家蚕核型多角体病毒lef-11基因RNA干扰策略的优化

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)lef-11基因作为杆状病毒高度保守的晚期表达因子之一,对病毒晚期基因的表达极其重要。因此对lef-11基因进行有效RNA干扰将提高宿主细胞对BmNPV的抗性。【方法】本文基于经典的sh-RNA-loop骨架及家蚕内源性的miRNA骨架,构建以BmNPV lef-11基因为靶标的干扰载体pIZ-shRNA1、pIZ-shRNA2和pIZ-Ds RedamiR279;分别构建基于A4、IE1、IE1-295、IE2、IE2-339、hr3A4和hsp70启动子驱动的干扰载体,用以评价不同启动子驱动的干扰载体的抗病毒效果,并对干扰载体进行优化。【结果】首先,本文通过比较miRNA-based和shRNA-based RNAi载体对同一靶基因同一位点的干扰效率,发现pIZ-Ds Red-amiR279对BmNPV lef11基因的干扰效率超过90%,远优于shRNA-based RNAi载体的干扰效果;其次,通过对干扰载体进行优化,发现IE1启动子的效果最优,由其驱动的pIZ-Ds Red-milef11干扰载体也是本研究中的最优干扰载体,对病毒的增殖抑制效果最为明显。【结论】对目的基因的干扰效果并非启动子的活性越高、miRNA积累得越多就越好,只有综合考虑干扰片段的选择、启动子的活性以及靶基因自身的功能等多方面的因素,才能提高干扰效率,达到干扰目的。     

Production of a Cellulase in Silkworm Larvae using a Recombinant Bombyx mori nucleopolyhedrovirus lacking the Virus-encoded Chitinase and Cathepsin Genes

The expression efficiency of an insect-derived cellulase was assayed in silkworm larvae infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) mutants lacking the virus-encoded chitinase (chiA) and/or cathepsin (v-cath) genes. Expression was increased by approx. 10% in mutants lacking chiA or v-cath and 17% in a mutant lacking both chiA and v-cath compared with that of the unmodified recombinant BmNPV. The recombinant BmNPV lacking both chiA and v-cath can therefore be used for a large-scale production of foreign proteins in silkworms. Revisions requested 27 September 2005 and 16 December 2005; Revisions received 6 December 2005 and 3 February 2006     

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.     

家蚕核型多角体病毒BmNPV泛素基因缺失显著降低病毒增殖效率北大核心

【目的】泛素是一类进化上高度保守的、由76个氨基酸组成的多肽,是蛋白质泛素化修饰过程中所必需的底物分子,蛋白质泛素化修饰异常会影响宿主的生长和发育。家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)基因orf26是一个编码病毒泛素的基因,其在病毒增殖中的具体功能尚不清楚。【方法】本研究利用同源重组的方法将氯霉素(chloramphenicol)基因(Cm)表达盒替换家蚕杆状病毒泛素基因序列3’端的50个碱基对序列,构建携带Cm表达盒的重组BmNPV病毒基因组(Bm-Bacmid^(Ub-KO)),利用转座原理将绿色荧光蛋白基因(green fluorescent protein,gfp)表达盒插入Bm-Bacmid^(Ub-KO)和野生型Bm-Bacmid^(WT)转座位点,构建重组质粒Bm-Bacmid^(Ub-KO-GFP)和Bm-Bacmid^(WT-GFP),以及异位互补型的质粒Bm-Bacmid^(GFP-Ub Rep)。这些重组病毒分别转染家蚕卵巢细胞(Bombyx mori ovarian cell,BmN)后,对转染后的细胞进行绿色荧光观察。【结果】泛素基因缺失后不影响感染性病毒粒子的产生,但与野生型相比,泛素缺失型重组病毒明显降低了感染的细胞中产生的绿色荧光数量。免疫印迹分析表明,泛素缺失后降低重组病毒结构蛋白GP64和VP39在细胞中的表达水平,显著降低病毒增殖效率。生物分析表明,泛素缺失的重组病毒能延缓家蚕半致死时间15 h。【结论】BmNPV泛素基因缺失不影响感染性病毒粒子的产生,但显著降低病毒增殖效率。本研究为阐明泛素在BmNPV增殖中的具体作用提供了实验依据。     

Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its host ortholog, BmChi-h

Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.     

Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L

Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.     

Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor

The effect of validoxylamine A (VAA), a potent and specific trehalase inhibitor, on the induction of non-diapause in Bombyx mori was examined. The VAA induced non-diapause eggs and prevented the glucogen accumulation in the eggs. Trehalase activity of the pupal ovary was effectively inhibited by the VAA injection.     

The Bombyx mori sex pheromone biosynthetic pathway is not mediated by cAMP

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

Quantitative measurement of general odorant using electroantennogram of male silkworm moth,Bombyx mori

The investigation of electroantennogram (EAG) using insect antennae has been primarily focused on the measurement of insect pheromone. Insect has highly specialized olfactory receptors inside their antennae. In this paper, EAG was applied to detect general odorants and the feasibility of this system for the olfactory biosensor was investigated. Electroantennogram measurement was carried out using the antennae of male silkworm moth,Bombyx mori, and ammonia gas as the model odorant. EAG parameters including peak amplitude, decay, and level were analyzed for the quantitative measurement. The peak amplitude increased linearly with the ammonia concentration and the reproducible electrical signals were generated at least for 2 hrs after the antenna was cut off from the silkworm moth.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.