孟氏隐唇瓢虫气味结合蛋白ComOBP1基因的克隆和时空表达

采用RT-PCR和RACE技术,从孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant中成功克隆出气味结合蛋白(Com OBP1)基因的全序列(Genbank登陆号:KU170686)。Com OBP1基因全长922 bp,包括5'端长为40 bp的非编码区域(UTR),及3'端长为462 bp的UTR,开放阅读框ORF长为420 bp,编码139个氨基酸,预测的分子量为15.516 k Da,等电点p I为6.57,存在AATAAA加尾信号。N-末端疏水区包含由20个氨基酸构成的信号肽,无跨膜结构,有4个保守的半胱氨酸,属于Minus-C OBP,也是在孟氏隐唇瓢虫中发现的第一个Minus-C OBP。氨基酸序列中有且仅有一个N-糖基化位点为62 NLSA,并存在2个潜在的磷酸化位点。与其它昆虫的Minus-C OBPs进行同源性比较并构建系统发育树,发现与同为鞘翅目Coleoptera昆虫的同源性较高。利用Real-time PCR、RT-PCR技术对Cmon OBP1基因在孟氏隐唇瓢虫不同发育阶段,不同组织,不同营养条件及不同食性下的表达水平进行了测定,结果显示,Cmon OBP1基因在整个发育阶段均有表达,雄性成虫期具有最高表达量,且多在成虫的头部及翅部表达。当营养条件发生变化时,表达丰度不会发生变化,当猎物由天然猎物柑橘粉蚧Planococcus citri Risso变成碗豆修尾蚜Megoura japonica Matsumura时,表达量会明显下降。该结果表明,孟氏隐唇瓢虫的不同发育阶段、不同组织及猎物种类会影响Cmon OBP1基因的表达,从而进一步影响其嗅觉行为。同时,Cmon OBP1基因可能在雄虫相关的信息素感受过程中发挥着重要作用。     

孟氏隐唇瓢虫成虫自残幼虫的研究

在室内对孟氏隐唇瓢虫成虫自残幼虫的行为以及雌虫取食幼虫后的生殖力进行了研究,结果表明,雌成虫在48h内残杀幼虫造成的死亡率为60%,显著高于雄成虫;与其他部位相比幼虫的腹部遭受更多地攻击,幼虫体表覆盖的蜡丝在一定程度上抵御了成虫的攻击。与取食粉蚧的雌虫相比,取食瓢虫幼虫的雌虫产卵率和平均产卵量均显著降低。     

孟氏隐唇瓢虫的触角感受器

本文使用场发射扫描电子显微镜对孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant雌雄成虫的触角和触角感受器进行了观察和研究，并对触角形态，感受器形态、类型、数量及分布进行了统计和分析。孟氏隐唇瓢虫雌雄成虫触角均由柄节，梗节及8个鞭小节组成。观察到7种触角感受器：四种锥形感受器（sba），四种刺形感受器（sch），四种毛形感受器（str），一种腔锥形感受器（sco），一种耳形感受器（sau），一种腔形感受器（scl），一种Bhm氏鬃毛（sbm）。毛形感受器和刺形感受器数量最多，其他类型感受器数量都较少。Bhm氏鬃毛只存在于柄节和梗节。第八鞭小节的顶端感受器种类最丰富，被六种感受器稠密覆盖。雌雄触角大小、感受器类型都没有明显差异。根据感受器的分布和以前的相关报道，推测孟氏隐唇瓢虫的毛形感受器可能是信息素接收者，刺形感受器可能是机械性刺激感受器和化学感受器，锥形感受器和腔锥形感受器可能都是植物挥发物接受者，化学和温湿度感受器，耳形感受器或许承担物理或者嗅觉器官的功能，而Bhm氏鬃毛或许能感知触角的位置和活动。     

温度对孟氏隐唇瓢虫实验种群的影响

在１４－３４℃范围内研究了温度对孟氏隐唇瓢虫实验种群生长发育的影响,结果表明,在１４－２９℃范围内,温度愈低,发育历期愈长,当温度达到３２℃后,发育历期又略有延长。孟氏隐唇瓢虫世代发育起点温度为１１．９℃,有效积温为４５２．３日度。孟氏隐唇瓢虫世代存活率为２６℃最高,以３４℃最低。在２０－３２℃范围内,孟氏隐唇瓢虫内禀增长率（ｒｍ）、周限增长率（λ）和世代净增长率（Ｒ０）均以２６℃为最高,分别为０     

