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

Abstract The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed 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 cDNAs encoding an aminopeptidase N (CsAPN) and a cadherin (CsCad) were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here (CsCad) showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region (CR7), which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin Cry1Ab. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.     

水稻螟虫神经肽PBAN及其受体序列的生物信息学分析

【目的】性信息素合成激活肽(PBAN)是控制昆虫产生性信息素的激素,本文旨在分析水稻螟虫神经肽PBAN及其受体的序列。【方法】通过t Blastn同源检索从水稻螟虫基因组和转录组数据库中鉴定水稻螟虫PBAN神经肽及其受体序列,在此基础上进行序列比对及系统发生分析。【结果】发现二化螟Chilo suppressalis、三化螟Tryporyza incertulas和大螟Sesamia inferens的PBAN成熟肽序列均含有33个氨基酸残基,其C端五肽序列完全相同,3种水稻螟虫PBAN多肽相似度为54.55%~63.64%;发现二化螟PBAN受体3个异构体全长氨基酸序列(PBANR-A、PBANR-B和PBANR-C),均含有7个跨膜区域。【结论】进化树分析发现不同昆虫PBAN神经肽及其受体存在一定的保守性和多样性,并且在进化树上的位置几乎与昆虫系统发育分类一致,推测PBAN神经肽和PBAN受体在昆虫系统进化过程中可能存在协同进化现象。本研究为水稻螟虫PBAN神经肽及其受体的结构和功能分析提供基础。     

Host status of wheat and corn for Chilo suppressalis (Lepidoptera: Crambidae)

Host plant specificity depends on recognition of the host and the ability to discriminate it from nonhost plants. Chilo suppressalis (Walker) (Lepidoptera: Crambidae), an important insect pest of rice, is considered to be polyphagous, although few papers have ever reported infestation of C. suppressalis on most of the recorded hosts. The present investigation was designed to test whether two important gramineous crops, wheat and corn, are host plants of C. suppressalis based on the host-plant finding process in cage and Y-tube olfactometer tests, oviposition and egg hatching, and larval feeding and survival. In the cage tests, gravid C. suppressalis females did not differentiate rice plants from wheat or corn plants when only visual cues were involved, but were more attracted to rice plants when only olfactory cues or both vision and olfaction were present. The Y-tube olfactometer tests further confirmed that the females did not prefer wheat or corn plants, and revealed that they responded equally to clean air and odors from wheat or corn plants. Under no-choice and choice condition alike, the females laid eggs on a lower proportion of wheat and corn plants and egg number and hatching rate were significantly reduced on wheat and/or corn plants than on rice plants. Larval feeding was not observed in wheat and lower in corn than in rice plants, and no pupae or surviving larvae were collected from wheat and corn plants. The results suggest that wheat and corn are not host plants of C. suppressalis. These findings are discussed in context of host-finding process in C. suppressalis and management of resistance to transgenic Bt rice.     

水稻品种(系)对二化螟抗性的初步研究

2000-2001年,对本省目前推广的55个主要水稻品种（系）进行二化螟抗性测定和田间调查,从中筛选出中抗品种（系）3个,耐虫品种（系）16个,且粳稻的抗虫性优于籼稻。田间试验进一步验证了各品种（系）对二化螟的抗耐性,为育种单位和生产部门提供理论依据。     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI-1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI-1B at more than 1 ng/10 g total protein was significantly retarded compared to that on non-transgenic control plants.     

二化螟种群动态及管理研究进展

本文综述了二化螟种群消长规律、性诱剂研究与应用、抗药性及综合治理的研究概况 ,并对如何提高科学治理二化螟水平作了展望     

二化螟乙酰胆碱受体α亚基的基因克隆与序列分析

烟碱型乙酰胆碱受体(nAChR)在昆虫的兴奋性突触传递中起着重要的作用,同时也是杀虫剂作用的重要靶标。近年来,二化螟对作用于昆虫nAChR的沙蚕毒素类杀虫剂杀虫单产生了高抗性。为了研究可能存在的靶标不敏感机制,我们采用RT-PCR技术,对二化螟nAChR-α亚 基全长cDNA进行了分子克隆。序分析表明,这是1个新的α亚基基因,定名为Cs α 1。基 因全长为1997个核苷酸,包含了1个开放阅读框,编码1个509氨基酸的成熟蛋白和1个24氨基酸的信号肽。Cs α 1与其他昆虫nAChR α亚基之间有52%-94%的同源性,高于与脊椎动物nAChR α亚基之间的同源性。     

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.     

Specific herbivore-induced volatiles defend plants and determine insect community composition in the field

In response to insect attack, plants release complex blends of volatile compounds. These volatiles serve as foraging cues for herbivores, predators and parasitoids, leading to plant-mediated interactions within and between trophic levels. Hence, plant volatiles may be important determinants of insect community composition. To test this, we created rice lines that are impaired in the emission of two major signals, S-linalool and (E)-β-caryophyllene. We found that inducible S-linalool attracted predators and parasitoids as well as chewing herbivores, but repelled the rice brown planthopper Nilaparvata lugens, a major pest. The constitutively produced (E)-β-caryophyllene on the other hand attracted both parasitoids and planthoppers, resulting in an increased herbivore load. Thus, silencing either signal resulted in specific insect assemblages in the field, highlighting the importance of plant volatiles in determining insect community structures. Moreover, the results imply that the manipulation of volatile emissions in crops has great potential for the control of pest populations.     

