Progress in Research on Silencing Mechanism of Genetically Modified Tobacco

The transgenic technology plays an important role in modern agricultural breeding. The phenomenon of transgene silencing is not conducive to the application of this technology, so this phenomenon is an important issue that transgenic workers need to consider. In the tobacco materials obtained by the researchers of the South China Botanical Garden of the Chinese Academy of Sciences in the early stage, 1/3 of the transgenic tobacco materials had transgene-silencing phenomenon and could not be used normally. If the target gene can be re-expressed in these materials, it will increase the efficiency of this technology.   Previous studies have shown that many factors are related to gene silencings, such as the insertion position of the transgene in the genome, the copy number of the transgene, the repeat sequence, the homozygous heterozygous state of the transgene, and DNA methylation. Among the materials previously obtained by the research group, there is a tobacco strain that successfully performed two rounds of gene-targeted superposition but the transgene was silent (23.C.7). This strain has two copies of the transgene, one for site-directed integration (S) and the other for random insertion (R). Whether the S copy is homozygous or heterozygous, the target transgene is silent. DNA methylation analysis showed that the target transgene promoter was hypermethylated. Is it possible to activate the expression of silent transgenes by removing one of the copies?     To answer this question, researchers conducted backcrossing of the transgenic material with wild-type tobacco and analyzed the gene expression of the offspring. The study found that gene silencing still occurs in the progeny of some plants backcrossing, while in the progeny of other plants backcrossing, the transgenes in the site-integrated copy have different levels of expression activation. Surprisingly, this activation phenomenon occurs not only in plants containing single copies (S) but also in plants containing double copies (RS).   Contrary to expectations, the activation of silent transgenes is not closely related to changes in copy number. The DNA methylation data showed that there was a significant difference in the degree of transgene methylation between the control lines with good gene expression and the lines with gene silencing 23.C.7. Researchers try to find the answer from the perspective of DNA methylation. The results showed that there was no significant difference in the level and pattern of DNA hypermethylation between plants with different levels of gene activation. Although DNA methylation is involved in gene silencing, DNA hypermethylation alone cannot "lock" the silent state.   In summary, this study shows that the gene silencing status of transgenic plants can be activated to a certain extent through backcrossing, but the appearance of this phenomenon does not depend on changes in transgene copy number and DNA hypermethylation, and the exact activation mechanism needs further exploration.