稻纵卷叶螟羧酸酯酶基因的鉴定与表达谱分析

【目的】鉴定稻纵卷叶螟Cnaphalocrocis medinalis的羧酸酯酶基因,并检测这些基因在成虫不同组织中的表达模式。【方法】从稻纵卷叶螟转录组中搜索羧酸酯酶基因,使用生物信息学软件对所获得的基因序列进行分析,使用荧光定量PCR检测这些基因在各组织中的相对表达水平。【结果】获得了15个羧酸酯酶基因,分别命名为Cm Car E1~Cm Car E15。其中Cm Car E12缺少3′区域,其余14个序列均含有完整的开放阅读框。除Cm Car E11外,其余14个基因编码的蛋白均具有羧酸酯酶的典型特征,如保守的五肽结构域,催化三联体和氧阴离子穴等。系统进化分析显示15个Cm Car Es被聚在不同的进化支内,Cm Car E13、Cm Car E14和Cm Car E15聚在"胞内催化类"进化支,其余12个Cm Car Es聚在"分泌催化类"进化支。Cm Car E1、Cm Car E2、Cm Car E3、Cm Car E7、Cm Car E8、Cm Car E10和Cm Car E13特异表达于成虫腹部,而Cm Car E9特异表达于雌雄成虫触角。其他基因的表达没有组织特异性。【结论】Cm Car E1、Cm Car E2、Cm Car E3、Cm Car E7、Cm Car E8、Cm Car E10和Cm Car E13编码的酯酶可能参与了内外源化合物的代谢,而Cm Car E9编码的酯酶可能参与了气味分子的降解。     

稻纵卷叶螟海藻糖酶基因的克隆、分子特性和表达分析

【目的】海藻糖酶(trehalase,Tre)是昆虫体内海藻糖代谢的一个关键酶,包括可溶型(Tre1)和膜结合型(Tre2)两种类型,在昆虫发育和能量调节中具有重要作用。本研究旨在克隆稻纵卷叶螟Cnaphalocrocis medinalis海藻糖酶基因(Cm Tre),解析其在稻纵卷叶螟不同发育阶段和不同组织中的表达模式,分析该基因及酶蛋白的分子特征。【方法】通过稻纵卷叶螟转录组数据结合RACE技术,克隆稻纵卷叶螟Cm Tre的全长c DNA序列,并对该基因进行生物信息学分析;采用实时荧光定量PCR(RT-q PCR)解析Cm Tre在稻纵卷叶螟不同发育阶段及成虫不同组织部位的m RNA表达模式。【结果】获得两种类型的稻纵卷叶螟Cm Tre基因,即可溶型海藻糖酶基因Cm Tre1和膜结合型海藻糖酶基因Cm Tre2。Cm Tre1的全长c DNA长度为2 364 bp,开放阅读框长1 704 bp,编码567个氨基酸;Cm Tre2的全长c DNA长度为2 079 bp,开放阅读框长1 923 bp,编码640个氨基酸。生物信息学分析表明,Cm Tre前端有一个信号肽,Cm Tre1无跨膜结构,Cm Tre2有一个跨膜结构。同源性和聚类分析表明,Cm Tre1和Cm Tre2的氨基酸序列与竹蠹螟Omphisa fuscidentalis海藻糖酶Tre1和Tre2氨基酸序列的一致性最高,分别为74%和79%。同源建模预测结果显示,Cm Tre1的三维分子结构包含19个α螺旋和2个β折叠片;Cm Tre2的三维分子结构含有23个α螺旋,没有β折叠片。RT-q PCR结果显示,Cm Tre在稻纵卷叶螟整个发育历期都有表达,在成虫期表达水平最高,在整个幼虫期都有相对稳定的表达;Cm Tre1在蛹期表达水平最低,Cm Tre2在5龄幼虫时期表达水平最低。Cm Tre在所检测的成虫组织(中肠、体壁、马氏管、头、卵巢、脂肪体、肌肉和精巢)中均有表达;Cm Tre1在中肠和体壁中的表达水平较高,Cm Tre2在肌肉和中肠中的表达水平较高。【结论】本研究克隆了稻纵卷叶螟两种类型的海藻糖酶基因,分析了其基因特征和表达模式。研究结果为阐明海藻糖酶基因的功能进而以海藻糖酶基因为靶标防治害虫奠定了基础。     

