Studies On The Fitness Of Cry1C Tolerant Strain Of Chilo Suppressalis And The Binding Affinity Of Midgut BBMV With Bt Toxin

Chilo suppressalis(Walker)is the main stem pests on rice,causing great damage to rice production in our country.The development of Bt transgenic rice has brought new strategies for the control of rice lepidopteran pests but also brought the potential resistance risk of target pests.The resistance of C.suppressalis is one of the key points of ecological safety evaluation of transgenic rice,and it is also a key factor to restrict the sustainable application of transgenic rice.At present,the resistance development and resistance mechanism of pests to Bt insecticidal protein is not clear.We obtained Cry 1C tolerant strain of C.suppressalis by laboratory selection.To investigate the fitness and the biochemical mechanism of Cry 1C tolerant strain is great significance to the risk assessment of the transgenic rice target pests,but also provide a scientific basis for the commercialization of transgenic rice and the resistance control strategy of transgenic rice target pests.At present,the laboratory has established a mature C.suppressalis laboratory rear technology.Cry 1C tolerant strain is obtained after continuous selection in the laboratory.we systematically studied the fitness of Cry 1C tolerant and susceptible strains on artificial diet without Bt insecticidal protein;And two strains were fed by using crylC+crylAb,cry1Ab,cry 1C three transgenic rice and parental MH63 rice in vitro leaves to compare the fitness;The binding ability of midgut BBMV and Cry 1C protein in Cry 1C susceptible and tolerant strains were compared by enzyme-linked immunosorbent assay(ELISA),explored the relationship between the resistance with the binding ability.The main research results are as follows:1.The Cry 1C tolerant and susceptible strains with the same genetic background were selected as the research object.Result shows,after successive 41 generations of resistance screening,the resistance ratio of Cry1C tolerant strain was 42.59 times.Studies the difference between the two strains in the artificial feed without Bt insecticidal protein,the result showes the larvae and pupal period of Cry1C tolerant strain are significantly prolong.At the same time,the 8-day-old and 12-day-old larvae body weight of Cry1C tolerant strain are significantly lower than susceptible strain.The adult duration of tolerant strain is significantly lower than susceptible strain.However,there are no significant differences in the pupation rate,emergence rate,single female fecundity,hatching rate,egg duration and 3 days age of pupal weight.2.Compare the Cry 1C tolerant and susceptible strains fitness in different growth stages of transgenic rice in vitro leaves.Result shows that the susceptible strain are highly sensitive to three transgenic rice.The mortality of Cry1C tolerant strain in cry1C rice is significantly higher than susceptible strain,and some larvae into the second age.The mortality of Cry 1C tolerant and susceptible strains in the cry1C+cry1Ab,crylAb transgenic rice five growth period are above 99%.It can be seen that the Cry 1C tolerant strain has obviously adapted to cry1C rice and has higher tolerant to Cry1C insecticidal protein.Also,Cry1C tolerant strain is highly sensitive to crylAb and cry1C+cry1Ab rice.It's demonstrated that Cry 1C tolerant strain did not have cross-resistance to CrylAb.The production of Bt protein in leaves of three transgenic rice lines 5 growth period was detected by ELISA.It was found that the mortality of Cry 1C tolerant strain on Bt rice was positive correlate with the production of Cry 1C insecticidal protein,indicating that with the Cry 1C protein production level increase,the insecticidal effect is gradually increase.3.The binding ability of Cry 1C tolerant and susceptible strains was studied by enzyme-linked immunosorbent assay(ELISA).Result shows the dissociation constant(Kd)and binding site concentration(Bmax)were 17.20±2.68 and 1.66±0.08 nM,respectively,for the Cry1C susceptible strain and 20.36±4.98 and 1.54±0.13nM,respectively,for the Cry 1C tolerant strain.There was no significant difference in the Kd and Bmax estimates were detected between the Cry 1C tolerant and susceptible strains.These findings indicate that reduced binding of Cry 1C to the midgut target sites is not a primary mechanism of resistance to Cry 1C in C.suppressalis at this resistance level.