Identification And Analysis Of Resistance Alleles Of Pink Bollworm Cadherin In The Yangtze River Valley

Pink bollworm?Pectinophora gossypiella?,one of the world's most destructive pests of cotton,had serious damage to cotton yield for many years in the Yangtze River Valley in China.The population of pink bollworm in this region has been effectively controlled since the planting of transgenic cotton expressing Bt toxins.However,with the increase of planting proportion of Bt cotton in this region,the susceptibility of pink bollworm field populations to Cry1Ac have been decreased.In order to search an effective approach to delaying the resistance of pink bollworm to Bt cotton,in this paper,several resistant strains of pink bollworm have been screened and purified from filed populations in the Yangtze River Valley by single-paired between individuals from field populations and individuals from a laboratory-resistant strain,and the resistance mechanism and the genetic characteristics of these strains to Cry1Ac have been illustrated using molecular biology techniques,and the molecular detection method for these resistance alleles have also been established.The main results are as follows:1)Four resistant strains of pink bollworm have been screened and purified,named AQ65,AQ47,AQ189 and JL88,respectively.The resistance rations of the four resistant strains to Cry1Ac were 303,220,254 and 289,and the inheritance of Cry1Ac resistance was autosomal and a partial recessive trait.AQ65,AQ47 and AQ189 strains exhibited low levels of cross-resistance to Cry2Ab toxin?with 2.60,2.09 and 1.82 of resistant ratio,respectively?,and JL88 strain exhibited no cross-resistance to this toxin.2)Pink bollworm in the four strains were resistant to Cry1Ac because of mutations in a gene encoding cadherin protein?PgCad?,and tight genetic linkage of PgCad allele with resistance to Cry1Ac.Compared with PgCad in susceptible strain,DNA sequence of PgCad189 and PgCad88 had a insertion of 234 and 3370 bases,respectively,and PgCad47 had a deletion of 314 bases,and PgCad65 not only had a deletion of 549 bp but also had a insertion of 198 bp.3)According to the difference of genome sequence of PgCad between susceptible strain and the four resistant strains,eight pairs of primers were designed for detecting the four resistance alleles and their genotypes.The frequency of resistance alleles PgCad47,PgCad189 and PgCad88 was detected in the field from 2012 to 2015.It was 0.0003,0.0002 and 0.0003,respectively,and there was no significant difference among the four years for each allele.4)Pink bollworm from all strains could complete their life cycles on conventional cotton.Pink bollworm from the four resistant strains could also complete their life cycles on Bt cotton expressing Cry1Ac toxin,rather than susceptible strain.The larval development time for resistant strains was significantly longer on Bt cotton bolls than resistant and susceptible strain on non-Bt cotton bolls.The pupal weigh for resistant strains were also significantly smaller on Bt cotton bolls than both resistant and susceptible strain on non-Bt cotton bolls.5)Expression vectors to get fusion protein of PgCad and GFP were constructed,and these vectors were expressed and localized in an insect cell line TnH5.The concentration for 50%of maximal effect(EC₅₀)of the transfected cells expressed PgCad-GFP was 6.79?g/ml,whereas EC₅₀ of the transfected cells expressed PgCad65/47/189/88-GFP was more than 40?g/ml.PgCad-GFP was mainly located on the cell membrane,whereas PgCad65-GFP and PgCad47-GFP were retained in the endoplasmic reticulum,and PgCad189-GFP located on the membrane of vesicles,only subcellular localization of PgCad88-GFP was correct.6)Pink bollworm adults feeding Cry1Ac showed that 10,40 and 160?g Cry1Ac per ml reduced the duration of the oviposition period as well as the number of eggs laid by both strains,but did not affect the percentage of pairs producing eggs,the duration of the pre-oviposition period,or the percentage of eggs hatching for both resistant and susceptible strain.The highest concentrations of Cry1Ac tested?160 and 640?g Cry1Ac per ml of 5%honey water?reduced lifespan for both strains,and it was significantly greater for the resistant strain than the susceptible strain.The results above showed that the pink bollworm in the Yangtze River Valley carried Bt resistance genes,and the resistance genes were mainly cadherin alleles,which were autosomal recessive inheritance.However,its resistance gene frequency was relatively low,and there was low or even no cross-resistance to Cry2Ab toxin.These results provide technological support for making a reasonable strategy for delaying the development of resistance of pink bollworm to Bt cotton in the Yangtze River Valley.