| Bombyx mori is a lepidopteran model and an important economic insect, which facing serious threat of diseases. B. mori nucleopolyhedrovirus (BmNPV) is the primary pathogen to silkworm, which causes serious economic loss every year in sericulture. The conventional sterilization methods could prevent the disease of silkworm to some extent but have many limitations. However, cultivation of resistance strains is an available way to copy with the silkworm disease. In the present study, we enhance the resistance to BmNPV by transgenic technology in silkworm. Firstly, we established the dectection platform of silkworm resistance. Afterward, according to the infection process of BmNPV, different genes were targeted to inhibit BmNPV in four key sites of infection, respectively. And then, four antiviral strategies were optimized and integrated to further increase the resistance of transgenic silkworm. Finally, the existing practical silkworm strains were large-scale genetically improved. The main results are as follows:1. Establishment of the detection platform of silkworm resistanceBased on development of the standards of preparation of BmNPV, cutting of mulberry leaves, individual infection of BmNPV, and investigation, the dectection platform of silkworm resistance was established. The operation process as follows:the blood of silkworm with typical symptoms of BmNPV infection was collected, which was used to infect health silkworms at fifth instar via the wound infection, once there were typical infection symptoms in these silkworm, of which the blood were collected for extraction of BmNPV. The fresh mulberry leaves were cut into standard pieces and then were neatly placed, which were coated with the same amount of BmNPV solution. Once the virus solution on leaves was almost dry, the newly exuviated larvae that have been starved overnight were placed on the leaves. Those larvae which ate a whole piece of leaf were selected for the subsequent assay. We set up the experiments with three individual repeats, so as the non-infected controls. When the larvae developed typical symptoms of BmNPV infection, such as prominent somites and manic crawl, they were recorded as dead and put aside. It can avoid the contamination of other pathogens and ensure the reliability of the phenotypes of infected silkworm by standardized purification process of BmNPV. Each larva ingested the same quantity virus by individual infection, which eliminated the impact of different viral intake. The second infection can be prevented by timely separation of infected larvae. These advantages of the detection platform can ensure the authenticity and repeatability of the resistance results.2. Inhibition of BmNPV at the initial infection stage via overexpression of Bmlipase-1in transgenic silkwormBmlipase-1has strong antiviral activity and inhibits BmNPV at the initial infection stage. We analyze the transgenic lines LI, in which the endogenous antiviral gene Bmlipase-1was overexpressed under the control of BmNPV IE1promotor (IE1P). The results of Southern blot and inverse PCR showed that there is a single copy insertion in each transgenic silkworm. qPCR revealed that the mRNA level of Bmlipase-1in the midgut of LI-A was27.3%higher than that of Nm DZ at fourth instar. Western blot suggested that the content of Bmlipase-1in the gut juice of LI-A was higher than that of Nm DZ at four and fifth instars. After oral infection of BmNPV with106OB/larva using fourth instar larvae, the mortality of LI-A decreased to approximately33%compared with Nm DZ. BmNPV DNA content in LI-A was37%of that in Nm DZ. Results of larvae weights analysis showed that the normal growth and development of larvae were not affected. There were no obvious differences in whole cocoon weights and cocoon shell rates of LI-A and Nm DZ. These results revealed that it can enhance resistance without affecting economic characteristics of transgenic silkworms by overexpression of Bmlipase-1.3. Suppression of BmNPV at the mRNA level via silence of virus genes in transgenic silkworm RNAi is an effective antivirus strategy. The viral proliferation would be inhibited by silence of BmNPV genes at the level of mRNA transcription. Some factors may affect the antiviral capacity of transgenic RNAi silkworms, which were comprehensively analyzed in this study. The transgenic RNAi vector contains promoter, sense fragment of target gene, spacer, antisense fragment of the target gene, and termination signal. The connection patterns of gene fragments and spacer contain "head to head" and "tail to tail". To determine the best option, we constructed transgenic lines, in which the dsRNA of iel was controlled by IE1P and the dsRNA of gp64was driven by A4P using "head to head" or "tail to tail", respectively. There was less viral mRNA and DNA after infection in the transgenic lines using "head to head", which had higher resistance. Promoter selection is also very important for the efficiency of transgenic RNAi. We