Effects of Various Degrees of Antennal Ablation on Mating and Oviposition Preferences of the Diamondback Moth,Plutella xylostella L.

Using scanning electron microscopy, we investigated the distribution of the trichoid, basiconic, and coeloconic sensilla on the antennae of the diamondback moth （DBM;Plutella xylostella）. The trichoid sensilla were the most abundant sensory organ, and the male moth antennae host signiifcantly more trichoid sensilla than female moth antennae. Conversely, basiconic and coeloconic sensilla were found more frequently on female than on male antennae. We performed experiments with various degrees of antennal ablation and demonstrated that DBM antennae played a key role in the control of mating and oviposition. We found that neither oviposition preference nor mating behaviors changed signiifcantly when less than 1/4 of both antennae were removed. However, there was a signiifcant behavioral change when the antennae were ablated by more than half. As the length of the antenna was shortened, the successful mating rate decreased and mating peak was delayed. An otherwise consistent host preference for oviposition was eliminated when both antennae were completely removed. Furthermore, we found that the number of trichoid sensilla was positively correlated with mating rate and oviposition preference. However, the numbers of basiconic and coeloconic sensilla were not correlated with mating rate and mating peak, but highly correlated with oviposition preference. Taken together, our results indicate that antennal sensory information plays a critical role in the mating and oviposition behaviors of this economically important pest.     

A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua(Hübner)

Odorant binding proteins(OBPs) in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors(ORs) located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP c DNA, namely Sexi OBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR(q PCR) measurement indicated that Sexi OBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of Sexi OBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, Sexi OBP13 displayed a high binding affinity(Ki=3.82 μmol L–1) to Z9,E12–14:Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles(Ki27 μmol L–1). The behavioral tests further confirmed that Z9,E12–14:Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that Sexi OBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed.     

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.     

Study on the Relationship Between Sex Differentiation and Ethylene in Ramie

To explore the role of ethylene in sexual determination in ramie, the ethylene release rates in ramie stem apex of different sex, bud of the same node in female and hermaphrodite ramie in the second crop, and single inflorescence of different sex in hermaphrodite ramie were measured by gas chromatography. Effects of two ethylene inhibitors on sex expression in ramie were investigated. The ethylene release rate of stem apex was higher in the second crop or female ramie than that in the third crop or hermaphrodite ramie during growth. Although ethylene release rates between lower nodes and higher nodes were little different, it was lower in the middle nodes in hermaphrodite ramie. The ethylene release rates were higher in the higher nodes of female ramie at the second crop. At the third crop, the ethylene release rates were higher in lower nodes, lower in middle nodes and then highest in higher nodes in female ramie. However, an opposite ethylene release pattern was observed in hermaphrodite ramie. The ethylene release rate was higher in female flower than male flower and mixed inflorescence in hermaphrodite ramie. The male flower could be distinctly induced by AVG （aminooethoxyvinylglycine）. The node of the first male flower, percentage of female flowers and ratio of female flowers to male in ramie were evidently depressed by AVG in contrast to water. The percentage of mixed male and female flowers was also increased and the percentage of female flower decreased by spraying AgNO3. There was a close relationship between sexual differentiation and ethylene release rate in ramie. The female ramie could be induced by high ethylene release rate. The female flower could be inhibited by AVG and AgNO3. AVG at a concentration of 300 mg L^-1 was most effective.     

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera （Hvbner）

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera(Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli.The real time quantitative PCR(RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings.Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles.The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde,whereas methyl phenylacetate,2-decanone,1-pentanol,carvenol,isoborneol,nerolidol,2nonanone and ethyl heptanoate have relatively weak binding affinity.Moreover,the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38(α1),40-48(α2),62-72(α3),80-96(α4),98-108(α5),and 116-119(α6),two pairs of disulfide bridges Cys49-Cys55,Cys75-Cys78.The two amino acid residues,Ile94 and Trp101,may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.     

