细菌表达dsRNA介导的家蚕FTZ-F1基因的RNA干扰

为探索细菌表达目标基因dsRNA介导的RNAi技术是否在家蚕Bombyx mori可行, 本研究引入了在其他物种中广泛应用的细菌表达dsRNA的RNAi系统: HT115细菌株和L4440质粒。利用L4440载体两端含有T7启动子的特点, 设计并构建了针对家蚕核受体FTZ-F1基因的RNA干扰（RNA interference）载体, 将构建好的质粒转入大肠杆菌Escherichia coli HT115, 在IPTG诱导下成功获得目标基因对应双链RNA（dsRNA）。 结果显示: 通过对5龄第7天家蚕幼虫注射IPTG诱导后提取的FTZ F1基因对应的dsRNA 25 μg, 85%的蛹变态发育过程明显延迟, 不能实现幼虫到蛹的形态完全转变。荧光定量PCR分析显示目标基因的表达得到了特异的抑制。实验结果初步表明, 通过细菌表达目标基因dsRNA介导的RNAi策略, 以其经济、高效的特点, 具有广泛应用于家蚕基因功能研究中的潜力。     

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.     

Isolation and Identification of a Pathogen of Silkworm Bombyx mori

A pathogenic bacterial strain, ST-1, was isolated from a naturally infected silkworm. The strain was identified on the basis of its physiological and biochemical properties and the results of sequence analysis of its 16S rRNA gene. The results of the 16S rRNA gene sequence analysis revealed that ST-1 shared the highest sequence identity (more than 99%) with Pseudomonas chlororaphis subsp. aurantiaca. ST-1 bacteria were gram-negative and 0.7-0.9 × 1.3-1.5 μm long, short rods with rounded ends. The strain could utilize sodium citrate, malonate, D-glucose, sucrose, D-fructose, D-mannose, and L-arabinose. Pathogenicity of ST-1 for silkworm could be depicted as a linear regression of the logarithm (y) of ST-1 concentration against probability (x) (y = 0.4040 + 0.0600x). The median lethal concentration (LC(50)) was 2.12 × 10(4) cfu/ml. In conclusion, ST-1 was identified as Ps. chlororaphis subsp. aurantiaca. This is the first report that Ps. aurantiaca is a pathogen for silkworm Bombyx mori.     

Partial purification of prostaglandin synthetase from silkmoth Bombyx mori L.

Prostaglandin synthetase has been isolated and purified from the post mitochondrial supernatant of the testis of male silkmoth Bombyx mori L. A 3-fold purification was achieved compared to that of post mitochondrial supernatant. The partially purified enzyme converted arachidonic acid to PGE₂in vitro in the presence of haemin and hydroquinone.     

家蚕吡哆醛激酶的融合表达与纯化

【目的】吡哆醛激酶（pyridoxal kinase, PLK, EC 2.7.1.35）是维生素B6的关键代谢酶。本研究原核表达家蚕Bombyx mori重组PLK, 为进一步开展家蚕PLK的催化作用机制和表达调控机制的研究奠定基础。【方法】构建家蚕PLK基因融合表达质粒, 转化大肠杆菌Escherichia coli诱导表达, 经Ni²⁺ 亲和层析纯化后, 对融合蛋白的催化活性进行分析。【结果】纯化后的家蚕重组PLK经SDS-PAGE鉴定为单一条带, 比活力为1 800 U/mg, 纯化倍数为40倍。在底物过量的条件下, 该重组酶的体外最适反应温度是50℃; 最适pH为5.5~6; Zn²⁺ 是酶促反应有效的激活剂。【结论】重组家蚕PLK与来源于家蚕组织的PLK具有相同的催化性质。     

内蒙古鄂尔多斯一古盐湖真菌多样性及其功能酶的初步筛选

【目的】探索内蒙古鄂尔多斯一古盐湖土壤沉积物和湖水样品中的真菌多样性,并初步筛选出产功能酶的活性菌株。【方法】采用含有2.5%、5%、10%及15%NaCl的3种分离培养基,利用稀释平板法分离可培养真菌,基于形态学和ITS序列分析对获得菌株进行系统分类学分析。利用6种筛选培养基定性检测盐湖真菌的产酶活性。【结果】共分离到2 121株真菌,共45个形态种,根据形态学研究和ITS序列的系统分类学分析将其归类为27个属,优势属为曲霉属(Aspergillus)。功能酶筛选结果表明,45个形态种中有22个形态种具有产酶活性,其中6个形态种只产蛋白酶,6个只产纤维素酶,2个只产复合酶,1个只产淀粉酶。有4个形态种可同时产3种酶,有3个可同时产2种酶。【结论】内蒙古鄂尔多斯地区盐湖中真菌多样性丰富,产酶特性良好,研究工作为耐(嗜)盐真菌资源的进一步开发和利用提供依据。     

一种寄生家蚕被毛孢的鉴定及产孢条件优化

【目的】对寄生家蚕的真菌被毛孢进行鉴定和孢子培养进行研究。【方法】采用形态特征比较和内转录间隔区(ITS)序列构建系统树进行鉴定,通过单因素筛选和正交试验进行产孢条件优化。【结果】根据形态特征比较和系统发育分析,该真菌为鹿儿岛被毛孢Hirsutella satumaensis Aoki。较优产孢条件为(质量体积比):蛋白胨3%,葡萄糖1%,蚕蛹粉1.5%,维生素B1 1%,硫酸镁0.05%,磷酸二氢钾0.1%,琼脂2%,蒸馏水1 000 mL,25°C。【结论】鹿儿岛被毛孢为已知种,文中对其显微特征进行了重新描述并补充相关分子系统学资料;产孢条件的优化可为该类群真菌孢子的获得与应用提供参考。     

Characterization and affinity purification of juvenile hormone esterase from Bombyx mori

Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.     