二化螟成虫雄性先羽化现象

二化螟Chilos uppressalis(Walker)是水稻一种重要的鳞翅目害虫,作者在室内人工饲养的过程中注意到二化螟的雄虫羽化较雌虫早,因此对二化螟的雄性先熟现象进行了初步的研究,结果发现雌雄二化螟从初孵幼虫到成虫羽化阶段的历期平均长度存在显著差异,雄虫的历期为38.8d,而雌虫的历期为40.6d,即雄虫较雌虫要早1.8d羽化。本文对雄性先熟现象的生物学意义进行了简要讨论,同时,就利用性诱方法对二化螟进行监测和防治进行了讨论。     

The broad-leaf herbicide 2,4-dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.     

敲除ebony基因导致二化螟体表黑色素沉积(英文)

【目的】二化螟Chilo suppressalis是中国和其他亚洲地区为害水稻的重要害虫。由于缺乏遗传学操作工具,对二化螟的功能基因组学研究受到严重限制。本研究的目的是借助一个标记基因ebony,在二化螟体内建立CRISPR/Cas9介导的基因编辑体系。【方法】以家蚕Bombyx mori ebony蛋白作为参考序列,通过TBLASTN程序在二化螟基因组数据库获得二化螟ebony基因的cDNA序列;通过PCR技术克隆二化螟ebony全长序列并进行生物信息学分析;运用qRT-PCR技术分析二化螟ebony基因在不同发育阶段(卵、幼虫、预蛹、蛹和雌雄成虫)和4龄幼虫不同组织(头、表皮、脂肪体、肠道和马氏管)中的表达模式;借助基于CRISPR/Cas9的基因编辑技术,把sgRNA/Cas9蛋白复合物显微注射到二化螟新鲜的卵内(产卵后2 h内),靶向敲除二化螟ebony基因。【结果】克隆得到二化螟Csebony基因(GenBank登录号: MZ846208)的全长cDNA序列,开放阅读框长为2 586 bp,共编码861个氨基酸。Csebony蛋白质的分子质量为9.5 kD,理论等电点为5.10,且在N端没有信号肽。结构域分析显示,Csebony有3个保守的结构域。系统发育树显示,Csebony与玉米螟Ostrinia furnacalis的ebony亲缘关系最近。qPT-PCR结果显示Csebony基因在蛹期和头部高表达。敲除Csebony导致二化螟幼虫、蛹以及成虫黑色素沉积。【结论】本研究结果表明,Csebony参与了二化螟表皮颜色模式的调控;CRISPR/Cas9基因组编辑技术可在二化螟中有效应用。借助标记基因在非模式生物中快速建立CRISRP/Cas9基因编辑体系,可为二化螟的功能基因组学研究提供有价值的遗传学工具。     

二化螟雄蛾交配行为与精巢大小的关系

【目的】为了明确二化螟Chilo suppressalis雄蛾交配行为与精巢大小的关系,阐明信息素群集诱杀防治二化螟的有效性。【方法】本研究通过测定和比较不同日龄、交配状态以及性信息素诱捕的二化螟雄蛾精巢体积,建立精巢体积与雄蛾发育及交配状态的相关性。【结果】雄蛾日龄显著影响二化螟的交配,刚羽化(0日龄)雄蛾的交配率较低,1日龄雄蛾的交配率最高,之后随着日龄增加交配率逐渐降低。精巢体积与二化螟雄蛾日龄之间存在显著的负相关性。同一日龄交配的二化螟雄蛾的精巢体积显著大于未交配雄蛾;性诱捕器诱捕雄蛾的精巢体积显著大于未被性诱捕器诱捕雄蛾,与已交配雄蛾的精巢体积相似。交配之后,精巢的发育过程和未交配雄蛾相似。田间性信息素引诱二化螟雄蛾精巢体积小于0-1日龄未交配雄蛾,大于2-6日龄未交配雄蛾。【结论】本研究结果表明,二化螟雄蛾对雌蛾性信息素的反应与其精巢大小存在正相关性;性信息素引诱的二化螟雄蛾具有更强的性信息素反应能力,同时性信息素引诱的大部分雄蛾为未交配状态;交配过程并不影响二化螟雄蛾精巢体积,也不影响精巢发育。二化螟交配能力与精巢大小的关系在理论上解释了性信息素群集诱杀二化螟的防控有效性,并为其他蛾类昆虫的交配研究提供依据。     

一种采集水稻二化螟卵(块)的高效简便新方法

比较了二化螟Chilosuppressalis卵块的塑料袋收集法和稻株法。结果表明 ,塑料袋法和稻株法收集的卵粒总数及卵孵化率都没有显著的差异 ,均为 2 79粒卵 雌蛾和 95 %。塑料袋法收集的卵块数较稻株法的多 ,但小卵块比例较高 ( <1 0 0粒卵 卵块 )。还讨论了采用塑料袋法收集二化螟及其它昆虫卵的优点。     

水稻黑条矮缩病传毒昆虫的防治实践与研究

水稻黑条矮缩病 (RBSDV)是由传毒媒介灰飞虱Laodelphaxstriatellus (Fall n)传播所致 ,治虫防病是目前防治水稻黑条矮缩病的重要手段。生产实践证明 ,防治该病应以控制灰飞虱种群数量增长为首选目标 ,把带毒灰飞虱尽可能地扑灭在迁移到水稻秧苗进行传毒侵染之前。因此掌握防治适期 ,选用高效低毒农药 ,切断初次侵染来源 ,才能达到控制该病发生的目的。