东亚飞蝗细胞色素P450基因的克隆及表达分析

本文克隆了东亚飞蝗Locusta migratoria manilensis(Meyen)细胞色素P450(cytochrome P450)基因全长,表达重组蛋白,并对其可溶性进行了分析。通过提取东亚飞蝗总的RNA,反转录成cDNA,设计特异性引物,PCR克隆东亚飞蝗细胞色素P450基因,将测序正确的目的片段克隆至原核表达载体pET-28a中,在大肠埃希菌Escherichia coli Rosetta中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重组蛋白表达结果。结果表明:东亚飞蝗细胞色素P450基因开放阅读框全长为1 551 bp,编码516个氨基酸,与GenBank中已登录的东亚飞蝗细胞色素P450基因(HM153426)的同源性为99%,重组质粒pET-28a-P450在E.coli Rosetta中获得高效表达,重组蛋白相对分子质量(Mr)约为53 000,主要以包涵体的形式存在。     

棉铃虫细胞色素P450 cDNA片段的克隆与序列分析

以 5龄实验室敏感品系棉铃虫Helicoverpaarmigera的总RNA为模板 ,采用简并性引物 ,利用反转录 -多聚酶链式反应 (RT PCR)扩增出了 2个新的长度分别为 2 3 7bp和 2 40bp的cDNA片段。序列分析表明 ,2 3 7bp的cDNA片段与棉铃虫P45 0CYP4家族有较高的相似性 ,最高达 73 %;而 2 40bp的cDNA片段与CYP6家族有较高的相似性 ,最高达 49%。     

孟氏隐唇瓢虫对产卵基质的颜色选择行为研究

在室内26±2℃,RH=50±10%,L:D=16h:8h环境下,使用白色、红色、蓝色、紫色、黑色5种棉花作为孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant的产卵基质供其产卵,研究该瓢虫对产卵基质的颜色偏好性.结果表明连续10日内该瓢虫在红色棉花上的产卵次数和总产卵量最多,产卵次数与总产...     

孟氏隐唇瓢虫研究现状及其种质资源描述规范的建立

孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant原产于澳大利亚,是粉蚧的重要捕食性天敌,作为生物防治重要的天敌资源被广泛引进世界各地.本文介绍了国内外近几十年来对孟氏隐唇瓢虫在生活史、行为、抗药性以及生物防治等方面的研究概况,并提出建立孟氏隐唇瓢虫种质资源描述规范的必要性,为充分发挥其控害潜能提供理论依据.     

孟氏隐唇瓢虫生殖系统结构和卵子发生的研究

【目的】孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant生殖系统结构和卵细胞发生将为昆虫的系统进化关系及瓢虫分类提供依据,同时可作为瓢虫人工饲料研究开发的参考。【方法】利用组织石蜡切片技术和光学显微镜,观察孟氏隐唇瓢虫生殖系统结构,以及自成虫羽化后不同发育阶段卵巢发育状况和成熟卵巢管卵子发生过程。【结果】孟氏隐唇瓢虫雄性生殖系统包括2对附腺、1对精巢、1对输精管、1对贮精囊、射精管、弯管和阳基。雌性生殖系统包括2片生殖板、生殖腔、受精囊、中输卵管、1对侧输卵管和1对卵巢。单侧卵巢管数量在11~14根之间,卵巢管端部延伸出细长的端丝。卵巢管属于端滋式,分为原卵区和生长区。滋养细胞分散且细胞核几乎充满整个细胞,未见合胞体。卵细胞稀疏地集中在原卵区下端,并且可见营养索向卵巢管顶端延伸。根据卵细胞位置和形态,卵黄积累情况,滤泡细胞形态变化,将卵细胞发生分为前期,中期,中后期和后期。卵细胞发育后期,营养索消失,滤泡细胞排列疏松,细胞间隙增大。【结论】孟氏隐唇瓢虫卵巢管的滋养细胞是端滋式卵巢管滋养细胞中的原始类型,且推测瓢虫科昆虫卵巢管滋养细胞均属于此类。卵细胞早期发育过程中,卵细胞通过营养索从滋养细胞获取营养物质。     

孟氏隐唇瓢虫和台毛艳瓢虫对茶椰圆蚧的捕食作用

本首次报道,孟氏隐唇瓢虫和志艳瓢虫是茶椰圆蚧的重要捕食性天敌。1998－1999年我们研究了这两种瓢虫的生活习性及其对茶椰圆蚧二龄若虫的捕食作用。这两种瓢虫成6虫对共椰圆蚧二龄若虫的功能反应均可用HollingⅡ型模型模拟。孟氏隐唇瓢虫的模型参数为：α'=1.1302,Th=0.0093,maxNa=107.6233;台毛艳主虫的模型参数为：α'=1.0005,Th=0.0126,maxNa=79.3741。温度对孟氏隐唇瓢虫捕食作用的影响大于对台毛艳瓢虫的影响,个体间相互干扰对捕食作用的影响,孟氏隐唇瓢虫大于台毛艳瓢虫孟氏隐唇瓢虫干扰反应模拟模型参数为：Q＝0.4045,m=0.4359;台毛艳瓢虫干扰反应模拟模型参数为：0.3335m=0.4042。     

孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选

选择合适的内参基因是qRT-PCR研究的关键。本文以孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant为研究材料,利用qRT-PCR技术,对孟氏隐唇瓢虫4个候选内参基因Actin、RPS23、GAPDH和β-tubulin的mRNA的表达量进行了分析,并用Ge Norm、Norm Finder和Best Keeper软件分析它们在孟氏隐唇瓢虫不同发育阶段及成虫不同组织中的表达稳定性。结果表明,以成虫不同组织为材料时,综合三种软件分析结果显示4个候选基因表达稳定性平均等级值排名为RPS23(rank=1)β-tubulin(rank=2.3)GAPDH(rank=3)Actin(rank=3.7),以不同发育时期虫体为材料时,综合分析结果显示4个候选内参基因表达稳定性平均等级值排名为RPS23(rank=1.7)Actin(rank=2)GAPDH(rank=2.7)β-tubulin(rank=3.7)。综合分析在瓢虫不同发育阶段及成虫不同组织两种处理下,三种软件的评价效果,4个候选基因表达稳定性等级值的总平均排名为RPS23(rank=1.3)Actin(rank=2.8)=GAPDH(rank=2.8)β-tubulin(rank=3)。RPS23在瓢虫不同发育阶段及成虫不同组织中均显示出较高的表达稳定性及与其它基因之间极大的相关性,可以确定为孟氏隐唇虫不同发育阶段及成虫不同组织基因表达分析中一个稳定表达的基因,可作为单个内参基因或者其它内参基因的协同基因,本实验为开展孟氏隐唇瓢虫功能基因表达分析奠定了方法学基础。     

Cloning and characterization of the rat cytochrome P450 4F5 (CYP4F5) gene

The analysis of a non-redundant set of human proteins, for which both the crystallographic structures and the corresponding gene sequences are available, show that bases at third codon position are non-uniformly distributed along the coding sequences. Significant compositional differences are found by comparing the gene regions corresponding to the different secondary structures of the proteins. Inter-and intra-structure differences were most pronounced in the GC-richest genes. These results are not compatible with any proposed hypotheses based on a neutral process of formation/maintenance of the high GC₃ levels of the genes localized in the GC-richest isochores of the human genome.     

Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) from Candida bombicola

Candida bombicola is a yeast with at least two appealing features. The species can grow on alkanes when provided as the sole carbon source, and it produces glycolipids, which have several industrial, cosmetic and pharmaceutical applications. Both metabolic processes require in their pathway the activity of cytochrome P450 monooxygenase. This enzyme needs and gets reducing equivalents from NADPH cytochrome P450 reductase (CPR). The CPR gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes an enzyme of 687 amino acids, which shows homology with known CPRs of other species. The functionality of the gene was proven by heterologous expression in Escherichia coli. The recombinant protein exhibited NADPH-dependent cytochrome c reducing activity. Cloning and characterization of this enzyme is an important step in the study of the cytochrome P450 monooxygenase system of Candida bombicola. The GenBank accession number of the sequence described in this article is EF050789.     

Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal,putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites.Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1 st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells.These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.     

Influence of cGMP on Feeding Potential of Predatory Coccinellid,Cryptolaemus montrouzieri Mulsant and Isolation of Partial foraging Gene

The cyclic guanomonophosphate (cGMP) dependent protein kinase (PKG) plays an important role in the food related behaviours of several insect species. Here we report the influence of cGMP dependent PKG on prey consumption of adult predatory coccinellid, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae). The oral cGMP treatment (which increases PKG activity) enhanced the feeding potential of C. montrouzieri. The good foragers responded more positively to the cGMP treatment compared to the poor foragers. The cGMP levels estimated through ELISA were significantly (P?<?0.001) high in the digestive tissues of unfed as well as cGMP treated C. montrouzieri compared to normal fed beetles. This finding suggests that cGMP is involved in the higher feeding rates of C. montrouzieri and the partial foraging gene (~455 bp) which encodes the cGMP dependent PKG was isolated from genomic DNA of C. montrouzieri using gene specific primers.     

Expression,purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.