Bt Research 2025, Vol.16, No.5, 224-233 http://microbescipublisher.com/index.php/bt 230 plasmid loss. By sequencing and aligning different generations of strains, it is possible to accurately determine whether or not the deletion of fragments of the plasmid still exists or occurs (Ye et al., 2012). 6.2 Protein level detection (Western blot, ELISA, etc.) The ultimate influence of plasmid loss is the yield and composition of toxin proteins, so the detection of protein levels can indirectly analyze whether plasmid is lost and its effects. Commonly used methods include SDS-PAGE analysis of crystal proteins, Western blot and ELISA quantification, etc. First, the Bt strain was cultured in spore production medium, the spore crystal mixture was collected, and the crystal protein was purified by NaBr density centrifugation, and the protein band was analyzed by SDS-PAGE electrophoresis. Intact plasmid strains usually present multiple major Cry toxin bands (such as 130 kDa, 65 kDa, etc.) and secondary toxin bands, while plasmid-loss strains may lose some bands or attenuated band strength (Benfarhat-Touzri et al., 2019). Western blot uses specific antibodies to detect the presence and abundance of toxin proteins. After the strain crystal protein was isolated by SDS-PAGE, Western hybridization was performed with antibody probes against Cry1, Cry2, Vip3, etc. If the Western signal of a strain disappears in Western signals, it indicates a corresponding toxin loss (possibly due to plasmid loss); if the signal is significantly weakened, it means that the toxin expression decreases, which may be related to plasmid instability (Daffrose et al., 2024). Western blot is sensitive and specific and is widely used in plasmid loss analysis. 6.3 Bioassay and virulence experiment methods The final evaluation of the effect of plasmid loss is the biological determination result of the Bt strain on the target pest. Through systematic bioassay experiments, the virulence differences between strains before and after plasmid loss can be directly compared. Commonly used biometric indicators include half of lethal concentration (LC50), corrected mortality rate, and lethality (LT50). The specific method is: select typical target insects, feed the larvae with the same dose of the original Bt strain and the spore suspension of the plasmid-loss mutant strain, record the insect mortality rate at each dose, and determine the LC50 by using probability unit analysis. If the LC50 of the plasmid lost strain is significantly higher than that of the original strain, it indicates a significant decrease in virility (Garbutt et al., 2025). Garbutt et al. studied that by comparing LC50 of plasmid cured strains with wild strains, they found that the former increased LC50 of phytonia worms hundreds of times, confirming that plasmid loss weakens virility. In addition to LC50, LT50 can be calculated by treating insects with a certain dose and recording the cumulative death curve. Plasmid loss usually leads to LT50 prolongation, which means that insecticidal speed is slower. Corrected mortality (Abbot formula) can also be used for intuitive comparison of virulence in different strains: the complete plasmid strain often reaches more than 90% of the corrected mortality 48 hours after treatment, while the plasmid deletion strain may only be 30%~40%. 7 Strategies to Deal With Plasmid Loss 7.1 Genetic improvement method to improve plasmid stability Given that plasmid loss can weaken the virility of Bt strains, researchers have developed a variety of genetic improvement methods to improve the stability of the plasmid. The first is to strengthen the plasmid's own maintenance system through genetic engineering. Overexpression or optimization of plasmid key allocation genes (such as tubZ, parA) can enhance the equalization efficiency of plasmids during cell division (Larsen et al., 2007). A Bt plasmid containing a double copy allocation sequence was constructed, and the plasmid loss rate was significantly reduced, indicating that enhanced allocation function contributed to plasmid stability. Secondly, manual screening marks are introduced to maintain selection pressure. The classic method is to carry antibiotic resistance genes or necessary nutrient genes on the plasmid, and add corresponding antibiotics or defective medium during culture to ensure that plasmid-free cell growth is restricted, thereby forcing plasmid retention. Modification or addition of plasmid toxin-antitoxin systems is also a strategy to improve stability (Liu et al., 2008). Some studies have caused the plasmid-lost cells to die quickly by inserting exogenously potent TA modules into the Bt plasmid, thus leaving only plasmid cells alive. This method has been shown to significantly reduce the plasmid loss rate in E. coli, etc., and is also suitable for Bt strains.
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