constructed transgenic silkworms, in which gp64dsRNA was driven by IE1P, IE1P combined with hr3, and A4P, respectively. In the transgenic silkworm using IE1P combined with hr3, the gp64mRNA, BmNPV DNA content, and mortality were lowest. The efficiency of RNAi is also affected by the target gene selection. BmNPV contains essential and unessential genes, which falled into immediate early, delayed early, late, and very late genes. We selected essential immediate early gene ie-1, essentially delayed early gene helicase, essential late genes gp64and vp39, and unessential immediate early gene ie-2as the targets. The results showed that the inhibitory effect was better when knocking down of essential gene, and the immediate early gene was the best target. These results suggested that transgenic silkworm with higher resistance can be constructed if using "head to head" and IE1P combined with hr3, and targeting essential immediate early gene.4. Inhibition of BmNPV at the protein synthesis stage via overexpression of hycu-ep32in transgenic silkwormThe hycu-ep32gene of HycuNPV inhibits BmNPV multiplication by inducing a global protein synthesis shutdown in co-infected cells, but it is not known whether hycu-ep32has an antiviral effect in silkworm larvae. Thus, we constructed transgenic silkworms EKG, HEKG, EAG, and HEAG, in which hycu-ep32was under the control of an inducible promoter (39KP), an inducible promoter combined with an enhancer (hr3＋39KP), a constitutive promoter (A4P), and a constitutive promoter combined with an enhancer (hr3＋A4P), respectively. RT-PCR results showed that hycu-ep32was successfully expressed in the transgenic lines, of which the expression level in HEKG-B was the highest. Mortality statistics indicated that HEKG and HEAG had significantly increased resistance; the antiviral capacity of HEKG-B was the highest. qPCR showed that the expression levels of hycu-ep32in HEKG-B were the highest regardless of whether infection of BmNPV, which suggested that the higher transcription of hycu-ep32mRNAs led to a higher anti-BmNPV capacity in the transgenic silkworms. Hr3could significantly increase the transcription activity of39KP in normal silkworm larvae; while significantly enhance the transcription capacity of39KP and A4P after BmNPV infection. BmNPV content of all the transgenic lines was significantly lower than that of the control, suggesting transgenic lines could inhibit BmNPV proliferation. As with our expectation, hr3combined with39KP was the best of all the tested promoters, and the expression level of hycu-ep32in HEKG-B was moderate in the normal state while significantly upregulated with an increase in the viral content after infection, so the transgenic silkworms had significantly enhanced anti-BmNPV capacity with no negative effects on economic characteristics.5. Activation of the host immune response to suppress BmNPV via RNAi of BmPGRP2in transgenic silkwormPeptidoglycan recognition protein (PGRP) is one of the primary pattern-recognition receptors (PRRs) in insect, which activates the humoral immune signaling pathways and is involved in prophenoloxidase cascading reaction. BmPGRP2was cloned after bioinformatical analyses. BmPGRP2had two alternative splicing forms BmPGRP2and BmPGRP2a, which was the firstly cloned PRRs with alternative splicing in silkworm.5’UTR of BmPGRP2a contains that of BmPGRP2and is longer, which revealed that the two splice forms are due to the different transcription initiation sites.Results of subcellular localization showed that the BmPGRP2proteins were distributed throughout the whole cell while the BmPGRP2a proteins were localized in the cell membrane. Expression level of BmPGRP2a was lower than that of BmPGRP2in the larvae stage but higher in the egg, pupa, and moth stages. Expression levels of BmPGRP2and BmPGRP2a in the female silkworm were higher than that of male silkworm, which in midguts were the lowest. Expression of BmPGRP2was higher than that of BmPGRP2a in all the tissue except for midgut. BmPGRP2has alternative splicing and is localized in the intracellular, suggesting it is a member of PGRP-L subfamily. The difference of subcellular localization and expression profiles between BmPGRP2and BmPGRP2a suggested that they may have different functions.The blast results showed that there was no gene similar to BmPGRP2in NCBI, suggesting it is a new gene. Result of phylogenetic analysis revealed that BmPGRP2was individually clustered into a class, which implied its function may be different from the other genes. BmPGRP2was related to the antiviral capacity in silkworm to some extent, the expression level of BmPGRP2was lower in the silkworm strains with higher resistance. To demonstrate whether improvement of the silkworm resistance by reducing the expression of BmPGRP2, we constructed the transgenic RNAi silkworm PGRP2I targeting BmPGRP2utilizing932strain. qPCR showed that the expression of BmPGRP2was decreased in PGRP2I. Mortality of PGRP2I-2was decreased30%to932, which