| Silkworm (Bombyx mori) is the representative of the Lepidopteran insects. As the second largest model insect, it can either bring research value and economic value. The development of transgenic technology constituted a crucial milestone in the history of life science, and is frequently used to alter biological traits and produce valuable foreign proteins in various organisms. The piggyBac transposon is one of the most useful transposable elements, and has been successfully used for genetic manipulation in more than a dozen species. The piggyBac transposon-mediated transgenic technology has made a great contribution to silkworm transgenesis. The silkglands of the silkworm is considered as an idea bioreactor for producing exogenous proteins because it has been proved with an amazing protein synthesis and secretion ability. After decades of effort, researchers have established middle silkgland and posterior silkgland bioreactors in silkworm, and more than dozens of exogenous proteins have been successfully expressed in these bioreactors. However, the expression efficiency is still insufficient, mainly reflects in low and unstable transgenic efficiency of piggyBac-mediated transgenic technology, the low expression efficiency of exogenous proteins, and the complexity of screening methods for transgene-positive silkworms. Therefore, improving transgenic efficiency and exogenous genes expression level, and establishing a high-efficiency screening method for transgene-positive individuals are particularly important. In view of these scientific problems, a series of studies have been performed, and some main achievements are concluded as follows:1. Transcription activator-like effector mediates high-efficiency transposition of piggyBac transposonTo improve the efficiency of piggy Bac-mediated transposition, we present a new strategy using a fusion of transcription activator-like effector (TALE) and the piggyBac transposase (PBase), TALE-PBase. The results demonstrate that the TALE-PBase fusion protein can produce a significantly improved stable transposition efficiency of up to 63.9%, which is at least 7 times higher than the current transposition efficiency in silkworm. Moreover, the average number of transgene-positive individuals increased up to 5.7-fold, with each positive brood containing an average of 18 transgenic silkworms. The breakthrough of transgenic efficiency in these two aspects will provide a good technical support for transgenic researches in the future.2. Analysis of structure and activity of sericinl promoters in silkwormThe promoter is one of the most important regulatory elements controlling the spatiotemporal expression of a target gene. However, most studies have focused on core or proximal promoter regions, and information on regions that are more distant from the 5’-flanking region of the proximal promoter is often lacking. Here, approximately 4 kb length of the sericinl (Serl) promoter was analyzed to contain many potential transcriptional factor binding sites (TFBSs) by bioinformatics methods. Transgenic experiments further revealed that improving the number and variety of TFBSs included in the promoter could enhance gene transcription. However, multi-copy proximal Serl promoter combinations did not improve gene expression at the transcriptional level. Instead, increasing the promoter copy number repressed transcription. In a word,4 kb length Serl promoter is more active than others, and multi-copy proximal Serl promoter combinations are not effective ways to improve the transcriptional level.3. Site-specific transposition of piggyBac transposon in the silkworm genomeThe random transposition of transposon is adverse to exogenous genes expression, and sometimes will induce endogenous genes mutation. In theory, TALE-PBase fusion protein may achieve site-directed transposition. Our results suggested that TALE-PBase-mediated transposition exhibited a selective integration into certain positions in the genome instead of original completely random insertion of piggyBac transposon, but no site-directed transposition events were found Two pairs of integration sites were found at the same position from only 71 insertion events, a phenomenon that is rare in the random transposition of native PBase. Therefore, TALE presents a guiding function in some degree during transposition, it provides a valuable reference for achieving site-directed transposition at the individual level in future.4. TALEN-mediated homologous recombination in silkwormSilkworm zygotes are the early-stage individuals, compared with cells, they are more suitable to reflect the real situations of lifes. So, using silkworm zygotes as the receptor for homologous recombination (HR) studies can real reflect the probability of HR occurrence in the individual level.The precise genome editing capability of HR can remedy the shortages of other genome targeting tools, as well as the random insertion of piggyBac transposon. The extremely low-efficiency of the HR is the main limiting factor hinders its applications. Here, a TALEN-mediated HR study was performed in the zygotes of silkworm. The results suggested that TALEN could efficiently break the target genome location after 72 h of microinjection, which provides a favorable guarantee for inducing HR genesis. From 15 detected groups of zygotes,12 groups were observed significant gene targeting, targeting efficiency reached 80%. At the same time, we also detected positive HR individuals in the zygotes,20%(3/15) groups were determinated containing HR positive individuals. Such early-stage detection of targeting efficiency and HR efficiency can evaluate the feasibility of the experiment in the short term. This evaluation method helps us saving a lot of time and ensuring the success of the experiments.5. The assisted detection of exogenous genes expression efficiencyFrom our results we found that if the two expression cassettes are constructed close to one another with the same transcriptional orientation, a significant linear correlation will be observed between their transcriptional levels, even if they are in different insertion sites. A correlation analysis between two contiguous genes, firefly luciferase (FLuc) and EGFP, was conducted at the transcriptional level; a significant correlation was obtained regardless of the insertion site. The ELISA results also revealed a significant correlation between the transcriptional and translational EGFP levels. Therefore, the exogenous gene expression level can be predicted by simply detecting an adjacent EGFP.In conclusion, the study is carried out from three aspects, improving transgenic efficiency, enhancing exogenous genes expression efficiency and establishing an assisted detection method for exogenous genes expression efficiency, to optimizethe silkglands bioreactor of silkworm. These results lay a solid foundation for establishing high-efficiency silkglands bioreactors. |
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