The Construction Of ABCC2 And Cadherin Mutant Strains Of Helicoverpa Armigera And The Research Of Their Bt Resistance

Bt resistance of target pests will restrict the long-term effective plant of Bt crops.The research of Bt resistance mechanism will delay the evolution of resistance.Previous researches mainly focused on the study of a single resistance gene mutation,but there are many factors influencing the resistance evolution in the field,and two or more genes mutation may happen at the same time in one pest.In this study,we studied the Bt resistance of ABCC2 and Cadherin mutant strains by methods of vitro cell experiment,construction of resistance strains,bioassay and life table.The main results are as follows:1.HaABCC2 was located on the cell membrane,and the insect cells transfected with ABCC2 showed cell lysis at low concentration of Cry1Ac,the EC₅₀?Cry1Ac concentration of 50%transfected cells lysis?was 0.109?g/mL.The resistance ratio to Cry1Ac of ABCC2 mutant near-isogenic strain?LFC2?was 512times.The mortality rate of LFC2 on Bt cotton 33B of 274.06±27.05 ng/g Cry1Ac was 27.33±5.00%,significantly lower than sensitive strain?46.00±5.29%?,and there was no significant difference with its mortality rate on non-Bt cotton?19.33±4.00%?.Compared with the sensitive strain,the developmental duration of 2^nd-5^thh larvae,female pupae and pre-male,pre-female of LFC2 was significantly prolonged,the survival rate of pre-adult was significantly reduced,the intrinsic growth rate,the finite rate of increase and the net reproductive rate were significantly reduced,and the mean of generation time was significantly extended.However,the developmental duration of 1^stt larvae and the male pupa,the longevity of the male and female adults,the egg laying period of the adult and the oviposition rate per female had no significant difference.2.HaCadherin was located on the cell membrane,and the EC₅₀0 to Cry1Ac of cells was 9.212?g/mL.The resistance ratio to Cry1Ac of Cadherin mutant strain?96CAD?was 396 times.The mortality rate of96CAD on 33B was 27.44±1.28%,significantly lower than the sensitive strain?46.00±5.29%?,and significantly higher than its mortality rate on non-Bt cotton?15.08±2.96%?.Compared with sensitive strain,the developmental duration of 2^nd,4^th,5^thh larvae,female pupa and pre-male,pre-female were significantly longer,egg laying period was significant shortened,oviposition rate per female was significantly reduced,the survival rate of pre-adult was decreased significantly,the intrinsic growth rate,the finite rate of increase and net reproductive rate were decreased significantly,the mean of generation time was significantly prolonged;However,the developmental duration of 1^stt and 3^rdd larvae,male pupae and the longevity of male and female adults had no significantly difference.3.The EC₅₀0 to Cry1Ac of HaABCC2 and HaCadherin co-transfected cells was 0.017?g/mL,which was significantly lower than the cells transferred HaABCC2 and HaCadherin separately.The resistance ratio of ABCC2 and Cadherin co-mutant strain?R1?was 6273 times.The mortality rate of the R1 on 33B was 18.16±1.51%,significantly lower than the sensitive strain?46.00±5.29%?,and had no significantly difference with its mortality rate on conventional cotton?14.33±2.33%?.Compared with the sensitive strain,the developmental duration of the 2^nd,4^thh and 5^thh larvae,female pupae,male and female adults of the R1 were significantly prolonged,the survival rate of the pre-adult was significantly reduced,the intrinsic growth rate and the finite rate of increase were significantly reduced,and the mean of generation time was significantly prolonged.There was no significant difference in the developmental duration of 1^stt and 3^rdd larvae,male pupae,the longevity of the male and female adults,the egg laying period and the net reproductive rate of the adults.However,the oviposition rate per female was increased significantly.4.From 2016 to 2018,the Cry1Ac resistance level of 22 field populations were measured.The IC₅₀were between 0.004?g/mL?Yinchuan population,2017?and 0.403?g/mL?Changdao population,2017?.Survival rates on diagnostic concentration 1.0?g/mL were ranged from 0 to 16.67%?Handan population 1and Jining population,2015?,and the percentage of the population with survival on diagnostic concentration in the total number of monitored populations in that year increased from 60%in 2016 to100%in 2018.DNA molecular detection technology was used to detect 254 samples,but found no individual with mutated ABCC2 and Cadherin gene.The study showed that the HaABCC2 and HaCadherin had synergistic effect in the action of Cry1Ac.ABCC2 and Cadherin mutant genes had“1+1>2”effect in Bt resistance.The ABCC2 and Cadherin mutant strains generally showed resistance fitness cost and negative cross-resistance to other insecticidal substances.The resistance level of cotton bollworm field populations was significantly increased,ABCC2and Cadherin gene mutation frequency were still at low level.