暗黑鳃金龟气味结合蛋白HparOBP15a基因的克隆及功能分析

【目的】暗黑鳃金龟Holotrichia parallela通过气味结合蛋白(odorant binding protein,OBP)识别性信息素和植物挥发物准确而迅速地定位配偶、寄主植物。本研究通过克隆暗黑鳃金龟气味结合蛋白15a(Hpar OBP15a)基因,解析该基因的编码蛋白特征、组织表达模式及与寄主植物气味等化合物的结合特性方面的研究,为阐明暗黑鳃金龟基于嗅觉识别的寄主植物选择机理奠定理论基础。【方法】根据暗黑鳃金龟成虫触角转录组测序的结果,利用RT-PCR克隆了Hpar OBP15a基因;Real-time PCR方法分析了该基因在成虫不同部位的表达量差异;荧光竞争结合测定了Hpar OBP15a蛋白和58种候选化合物的结合特征。【结果】暗黑鳃金龟Hpar OBP15a基因全长534 bp,编码147个氨基酸,Gen Bank登录号为AK1834747。Hpar OBP15a在触角中特异表达,且在雌虫触角中表达量显著高于雄虫。在被测的58种化合物中,Hpar OBP15a与46种气味化合物具有较好的亲和性,其中与十二烷、十二醇结合能力最强,其解离常数分别为8.5和11.3μmol/L;同时,对性信息素(L-异亮氨酸甲酯和R-芳樟醇)也有一定的结合能力(解离常数分别为21.0和18.5μmol/L)。【结论】Hpar OBP15a具有广泛的气味结合谱,其中对榆树挥发物十二烷的结合能力最强,因此该蛋白可能在暗黑鳃金龟对榆树的定位过程中具有重要作用。     

暗黑鳃金龟UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UAP)基因的克隆、原核表达及功能分析

【目的】本研究旨在阐明暗黑鳃金龟Holotrichia parallela UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UAP)基因HpUAP的序列特征和功能。【方法】通过PCR方法从暗黑鳃金龟2龄幼虫中扩增HpUAP全长cDNA序列,并进行生物信息学分析;构建pET30a-HpUAP重组表达载体,转化大肠杆菌Escherichia coli BL21(DE3),IPTG诱导蛋白表达;利用qRT-PCR检测HpUAP在暗黑鳃金龟幼虫不同发育阶段(1-3龄幼虫)和3龄第2天幼虫不同组织(体壁、中肠、直肠、回肠、马氏管和脂肪体)中的表达量。利用RNAi沉默暗黑鳃金龟2龄幼虫体内HpUAP基因后,观察其生长发育和存活情况,并测定RNAi 72 h后其HpUAP表达量和体壁几丁质含量。【结果】PCR扩增获得暗黑鳃金龟HpUAP 全长cDNA序列(GenBank登录号: MW676788),开放阅读框长1 461 bp,编码486个氨基酸残基,蛋白分子量约为53.9 kD。系统进化分析发现HpUAP与似牛嗡蜣螂Onthophagus taurinus UAP的氨基酸序列以较高的置信度聚为一个分支。经IPTG诱导可表达53.9 kD的HpUAP蛋白,与预期大小一致。发育表达谱结果表明HpUAP在1龄第1天和3龄第1天暗黑鳃金龟幼虫中表达量较高,组织表达谱结果表明HpUAP在暗黑鳃金龟3龄第2天幼虫中肠和体壁中表达量较高。HpUAP RNAi导致暗黑鳃金龟2龄幼虫生长与行动缓慢,体表颜色加深并皱缩;RNAi处理72 h后,与对照组（dsGFP注射组）相比,dsHpUAP注射组HpUAP表达量下降了93.06%,死亡率增加了40%左右,表皮几丁质含量下降了约29%。【结论】结果说明HpUAP参与几丁质代谢,在暗黑鳃金龟幼虫的生长发育过程中起关键作用。     

