Dynamics model in the system of Tessaratoma papillosa-parasitoid wasp and its application

研究荔枝蝽 -寄生蜂系统的数量变动规律 ,得到一个差分 -积分方程组及求解的递推公式 ,指出了公式中参数的确定方法。推导出荔枝蝽 -寄生蜂系统长期演变的特性 ,用模型证明了人工放蜂防治荔枝蝽的优越性及滥用农药的不良后果 ,给出了人工放蜂的最佳次数 ,最佳时刻及合适的放蜂量的计算公式 ,导出的结论与实验结果相符It is well known that Anastatus sp. and Ooencyrtus sp. are effective in killing Tessaratoma papillosa .So it is important to investigate the population dynamics in parasitoid wasp Τessaratoma papillosa system and to develop strategy of releasing of artificially reared parasitoid to the field.It is a common method to predicate the current pest quantity by its last generation dynamics combining with the analysis of environment factors. Different generations of Tessaratoma papillosas can be easily distinguished but that of parasitoid wasps would not be done. In fact, it is quite difficult to check which generations the parasitoid wasps should belong to.Thus we could not describe exactly the population of parasitoid wasp Tessaratoma papillosa by only differential or difference equations. A coupled equations consisting of both differential equation and difference equation modeling Tessaratoma papillosa parasitoid wasp interaction is necessary. A difference integral equation model for Tessaratoma papillosa parasitoid wasp system using the Lotka Volterra equation and a recurrence formula for solving the equation are presented in this paper. We modelled the population dynamics with the following coupled equations. Let X i (t) denote the egg number of new generation of Tessaratoma papillosa at time t of the i 'th year in a certain space (0≤ t≤T), i =1, 2,3…. The function X 1 (t) (0≤t≤T )can be obtained by fitting actual data. Using Y 1(t) to figure the amount of parasitoid wasps at time t (0≤t≤T) of the i 'th year ( i =1, 2,…) within the same space. Suppose Y i (0) is the number of parasitoid at time 0 of the i 'th year. According to the Lotka Volterra equation, we have dY\-1(t)dt=Y\-1(t)[-α+βX 1(t)] (0≤t≤T) where both α and β are two positive constants.Since X 1 (t) and Y 1 (0) are known, we have Y 1(t)=Y 1(0)e ∫ t o[-α+βX 1(σ)] d σ ,(0≤t≤T) Let k be the average parasitism rate per parasitoid. We use X 1(t)=X 1(t)-kY 1=X 1(t)-kY 1(o)e ∫ t 0[-α+βX 1(σ)] d σ to denote the amount of pest eggs which are not parasitoid in the first year and suppose they can all grow up and lay eggs, where λ serve as the average ovipositor quantity per adult pest. Thus we have: X 2(t)=λX 1(t)=λ[X 1(t)-kY 1(t)]=λX 1(t)-λkY 1(0)e ∫ t 0[-α+βX 1(σ)] d σ Inductively, we obtain the following model:X n(t)=λ[X n-1 (t)-kY n-1 (t)] Y n(t)=Y 1(o)n-1 i=1e ∫ t 0[-α+βX i(σ)] d σ e ∫ t 0[-α+βX n(σ)] d σ (1)Using the integral difference equations above,we can predicate the number of the pest eggs and parasitoid at time t of each year by means of recurrence. The parameter κ , denoting the average number of porosities eggs per parasitoid, is transformed from mean ovipositor number per parasitoid combined with parasitism rates, and λ , denoting the average fecundity value per pest, which can be determined from laboratory rearing experiment respectively. Parameter α and β are confirmed based on model (1), one can obtain. ln (y n(0)/y n-1 (0))=-αT+β∫ T 0x n-1 (σ) d σ (n=1,2,3,…) where ∫ T 0X n(σ) d σ is the ovipositor amount of the pest during the n 'th year. When the amount of pest ovipositor in the i 'th year and the amount of parasitoid wasps at time 0 in the corresponding year, namely ∫ T 0X i(σ) d σand Y i (0), ( i =1,2,… m ) are known, we can obtain the parameters of α and β by linear regression. Long term dynamic characterization of the system is deduced base on the model. In Model (1), assume t=T . Then it follows: X n(T)=λ[X n-1 (T)-kY n-1 (T)] Y n(T)=Y 1(O)ni=1e ∫ T 0[-α+βX i(σ)] d σ (2)when n →∞, Y n(t) becomes a infinite product. We get the following results by using the infinite product theory: Proposition 1 The prerequisi

