Down Regulation of a Gene for Cadherin, but Not Alkaline Phosphatase, Associated with Cry1Ab Resistance in the Sugarcane Borer Diatraea saccharalis

The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt) proteins (i.e., Cry1Ab) in South America and the mid-southern region of the United States. Evolution of insecticide resistance in such target pests is a major threat to the durability of transgenic Bt crops. Understanding the pests' resistance mechanisms will facilitate development of effective strategies for delaying or countering resistance. Alterations in expression of cadherin- and alkaline phosphatase (ALP) have been associated with Bt resistance in several species of pest insects. In this study, neither the activity nor gene regulation of ALP was associated with Cry1Ab resistance in D. saccharalis. Total ALP enzymatic activity was similar between Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of D. saccharalis. In addition, expression levels of three ALP genes were also similar between Cry1Ab-SS and -RR, and cDNA sequences did not differ between susceptible and resistant larvae. In contrast, altered expression of a midgut cadherin (DsCAD1) was associated with the Cry1Ab resistance. Whereas cDNA sequences of DsCAD1 were identical between the two strains, the transcript abundance of DsCAD1 was significantly lower in Cry1Ab-RR. To verify the involvement of DsCAD1 in susceptibility to Cry1Ab, RNA interference (RNAi) was employed to knock-down DsCAD1 expression in the susceptible larvae. Down-regulation of DsCAD1 expression by RNAi was functionally correlated with a decrease in Cry1Ab susceptibility. These results suggest that down-regulation of DsCAD1 is associated with resistance to Cry1Ab in D. saccharalis.

甘蔗螟虫（Diatraea saccharalis）是南美洲以及美国中南部地区表达苏云金杆菌（Bacillus thuringiensis，Bt）蛋白（即Cry1Ab）的转基因玉米的主要靶标害虫。此类靶标害虫的抗药性演化是Bt转基因作物持效性面临的主要威胁。解析该害虫的抗药性机制，将有助于开发延缓或逆转抗药性的有效策略。已有研究证实，多种害虫体内钙粘蛋白与碱性磷酸酶（alkaline phosphatase，ALP）的表达改变与Bt抗药性相关。本研究中，ALP的活性与基因调控均与甘蔗螟虫的Cry1Ab抗药性无关。甘蔗螟虫Cry1Ab敏感品系（Cry1Ab-SS）与抗药品系（Cry1Ab-RR）的总ALP酶活性无显著差异；此外，3个ALP基因的表达水平在两个品系间亦无明显差异，敏感与抗药性幼虫的cDNA序列也无区别。与之相反，中肠钙粘蛋白DsCAD1的表达改变与Cry1Ab抗药性相关。尽管两个品系的DsCAD1 cDNA序列完全一致，但Cry1Ab-RR品系中DsCAD1的转录本丰度显著降低。为验证DsCAD1在Cry1Ab敏感性中的作用，本研究通过RNA干扰（RNA interference，RNAi）技术对敏感幼虫的DsCAD1表达进行敲低。RNAi介导的DsCAD1表达下调与Cry1Ab敏感性降低存在功能相关性。上述结果表明，甘蔗螟虫体内DsCAD1的表达下调与其对Cry1Ab的抗药性相关。