Bt抗、感种群亚洲玉米螟幼虫取食Bt玉米后Cry1Ab杀虫蛋白在其体内的组织分布与含量

采用ELISA方法检测了实验室汰选的对Cry1Ab产生107倍抗性的亚洲玉米螟Ostrinia furnacalis (Guenée)种群与敏感种群3龄幼虫取食表达Cry1Ab杀虫蛋白的Bt玉米心叶后,杀虫蛋白在幼虫体内的分布情况。结果表明：Cry1Ab杀虫蛋白在抗性种群幼虫中的组织分布情况与敏感种群相近,主要存在于中肠组织和血淋巴中。抗、感种群中均以含有内含物的中肠组织中含量最高,分别为277.2 ng/g 和104.9 ng/g;其次为血淋巴,分别为93.7 ng/g 和69.5 ng/g;不含内含物的中肠组织中52.7 ng/g 和40.1 ng/g;在丝腺和马氏管组织的含量很低,丝腺中分别为8.5 ng/g和11.7ng/g,而马氏管中分别为6.7 ng/g和6.5 ng/g。脂肪体、生殖器官中未检测到杀虫蛋白。抗性种群中肠组织(含有内含物和不含内含物)中Cry1Ab的含量显著高于敏感种群。幼虫期取食过Bt玉米的亚洲玉米螟发育的蛹、成虫及其卵中均不含杀虫蛋白,说明Bt杀虫蛋白不会通过幼虫取食向蛹、成虫及卵传递。     

二化螟APN1的原核表达及其与Cry2Aa蛋白的结合特性研究

【目的】Bt杀虫蛋白发挥杀虫活性的重要前提是Cry蛋白能够与昆虫中肠上皮细胞刷状缘膜囊(BBMVs)上的受体蛋白结合。在前期获得二化螟氨肽酶N1(Aminopeptidase N,APN1)基因全长序列的基础上,明确二化螟APN1多肽片段与Cry2Aa的结合能力。【方法】将二化螟APN1序列片段在大肠杆菌BL21(DE3)中表达,利用蛋白质单向电泳和ligand blotting技术分析二化螟APN1多肽片段与Cry2Aa的结合能力。【结果】重组载体可在表达菌株BL21(DE3)中表达一个约70 ku的蛋白,纯化后的多肽条带单一,纯度较好。Ligand blot分析结果显示,表达的二化螟APN1多肽片段可以与活化的Cry2Aa杀虫蛋白结合,且结合条带随着重组蛋白上样量的降低而减弱。【结论】APN1多肽片段可以与Cry2Aa结合,为阐明APN1基因的功能奠定基础,也为其他Bt蛋白的受体蛋白相关研究提供新的借鉴。     

儿童运动发育迟缓与血碱性磷酸酶、血25-羟维生素D3表达水平的相关性

摘要 目的：探讨儿童运动发育迟缓与血碱性磷酸酶(Alkaline phosphatase,ALP)、血25-羟维生素D3[25(OH)D3]表达水平的相关性。方法：2016年10月到2018年6月选择在本院儿保科门诊就诊500例(6~12月龄)的儿童作为研究对象,诊断儿童发育迟缓的发生率,检测发育迟缓患儿血清ALP与25(OH)D3水平,Gesell测评评定小儿的运动发育状况,所有患儿每天均给予了维生素D3 400 IU,对于发育迟缓患儿每天给予维生素D 800 IU~1200 IU补充,治疗3个月,再做Gesell测评评估其运动发育水平,对比治疗后运动发育情况,并分析影响儿童运动发育的相关因素。结果：在500例小儿中,判断为运动发育迟缓120例(迟缓组),占比24.0 %。两组小儿的性别、胎龄、分娩方式、出生体重、头围、身长等对比差异无统计学意义(P>0.05)。迟缓组的血清ALP水平高于非迟缓组(P<0.05),25(OH)D3水平低于非迟缓组(P<0.05)。迟缓组的大动作、精细运动、适应性行为、语言、个人社交评分都低于非迟缓组(P<0.05),迟缓组治疗后,大动作、精细运动、适应性行为、语言、个人社交评分均显著升高(P<0.05)。在120例发育迟缓中,Pearson分析显示ALP、25(OH)D3与小儿运动迟缓发育具有相关性(P<0.05);二分类多因素条件Logistic分析结果显示ALP、25(OH)D3都影响儿童运动发育迟缓的主要因素(P<0.05)。结论：儿童运动发育迟缓与血清ALP、25(OH)D3水平存在相关性,两者的联合检测可为儿童发育迟缓的早期诊断提供实验依据,经过维生素D治疗后,能显著的改善其患儿的运动发育,有很好的应用价值。     