粤北稻区稻纵卷叶螟的虫源地分析

根据曲江地区12年稻纵卷叶螟Cnaphalocrocis medinalis Guenée的历史虫情资料,挑选了31个代表性的发蛾高峰日,利用HYSPLIT轨迹分析平台对其迁飞峰次进行轨迹分析,结果表明:粤北稻区稻纵卷叶螟早期迁入虫源地主要分布在海南及两广南部稻区,其中5月大多分布在海南及雷州半岛,6月主要分布在两广南部稻区;夏季迁出虫源的降落地分布在长三角稻区及安徽稻区;秋季回迁虫源的虫源地主要分布在安徽南部及长三角稻区,少部分来自浙南及闽北;秋季迁出虫源的降落地主要集中在海南南部和越南北部稻区;随着时间的推移,其早期迁入虫源地的时空分布由南向北依次偏移,存在明显的季节性差异,而夏秋季迁出虫源降落地和秋季回迁虫源地的时空分布没有明显的季节性差异。本研究初步明确了粤北地区稻纵卷叶螟南北往返迁飞的虫源衔接关系。     

纵卷叶螟绒茧蜂对稻纵卷叶螟幼虫的功能反应

在室内测定了纵卷叶螟绒茧蜂Apanteles cypris对稻纵卷叶螟Cnaphalocrocis medinalis幼虫的功能反应和寻找效应.结果表明,纵卷叶螟绒茧蜂对稻纵卷叶螟幼虫的功能反应可用Michaelis-Menten-Ⅱ型功能反应模型Na=AN0/（F+N0）拟合,一头纵卷叶螟绒茧蜂雌蜂在24h内对稻纵卷...     

稻纵卷叶螟发生程度的神经网络预警

利用神经网络的原理,结合500pb西太平洋副热带高压以及江苏省通州市稻纵卷叶螟发生程度的数据,建立了该地的神经网络中期预警模型,结果表明,这一方法是可行的,同时也说明了对分级量这样的离散数据可以直接进行建模预测。文中还讨论了其研究和应用前景。     

稻纵卷叶螟氨肽酶N基因的克隆及时空表达分析

【目的】昆虫中肠氨肽酶N(Aminopeptidase N,APN)是Bt杀虫蛋白的重要受体之一,与Bt蛋白的杀虫机制及昆虫对Bt蛋白的抗性密切相关。为阐明稻纵卷叶螟Cnaphalocrocis medinalis(Guenee)APN基因的功能及明确Bt蛋白对稻纵卷叶螟的毒力机制,本研究系统开展了稻纵卷叶螟中肠APN基因的克隆及时空表达分析。【方法】通过简并引物PCR结合RACE技术克隆并获得4条稻纵卷叶螟APN基因的cDNA序列全长,采用实时定量PCR技术研究了APN基因在稻纵卷叶螟不同虫态及幼虫不同组织中的时空表达情况。【结果】经NCBI同源比对分析,认为这4个基因分属于。4PN基因家族的不同类别,分别将其命名为CmAPN1(GenBank登录号:HQ853294)、CmAPN2(GenBank登录号:HQ853295)、CmAPN3(GenBank登录号:KJ143755)、CmAPN4(GenBank登录号:HQ853296)。序列分析表明,CmAPN1-4 cDNA序列全长分别为:3 698、3 478、3 150和3 149 bp,开放阅读框分别为:3 045、2 877、3 045和2 862 bp,分别编码965、958、1 014和952个氨基酸。其推导的氨基酸序列具有鳞翅目昆虫氨肽酶N的典型结构特征,即含有糖基化位点、N-末端信号肽序列、谷氨酸锌化氨肽酶保守结构GAMEN、锌结合位点HEX_2HX_(18)E、C-末端糖基磷脂酰肌醇(GPI)锚信号肽。实时定量研究表明,CmAPNs在幼虫中的表达量显著高于卵、蛹和成虫;在幼虫中,CmAPN7的表达水平明显低于CmAPN22-4,且其在不同龄期中的表达差异显著;CmAPN2-4的表达量随幼虫龄期的增加而增加;CmAPNs在幼虫肠道组织中的表达量显著高于其它组织器官,且CmAPN1和CmAPN2分别在中肠和后肠中呈现高水平表达,CmAPN3在前、中肠内均高水平表达;但CmAPN4在各个组织器官中的表达均保持较低水平。【结论】CmAPNs基因在稻纵卷叶螟的不同虫态和不同组织中呈现了差异显著的时空表达,采用RNA干扰方法进行CmAPNs基因功能研究时,要选择适宜的虫态和虫龄进行干扰。     

稻纵卷叶螟的人工饲养技术

稻纵卷叶螟Cnaphalocrocis medinalis的人工饲养技术是科研人员顺利开展相关研究工作的前题.稻纵卷叶螟人工饲养所要解决和重视的关键问题是食料和饲养条件.目前主要以天然食料、人工饲料以及2种食料相结合的方法饲养稻纵卷叶螟.而饲养条件,如温湿度、饲养密度、化蛹介质、产卵介质等会对对稻纵卷叶螟的生长发育产生影响.本文对目前我国稻纵卷叶螟人工饲料及人工饲养技术进行综述,有助于厘清该虫人工饲养技术的发展脉络,促进人工饲养技术的改善和提高,推动我国稻纵卷叶螟的研究.     

