Functional Analysis Of ALP Gene Family As The Receptor Of Cry Proteins In Chilo Suppressalis

The rice stem borer, Chilo suppressalis(Walker)(Lepidoptera: Pyralidae) is one of the major rice pests worldwide. Transgenic Bt rice offers a new effective pest control tactic, which can reduce the use of chemical pesticides,the study of the potential target pest resistance risk and mechanism is necessary. In this study, full lengths of ALPs cDNA sequences were amplified and molecular characterization and function of ALPs were analyzed further. The main results are as follows:1. We use the transcriptome database and genomic database of Chilo suppressalis to ensure the integrity of the clone of ALP gene family. cDNAs of C. suppressalis ALP isoforms were cloned and sequenced, including 3 ALPs which have been reported in GenBank and 3 new ALPs named as CsALP4, Cs ALP5 and CsALP6. The molecular weights of 3 new proteins were 56.79, 55.68 and 59.77 kD respectively, all the 3 new ALPs have N-, O- glycosylation sites and GPI anchor sites.2. The binding of alkaline phosphatase with Cry1 Ac, Cry2 Aa and Cry1 Ca, respectively: Expressed target proteins in E. coli and purified by Ni2+ column,dot blot analysis the binding with Cry1 Ac, Cry2 Aa and Cry1 Ca,the results showed that Cry1 Ac, Cry2 Aa and Cry1 Ca toxins have binding capacity with Cs ALP1, CsALP2, CsALP3, CsALP4, CsALP5 and CsALP6.3. CsALP1, CsALP2, CsALP3, CsALP4 and CsALP5 transcription was significantly reduced in C. suppressalis larvae continuously fed on diet overlaid with ds ALPs for 24 h compared with those fed on dsGFP or water. Our results indicate that the CsALP1, CsALP2, CsALP4 function as receptor for Cry1 Ac, Cry2 Aa and Cry1Ca; CsALP3 function as receptor for Cry1 Ac and Cry1Ca; CsALP5 and CsALP6 function as receptor for Cry1 Ca. Different members of the ALP family play different specific roles in zhe mode of action of Cry proteins.