The Roles Of Receptors In The Mechanism Of Cross-resistance Between Cry1Ac And Cry2Ab In Cotton Bollworm

The 1996 launch of transgenic crops that produce crystal insecticidal toxin proteins(Cry toxins) from the bacterium Bacillus thuringiensis(Bt) marked the adoption of a new pest management strategy that largely relies on in planta production of pest-killing traits to protect crops from pest damages. These economic and environmental benefits have led to rapidly increase in the acreage of Bt transgenic crops in last 20 years. To delay pest adaptation, farmers in many countries now grow Bt crop “pyramids” that produce two or more toxins that kill the same pest, rather than first generation Bt crops that each produce a single toxin. The rationale for such pyramids is that insects resistant to one toxin will be killed by the other toxin in the pyramid. For example, transgenic cotton producing Bt toxins Cry1 Ac and Cry2Ab(Bollgard II) is the only type of Bt cotton grown in Australia, and the predominant type of Bt cotton grown in India and the United States. However, only first generation Bt cotton producing one toxin(Cry1Ac) is grown in China, the world’s leading producer of cotton. After nearly two decades of exposure to transgenic cotton plants producing Cry1 Ac, field populations of H. armigera have shown significant increases in resistance to Cry1 Ac in China. Although two-toxin Bt cotton is generally expected to be more durable than Bt cotton producing only Cry1 Ac, the cross-resistance between Cry1 Ac and Cry2 Ab is one of the key factors, which can restrict the application of Bollgard II cotton in China. Our previous study indicated there is some cross-resistance between Cry1 Ac and Cry2 Ab in cotton bollworm. Meanwhile, the mode of action and the mechanism of resistance of Cry1 A toxin are complex; and little known of that of Cry2 Ab toxin. In order to solve these problems and better to use Bollgard II, it is urgent to study mechanism of cross-resistance between Cry1 Ac and Cry2 Ab in cotton bollworm.In this study, firstly, we analyzed the candidate resistant gene by genomics and proteomics; secondly, we build a method to study the functional of the receptor in three cell lines; later, we compared the receptors between Cry1 Ac and Cry2 Ab and study the mode of action of Cry1 Ac. The results are as following:1: We compared the different genes between LF susceptible and LF resistant strains(LF5, LF10, LF20, LF30, LF60 and LF120) by RNA-seq and DGE and the different proteins expression levels among LF,LF5,LF10,96 S,96+2Ab,96-2Ab,96+1Ac and 96-1Ac by Itraq. The results showed cadherin, APN1, ALP2, ABCC2, V-ATPase a, V-ATPase c and ATP synthase have different proteins levels in both Cry1 Ac resistant strains and Cry2 Ab resistant strains. 2: In order to quickly evaluate whether a gene evolve in the mode of action of Cry1 Ac or Cry2 Ab or not, and whether a gene lead to cross-resistance between Cry1 Ac and Cry2 Ab or not, we first developed a cell bioassay method for testing the cytotoxicity of the two Bt toxins. The results showed the cell culture medium is the suitable solvent for measuring the cytotoxicities of the two toxins and 4.5 and 5.5 h are the best observation time points for measuring cytotoxicitis of Cry1 Ac and Cry2 Ab, respectively. The cytotoxicity of the two Bt toxins indicated that the major receptors of the two Bt toxins are different.3: The heterologously expressing H. zea receptors the double strand RNA(dsRNA) and siRNA, knocking down the endogenous receptors’ gene showed Cadherin、APN1、ALP2 and V-ATPa are the functional receptors of Cry1 Ac, but not for Cry2 Ab.4: Our data demonstrated that HzABCC2 may be a receptor for Cry1 Ac and Cry2 Ab. Meanwhile, the same receptor- HzABCC2 may play a role in the mechanism of cross-resistance between Cry1 Ac and Cry2 Ab.5: We studied the interactions of Cadherin、APN1and ALP2 in the mode of action of Cry1 Ac, the results showed there were no significant dependence and work order, and we speculated there were at least four pathways n the mode of action of Cry1 Ac.