家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

家蝇幼虫血细胞类型及免疫功能的初步研究

目的用不同方法观察家蝇3龄幼虫血细胞的形态,并对血细胞进行分类和免疫功能研究,为昆虫血细胞形态、分类及免疫研究提供实验依据。方法 (1)应用姬氏染色结合相差显微镜及荧光染色方法观察家蝇3龄幼虫血细胞形态,并对家蝇3龄幼虫血细胞进行分类;(2)观察家蝇3龄幼虫感染大肠杆菌后不同时间血细胞总数(THC)、各类血细胞数量(DHC)及形态的变化;(3)应用倒置显微镜观察家蝇3龄幼虫离体血细胞感染大肠杆菌后的形态变化;(4)采用酶细胞化学技术测定感染前后家蝇3龄幼虫血细胞中ACP、POD活性的变化。结果 (1)家蝇3龄幼虫血细胞可分为原血胞、浆血胞、粒血胞、珠血胞、类绛血胞5类,其中浆血胞又分为大核浆血胞和小核浆血胞两种;(2)感染后各时间组血细胞总数、浆血胞和粒血胞数量均显著升高,且浆血胞和粒血胞聚集成堆,出现细胞变形、空泡等形态变化;感染后16h、24h组的珠血胞数显著升高;原血胞和类绛血胞数量和形态无明显变化;(3)家蝇幼虫离体血细胞感染大肠杆菌后粒血胞周围见大量细菌聚集,浆血胞、粒血胞聚集成团将细菌包裹形成包囊,未见原血胞、珠血胞、类绛血胞形态的变化;(4)感染后浆血胞和粒血胞中ACP、POD的活性增强,感染前后原血胞、珠血胞、类绛血胞中均未见ACP、POD的阳性反应物。结论通过3种方法能很好地将家蝇3龄幼虫血细胞分为5类,其中浆血胞和粒血胞是家蝇幼虫参与免疫反应的主要细胞类型,珠血胞不参与感染后的早期细胞免疫反应。     

Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica

Abstract A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly ( Musca domestica ) as well as other Diptera and Lepidoptera . Crystal δ-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 μg/ml, and β-exotoxin was not detected. Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination. Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cry IA(b), one cry IIA, and two cry IIB genes.     

基因芯片技术筛选家蝇抗菌肽相关基因

用生物学软件对GenBank中部分昆虫抗菌肽基因编码区保守域设计探针, 用直接点样法将探针点印在特制玻片上构建寡核苷酸(Oligonucleotide, oligo)探针微阵列; 提取诱导后24 h的家蝇三龄幼虫脂肪体总RNA, 逆转录成cDNA并标记上荧光标记物Cy3, 与构建的oligo探针微阵列杂交, 经洗片、扫描处理后进行数据分析。结果在两次重复实验中均检测到有效杂交信号的基因点有15个(不包括阳性对照基因), 为进一步发现其新基因提供了依据。     

大肠杆菌感染后家蝇幼虫的细胞免疫反应

目的探讨家蝇3龄幼虫被大肠杆菌感染后的细胞免疫反应.方法 (1)观察家蝇3龄幼虫感染大肠杆菌后不同时间血淋巴细胞总数(THC)、各类血细胞比例及形态的变化.(2)用比色法分别测定感染后不同时间家蝇3龄幼虫血清中酸性磷酸酶(ACP)及过氧化氢酶(CAT)活性的变化.(3)用聚丙烯酰胺凝胶电泳法测定感染后不同时间家蝇3龄幼虫血清中二酚氧化酶(DPO)活性的变化.结果 (1)感染后4 h、8 h、16 h、24 h THC均有显著增加(P<0.01),(2)感染后4 h、8 h、16 h组的浆血胞和粒血胞的比例明显增加(P<0.01),而珠血胞在感染后各时间组的比例均明显减少(P<0.01),各时间组原血胞和类绛血胞的比例均无明显变化(P>0.05).(3)浆血胞出现细胞变形、空泡等形态变化.(4)大肠杆菌感染后各时间组血清中ACP及CAT活性显著高于对照组(P<0.01),活性分别在感染后8 h、16 h达高峰后逐渐下降.(5)家蝇幼虫血清中二酚氧化酶(DPO)活性在感染后4 h上升,16 h达高峰后下降.结论家蝇幼虫感染大肠杆菌后诱发体内细胞免疫反应,不仅出现血淋巴细胞形态和数量的变化,而且还有血细胞合成、释放多种参与微生物杀灭、清除的酶.     

