest control.The full-length cDNAsencoding an aminopeptidase N (CsAPN) and a cadherin (CsCad) were cloned from C.suppressalis.CsAPN showed common features of,and high identities to,other insect AP...     

Impact of insect-resistant transgenic rice on target insect pests and non-target arthropods in China

Progress on the research and development of insect-resistant transgenic rice, especially expressing insecticidal proteins from Bacillus thuringiensis （Bt）, in China has been rapid in recent years. A number of insect-resistant transgenic rice lines/varieties have passed restricted and enlarged field testing, and several have been approved for productive testing since 2002 in China, although none was approved for commercial use until 2006. Extensive laboratory and field trials have been conducted for evaluation of the efficiency of transgenic rice on target lepidoteran pests and potential ecological risks on non-target arthropods. The efficacy of a number of transgenic rice lines currently tested in China was excellent for control of the major target insect pests, the rice stem borers （Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens） and leaffolder （ Cnaphalocrocis medinalis）, and was better than most insecticides extensively used by millions of farmers at present in China. No significantly negative or unintended effects of transgenic rice on non-target arthropods were found compared with non-transgenic rice. In contrast, most of the current insecticides used for the control of rice stem borers and leaffolders proved harmful to natural enemies, and some insecticides may directly induce resurgence of rice planthoppers. Studies for developing a proactive insect resistance management of transgenic rice in the future are discussed to ensure the sustainable use of transgenic rice.     

Molecular diversity of PBAN family peptides from fire ants

The PBAN/Pyrokinin peptide family is a major neuropeptide family characterized with a common FXPRLamide in the C‐termini. These peptides are ubiquitously distributed in the Insecta and are involved in many essential endocrinal functions, e.g., pheromone production. Previous work demonstrated the localization of PBAN in the fire ant central nervous system, and identified a new family of PBAN from the red imported fire ant, Solenopsis invicta. In this study, we identified five more PBAN/Pyrokinin genes from S. geminata, S. richteri, S. pergandii, S. carolinensis, and a hybrid of S. invicta and S. richteri. The gene sequences were used to determine the phylogenetic relationships of these species and hybrid, which compared well to the morphologically defined fire ant subgroup complexes. The putative PBAN and other peptides were determined from the amino acid sequences of the PBAN/pyrokinin genes. We summarized all known insect PBAN family neuropeptides, and for the first time constructed a phylogenetic tree based on the full amino acid sequences translated from representative PBAN cDNAs. The PBAN/pyrokinin gene is well conserved in Insecta and probably extends into the Arthropod phylum; however, translated pre‐propeptides may vary and functional diversity may be retained, lost, or modified during the evolutionary process. Published 2010 Wiley Periodicals, Inc.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.     

稻螟赤眼蜂对二化螟和台湾稻螟的控制潜能评价

为了明确稻螟赤眼蜂种群增长力与水稻螟虫种群增长力之间的关系和评价稻螟赤眼蜂的控害能力,通过生命表方法,组建了二化螟和台湾稻螟在水稻上的实
验种群生命表,以及稻螟赤眼蜂在二化螟卵和台湾稻螟卵上的生殖力表,测定了稻螟赤眼蜂对两种螟虫卵的寄生能力.结果表明：二化螟和台湾稻螟的世代历期（T）分别为56.40 d和47.80 d,内禀增长率（r_m）分别为0.0489和0.072.稻螟赤眼蜂在二化螟卵和台湾稻螟卵上的平均T分别为9.75 d和9.78 d,r_m分别为0.3161和0.3154.通过比较分析稻螟赤眼蜂与两种螟虫的实验种群生命表参数,可知稻螟赤眼蜂能够有效控制两种水稻螟虫,赤眼蜂种群增长力相对于螟虫种群是超前而不是跟随关系.     

Differential fipronil susceptibility and metabolism in two rice stem borers from China

The susceptibilities of larvae of two rice stem borers, namely, Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Sesamia inferens (Walker) (Lepidoptera: Nocutidae) to fipronil and its metabolites were investigated, and then the activities of microsomal O-demethylase, and glutathione transferase (GST) in two species were measured. The metabolism of fipronil in both stem borers was determined in vivo and in vitro. The LD50 value of fipronil to S. inferens was 118.5-fold higher than that of C. suppressalis. The bioassay results offipronil metabolites showed that the toxicities of sulfone and sulfide were higher than fipronil for both species, and the differential toxicity between sulfone and fipronil was remarkable. Alternatively, the activities of microsomal O-demethylase and GST of C. suppressalis were 1.35- and 2.06-fold higher than S. inferens, respectively. The in vivo and in vitro studies on metabolism of fipronil showed that all of fipronil, sulfone, and sulfide were detected and the content of sulfone was higher than sulfide in both stem borers. The residue of sulfone in C. suppressalis was significantly higher than that in S. inferens. These results suggest that the higher activity of mixed function oxidases may cause the higher capacity of C. suppressalis to produce fipronil-sulfone, which is more toxic than fipronil leading to the higher susceptibility of this species.     

Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers

Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.     

Enhanced oral availability/pheromonotropic activity of peptidase-resistant topical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides

The peptide bond between active core residues Pro and Arg is the primary site of susceptibility for the pyrokinin/PBAN neuropeptides to insect tissue-bound peptidases, and incorporation of modified Pro residues can enhance resistance to peptidase hydrolysis. An Hyp-containing amphiphilic analog (Hex-FT[Hyp]RLa) is shown to operate as a topically active tissue-bound peptidase-resistant analog of the pyrokinin/PBAN class of insect neuropeptides in adult Heliothis virescens moths. An Oic amphiphilic analog (Hex-FT[Oic]RLa) is ineffective topically, but proves to be a superior tissue-bound, peptidase-resistant pyrokinin/PBAN analog for oral administration; outperforming both the Hyp analog and the orally inactive natural hormone PBAN in the moths. The Oic analog is effective in penetrating an isolated, ligated foregut preparation, but less successful in transmigrating an isolated midgut preparation; whereas the opposite behavior is observed for the Hyp analog. The success of the Oic analog via oral administration may be related to its ability to effectively penetrate the foregut, thereby bypassing the hostile environment of the midgut region.