冠心病患者血清成纤维细胞生长因子23、碱性磷酸酶及胎球蛋白A水平与冠状动脉钙化的关系及其预测价值分析

摘要 目的：分析冠心病（CHD）患者血清成纤维细胞生长因子23（FGF23）、碱性磷酸酶（ALP）、胎球蛋白A（FA）水平与冠状动脉钙化（CAC）的关系并探讨其对CAC的预测价值。方法：选取2021年2月～2022年2月本院收治的165例CHD患者,根据是否伴有CAC分为CAC组（n=73）和非CAC组（n=92）。收集患者临床资料,采用酶联免疫吸附法（ELISA）检测血清FGF23、ALP、FA水平。通过多因素Logistic回归分析CHD患者CAC的影响因素,绘制受试者工作特征（ROC）曲线分析血清FGF23、ALP、FA水平对CHD患者CAC的预测价值。结果：CAC组血清FGF23、ALP水平高于非CAC组,血清FA水平低于非CAC组（均P＜0.01）。多因素Logistic回归分析显示,年龄（较大）（OR=1.220,95％CI：1.087～1.369）、高血压病（OR=1.461,95％CI：1.062～2.010）、血钙（较高）（OR=1.532,95％CI：1.042～2.251）、血磷（较高）（OR=1.209,95％CI：1.097～1.333）、FGF23（较高）（OR=1.012,95％CI：1.007～1.018）、ALP（较高）（OR=1.046,95％CI：1.023～1.070）为CHD患者CAC的独立危险因素,FA（较高）（OR=0.827,95％CI：0.750～0.912）为独立保护因素（均P＜0.05）。ROC曲线分析显示,血清FGF23、ALP、FA单独与联合预测CHD患者CAC的曲线下面积（AUC）分别为0.790、0.773、0.786、0.915,联合预测CHD患者CAC的AUC大于各指标单独预测。结论：血清FGF23、ALP水平升高和FA水平降低与CHD患者发生CAC密切相关,可作为CHD患者发生CAC的辅助预测指标,且三个指标联合预测CHD患者CAC发生风险的价值较高。     

暗黑鳃金龟甲幼虫肠道细菌分离及鉴定

目的从微生态学角度研究暗黑鳃金龟甲幼虫营养生理活动,探讨其肠道菌群的构成,为其资源开发及生物防治提供理论依据。方法按传统分离方法,从暗黑鳃金龟甲幼虫肠道环境中分离纯化获得10个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行了系统研究。结果研究结果表明,上述10个细菌菌株分别属于鲁氏耶尔森菌（Yersinia．ruckeri）、侧胞芽胞杆菌（Bacilluslaterosporus）、坚强芽胞杆菌（Bacillus．firmus）、放线杆菌属（Actinobacillus）、飞虫杀雄菌（Arsenophonusllasoniae）、不动杆菌属（Acinetobacter）、气单胞菌Aeromonas）、沙门菌属（Sal—moneUa）、短芽胞杆菌（Bacillusbrevis）、变形菌属（Proteus）。结论通过对暗黑鳃金龟甲幼虫肠道细菌的鉴定,其肠道细菌在培养性状、生理性状、生理生化测定等方面存在较多差异。     

暗黑鳃金龟对性信息素的触角电生理及行为反应

为了分离鉴定暗黑鳃金龟的性信息素成分并对其功能进行验证,本研究对比摸索了3种触角的处理方法,并进行同一浓度（30 ng·μL^-1）二元混合物、不同配比的触角EAG测试。结果包括: 建立了一种鳃叶状触角的触角电位（EAG）测定方法,即将暗黑鳃金龟Holotrichia parallela触角的各鳃叶用针分离开后进行测定,这种方法测得的触角电位反应值较高。雄虫触角对L-异亮氨酸甲酯和(R)-(-)-芳樟醇为7∶1的二元混合物的反应值最高,和暗黑鳃金龟雌虫性信息素25个腺体提取液的EAG反应相当;对同一配比（7∶1）不同剂量刺激液的EAG测试表明,雄虫对浓度为30 ng·μL^-1的二元混合物刺激液的反应值最高。嗅觉反应结果进一步证实,试虫对L-异亮氨酸甲酯和(R)-(-)-芳樟醇7∶1的选择最高,选择系数达93.3%。研究结果为利用性信息素防治暗黑鳃金龟技术的开发提供了基础。     

The role of DNA binding with the Cry8Ea1 toxin of Bacillus thuringiensis

The Cry8Ea1 protoxin is a DNA-protein complex. Both forms of the Cry8Ea1 toxin (with or without DNA binding) were obtained separately, and their stability and ability to insert into a phospholipid monolayer in vitro were compared. The presence of DNA can prevent the toxin from aggregation. Data regarding the penetration of the Cry8Ea1 toxin and Cry8Ea1 toxin-DNA complex into the air/water interface without a phospholipid monolayer show that the Cry8Ea1 toxin-DNA complex is more likely to move towards the air/water interface and is more hydrophobic. Experiments examining the protein interaction with the phospholipid monolayer show that the ability of the Cry8Ea1 toxin-DNA complex to insert into the lipid bilayer is much greater than that of the Cry8Ea1 toxin without DNA.     

