| As a selfish DNA elements that can move independently within the genome, transposon is one of the most abundant genetic types in nature. The presence and activity of transposons makes the genome a dynamic body. The transposon plays an important role in evolution, individual differences, and biodiversity. However, transposon’s hyperactivity can also cause disorder in the genome, resulting in excessive mutations and many kinds of diseases. Therefore, the organism has evolved a new mechanism called piRNA to balance the dynamic changes of genome. Studying the dynamic balance mechanism between transposons and piRNA has a vital significance for revealing molecular basis of the evolution and genetic variation. The sex determination of silkworm is ZW type, with a strong female determines-W chromosome that can be transcribed to produce large numbers of piRNA. Silkworm is an ideal research model for studying the interaction between transposons and piRNA. In this study, we investigate the exogenous piggyBac transposition activity in W and Z chromosome, combined with the methods of multilayer transcriptome, to reveal the relationship between transposon activity and piRNA expression, and the main results and conclusions obtained are as follows:(1) The establishment of a single copy transgenic strains in Z chromosomesPreviously, we have constructed two genetically modified strains that the insertion site is located in W chromosome, respectively named TG-W1 and TG-W2 to determine whether the stability of piggyback on chromosome W will be affected by the piRNA. We screen more than 100 transgenic strains to get a strain whose insertion locus is located on Z chromosome, which we named TG-Z. Wild silkworm (♂) are crossed to TG-Z transgenic silkworm (♀), and green fluorescence can be observed only in the compound eye and nerve of the male silkworm of F1 generations. After five generation of TG-Z strain with wild type hybrid, the results of offspring is that males have a green fluorescent expression, females have no fluorescence expression. In order to confirm the insertion site in the transgenic strains, we carry out reverse PCR on the silkworm individuals, according to the results the insertion site is located on 11.5Mb chromosome 1, genetic linkage position is 23.7 cM.(2) piggyBac stability on Z and W chromosomeIn order to detect the piggyBac re-translocation activity in transgenic silkworm, we develop a methods using the sex chromosome linked and stable expressed of exogenous transposon. Because W chromosome exists only in the female silkworm, so genetically modified tags of W-linked transgenes can only be detected in female. The female silkworm has only one Z chromosome, therefore transgenic tag can be detected in only males from the offspring of Z chain female silkworm with wild male silkworm individuals. We cross TG-W1, TG-W2 and TG-Z female silkworm to TG-R, in which piggyBac transposase is stably expressed in whole body, respectivly, the offspring are named T-W1/W2/Z-R. To make the F1 individuals which contain green and red fluorescence with wild silkworm hybrid, according to the number of fluorescence expression of offspring, calculation of piggyBac re-transposition rate. Results show that piggyBac re-transposition efficiency in the TG-W1 strains is 2.77%; In the TG-W2 is 5.68%; In the TG-Z is 16.73%. indicating that re-transposition efficiency of piggyBac on the W chromosome is significantly lower than the Z chromosome.(3) Different analysis of differentially expressed genes in transgenic strainsTo explore the molecular mechanism of piggyBac re-translocation rate in three transgenic strains,we extracted RNA from the ovaries and brain tissue of different individuals, and carry on the analysis of the transcriptome sequencing. The concentration of samples are all more than 200 ng/μl,the extracted RNA detected by agilent and the integrity of RNA (RNA Integrity Number) are all above 6.0. The transcriptome data are analyzed in three aspects, differentially expressed genes (DEGs), Gene Ontology function significant enrichment analysis, Pathway significant enrichment analysis, differential gene expressions between different genetically modified individuals have great difference. GO annotation is divided into three categories:molecular function, cellular component, biological process. In different functional level have different genes annotation. Pathway main enrich gene to the cellular processes, environmental information processing, genetic information processing pathway, metabolism, and biological systems. Although many differentially expressed genes are identified, the indepth analysis of these genes have not found a direct relationship with the transposon activity, we hypothesized that these differences may be caused by genetically modified insertion sites.(4) Identification and analysis of the small RNA between different transgenic strainsFrom the transcriptome data analysis results, we speculate that the difference of piggyBac again translocation efficiency in different genetically modified individuals is not related with specific gene expression. Along with more and more clues indicate that the again translocation activity of transposon may have larger relationship with the expression of piRNA, therefore, we sequence and analyze small RNA in different genetically modified strains. For miRNA we also do the same analysis with the transcriptome in three aspects, find no significant differences, suggested miRNA has no direct relevance with transposon translocation activity. Through analysis method of piRNA introduced in literature, found that in the brain the differential transposon expression significantly lower than the amount of the ovary, about 76% piRNA expression in ovarian, and 24% piRNA expression in the brain. To align piRNA with upstream and downstream insertional locus sequence of genetically modified carrier, the results show that TG-W1/W2/Z piRNA quantity in ovary is greater than the quantity in brain. Also found that upstream and downstream sequence of the ovaries transgenic insertional locus piRNA enrichment with high quantity, near the insertion site the number of TG-Z piRNA s significantly lower than the number of TG-W1/W2 piRNA. The results indicated that piggyBac re-translocation efficiency on W and Z chromosome difference may be due to the difference piRNA content near insertion site.In conclusion, this thesis for the first time reveals the relationship between piRNA with piggyBac re-translocation efficiency which is widely used in animal and plant transgenic transposon by genetic analysis, analysis of the transcriptome and small RNA sequencing.This research result is not only provided important reference for transposon basic study and piRNA action mechanism, but also established important foundation for the study of the stability and safety of genetically modified. |
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