美洲大蠊胸腺素THY3的表达模式分析

美洲大蠊Periplaneta americana胸腺素基因具有THY1、THY2和THY3三个不同剪接体,其中THY3结构域最多。本研究使用实时荧光定量PCR技术比较分析了THY3在美洲大蠊不同性别、不同发育阶段及不同组织中的表达差异,以及大肠杆菌Escherichia coli诱导对美洲大蠊血淋巴和脂肪体中THY3表达的影响。结果表明:THY3在成虫期的表达量显著高于其他虫期,雌虫表达量显著高于雄虫,脂肪体表达量显著高于血淋巴、头部、肌肉、体壁组织。雌性成虫体腔注射大肠杆菌3 h后血淋巴中THY3表达量显著增高,而在脂肪体中12 h后才检测到THY3表达明显上调,研究结果为进一步研究美洲大蠊胸腺素的功能打下了基础。     

美洲大蠊成虫肠道菌群多样性分析

美洲大蠊Periplaneta americana既是卫生害虫,也是重要的药用昆虫。许多研究表明,其肠道菌群种类和数量与宿主健康密切相关。本实验利用16S r RNA高通量测序技术分析了人工饲养的美洲大蠊成虫肠道菌群。结果得到有效序列67 492条,归类为581个操作分类单元,注释到21门79目193属。厚壁菌门Firmicutes(56.96%)和拟杆菌门Bacteroidetes(33.13%)为美洲大蠊成虫肠道的优势菌门。葡萄球菌属Staphylococcus(16.59%)、芽孢杆菌属Bacillus(13.26%)和副杆菌属Parabacteroides(10.26%)为美洲大蠊成虫肠道的优势菌属。研究结果为进一步探讨美洲大蠊肠道菌群多样性以及与宿主之间的关系打下了基础。     

冈比亚按蚊性别决定基因 doublesex 的克隆、序列分析及表达谱

【目的】doublesex 是控制昆虫性别分化的关键基因,决定了昆虫体细胞与生殖细胞的性别。本研究旨在克隆、鉴定重要疟疾媒介冈比亚按蚊 Anopheles gambiae 性别决定基因 doublesex（Angdsx）,分析其在雌雄个体内的剪切体及在不同发育时期的表达模式。【方法】基于冈比亚按蚊转录组数据库,比对到 Angdsx 相关片段,分别以雌雄成蚊cDNA为模板,采用RT-PCR与RACE方法克隆分别获得雌雄个体内 Angdsx 全长基因,利用生物信息软件对所得序列进行结构域预测、氨基酸序列比对和进化树分析。根据 Angdsx 特异性表达引物,利用RT-PCR方法研究其在冈比亚按蚊雌雄个体及不同发育时期的表达谱。【结果】分别从冈比亚按蚊雌雄成虫中克隆获得 Angdsx cDNA全长序列,分别命名为Angdsx^F（GenBank登录号：KM978937）和 Angdsx ^M（GenBank登录号：KM978938）。Angdsx 位于2号常染色体右臂,基因横跨接近80 kb基因组长度。Angdsx^F 长度为4 874 nt,编码长度为265 个氨基酸的雌性特异性蛋白DSX^F;Angdsx ^M 长度为3 183 nt,编码长度为633个氨基酸的雄性特异性蛋白DSX^M。结构域分析发现 Angdsx 包括 doublesex 保守的TRA/TRA-2结合位点、dsx 重复序列、富含精氨酸/丝氨酸双肽区、多聚嘌呤增强子序列和RNA结合蛋白结合序列,以及连续的双核苷酸GT为主的重复序列。与Angdsx^F 相比, Angdsx ^M具有一个雌性特异性的外显子。Angdsx ^M 在0-2 h卵中高表达,随后逐渐减少,在12-24 h卵中降至最低,之后再次升高;Angdsx^F 则在6-8 h卵中开始表达。【结论】本研究获得了冈比亚按蚊性别决定基因 Angdsx 在雌雄个体内的全长序列,Angdsx 具有保守的结构域与表达特征。本研究结果为蚊虫性别分化的分子机制及将其最终应用于显性致死昆虫施放技术进行蚊媒的防制提供了理论基础。     

