Enhancement of antiviral capacity of transgenic silkworms against cytoplasmic polyhedrosis virus via knockdown of multiple viral genes.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a major pathogen of silkworms, causes serious economic losses in sericulture. The BmCPV genome contains 10 discrete dsRNA segments; among these, S1, S2, S3, S4, S6, and S7 encode virus structural proteins, whereas S5, S8, S9, and S10 encode nonstructural proteins. In an attempt to create an anti-BmCPV silkworm strain, we constructed transgenic RNAi vector pb-CNS for knockdown of S5, S8, S9, and S10, and pb-SNS targeting S1, S2, S4, S5, and S8. Transgenic silkworm line CNS and SNS were generated via microinjection of the practical diapause silkworm strain Furong. Following infection via the oral administration of a high dose of BmCPV, the mortality rates of the nontransgenic control, CNS, and SNS were 91%, 37%, and 41%, respectively. qPCR showed that the viral mRNA content in CNS and SNS was significantly lower than that in the nontransgenic line. The economic traits of CNS and SNS were not affected. These results suggest that the knockdown of multiple BmCPV genes significantly enhances the antiviral capacity of the silkworm.

Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in FB but not in MG. BGI001299, BGI014434, BGI012068, and BGI009201 were MG-specific genes with transmembrane transport function, the expression of which were induced by BmCPV. BGI001299, BGI014434, and BGI012068 expressed in entire MG and may be involved in BmCPV invasion. BGI009201 expressed only in PM and may be necessary for BmCPV proliferation. BmPGRP-S2 and BGI012452 (a putative serine protease) were induced by BmCPV and may be involved in immune defense against BmCPV. The expression level of BmCPV S1, S2, S3, S6, and S7 was high and there was no expression of S9 in MG 72 h, implying that the expression time of structural protein coding genes is earlier. These results provide insights into the mechanism of BmCPV infection and host defense.

The 5'-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms.

Introns are important for regulating gene expression. BmAPN4, which has a 5'-UTR upstream intron (5 UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5 UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5 UI was cloned and named as P3P+5 UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5 UI. Transgenic P3+5 UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5 UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5 UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5 UI silkworms was 64% higher than in P3 silkworms, indicating the 5 UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5 UI affected the level and site of expression. The 5 UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5 UI and transgenic P2+5 UI silkworms. Expression patterns were the same for P2P+5 UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5 UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.

Overexpression of host plant urease in transgenic silkworms.

Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms.

Postintegration stability of the silkworm piggyBac transposon.

The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3 × p3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome.

A transgenic animal with antiviral properties that might inhibit multiple stages of infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms, causing severe economic losses in sericulture. To create antiviral silkworm strains, we constructed a transgenic vector in which the dsRNA for five tandem BmNPV genes was controlled by the BmNPV hr3 enhancer and IE1 promoter. The antivirus gene Bmlipase-1 was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. After infection with a high dose of BmNPV, the survival rates of SW-H and non-transgenic SW were 64% and 13%, respectively. SW-H could be the first transgenic animal that is highly antiviral and that might inhibit the virus at multiple stages of infection.

Identification of a midgut-specific promoter in the silkworm Bombyx mori.

The midgut is an important organ for digestion and absorption of nutrients and immune defense in the silkworm Bombyx mori. In an attempt to create a tool for midgut research, we cloned the 1080 bp P2 promoter sequence (P2P) of a highly expressed midgut-specific gene in the silkworm. The transgenic line (P2) was generated via embryo microinjection, in which the expression of EGFP was driven by P2P. There was strong green fluorescence only in the midgut of P2. RT-PCR and Western blot showed that P2P was a midgut-specific promoter with activity throughout the larval stage. A transgenic truncation experiment suggested that regions -305 to -214 and +107 to +181 were very important for P2P activity. The results of this study revealed that we have identified a midgut-specific promoter with a high level of activity in the silkworm that will aid future research and application of silkworm genes.