桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

doi:10.16380/j.kcxb.2022.09.007

昆虫学报 ›› 2022, Vol. 65 ›› Issue (9): 1136-1143.doi: 10.16380/j.kcxb.2022.09.007

桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

静大鹏¹, 黄晓丹^1,2, 张天涛¹, 王振营^1,*

(1. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室，北京 100193; 2. 吉林农业大学生物防治研究所, 天敌昆虫应用技术工程研究中心，长春 130118)

出版日期:2022-09-20 发布日期:2022-10-16

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

JING Da-Peng¹, HUANG Xiao-Dan^1,2, ZHANG Tian-Tao¹, WANG Zhen-Ying^1,*

(1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Engineering Research Center of Natural Enemy Insects, Institute of Biological Control, Jilin Agricultural University, Changchun 130118, China)

Online:2022-09-20 Published:2022-10-16

摘要/Abstract

摘要： 【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。

关键词: 桃蛀螟, 细胞色素P450, Cry1Ab蛋白, 存活率, RNAi, 基因表达量

Abstract: 【Aim】 To clarify the overexpression and detoxification effect of CYP6AE76 in vivo 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 and hemolymph of the 4th instar larvae of C. punctiferalis fed with Cry1Ab protein (LC₅₀=1.08 ng/cm²) for 3 d were detected by RT-qPCR. The expression level of CYP6AE76 in the midgut of the 4th instar larvae was detected and the larval weight and larval survival rate at 120 h after silencing the expression of CYP6AE76 in the 4th instar larvae of C. punctiferalis by 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 in the newly hatched larvae followed by feeding on the Cry1Ab-containing (1.08 ng/cm²) diet were calculated. 【Results】 The open reading frame of the CYP6AE76 of C. punctiferalis is 1 572 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 that CYP6AE76 is expressed throughout the larval stage of C. punctiferalis, with the highest expression level in the 1st instar larval stage and its expression level gradually decreasing with increasing larval instar. Tissue expression profiling revealed that the expression level of CYP6AE76 in the midgut of the 4th instar larvae was the highest. After the 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 significantly up-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 Cry1Ab protein significantly decreased. 【Conclusion】 CYP6AE76 may participate in detoxifying the Cry1Ab protein ingested by C. punctiferalis larvae.

Key words: Conogethes punctiferalis, cytochrome P450, Cry1Ab protein, survival rate, RNAi; gene expression level

静大鹏, 黄晓丹, 张天涛, 王振营. 桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用[J]. 昆虫学报, 2022, 65(9): 1136-1143.

JING Da-Peng, HUANG Xiao-Dan, ZHANG Tian-Tao, WANG Zhen-Ying. Involvement of CYP6AE76 gene in detoxifying the Cry1Ab protein in Conogethes punctiferalis (Lepidoptera: Crambidae)[J]. Acta Entomologica Sinica, 2022, 65(9): 1136-1143.

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桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

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

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