Assigning Archaeal Sequences to HelmCoP Groups

含有古菌序列的直系同源组可以在很大程度上便利各种基于HElMCOP数据的研究,但是目前HElMCOP直系同源组不含有任何古菌序列.为了向HElMCOP直系同源组添加古菌序列,以1个OrTHOMCl-db网站工具的分配方法为依据,我们将29种古菌物种模式菌株的蛋白质组数据,通过blAST比对和OrTHOMCl分析,逐步添加至各个HElMCOP直系同源组中.结果显示,21 326条古菌序列被成功地分配给了1 954个HElMCOP直系同源组中,而其他2 821条古菌序列被归类至nO_grOuP类别.这些分配结果得到了良好blAST匹配数据的支持.一些自由生活物种包括CAEnOrHAbdITIS JAPOnICA、大豆和拟南芥在研究中也受到了关注,因为这些物种相当多的基因在古菌物种内有同源基因.Orthologous groups containing archaeal sequences could facilitate many researches based on HelmCoP data,but unfortunately there were no archaeal sequences in HelmCoP groups.To assign archaeal sequences to HelmCoP groups,we assigned sequences in proteomes of 29 type strains of multiple archaeal species to HelmCoP groups through both BLAST search and OrthoMCL analysis,according to the protocol for assigning sequences by a OrthoMCL-DB web tool.The results showed that 21 326 archaeal sequences were successfully assigned to 1 954 HelmCoP groups,and other2 821 sequences were assigned to NO_GROUP.These assignments gained support from quite good BLAST hits.In addition,some free-living species including Caenorhabditis japonica,soybean,and thale cress were also highlighted in this study,because many genes of these species had homologs in archaeal species.国家自然科学基金(81171595); 福建省自然科学基金(2010J01229

Expressed Sequence Tags(ESTs) Analysis of Angiostrongylus cantonensis

从gEnbAnk下载1277条广州管圆线虫的表达序列标签(EST),用blASTX比对分析,并用SIgnAlPV3.0预测潜在的抗原或过敏原相关蛋白n端是否具有分泌信号肽或信号锚定肽。结果显示,得分值>100有614条,其中14条与广州管圆线虫一致,540条与其他物种的相关蛋白相匹配,60条与数据库中序列无同源性。614条序列可分为10类,抗原或过敏原相关蛋白有80条,编码22种蛋白,其中12种具有分泌信号肽,3种具有信号锚定肽。A total of 1 277 ESTs of Angiostrongylus cantonensis were downloaded from GenBank and analyzed with BlastX.SignalP V3.0 analysis was applied to predict potential putative antigen or allergen relative proteins with N-terminal secreted signal peptides or signal anchors.BlastX analysis showed that there were 614 ESTs scored more than 100, of which 14 were identical with A.cantonensis, 60 ESTs did not match any proteins in the databases.The identified 614 ESTs could be grouped into 10 categories, 80 ESTs expressed 22 antigen or allergen relative proteins, in which 12 had N-terminal secreted signal peptides and 3 had signal anchors.福建省科技计划项目(No.2008N2005);厦门市科技计划项目(No.3502Z20074036)---

Histopathological Response of Giant Cell Induced by Root-knot Nematode,Meloidogyne javanica,in Tomato Roots under Potassium Stress

Comparison of histopathological response and quantitative measurement of giant cell(GC)induced by Meloidogyne javanica in tomato root were studied under potassium-deficient(0.2 mmol/L K+)and replete conditions(control,6.0 mmol/L K+).K+-deficient stress did not impede the formation and maintenance of GC.The mean number of GC per feeding site as well as the mean diameter of GC did not differ between the treatments.However,the thickness of cell wall including components resulted from the accumulated polysaccharide and the length of cell-wall ingrowth increased 5-25 d after inoculation in K+-deficient as compared with K+-replete conditions.An increase of cell-wall ingrowth suggested a kind of compensational response to the potassium stress.Comparison of histopathological response and quantitative measurement of giant cell(GC)induced by Meloidogyne javanica in tomato root were studied under potassium-deficient(0.2 mmol/L K+)and replete conditions(control,6.0 mmol/L K+).K+-deficient stress did not impede the formation and maintenance of GC.The mean number of GC per feeding site as well as the mean diameter of GC did not differ between the treatments.However,the thickness of cell wall including components resulted from the accumulated polysaccharide and the length of cell-wall ingrowth increased 5-25 d after inoculation in K+-deficient as compared with K+-replete conditions.An increase of cell-wall ingrowth suggested a kind of compensational response to the potassium stress.This study was supported by grants from the Science and Technology Commission of Shanghai Municipality (06DZ22110