神农洁蜣螂两性成虫肠道微生物多样性分析

昆虫肠道微生物多样性对宿主的生长发育以及营养物质的消化和吸收等起着非常重要的作用。本研究利用16S rRNA基因序列构建神农洁蜣螂Catharsius molossus雌性成虫和雄性成虫肠道微生物的克隆文库,旨在探究其菌群的结构并加以对比和分析,采用Illumina NovaSeq高通量测序的方法揭示两种菌群组成方面的相似性和差异性。结果表明：神农洁蜣螂雌性成虫和雄性成虫肠道微生物在结构和组成方面存在着一定的差异。雌性成虫肠道菌群的物种丰富度和物种多样性都高于雄性成虫。雌性成虫、雄性成虫体内肠道菌群共有1 253个ASVs,归属于20个门、35个纲、54个目、134个科、253个属,其中雌性成虫肠道菌群的ASVs有1 024个,而雄性成虫肠道菌群的ASVs有410个,二者优势菌群虽然都为阴道球菌属Vagococcus,但二者所占的比例不同。本研究分析了神农洁蜣螂雌雄成虫肠道微生物多样性及群落在结构和组成方面的差异,为探究神农洁蜣螂雌雄成虫的肠道微生物的潜在功能,以及其肠道菌群的开发应用提供了理论支持。     

异斑酷大蚕蛾幼虫、蛹和成虫的性别鉴定

本文描述了一种准确、快速鉴定异斑酷大蚕蛾末龄幼虫、蛹和成虫性别的方法。通过对蛹和成虫性别特征比较可知,雌虫生殖孔位于第8和第9腹节,且第8腹节不愈合,雄虫生殖孔仅位于第9腹节上,且生殖孔两侧有瘤状突起。雌性末龄幼虫头壳的蜕裂线附近浅色区域较多,雄性浅色区域较少,且雌性后唇基区域的颜色较雄性更浅。雌雄成虫差异较大,雄虫触角羽状,前翅一般无透明斑或透明斑很小;雌虫为近丝状的羽状触角,前翅有3个较大且连续排列的透明斑。经过对30对雌雄蛹的测量后,发现在蛹长、蛹宽与蛹重等体型数据上,雌雄蛹之间都存在显著性差异（P＜0.05）。     

花蓟马雄虫释放的聚集信息素的分离和鉴定

明确花蓟马Frankliniella intonsa雄虫释放的聚集信息素对于花蓟马生物防治具有重要意义。但目前除了西花蓟马F. occidentalis, 关于其他蓟马雄虫释放的聚集信息素及其成分鉴定的研究未有报道。本研究采用Y型嗅觉仪测定花蓟马雌雄成虫和西花蓟马雌雄成虫对花蓟马雄虫和西花蓟马雄虫气味源的嗅觉反应; 用固相微萃取（solid-phase microextraction,SPME）方法提取花蓟马雄虫挥发物, 利用含有BGB-176 SE或CP-chirasil-Dex CB 2种不同手性柱的气-质联用仪（gas chromatography-mass spectrometry, GC-MS）对挥发物进行分离和鉴定。行为学试验结果表明： 花蓟马雄虫气味源对其雄性及雌性成虫均具有明显的吸引作用。经GC-MS分析, 花蓟马雄虫挥发物中含有2种主要的化合物, 分别为(R)-lavandulyl acetate和neryl (S)-2-methylbutanoate, 其与已报道的西花蓟马雄虫释放的聚集信息素成分相同。但定量分析结果表明, (R)-lavandulyl acetate与neryl (S)-2-methylbutanoate在花蓟马和西花蓟马雄虫中释放的比例不同; 行为学试验进一步证明, 2种组分比例的不同在花蓟马和西花蓟马种间识别过程中发挥重要作用。本研究推测花蓟马属的昆虫释放的聚集信息素组分可能是相同的。     

Isolation and identification of the aggregation pheromone released by male adults of Frankliniella intonsa (Thysanoptera: Thripidae)