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. We have isolated the mouse orthologue, Crb1, and analyzed its expression pattern in embryonic and post-natal stages. Crb1 is expressed exclusively in the eye, and the central nervous system. In the developing eye, expression of Crb1 is detected in the retinal progenitors, and later on becomes restricted to the differentiated photoreceptor cells where it remains active up to the adult stage. In the developing neural tube, expression of Crb1 is restricted to its most ventral structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1 expression is defined to areas where the production and migration of neurons occurs in adulthood.     

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down‐regulation of BmPP2A expression. Furthermore, up‐regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down‐regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti‐BmNPV mechanism, and a possible target for the breeding of silkworm‐resistant strains.     

Identification and characterization of a silkgland-related matrix association region in Bombyx mori

From DNA fragments in vivo attached to the nuclear matrix in silkglands of Bombyx mori 5th instar larvae, we have screened a matrix association region (MAR), termed BmMAR1, by means of in vitro binding assay. BmMAR1 was identified to be specifically in vivo attached to the nuclear matrix only in the silkglands, neither in other tissues nor in the silkworm cell line Bm5, indicating its silkgland-relatedness. This 1983-bp DNA fragment contains a 1.1-kb core necessary for the effective in vitro binding although it is of relatively lower A/T composition (61%) compared to the 5' and 3' flanking regions (73 and 69%, respectively). Two degenerate sequences derived from Bm1 and L1Bm repetitive elements are located in the core region. BmMAR1 shares the widely considered typical MAR's features, DNA unwinding motif, A-box, T-box, H-box, replication origin, MAR recognition signature (MRS), the 90%AT box and Drosophila topoisomerase II consensus sequence. Furthermore we compared the occurrences of these patterns in BmMAR1 and some MARs from other organisms.     

家蚕精氨酸激酶原核表达纯化、结构与活性分析

精氨酸激酶(Arginine kinase,AK)是无脊椎动物体内能量代谢的关键酶,在生长发育、营养利用、免疫抗性、胁迫应答等生命活动过程中发挥着重要的调控作用。家蚕精氨酸激酶BmAK与能量平衡、抗NPV病毒过程相关,但目前关于其分子结构和酶学性质的研究不多。克隆了BmAK基因ORF序列,分析了其染色体定位、基因组结构、mRNA结构、二级结构和三级结构。进化分析表明AK在进化过程中高度保守。原核表达获得了可溶性的BmAK重组蛋白,通过Ni-NTA亲和层析纯化了BmAK。圆二色光谱分析显示BmAK包含α螺旋结构,其α螺旋结构在pH 5–10范围内相对稳定。酶活分析表明BmAK的最适温度为30℃,最适pH为7.5。25℃时BmAK的催化活性最大,在15–30℃范围内,BmAK的结构相对稳定,活性差别不大。BmAK的结构在pH 7.0左右相对稳定。这些研究为揭示BmAK的结构和功能提供了基础,有助于开发以AK为分子靶标的绿色安全环保的新型杀虫剂。     

家蚕hsp70启动子的克隆及功能研究

该研究通过PCR的方法克隆得到家蚕热激蛋白70基因(Bombyx mori hsp70)的5'侧翼的两个长度分别为538 bp和305 bp的序列hsp70-538和hsp70-305。生物信息分析结果表明这两段序列在TATA序列的上游存在保守的热激元件HSE(heat shock element)CTnGAAnnTTCnAG。采用双荧光报告基因技术研究表明这两段序列在BmN细胞中都表现出热激活性,转基因家蚕实验证明hsp70-305在家蚕个体中也具有热激活性,可以认为这两个片段具有hsp70热激启动子特性。     

家蚕半乳糖凝集素BmGalectin-4的表达、纯化及性质分析

【目的】鉴定一种新的家蚕Bombyx mori半乳糖凝集素(galectin)基因,分析其序列和结构特征,测定其在微生物感染后的表达变化,分析其重组表达蛋白与微生物及微生物表面多糖分子的结合特性。【方法】利用TBlastN从家蚕基因组数据库中搜索得到一种新的半乳糖凝集素基因,命名为BmGalectin-4。利用生物信息学分析其序列和结构特征,用半定量RT-PCR检测家蚕分别感染病原菌绿脓杆菌Pseudomonas aeruginosa、金黄色葡萄球菌Staphylococcus aureus和白色念珠菌Candidiasis albicans后BmGalectin-4在不同组织中的表达变化,采用原核表达技术对该基因进行重组表达,并利用亲和层析得到纯化的蛋白。通过ELISA检测BmGalectin-4与细菌和真菌的结合;通过Western blot检测它与多糖的结合。【结果】序列分析显示,BmGalectin-4是一种串联重复型半乳糖凝集素,含有2个糖识别结构域,其结构非常保守。RT-PCR检测表明,BmGalectin-4在家蚕脂肪体、血细胞和中肠中都有表达,且感染细菌和真菌后其表达有显著变化。纯化的重组BmGalectin-4与革兰氏阳性菌、阴性菌和真菌有较强的结合,与微生物表面多糖的结合试验表明其识别有较高的特异性。【结论】BmGalectin-4 是一个典型的串联重复型半乳糖凝集素,可能参与家蚕对病原微生物的免疫防御反应。本研究为进一步研究昆虫中半乳糖凝集素的功能奠定了基础。