Sequence anlysis of rDNA of Anisakid nematodes with zoonotic potential from Taiwan strait

目的分析台湾海峡异尖线虫病潜在病原线虫的rdnA序列,为建立病原分子生物学鉴别提供依据。方法剖检台湾海峡常见鱼类的寄生线虫幼虫,镜检分类。提取虫体dnA、用通用引物对rdnA进行PCr扩增、克隆、双向测序及blAST比对。结果获得6条来自6种线虫幼虫的rdnA序列,其中的3种在gEnbAnk数据库中没有发现匹配的序列;证实在台湾海峡分布的灰海鳗对盲囊线虫成虫和疑似其第三期幼虫实属同种;构建了基于ITS-2的序列间内部相似性程度关系树。结论异尖线虫病原幼虫所具有的分子生物学鉴别信息位点主要集中在ITS-2间隔区。To analyze the rDNA sequence of the Anisakid nematodes with zoonotic potential from Taiwan straits,Anisakid nematodes were obtained from the gut of marine fish and identified chiefly based on the morphological characteristics.The genomic DNA of nematodes were extracted,and the sequence of rDNA ITS were amplified by PCR using universal primers,then cloned and sequenced bidirectionally.Sequences analysis was conducted by blasting database and software DNAMAN.Results of the blasting database showed that no match could be demonstrated in 3 of 6 rDNA sequences obtained from 6 different nematode species;the new sequences were submitted to the database and the accession numbers in GenBank were obtained.The relationship between adult Contracaecum muraenesoxi and its putative third stage larva was validated by molecular evidences.Sequences relationship based on the inner similarity of the internal transcribed spacer sequences was constructed.It was found that the phylogenetic informative loci were mainly gathered in the second internal transcribed spacer(ITS2) for these anisakid nematodes because of the ITS2 has higher transition ratio than that of the ITS1.It is evident that the sequence in this region could provide valuable information for the molecular distinction of anisakid nematodes with zoonotic potential from Taiwan straits.福建省科技计划项目(2008N2005);厦门市科技计划项目(3502Z20074036

植物线虫的分泌蛋白质及其功能研究进展

植物体内寄生的植物线虫必须寄生于宿主体内并获取充足的营养才能完成完整的生活史。植物线虫的寄生过程主要是依靠线虫分泌蛋白质的作用来完成。近年来,对植物线虫分泌蛋白质的研究取得了重大进展,分离出一系列由线虫表皮、侧器、食道腺等分泌的蛋白质,这些蛋白在线虫与植物相互作用的过程中起重要的作用:破坏宿主细胞壁,影响宿主细胞周期,改变宿主细胞代谢过程和传导信号,选择性蛋白质降解、抵抗宿主细胞对线虫的防御等。国家基础科学人才培养基金项目(J0630649);; 上海市科学技术委员会资助项目(06DZ22110