明确花蓟马Frankliniella intonsa雄虫释放的聚集信息素对于花蓟马生物防治具有重要意义.但目前除了西花蓟马F.occidentalis,关于其他蓟马雄虫释放的聚集信息素及其成分鉴定的研究未有报道.本研究采用Y型嗅觉仪测定花蓟马雌雄成虫和西花蓟马雌雄成虫对花蓟马雄虫和西花蓟马雄虫气味源的嗅觉反应;用固相微萃取( solid-phase microextraction,SPME)方法提取花蓟马雄虫挥发物,利用含有BGB-176 SE或CP-chirasil-Dex CB 2种不同手性柱的气-质联用仪(gas chromatography-mass spectrometry,GC-MS)对挥发物进行分离和鉴定.行为学试验结果表明:花蓟马雄虫气味源对其雄性及雌性成虫均具有明显的吸引作用.经GC-MS分析,花蓟马雄虫挥发物中含有2种主要的化合物,分别为(R)-lavandulyl acetate和neryl (S) -2-methylbutanoate,其与已报道的西花蓟马雄虫释放的聚集信息素成分相同.但定量分析结果表明,(R)-lavandulyl acetate与neryl (S) -2-methylbutanoate在花蓟马和西花蓟马雄虫中释放的比例不同;行为学试验进一步证明,2种组分比例的不同在花蓟马和西花蓟马种间识别过程中发挥重要作用.本研究推测花蓟马属的昆虫释放的聚集信息素组分可能是相同的.     

药用美洲大蠊氨基酸和维生素含量分析

以人工饲养条件下不同发育阶段的美洲大蠊Periplaneta americana为材料,测定了其氨基酸和维生素的含量。结果表明,美洲大蠊各虫态均含有18种氨基酸,氨基酸总量为23. 27%～29. 44%,人体必需氨基酸含量占44. 04%～49. 55%,优于联合国粮农组织/世界卫生组织推荐的40%的标准,属于优质蛋白质资源。与成虫期相比,若虫期氨基酸含量较高,色氨酸、谷氨酸、丙氨酸、天门冬氨酸和甘氨酸较为丰富。所检测的5种维生素中,维生素E含量最高,刚蜕皮成虫及雌成虫含量均超过12 mg·kg^-1。美洲大蠊相关产品促睡眠、抗氧化功能可能与其富含色氨酸、谷氨酸和维生素E有关。     

美洲大蠊粪便粗提物的聚集活性

用不同溶剂不同方法提取美洲大蠊Periplaneta americana(L.)粪便,测定美洲大蠊各虫态的诱集活性。结果表明,采用直接浸泡提取方法,丙酮、乙醇、正己烷和二氯甲烷4种粗提物对美洲大蠊各虫态都具有明显的诱集作用,其中乙醇和丙酮粗提物的引诱效果最好,正己烷次之,二氯甲烷最弱。4种溶剂粗提物对美洲大蠊雄成虫和高龄若虫聚集活性最强,对低龄若虫聚集活性最弱。用乙醇溶剂对粪便粗提,3种提取方法均对美洲大蠊有很强的诱集效果,其中索氏抽提诱集效果最弱,直接浸泡和超声波提取效果好,且差异不显著,但直接浸泡提取效果更好。     

美洲大蠊抗菌肽AMPPA13的表达及纯化

[目的]预测美洲大蠊新的抗菌肽基因,构建原核表达体系并纯化表达产物。[方法]通过生物信息学方法,预测分析出潜在的美洲大蠊抗菌肽。构建pET32a重组质粒,优化诱导表达条件,通过亲和层析,分子筛等手段获得纯化的抗菌肽,并进行Western Blot鉴定。[结果]预测出美洲大蠊新的抗菌肽基因AMPPA13,成功构建重组质粒pET32aAMPPA13。优化诱导表达条件得最佳IPTG浓度为0.1 mmol/L,最佳诱导时间为4 h,最佳诱导温度为37℃。纯化后AMPPA13浓度为268μg/m L。[结论]克隆出美洲大蠊抗菌肽基因,并成功构建原核表达体系,得到1 m L纯化的AMPPA13,经Western Blot鉴定,表达产物正确。     

Olfactory soluble proteins of cockroaches

We have characterized antennae-specific proteins from three species of cockroaches Periplaneta americana, P. fuliginosa and Blattella germanica. Based on the N-terminal sequences, cockroach antennal proteins can be divided into three groups: (i) proteins with amino acid similarity to OS-D, a product of gene cloning from Drosophila melanogaster, (ii) proteins similar to a phasmid, Sipyloidea sipylus, olfactory protein, and (iii) cockroach specific proteins without any relevant similarity to other known proteins. Whereas most of the antennae-specific proteins were detected in extracts from male and female antennae, some proteins are sex specific.     

