Analysis of mitochondrial protein‐coding genes of Antheraea assamensis: Muga silkworm of Assam

To understand the synonymous codon usage pattern in mitochondrial genome of Antheraea assamensis, we analyzed the 13 mitochondrial protein‐coding genes of this species using a bioinformatic approach as no work was reported yet. The nucleotide composition analysis suggested that the percentages of A, T, G,and C were 33.73, 46.39, 9.7 and 10.17, respectively and the overall GC content was 19.86, that is, lower than 50% and the genes were AT rich. The mean effective number of codons of mitochondrial protein‐coding genes was 36.30 and it indicated low codon usage bias (CUB). Relative synonymous codon usage analysis suggested overrepresented and underrepresented codons in each gene and the pattern of codon usage was different among genes. Neutrality plot analysis revealed a narrow range of distribution for GC content at the third codon position and some points were diagonally distributed, suggesting both mutation pressure and natural selection influenced the CUB.     

短梗长尾啮小蜂的生物学特性

短梗长尾啮小蜂Aprostocetus brevipedicellus是林业害虫的一种重要卵寄生蜂,目前国内外对其生物学特性尚没有报道.本研究在温度25±1℃、相对湿度70％ ±5％、光周期14 L:10 D条件下对该蜂的生物学特性进行了观察.结果表明,以柞蚕卵为寄主,该蜂的发育历期为23.58 d,其中卵期、幼虫期、预蛹-蛹期分别为2.73 d、8.96 d、11.50 d;在无营养补充条件下,雌、雄蜂平均寿命分别为2.92 d和1.96 d;单卵平均出蜂量为7.04头,后代雌蜂比为87.49％.该蜂羽化主要发生在白天,日羽化最高峰在10:00-12:00,占全天羽化总数的33.33％.此外,成蜂交配行为均发生在寄主卵外,且雄蜂可多次交配,但雌蜂一般只交配1次,平均交配时间为43.60 s.本研究基本明确了短梗长尾啮小蜂的生物学特性,为进一步开发和利用该蜂防治林业害虫提供了理论依据.     

柞蚕14-3-3基因的鉴定及表达分析

14-3-3蛋白是一种可以改变其结合蛋白构象的酸性蛋白质.柞蚕14-3-3 cDNA序列全长1 220 bp,包括一个126 bp的5'非编码区和一个350 bp的3'非编码区.该基因的开放读码框长度为744 bp,编码247个氨基酸.序列比对结果表明,柞蚕14-3-3蛋白与家蚕的14-3-3蛋白具有高度同源性.此外对柞蚕14-3-3基因进行了原核表达和重组蛋白纯化.SDS-PAGE和免疫印迹结果表明,分子量大小约32 kD的重组蛋白在大肠杆菌中得到了成功表达.     

A novel pattern recognition protein of the Chinese oak silkmoth,Antheraea pernyi,is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]     

密码子优化后的柞蚕溶菌酶在酵母中的表达及活性测定

【目的】提高柞蚕溶菌酶在毕赤酵母中的表达量,对柞蚕溶菌酶活性进行测定。【方法】根据毕赤酵母密码子偏爱性对柞蚕溶菌酶基因进行优化,并在目的基因3′端加上6个组氨酸标签,优化后的基因克隆至表达p PIC9K载体中,并通过电转化的方法导入毕赤酵母GS115中。利用不同浓度梯度的G418进行高拷贝转化子的筛选,经甲醇诱导实现分泌表达。通过硫酸铵盐析、镍柱亲和层析等工艺分离纯化柞蚕溶菌酶,利用琼脂扩散方法测定柞蚕溶菌酶对溶壁微球菌、金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、苍白杆菌及过氧化醋杆菌的抑菌作用,利用比浊法测定酶活大小。【结果】密码子优化后的柞蚕溶菌酶在诱导温度25°C、甲醇量0.75%和p H 7.0条件下实现了最高表达,表达量达到了2.4 g/L,并且纯化后的柞蚕溶菌酶酶活达到23 970 U/mg。【结论】密码子优化后的柞蚕溶菌酶在毕赤酵母中实现了高效表达,纯化后的柞蚕溶菌酶对溶壁微球菌、金黄色葡萄球菌、枯草杆菌、大肠杆菌、苍白杆菌及过氧化醋杆菌均有抑菌作用。     

Species-specific pheromonal compounds induce distinct conformational changes of pheromone binding protein subtypes from Antheraea polyphemus