Cloning and Expression of L-like Cysteine Protease of Anisakis simplex

目的克隆简单异尖线虫l-样半胱氨酸蛋白酶基因(ASCP)全长,研究其表达特性。方法根据gEnbAnk中简单异尖线虫表达序列标签l-样半胱氨酸蛋白酶基因的部分信息,设计特异引物,用CdnA末端快速扩增技术扩增3′端部分,获得基因全长序列。根据基因全长序列设计引物,以简单异尖线虫总rnA为模板,rT-PCr扩增ASCP基因编码序列,产物经ECOrⅠ和SAlⅠ双酶切,克隆至表达载体PET32а(+),转化大肠埃希菌bl21(dE3)株,以异丙基-β-d-硫代半乳糖苷(IPTg)诱导表达,表达效果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SdS-PAgE)检测。结果3′端扩增片段大小为1211bP,拼接完整后基因全长1462bP,编码411个氨基酸,与秀丽隐杆线虫的l-半胱氨酸蛋白酶相似性达36.4%;重组载体PET32A(+)-ASCP经ECOrⅠ和SAlⅠ双酶切后有一条约1150bP的条带,测序结果显示重组载体构建成功。SdS-PAgE结果表明,重组蛋白相对分子质量约为Mr60000(含6个组氨酸的标签),与目的蛋白相符。用不同浓度的IPTg诱导对表达量的影响很小,1MMOl/lIPTg诱导2H后表达量达到最高水平。结论成功克隆并表达了l-样半胱氨酸蛋白酶。Objective To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex (AsCP) .Methods According to L-like cysteine protease encoding gene of A.simplex from GenBank EST database, specific primers were designed to amplify 3′-end of AsCP gene using rapid-amplification of cDNA ends (RACE), and the full lenth of the L-like cysteine protease gene was obtained.Specific primers were designed according to the full length of the gene.Using total RNA of A.simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT- PCR.The PCR product was digested by EcoR I and Sal I, and cloned into pET32а(+) vector.The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21(DE3).Expression of the protein induced by IPTG of gradient concentration (0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L) and by the same concentration (1 mmol/L) of IPTG at different time(0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) was conducted.The expression situation of recombinant protein was analyzed by SDS-PAGE.Results A 1 211 bp of 3′-end of AsCP gene was amplified by 3′RACE, full length of the gene was 1 462 bp and coding 411 amino acids.It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans.Double enzyme digestion of the constructed recombinant plasimid pET32a(+)-AsCP showed that there was about 1 150 bp fragment, the constructed recom- binant plasmid was then identified by sequencing.SDS-PAGE showed that the recombinant protein (Mr 60 000) was identical with the target.IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction.Conclusion The AsCP gene has been cloned and expressed.福建省科技计划项目(No.2008N2005)---

Cloning and Expression of D-like Aspartic Protease of Anisakis simplex

作者单位： 厦门大学生命科学院， 厦门361005 通讯作者， E-mail:[email protected][中文文摘]目的克隆简单异尖线虫Ⅲ期幼虫(L3)的D-天冬氨酸蛋白酶基因(AsAP)全长,研究其表达蛋白的特性。方法根据GenBank中简单异尖线虫D-天冬氨酸蛋白酶基因表达序列标签的部分信息,设计特异引物并用cDNA末端快速扩增技术得到AsAP全长序列,分析推导的蛋白序列特征,并预测其三级结构。用RT-PCR扩增简单异尖线虫L3的AsAP基因编码序列,产物用EcoRⅠ和SalⅠ双酶切,连入表达载体pET32а(+),转化大肠埃希菌(E.coli)BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果简单异尖线虫L3的AsAP基因全长1 753 bp,编码453个氨基酸,与锡兰钩虫(Ancylostoma ceylanicum)的D-天冬氨酸蛋白酶相似性达65%。该蛋白具有两个保守的催化域,1个活性中心翼环,S2和S3亚位点各1个;具有由20个氨基酸组成的N端信号肽,构成疏水性强的跨膜域。不同浓度的IPTG(0.2～1.6 mmol/L)诱导对AsAP表达的影响较小,1.0 mmol/LIPTG诱导2 h后表达量达到最高水平。结论克隆并表达了简单异尖线虫的D-天冬氨酸蛋白酶。[英文文摘]Objective To clone and express the full length of D-like aspartic protease gene(AsAP) of the third stage larvae of Anisakis simplex.Methods According to the partial information of D-like aspartic protease encoding gene of A.simplex from GenBank,specific primers were designed to amplify 3′end and 5′ end of AsAP gene using rapid amplification of cDNA ends(RACE),and the full length of the D-like aspartic protease gene was obtained.Using total RNA of the third-stage larvae of A.simplex,coding sequence of the AsAP gene was amplified by reverse transcription-PCR(RT-PCR).The PCR product was digested by EcoRⅠ and SalⅠ,and cloned into pET32 vector.The recombinant plasmid was checked by double enzyme digestion and sequencing,and the positive recombinant plasmid was transformed into E.coli BL21(DE3).Expression of the protein induced by IPTG under gradient concentration and different time was conducted.Result A 1 753 bp full length of AsAP was obtained,which contained 30 bp 5′UTR,361 bp 3′UTR and a 1 362 bp open reading frame(ORF) encoding 453 amino acids with a predicted molecular mass of Mr 50 726.It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum.The predicted amino acid sequence contains two conserved catalytic motif,an active site flap,an S2 subsite and an S3 subsite.A 20 amino acids signal peptide was found in the N-terminus,with significant hydrophobic property.Different concentration of the IPTG(0.2~1.6 mmol/L) showed little effect on the expression,and the production of the protein was up to maximum after 2 hours induction.Conclusion The AsAP gene has been cloned and expressed.国家自然科学基金(No.81171595);福建省自然科学基金(No.2010J01229

r DNA sequence analysis and morphological redescription of Empoasca onukii from the tea growing area of Fujian

