Proliferation And Differentiation Of Midgut Stem Cell And Its Relationship With Chilo Suppressalis Tolerance To Cry1Ab

Chilo suppressalis Walker is an important rice pest which widely distributed in the main rice-growing areas of China. An alternatively attractive strategy for control of C. suppressalis is to produce insecticidal proteins by introducing the corresponding insecticidal genes into the rice. Stem cell is one kind of self-renewal and multipotential differentiation cells and its proliferation and differentiation is closely related to resistance of pest. In this paper, we study the influence of different factors including larval instar, medium additives on the proliferation and differentiation of midgut stem cell from C. suppressalis and its relationship with Cry1Ab-tolerance. The main results are as the following:1. Stem cell isolation and collagen I enzyme digestion methods were used to extract stem cells from the midgut of C. suppressalis, respectively. The results showed that extracting cells using 0.1% collagenase I digestion method could acquire purer stem cells than the other methods. We also conducted a flow cytometry method to differentiate stem cells from mature cells and monitor proliferation and differentiation of primary midgut stem cell cultures from C. suppressalis larvae, based on differential light scattering and vital stain fluorescence properties between stem and mature cells.2. In this work, we demonstrated that 3rd instar C. suppressalis have much more stem cells compared to 4th and 5th instar larvae, so the 3rd instar was used for subsequent researches. We also confirmed that the prepupa C. suppressalis fat body extract and ecdysone could regulate the proliferation and differentiation of midgut stem cells from C. suppressalis3. Different concentrations of Cry1Ab had different influence on proliferation and differentiation of stem cells from the susceptible and tolerant C. suppressalis to Cry1Ab. strain are more strongly influenced by. The most effective sublethal concentrations of Cry1 Ab were 0.15 and 0.3 μg/mL to stimulate proliferation and differentiation of midgut stem cell from susceptible and Cry1Ab tolerant C. suppressalis, respectively.4. To confirm the influence of CrylAb on the midgut cells, we cut the midgut of C. suppressalis fed on LC10 (0.3 μg/mL) dose of Cry1Ab solution into slices to observe the features of apoptosis by transmission electron microscope (TEM). Obvious features were showed in induced apoptosisshowed, including the reduced numbers of microvilli and mitochondria, and the present of autophagy, heterochromatin and vortex membrane structure.5. The apoptosis in midgut cells exposed to different concentrations of Cry1Ab toxin was detected at different times. The number of apoptotic cells was increasing over time without changing medium. The most serious apoptosis was observed when exposed to 0.06 μg/mL Cry1Ab. The results showed that the Cry1Ab hadobvious effect on the midgut stem cell in vitro of C. suppressalis.