Cloning and Sequencing of a Gene Encoding GOBP2 in the Antenna of Spodoptera exigua

A pair of degenerate primers was designed, based on the comparison of five insects‘ GOBP2 gene sequences reported previously. A specific band (about 400bp in length) was amplified from cDNA of Spodoptera exigua antenna and another specific band (about 2kb in length) was amplified from genomic DNA.The two segments were cloned into T-easy vector, respectively. Results of sequencing and structural analysis showed that the full-length of GOBP2Sexi ORF is 426bp, 141 amino acid residues were encoded. The predicted MW and pI are 16.07ku and 5.09, respectively. There are six conservative Cys locus in the sequence, which is the typical characteristic of OBPs. GOBP2Sexi gene was inserted by two introns between amino acid residue 22 and 23 and between 82 and 83. The length of two introns is 160bp and 1403bp. Results of Northern blot showed that GOBP2 gene expressed specifically in the antenna of Spodoptera exigua, and the expression level is nearly equal in the antenna of male and female moths. The sequence was deposited in GenBank/EMBL and the accession number is AJ294809.     

斜纹夜蛾感受器超微形态特征

采用扫描电镜技术,系统研究了斜纹夜蛾触角、跗节和产卵器的感受器形态结构特征,发现触角感受器主要包括毛形、锥形、刺形、耳形和腔锥感受器.雌、雄虫触角锥形感受器形态特征区别明显,雄蛾锥形感受器长约10～15 μm,基部直径约4～5μm,显著比雌虫锥形感受器长、粗,雄蛾锥形感受器顶端有一小锥,雌蛾没有.雌成虫和雄成虫前足、中足、后足跗节均分布有毛形和刺形感受器,但数量明显少于触角同类感受器数量.雌成虫产卵器上主要为长毛形和短毛形感受器,这些感受器可能与其感受环境信息、产卵场所选择等行为有关.

Abstract：

The ultrastructures of the sensilla on the antennae, the tarsi and the ovipositor of Spodoptera litura were observed with scanning electron-microscopy. The antennae of both male and female moths were found to contain five kinds of antennal sensilla, namely, sensillum trichodeum, sensillum basiconicum,sensillum chaeticum, ear-shaped sensillum and sensillum coeloconicum. The characters of sensillums basiconicum on the antennae were different between the male and the female. The length was 10-15 μm, the diameter at the base was approximately 4-5 μm for the male antennae, which were significantly longer and bigger than the female antennae, and the male moths had onesmall bradawl at the bass of the antennae while the female moths did not. The tarsi of both male and female moths contained two kinds of sensilla:sensillum trichodeum and sensillum chaeticum; however, their numbers were significant less than those on the antennae. The long sensillun trichodeum and short sensillun trichodeum were mainly distributed on the ovipositor. These results provided basis for exploring the molecular mechanism of oviposition deterrent.     

Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera: Thripidae)

Heat shock protein 70(HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length c DNAs of HSP70s gene(Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of c DNA. Real-time PCR was used to analyze the HSP70 expression patterns. The c DNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids(aa) with a calculated molecular weight of 75 and 54 k Da, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported Fo HSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.     

Gene Cloning and Tissue-Specific Microplitis mediator （Hymenoptera： Expression of G Protein β Subunit in Braconidae）

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

The Establishment of Double-Transgenic Mice that Co-Express the appA and MxA Genes Mediated by Type A Spermatogonia In vivo

Type A spermatogonial stem cells are the only immortal diploid cells in the postnatal animal that undergo self-renewal through the lifetime of an animal and transmit genes to subsequent generations. In this paper, the generation and characterization of double-transgenic mice co-expressing the Escherichia coli appA gene and human MxA gene generated via the in vivo transfection of type A spermatogonial cells were reported for the ifrst time. The dicistronic expression vector pcDNA-appA-MxA（AMP） and ExGen500 transfection reagent were injected into the testicular tissue of 7-d-old male ICR mice. The mice that underwent testis-mediated gene transfer were mated with wild-type female mice, and the integration and expression of the foreign genes in the offspring were evaluated. Transgenic mice that co-expressed appA and MxA showed a gene integration rate of 8.89%（16/180）. The transgenic mice were environmentally friendly, as the amount of phosphorous remaining in the manure was reduced by as much as 11.1%by the appA gene （P〈0.05）;these animals also exhibited a strong anti-viral phenotype.     