revealed that the resistance of transgenic lines was significantly enhanced. qPCR results indicated that PGRP2I-2can significantly inhibit the BmNPV proliferation, and the virus DNA content in PGRP2I-2was almost45%of932. These results suggested that BmPGRP2is involved in the innate immune response to virus, and it can enhance the resistance while not affect the economic characteristics by RNAi of BmPGRP2in silkworm.BmPGRP2was induced effectively but the genes of Imd and Toll pathways were not after infection of BmNPV in932. Expression level of BmPGRP2was also increased after infection in PGRP2I-2, however, there was not obvious difference between the induced BmPGRP2expression of PGRP2I-2and the normal BmPGRP2expression of932, which resulted in the significantly increased expressions of Bmlmd and BmPelle in PGRP2I-2after BmNPV infection. These results suggested certain immune signal pathway was activated after BmNPV infection via RNAi of BmPGRP2in silkworm. Other genes of Imd and Toll pathways were not induced after BmNPV infection in PGRP2I-2, which suggested that the activated immune signal pathway may be a new pathway.6. Optimization and integration of different antiviral strategiesThe multiplication of BmNPV can be inhibited in silkworm by overexpression of Bmlipase-1, RNAi of virus genes, overexpression of hycu-ep32, and silence of BmPGRP2, which targeting of the initial stage of infection, virus mNRA transcription, virus protein synthesis, and regulation of host innate immune defense, respectively. In attempt to further enhance the resistance of silkworm, the four antivirus strategies were optimized and integrated. The homozygous IE1Pie1H-B, HEKG-B, and PGRP2I-2were used to generate the hybrids, the resistance results revealed that there was not obvious difference between hybrids and homozygous parents. So, an improved transgenic vector was constructed, in which the dsRNA for five tandem BmNPV genes (ie1, gp64, lef1, left, and DNA polymerase) was controlled by hr3and IE1P, while Bmlipase-1was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. The mRNA levels of Bmlipase-1in third instar larvae and the fifth instar silkworm midgut of SW-H were about40%and90%higher than SW, respectively. These results suggested that SW-H might suppress BmNPV at the initial stage of infection. After oral infection of BmNPV with5×105OB/larva at third instar, the mRNA of ie-1in SW-H was about17%and gp64was about25%compared to SW. This suggested that SW-H inhibited viruses at the multiplication stage by silencing BmNPV genes. The accumulated BmNPV DNA content in SW-H was less than20%of SW, suggesting SW-H significantly suppressed BmNPV proliferation. Resistance of SW-H1was significantly increased, of which the survival rate was increased by78%compared with SW. The economic traits of SW-H were not changed although resistance was significantly enhanced, which indicated that the resistance can be further improved by overexpression of antiviral gene and RNAi of virus gene in silkworm. SW-H is the first transgenic animal with strong antiviral properties that might suppress virus at multiple infection stages. As a further improvement, we constructed a new transgenic vector pb-KNT by optimization and integration of the four antiviral strategies, in which under the mediation of hr3, hycu-ep32was controlled by39KP, the dsRNA of five tandem genes (BmPGRP2, iel, gp64, helicase, and lef-1) was driven by IE1P, and Bmlipase-1was controlled by P2. The transgenic silkworms constructed by using pb-KNT will inhibit BmNPV at four different stages of infection, which would have very strong antiviral capacity.7. Transgenic improvement of current commercial silkworm strains and safety evaluationIn order to transform the basic research results to application profits, we have selected "CangHaiRiYue","781×7532","XiaFangQiuBai", and "LiangGuangErHao" for the first batch of transgenic improvement, which were widely used in the major sericulture areas of Chongqing, Sichuan, Guangdong, and Guangxi et al. The transgenic lines of most of these strains have been generated via embryo microinjection of pb-HFPL. Currently, many practical silkworm strains are being transgenic improved with pb-KNT for high antiviral capacity. Silkworm is a completely domesticated animal, which is only reared indoors. There is no unsafe record in the long history of sericulture. So, the safety class of transgenic silkworm and their products may be I. In order to assess the safety of transgenic silkworm, as the requirements of safety regulations of genetically modified organisms of Ministry of Agriculture, we are requesting for the intermediate detection of safety test. We will strictly comply with the laws, and perform rigorous detection and evaluation of these transgenic silkworms. We would do our best to achieve the security certificate and then apply these antiviral strains in sericulture as early as possible. |
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