稻纵卷叶螟的人工饲养技术

稻纵卷叶螟Cnaphalocrocis medinalis的人工饲养技术是科研人员顺利开展相关研究工作的前题.稻纵卷叶螟人工饲养所要解决和重视的关键问题是食料和饲养条件.目前主要以天然食料、人工饲料以及2种食料相结合的方法饲养稻纵卷叶螟.而饲养条件,如温湿度、饲养密度、化蛹介质、产卵介质等会对对稻纵卷叶螟的生长发育产生影响.本文对目前我国稻纵卷叶螟人工饲料及人工饲养技术进行综述,有助于厘清该虫人工饲养技术的发展脉络,促进人工饲养技术的改善和提高,推动我国稻纵卷叶螟的研究.     

两种组建稻纵卷叶螟种群生命表方法的比较

本文从1987～1988年在江苏句容,1988年在广东阳江海陵对稻纵卷叶螟自然种群采用系统调查法和分段接虫法比较了各组分的种群控制指数(Index of Population Control,IPC),初步的结果认为分段接虫法对捕食作用的估计明显偏大。在稻纵卷叶螟自然种群数量动态研究中拟采用系统调查法辅以天敌排除法测定自然存活率组建生命表为好。     

异常高温干旱对稻纵卷叶螟迁飞的影响

根据本县稻纵卷叶螟多年发生实况和气象资料,认为高温(29℃以上)促使稻纵卷螟迁飞,但天气异常高温(31℃以上)干旱(降水极少)反而迫使其在本地居留。迁飞代这种反常现象的发生,将较大程度影响下一代的发生程度。这一现象的发现将有助于进一步掌握水稻迁飞害虫的迁飞规律和预测预报技术。     

Cloning,characterization, and RNAi effect of the chitin synthase B gene in Cnaphalocrocis medinalis

The rice leaf folder (RLF), Cnaphalocrocis medinalis, is one of the major pests of rice, and chitin synthase is a key enzyme for the chitin synthesis pathway in insects. In this study, the chitin synthase B gene from C. medinalis (CmCHSB) was cloned and characterized. The cDNA of CmCHSB is 4824 bp in length, containing an open reading frame of 4578 nucleotides that encodes 1525 amino acids. The CmCHSB zymoprotein consists of 10 transmembrane domains (TMDs) in the N-terminus, a middle conserved catalytic domain, and 7 TMDs in the C-terminus. Homology and phylogenetic analyses revealed that CmCHSB possesses the closest relationship with its homolog in Ostrinia furnacalis. CmCHSB was expressed throughout development and in all of the adult tissues tested, with the highest expression level in the adult and in the midgut. Silencing of CmCHSB through RNA interference (RNAi) severely affected RLF larval growth and caused larval lethality. Our results revealed that CmCHSB is essential for RLF growth and development, which sheds new light on the characteristics and functions of this gene. These findings will be helpful for green control of RLF, by targeting the CmCHSB gene using RNAi technology.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.

Principal Findings

Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.

Conclusions

We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.     

Identification of three chitin synthase genes in the dimorphic fungal pathogenSporothrix schenckii

Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA ofSporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1. program showed thatS. schenckii consistently clustered most closely withNeurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungusS. schenckii with the Pyrenomycetes of the Ascomycota.     

Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803

The biosynthetic pathway for the pteridine moiety of cyanopterine, as well as tetrahydrobiopterine, has been investigated in Synechocystis sp. PCC 6803. Open reading frames slr0426, slr1626, slr0078 and sll0330 of the organism putatively encoding GTP cyclohydrolase I, dihydroneopterine aldolase, 6-pyruvoyltetrahydropterine synthase and sepiapterine reductase, respectively, have been cloned into T7-based vectors for expression in Escherichia coli. The recombinant proteins have been purified to homogeneity and demonstrated to possess expected genuine activities except that of sll0330. Our result is the first direct evidence for the functional assignment of the open reading frames in Synechocystis sp. PCC 6803. Furthermore, the 6-pyruvoyltetrahydropterine synthase gene is demonstrated for the first time in prokaryotes. Based on the result, biosynthesis of cyanopterine is discussed.     

Identification and characterization of chitin synthase genes in the postharvest citrus fruit pathogen Penicillium digitatum

In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77 kb intergenic region and arranged in opposite directions.