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达

[目的]克隆金黄色葡萄球菌FnBP配体结合区基因,并构建原核表达载体,进行原核表达：方法]设计引物,采用PCR方法扩增FnBP配体结合区基因,BA克隆后,构建了克隆质粒pMD18-FnBP。用BarnHI和EcoRI双酶切pMD18-FnBP和pET28a（＋）,将纯化的基因FnBP亚克隆至pET28a（＋）,构建重组表达质粒pET28a-FnBP,并将其转化至大肠杆茼感受态B121（DF3）中,IPTG诱导表达,并对表达产物进行分析。[结果]PCR扩增出1条约370hp的目的片段,表达产物经SDS-PAGE分析,在30kDa处出现了与目的蛋白一致的外源蛋白带．Westem blot分析表明该蛋白具有金黄色葡萄球菌的抗原性。[结论]已成功构建了FnBP配体结合区基因,并在原核细胞中表达。     

水貂阿留申病毒VP2基因主要抗原表位区的原核表达

根据GenBank中已发表的水貂阿留申病毒(ADV)VP2基因核苷酸序列分别设计合成两对引物,用PCR方法扩增ADV国内分离株VP2基因中主要抗原表位区的两个片段,分别将其克隆到原核表达载体pMAL-c2的多克隆位点中。经酶切、PCR扩增和测序分析证实其已正确插入到表达载体中,且阅读框是正确的,构建原核表达载体pMAL-VPa和pMAL-VPb。阳性重组质粒转化宿主菌TB1,用IPTG进行诱导表达,对表达产物进行SDS-PAGE检测和免疫印迹分析。结果表明两段蛋白均获得了表达,表达产物的分子质量分别约为63、65kD,与理论推测的分子质量一致;并在终浓度为1mmol/L的IPTG诱导下,4h时其表达量达到高峰;Western blot分析表明表达蛋白能被兔抗MBP抗体所识别,具有一定的抗原性。     

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达(英文)

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.     

斜纹夜蛾普通气味结合蛋白ⅠcDNA的克隆及在原核细胞中的表达

 【目的】克隆、序列分析和原核表达编码斜纹夜蛾（Spodoptera litura）GOBPⅠ（SlGOBPⅠ）的cDNA。【方法】采用同源克隆结合RACE的方法克隆SlGOBPⅠ基因的cDNA序列,并在 pET-32a/BL21（DE3）系统中进行原核表达。【结果】从斜纹夜蛾触角中克隆了GOBPⅠ的cDNA序列（GenBank登录号为EU086372）,序列分析表明,SlGOBPⅠ开放阅读框序列为495 bp,编码164个氨基酸,分子量为19.3 kD,等电点为5.54。SlGOBPⅠ具有昆虫气味结合蛋白的典型特征,即氨基酸序列中具有 6个半胱氨酸残基,呈酸性。SlGOBPⅠ氨基酸序列与鳞翅目昆虫的气味结合蛋白氨基酸序列具有较高的相似性,表明SlGOBPⅠ属于GOBP家族。将SlGOBPⅠ编码序列,克隆到表达载体pET-32 a上,构建原核表达载体pET-SlGOBPⅠ,在表达宿主菌BL21（DE3）中,经IPTG诱导成功表达了相对分子质量为32.0 kD的可溶性融合蛋白,利用Ni2+-NTA亲和柱一步纯化了SlGOBPⅠ融合蛋白,以此融合蛋白免疫新西兰大白兔制备了SlGOBPⅠ的抗血清,ELISA滴度为1﹕5 000,Western印迹检测结果显示,SlGOBPⅠ抗血清与表达的融合蛋白质呈阳性反应,表明所表达的融合蛋白保持原有的免疫原性。【结论】克隆、分析和表达了编码斜纹夜蛾气味结合蛋白GOBPⅠcDNA 序列,为今后深入研究斜纹夜蛾GOBPⅠ基因的结构和功能奠定了基础。     

大鼠热休克因子结合蛋白1原核表达载体的构建及表达

以重组克隆载体pGEM—T—hsbp1为模板,PCR扩增hsbp1,用限制性内切酶双酶切、T4连接酶连接、转化BL -21的方法构建pGEX-2TK-hsbp1原核表达载体,实现了hsbp1在BL21中的表达,SDS—PAGE检测表达的融合蛋白主要以可溶形式存在,并利用亲和层析得到融合蛋白。     

Identiifcation and Molecular Mapping of the RsDmR Locus Conferring Resistance to Downy Mildew at Seedling Stage in Radish （Raphanus sativus L.）

Downy mildew （DM）, caused by the fungus Peronospora parasitica, is a destructive disease of radish （Raphanus sativus L.） worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically iftted a 3：1 ratio in two F2 populations （F2（SR） and F2（RS））, and 1：1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1 972 primer pairs （1 036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR） were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers （Em9/ga24370, NAUISSR826700 and Me7/em10400） linked to the RsDmR locus within a 10.0 cM distance were identiifed using bulked segregant analysis （BSA）. The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.