重新描述了小贯小绿叶蝉的形态结构,尤其是头部色斑、翅脉、腹部内突、下生殖板及其刚毛着生位置,并增加了足部(刺毛列)特征描述。通过PCr技术克隆得到该物种的r dnA序列,包括部分18S(1 843bP)和28S序列(667 bP)以及完整的5.8S(155 bP)、ITS1(3 114 bP)和ITS2序列(1 008 bP)。序列分析表明,18S,5.8S,28S与其他物种间具有90%~95%的序列一致性;而ITS1和ITS2序列变异性非常大。碱基组成比率分析显示ITS1以及ITS2具有AT偏好性,前者A+T占66.0%,后者占65.1%。与亲缘种的比较分析显示ITS1和ITS2具有丰富的多态位点,并且ITS2更适用于近缘种的分子鉴定。The morphological structures of Empoasca onukii,the cephalic splashes,vein,abdominal apodemes,subgenital plate and the arrangement of its bristles,and the characteristics of legs were redescribed,especially the arrangement of spines were added.Ribosomal DNA( r DNA) including partial of 18S( 1 843 bp),28S( 667 bp) and complete 5.8S( 155 bp),ITS1( 3 114 bp),ITS2( 1 008 bp) were isolated by PCR technology.Sequence analysis indicated that 18 S,5.8S and 28 S had high sequence identity( 90%- 95%) with other insects; however,ITS1 and ITS2 showed much variability.The analysis of base composition showed that ITS1 and ITS2 were more preferences for AT( contained 66.0% and 65.1% A + T,respectively).Comparison with the phylogenetic species revealed that ITS1 and ITS2 from E.onukii had rich polymorphic loci and ITS2 was more suitable for molecular identification of closely related species.国家自然科学基金(81171595

Detection of Anisakid Nematodes by an SYBR Green Ⅰ Real-time PCR

目的运用荧光定量PCr法检测异尖线虫类病原体。方法于鱼类内脏中检获6种异尖线虫类幼虫:抹香鲸异尖线虫、简单异尖线虫、内弯对盲囊线虫、带鱼针蛔线虫、灰海鳗对盲囊线虫和台湾海峡鱼类中一优势种对盲囊线虫。提取各虫体dnA,PCr扩增ITS-2序列,测序并进行数据库比对。依据测序结果设计特异引物,常规PCr检验引物特异性。将ITS-2序列扩增产物回收、纯化后经T克隆转入大肠埃希菌dH5α,提取重组质粒,鉴定后作为标准品模板建立荧光定量PCr标准曲线,并做敏感性和重复性试验。结果构建的荧光定量PCr标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数均在0.998以上。重复性实验中,6种虫体对应的变异系数(CV)最小值为0.18%,最大值为2.80%,试验间平均CV最小值为0.55%,最大值为1.94%,无非特异性扩增,溶解曲线的特异性和重复性良好。灵敏度实验中,可检出的最低模板浓度为1x102拷贝/μl,比常规PCr灵敏度高100倍。结论初步建立了SybrgrEEnⅠ荧光定量PCr检测异尖线虫类病原体的方法 。Objective To establish an SYBR Green Ⅰ real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait.Methods Anisakid larvae of six species (Anisakis simplex, A.physeteris, Raphidascaris trichiuri, Contracaecum aduncum, C.muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait) were obtained from the guts of marine fishes and identified chiefly based on the morphological features.The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced.According to these sequences, six specific forward primers were designed and synthesized.Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E.coli DH5α.Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity and reproducibility were determined.Results All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle(Ct) and template concentration.Melt curves were specific and all the 6 correlation coefficients were above 0.998.In the reproducibility test, the coefficients of variation (cv) of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%.The sensitivity of the real-time PCR was 1×102 copies/μl, about 100 times higher than the conventional PCR assays.The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report.Conclusion An SYBR Green Ⅰ fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.福建省科技计划项目(No.2008N2005)---