桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

【目的】明确桃蛀螟Conogethes punctiferalis幼虫取食Cry1Ab蛋白后体内CYP6AE76的过表达及对Cry1Ab蛋白有解毒作用。【方法】分析桃蛀螟CYP6AE76序列特征；利用RT-qPCR检测CYP6AE76在不同发育阶段(1-5龄幼虫)和4龄幼虫不同组织(头、中肠、血淋巴和脂肪体)以及4龄幼虫取食含有Cry1Ab蛋白(LC₅₀=1.08 ng/cm2)的人工饲料3 d存活的幼虫中肠和血淋巴中的表达量；利用RNAi饲喂法沉默桃蛀螟4 龄幼虫CYP6AE76后检测中肠中CYP6AE76的表达量，并统计120 h后幼虫体重并计算幼虫存活率；利用RNAi饲喂法沉默桃蛀螟初孵幼虫CYP6AE76后饲喂含1.08 ng/cm² Cry1Ab蛋白的饲料，7 d后统计幼虫体重并计算幼虫存活率。【结果】桃蛀螟CYP6AE76基因开放阅读框长1 572 bp，编码524个氨基酸，分子量约为60.34 kD，属于CYP6家族基因。发育表达谱结果表明， CYP6AE76基因在桃蛀螟整个幼虫阶段均有表达且在1龄幼虫期表达量最高，随着幼虫龄期增大而表达量降低；组织表达谱结果表明,CYP6AE76在4龄幼虫中肠中表达量最高。4龄幼虫取食含有Cry1Ab蛋白(1.08 ng/cm²)的人工饲料后，CYP6AE76在中肠和血淋巴中的表达量相比对照显著上调。通过RNAi沉默CYP6AE76后，桃蛀螟初孵幼虫再取食含有Cry1Ab蛋白的人工饲料后体重显著降低。【结论】CYP6AE76可能参与对桃蛀螟幼虫摄入的Cry1Ab蛋白的解毒。

Involvement of CYP6AE76 gene in detoxifying the Cry1Ab protein inConogethes punctiferalis(Lepidoptera: Crambidae)

【Aim】 To clarify the overexpression and detoxification effect of CYP6AE76 invivo on Cry1Ab protein in Conogethes punctiferalis larvae after fed with Cry1Ab protein. 【Methods】 The CYP6AE76 sequence of C. punctiferalis was characterized. The expression level of CYP6AE76 in different developmental stages (1st-5th instar larvae) and tissues (head,midgut, hemolymph and fat body) of the 4th instar larvae as well as in the midgut andhemolymph of the 4th instar larvae of C. punctiferalis fed with Cry1Ab protein (LC₅₀=1.08ng/cm²) for 3 d were detected by RT-qPCR. The expression level of CYP6AE76 in the midgutof the 4th instar larvae was detected and the larval weight and larval survival rate at 120h after silencing the expression of CYP6AE76 in the 4th instar larvae of C. punctiferalisby RNAi through feeding were counted. The larval weight and the larval survival rate of C.punctiferalis at 7 d after silencing the expression of CYP6AE76 by RNAi through feeding inthe newly hatched larvae followed by feeding on the Cry1Ab-containing (1.08 ng/cm²) dietwere calculated. 【Results】 The open reading frame of the CYP6AE76 of C. punctiferalis is1 572 bp in length. It encodes 524 amino acids, with a molecular weight of about 60.34 kD,and belongs to the CYP6 family gene. Developmental expression profiling showed thatCYP6AE76 is expressed throughout the larval stage of C. punctiferalis, with the highestexpression level in the 1st instar larval stage and its expression level graduallydecreasing with increasing larval instar. Tissue expression profiling revealed that theexpression level of CYP6AE76 in the midgut of the 4th instar larvae was the highest. Afterthe 4th instar larvae were fed with the Cry1Ab-containing (1.08 ng/cm²) artificial diet,the expression levels of CYP6AE76 in the midgut and hemeolymph were significantlyup-regulated as compared to those in the control group. After silencing CYP6AE76 by RNAi,the body weight of the newly hatched larvae fed on the artificial diet containing Cry1Abprotein significantly decreased. 【Conclusion】 CYP6AE76 may participate in detoxifying theCry1Ab protein ingested by C. punctiferalis larvae.