Sensitivity of the cereal leaf beetle Oulema melanopus (Coleoptera: Chrysomelidae) to Bt maize-expressed Cry3Bb1 and Cry1Ab

The sensitivity of the cereal leaf beetle, Oulema melanopus (Coleoptera: Chrysomelidae), to maize-expressed Bacillus thuringiensis (Bt) proteins was investigated in the present study. Neonate larvae of O. melanopus were caged on leaves of Cry3Bb1-expressing (MON88017) or Cry1Ab-expressing (MON810) Bt maize, the corresponding near-isolines, or two non-related, conventional maize varieties. Larval survival was reduced on Cry3Bb1-expressing, but not on Cry1Ab-expressing maize compared with conventional varieties. Differences among conventional varieties were also present. The amount of eaten leaf material, developmental time to prepupal stage, and prepupal weight did not differ between Bt maize varieties and their corresponding near-isolines. In an additional feeding study with newly emerged adults, survival and beetle weight did not differ when leaves of Cry3Bb1-expressing maize or the near-isoline were offered as food over 3 weeks. ELISA measurements revealed that larvae feeding on Bt maize contained rather high Cry3Bb1 or Cry1Ab levels, which were in the same order of magnitude as the leaves. In contrast, concentrations in feces were one order, and concentrations in prepupae and adults two orders of magnitude lower.     

Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

Bacillus thuringiensis (Bt), a Gram-positive bacte-rium, produces insecticidal crystal proteins during sporulation. Bt has been used as biopesticides to con-trol a number of insect pests from Lepidoptera, Dip-tera and Hymenoptera and also has become so far the leading gene sources of transgenic plants resistant toinsect pests[1,2]. In China, the use of Bt cotton began in 1997 in Hebei, Shandong and Henan provinces, etc. and rapidly increased to more than 2 million ha in 2002, which is effe…     

多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析

【目的】本研究旨在克隆多异瓢虫Hippodamia variegata气味结合蛋白(odorant-binding proteins, OBPs)基因HvarOBP2,并解析这一蛋白的配体结合特征。【方法】采用BLAST和生物信息学技术从多异瓢虫成虫触角转录组中鉴定候选气味结合蛋白基因HvarOBP2,PCR特异扩增HvarOBP2基因全长cDNA序列;通过体外原核表达和亲和柱层析等手段获得HvarOBP2重组蛋白;运用荧光竞争结合实验测定重组蛋白HvarOBP2与40种植物挥发物组分以及24种蚜虫相关挥发物组分的结合能力;使用PyMOL1.9.0进行重组蛋白HvarOBP2与4种配体(β-紫罗兰酮、邻苯二甲酸二丁酯、油酸和橙花叔醇)的分子对接模拟。【结果】HvarOBP2(GenBank登录号：OK340816)开放阅读框(open reading frame, ORF)长447 bp,其编码蛋白N端有17个氨基酸组成的信号肽,具有6个保守的半胱氨酸位点,属于classical OBPs亚家族。荧光竞争结合实验结果表明,重组蛋白HvarOBP2与植物挥发物组分油酸(解离常数K     

Synopsis of Antitrogus Burmeister (Coleoptera: Scarabaeidae: Melolonthini)

Abstract The genus Antitrogus Burmeister is widely distributed in eastern Australia and, because some species are significant pests of sugarcane, pastures and pineapples, it has attracted considerable research interest. This paper reviews the systematics, biology, ecology and management of the genus. The morphology of the 22 known species is described and a further species, A. gubbi sp. n. from south-eastern Queensland which was previously confused with A. robertsi Britton, is described. These species clearly divide into two species-groups: (i) those from south-eastern Queensland, eastern New South Wales and south-eastern Australia, which sometimes have closely related species pairs; and (ii) a more diverse group of species from central and northern Queensland. The latter is linked to A. villosus Allsopp from western Victoria, and this may indicate that further species await discovery in western New South Wales. Known life cycles are of 1 or 2 years. Adults fly for short periods following rain in spring or summer and do not feed. Adult females tend to disperse poorly, and this, coupled with ephemeral and localised rainfall, has probably contributed to speciation. Larvae feed on the roots of grasses and other similar plants. Insecticides are widely used to control them in sugarcane, but are too expensive for use on pastures. Integrating new controls with an emphasis on cultural controls is seen as the best way of managing these pest species.