家蝇幼虫差异表达基因的克隆筛选与分析

目的是利用mRNA差异显示(DDRT-PCR)技术对诱导后家蝇幼虫差异表达基因进行克隆分析。取诱导和未诱导家蝇(Musca domestica vicina)三龄幼虫总RNA进行mRNA差异显示反应,产物经6％非变性聚丙烯酰胺凝胶电泳展开,银染分析后,回收差异显示条带。随机选取4条诱导家蝇三龄幼虫中上调表达的基因条带,进行Northern杂交验证,有2条被验证为真实带,命名为YD1和YD2。对这2条基因片段进行T-A克隆和测序,长度分别为495bp和265bp,登录NCBI运用Blastn程序进行同源性比较,发现两者与任何已知基因同源性都低于70％,提示为两个未报道的新基因,在家蝇免疫反应中可能起着一定的作用。     

滞育家蝇幼虫氨基酸、SOD、CAT的变化

目的 观察家蝇幼虫滞育前后血淋巴中氨基酸、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的变化,探讨家蝇幼虫滞育发生的生化机制.方法 低温诱导家蝇幼虫滞育,氨基酸自动分析仪测滞育组及非滞育组血淋巴氨基酸含量;紫外分光光度计测滞育组及非滞育组家蝇幼虫血淋巴SOD、CAT含量.结果 除滞育20 d组家蝇幼虫血淋巴内胱氨酸较非滞育组升高外,各滞育组家蝇幼虫血淋巴内氨基酸含量均低于非滞育组;各滞育组SOD、CAT均低于非滞育组,随着滞育时间的延长SOD、CAT含量逐渐增高.结论 家蝇滞育幼虫体内各氨基酸含量减少,SOD、CAT活力降低.     

家蝇变形期抗菌蛋白的提取

目的:分离纯化家蝇变形期体内抗菌蛋白并分析其抑菌作用。方法:对家蝇（Ｍusca domestica）5日龄幼虫进行针刺诱导,并于24h后挑取变形后的蛹,通过研磨、调酸、加热提取蛹体内的耐热水溶总蛋白,经过两步CM sepharose F.F.离子交换层析分离,以金黄色葡萄球菌作指示菌,检测其抑菌活性,并用SDS PAGE不连续电泳检测蛋白质的分子量。结果:经两步离子交换分离后,得到一种对金黄色葡萄球菌具有较强抑菌活性的蛋白,经SDS PAGE检测为单一条带,其分子量为43kDa。结论:家蝇处于变形期时,经过诱导后体内能产生一种抑菌活力较强的蛋白质。     

家蝇变形期抗菌蛋白的提取及其抑菌活性研究

目的:分离纯化家蝇变形期体内抗菌蛋白并分析其抑菌作用.方法:对家蝇(Muscadomestica)5日龄幼虫进行针刺诱导,并于24h后挑取变形后的蛹,通过研磨、调酸、加热提取蛹体内的耐热水溶总蛋白,经过两步CM-sepharose F.F.离子交换层析分离,以金黄色葡萄球菌作指示菌,检测其抑菌活性,并用SDS-PAGE不连续电泳检测蛋白质的分子量.结果:经两步离子交换分离后,得到一种对金黄色葡萄球菌具有较强抑菌活性的蛋白,经SDS-PAGE检测为单一条带,其分子量为43kDa.结论:家蝇处于变形期时,经过诱导后体内能产生一种抑菌活力较强的蛋白质.     

The clock gene period of the housefly,Musca domestica,rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.     

家蝇抗菌物质的诱导

针刺损伤诱导的家蝇三龄幼虫免疫血淋巴对动、植物病原菌有广谱的抗菌活性,同时,未经诱导的家蝇幼虫也具有抗菌活性。ＳＤＳ－ＰＡＧＥ电泳分析表明：外源诱导能增强原有抗菌物质的表达量,并能激活新的蛋白产生。家蝇免疫血淋巴中的抗菌物质具热稳定性和耐冷冻贮藏的特性     

Genetic consequences of linkage between malathion resistance and an autosomal male-determining factor in house fly (Diptera: Muscidae).

The pattern of inheritance of genes conferring resistance to malathion and genetic consequences of linkage between an autosomal male-determining factor and resistance genes on the second chromosome were investigated in a strain of house fly, Musca domestica L., selected for malathion resistance. The second and fifth chromosomes contribute significantly to malathion resistance. The presence of a male-determining factor linked with the resistance genes on the second chromosome resulted in a strong sexual dimorphism in malathion resistance. We also observed that the male-determining factor changed its linkage relationship from the third linkage group to the second linkage group during the selection experiments.     

弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究

家蝇抗菌肽多是碱性蛋白,目前尚无弱酸性家蝇抗菌肽的报道。通过稀醋酸低温浸提,海藻酸吸附,稀盐酸低温洗脱、盐析、Sephadex G25凝胶过滤和CMC23弱阳离子交换柱层析等方法,利用灵敏的杀菌活性检测手段,从家蝇蛆(Musca domesticalarvae)中分离纯化出一组弱酸性抗菌肽,对苏云金芽孢杆菌(Bacillus thuringiensis)等革兰氏阳性菌和几种革兰氏阴性菌有强烈的杀灭作用,有极强的耐热、耐冻融的特性。通过电洗脱方法进一步纯化出抗菌肽MD7095,质谱测定其分子量7095Da,IEF电泳测得其等电点5.59,经肽质量指纹谱(PMF)鉴定为一新肽。扫描电镜超微结构观察表明,弱酸性家蝇蛆抗菌肽对苏云金芽孢杆菌的杀菌机制主要是使细胞膜穿孔,内容物外泄,最终使细菌完全解体死亡。     

家蝇抗菌肽的抑菌动力学研究及其机理初探

利用鼠伤寒沙门氏菌针刺诱导家蝇幼虫表达抗菌肽,对抗菌肽的抑菌动力学进行研究,并通过抗菌肽样品对不同细菌动力学特性的研究出发对抗菌肽抑菌作用机制进行探讨。研究发现抗菌肽样品的活性与作用时间有关,24h内出现一到两个活性峰,同一抗菌肽样品对不同细菌的抑菌动力学有差异,抗菌肽的抑菌动力学机制应该与它的的抑菌作用机制有关。通过电镜观测、细胞磷代谢、紫外吸收物测定以及抗菌肽与细菌DNA相互作用结果可知,微生物诱导家蝇表达的抗菌肽样品不仅能够造成细菌细胞的快速坍塌破裂而且能够破坏细胞核心,与DNA结合作用。抗菌肽抑菌动力学的解释：微生物诱导产物中含有两类抗菌肽,一类抗菌肽能造成细胞膜的快速坍塌破裂形成第一个活性峰;另一类抗菌肽可进入细胞,破坏细胞核心,造成紫外吸收物大量外泄形成第二个活性峰。     

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.     

Nymphal Development and Reproduction of Podisus nigrispinus (Heteroptera: Pentatomidae) Fed With Combinations of Tenebrio molitor (Coleoptera: Tenebrionidae) Pupae and Musca domestica (Diptera: Muscidae) Larvae

The development and reproduction of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed on Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Musca domestica L. (Diptera: Muscidae) were studied in four treatments. P. nigrispinus was permitted to feed on: (1) T. molitor pupae (T 1 ); (2) Musca domestica larvae (T 2 ); (3) both prey supplied simultaneously (T 3 ); (4) both prey supplied on alternate days (T 4 ). Duration of the nymphal period of P. nigrispinus was similar in all diets studied with nymphal viability of approximately 75%. Heavier females were obtained in T 1 and T 4 , but no correlation between this factor and the reproductive rate of the predator was found. Therefore, the use of body weight as a parameter to evaluate rearing quality should be approached with caution. However, females of this predator showed higher egg and nymph production when they received both prey. For this reason P. nigrispinus should be reared with T. molitor and M. domestica simultaneously or on alternate days.     

Regional distribution and substrate specificity of digestive enzymes involved in terminal digestion in Musca domestica hind-midguts.

One membrane-bound alpha-glucosidase and two soluble alpha-glucosidases were isolated from homogenates of the hind-midgut, the main digestive region in Musca domestica larvae. The membrane-bound alpha-glucosidase and the low-Mr soluble alpha-glucosidase hydrolyze maltopentaose better than maltose, maltotriose, and maltotetraose, the reverse being true for the high-Mr soluble alpha-glucosidase. A membrane-bound glucoamylase previously described in Musca domestica midgut was shown by gradient centrifugation and dialysis against EDTA to result from the combined action of an amylase and an alpha-glucosidase. The determination of amylase, alpha-glucosidases, soluble and membrane-bound carboxypeptidase A, membrane-bound aminopeptidase and dipeptidase along the tissue and luminal contents of the hind-midgut is described. The data support a proposal concerned with how starch and protein are digested in Musca domestica larval hind-midguts and where and how midgut glycosidases and peptidases are secreted.