High throughput sequencing of microRNAs in chicken somites

High throughput Solexa sequencing technology was applied to identify microRNAs in somites of developing chicken embryos. We obtained 651 273 reads, from which 340 415 were mapped to the chicken genome representing 1701 distinct sequences. Eighty-five of these were known microRNAs and 42 novel miRNA candidates were identified. Accumulation of 18 of 42 sequences was confirmed by Northern blot analysis. Ten of the 18 sequences are new variants of known miRNAs and eight short RNAs are novel miRNAs. Six of these eight have not been reported by other deep sequencing projects. One of the six new miRNAs is highly enriched in somite tissue suggesting that deep sequencing of other specific tissues has the potential to identify novel tissue specific miRNAs.     

吉富罗非鱼IGF2基因分离及其单核苷酸多态性与体型、增重相关性

胰岛素样生长因子2(insulin-like growth factor2,IGF2)是控制动物生长和脂肪沉积的重要基因之一。本文采用PCR方法分离了吉富罗非鱼(GIFT strain Nile tilapia Oreochromis niloticus)IGF2基因5475bp,包含由4个外显子组成的整个阅读框669bp以及3个内含子。通过比对吉富罗非鱼10个个体IGF2序列,共发现11处单核苷酸多态性(single nucleotide polymorphism,SNP)位点,本文检测了内含子1的621nt(C/T)和外显子3的161nt(A/G)两位点在192尾吉富罗非鱼中的基因型分布,并分析不同基因型与体型、增重的相关性。使用四引物扩增受阻体系PCR检测内含子1的621nt基因型,结果显示,CC、CT、TT基因型频率在雄鱼中分别为0.32、0.32、0.36,在雌鱼中分别为0.38、0.38、0.24;与体型、增重的相关性分析表明,此位点不同基因型只与雄鱼体型(体高/体长)显著相关(P0.05),CC型个体显著高于CT和TT型个体。外显子3的A/G转换导致了MSPⅠ酶切位点改变,使用PCR-RFLP法检测该位点基因型,结果显示整个群体中不存在AA基因型,在雄鱼中,GG、AG基因型频率分别为0.71、0.29,而雌鱼中则为0.75和0.25;与体型、增重的相关性分析表明,此位点不同基因型只与雄鱼增重极显著相关(P0.01),GG型的雄鱼明显较AG型增重快。     

美洲大蠊成虫肠道抗细菌共生放线菌的筛选及鉴定

【目的】鉴于野外美洲大蠊Periplaneta americana对恶劣环境适应性强,本研究旨在从云南省大理州的野外美洲大蠊成虫肠道中分离、筛选出抗细菌活性放线菌,为抗生素开发提供菌种资源。【方法】采用涂布平板法和平板划线法对美洲大蠊成虫肠道放线菌进行分离;以金黄色葡萄球菌Staphylococcus aureus、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌Pseudomonas aeruginosa、粪肠球菌Enterococcus faecalis、大肠杆菌Escherichia coli和鼠伤寒沙门氏菌Salmonella typhimurium共6种人体病原细菌为指示菌株,采用牛津杯法对分离自这些放线菌的次生代谢产物进行抗菌活性测定;通过形态学特征和16S rRNA基因序列分析,对具有广谱和明显抗菌活性的放线菌进行鉴定,并经16SrRNA基因序列的BlAST同源性比对及系统发育分析确定它们的分类地位。【结果】从美洲大蠊成虫肠道共分离获得41株放线菌。抗菌活性测定结果表明,34株(82.9%)放线菌对至少1种指示病原细菌具有抑制作用,其中有7株对3种以上病原细菌具有抑制作用,9株表现...     

RNA-binding proteins in RNA interference

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.     

Hybridization studies in the Schistocerca americana complex. I. The specific status of the Central American Locust

The Central American Locust has previously been identified as Schistocerca paranensis (Burmeister), the Argentinian Locust, and it has been suggested that S. americana of North America represents its solitarious phase. Hybrids of locusts from the Yucatan peninsula of Mexico with locusts from Argentina and Florida were reared, their fertility measured and meiosis in the males examined. It was found that: (1) sex ratios in the F, hybrids were distorted; (2) hybrids had reduced fertility; (3) female hybrids were more fertile than male hybrids; (4) meiosis in hybrid males was disturbed; (5) these effects were more pronounced in die hybrids with the Argentinian Locust than in those with Florida locusts.
These results are discussed in relation to Haldane's rule and the biogeography of die group. It is concluded that the Central American and Argentinian Locusts are separate species, but that the relationship of the former to S. americana americana requires further investigation.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.