We have investigated the structural features of three pheromone binding protein (PBP) subtypes from Antheraea polyphemus and monitored possible changes induced upon interaction with the Antheraea pheromonal compounds 4E,9Z-14:Ac [(E4,Z9)-tetradecadienyl-1-acetate], 6E,11Z-16:Ac [(E6,Z11)-hexadecadienyl-1-acetate], and 6E,11Z-16:Al [(E6,Z11)-hexadecadienal]. Circular dichroism and second derivative UV-difference spectroscopy data demonstrate that the structure of subtype PBP1 significantly changes upon binding of 4E,9Z-14:Ac. The related 6E,11Z-16:Ac was less effective and 6E,11Z-16:Al showed only a small effect. In contrast, in subtype PBP2 pronounced structural changes were only induced by the 6E,11Z-16:Al, and the subtype PBP3 did not show any considerable changes in response to the pheromonal compounds. The UV-spectroscopic data suggest that histidine residues are likely to be involved in the ligand-induced structural changes of the proteins, and this notion was confirmed by site-directed mutagenesis experiments. These results demonstrate that appropriate ligands induce structural changes in PBPs and provide evidence for ligand specificity of these proteins. Electronic Publication     

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

柞蚕蛹期灵菌败血病Serratia marcescens C3菌株分离鉴定

以柞蚕(Antheraea pernyi)蛹为替代寄主繁殖白蛾周氏啮小蜂(Chouioia cunea)技术在辽宁、北京、天津、上海、河北、山东等地美国白蛾(Hyphantria cunea)的生物防治中发挥了重要作用。利用柞蚕蛹繁殖白蛾周氏啮小蜂时,柞蚕蛹期软化病是繁蜂的主要障碍。通过对利用柞蚕蛹繁蜂时蛹内组织液化后呈粉红色这一未知软化病的典型症状进行病原细菌的分离和纯化,得到C3菌株。经Biolog系统和16S rRNA序列分析,鉴定C3菌株为灵菌(Serratia marcescens),经过柯赫法则检验,确定灵菌C3菌株是导致柞蚕蛹期灵菌败血病的病原菌。描述了繁蜂时柞蚕蛹期灵菌败血病发病期的认别特征。     

柞蚕热休克蛋白70基因的克隆和序列分析

HSP70蛋白是受热等因素刺激后而诱导产生的蛋白质,是热休克蛋白家族中最重要的一员。采用RT-PCR方法克隆了柞蚕(Antheraea pernyi)热休克蛋白70基因(HSP70)的ORF序列(GenBank登录号:GU945199),该片段的序列长度为1905bp。生物信息学分析表明,该序列共编码634个氨基酸,预测蛋白的等电点和分子量大小分别为5.62kD和69.5kD。具有HSP70的保守性结构特征,与天蚕(Antheraea yamamai)、家蚕(Bombyx mor)、甘蓝夜蛾(Mamestra brassicae)、棉铃虫(Heliothis viriplaca)、甜菜夜蛾(Spodoptera exigua)、烟草夜蛾(Manduca sexta1)、膜翅目寄生蜂(Cotesia rubecula)的同源性分别为95.7%、78.5%、76.1%、77.3%、76.6%、74.7%、65.9%。根据它们的一级结构构建了系统进化树,进一步确立了它们之间的亲缘关系。     

Parasitization of fifth instar tasar silkworm, Antheraea mylitta, by the uzi fly, Blepharipa zebina; a host-parasitoid interaction and its effect on host's nutritional parameters and parasitoid development

The uzi fly, Blepharipa zebina, is a well-known larval endoparasitoid of the tropical tasar silkworm, Antheraea mylitta. The present study dealt with the effect of the number of maggots developing per host on host nutritional parameters, parasitoid development and reproduction. Nutritional indices for ingestion, digestion, approximate digestibility, relative consumption rate, relative growth rate, and gain in body weight declined significantly with the increase in parasitoid burden, but the efficiency of conversion of digested food recorded a significant increase. The efficiency of conversion of ingested food remained little affected. The developmental period was significantly extended in larvae parasitized with 5 and 10 maggots per larva (mpl). Cocoon shell weight decreased by 27-63.5% in parasitized groups (1, 2, and 5 mpl) while larvae parasitized with 10 mpl could not spin cocoons. The maggot development period, recovery percentage, and fecundity of the uzi fly declined significantly with the increase in number of maggots developing per host.