褐飞虱ABCB基因的克隆与表达分析

Genecloning and expression of ABCB in Nilaparvata lugens

三磷酸腺苷结合盒转运蛋白(ATP-binding cassette,ABC)广泛分布于各种生物体中,是最大的膜蛋白家族之一。在昆虫中,ABC转运子不仅在分子转运过程中有重要的功能,同时在杀虫剂抗性、代谢和发育中都起到重要的作用。本研究克隆和鉴定了褐飞虱Nilaparvata lugens St l ABCB基因(NlABCB),结果表明NlABCB基因全长1468 bp,含有一个长度为459 bp的开放阅读框,编码153个氨基酸。利用定量PCR检测NlABCB基因在褐飞虱体内的表达,发现NlABCB转录本在褐飞虱的所有发育阶段都有表达,在1龄若虫中表达量最低,随着生长发育表达量逐渐升高。NlABCB在褐飞虱5龄若虫的中肠中表达量最高。研究发现该基因与褐飞虱的生理活动有关。构建NlABCB-dsRNA载体,转化大肠杆菌Escherichia coli HT115后,经IPTG诱导,形成NlABCB-dsRNA,喂养褐飞虱取食含NlABCB-dsRNA人工饲料,发现褐飞虱的存活率明显降低。因此该基因的克隆和功能鉴定,对于作物的抗虫育种,尤其对水稻害虫的生物防治将起到重要的促进作用。

ATP-binding cassette(ABC)transporter protein is one of the largest protein superfamilies and widely exists in all organisms.In insects,ABC transporter has important functions in molecule transport and insecticide resistance,metabolism and development.The Nilaparvata lugens(Hemiptera:Delphacidae)ABCB(NlABCB)gene was identified and cloned.The complete mRNA sequence of NlABCB was 1468 bp with an open reading frame of 459 bp encoding a protein of 153 amino acids.The developmental expression analysis using quantitative real-time reverse transcriptase PCR showed that the NlABCB transcript was expressed at all developmental stages of N.lugens.The relative expression level of NlABCB was the lowest in 1 st instar and increased with larval growth.The transcript profiles of NlABCB were analyzed in different tissues of 5 th instar nymph.The highest expression was observed in the midgut.It was found that the gene was related to the digestive feeding and survival of brown planthopper.After transforming L4440-NlABCB dsRNA into Escherichia coli HT115,NlABCB-dsRNA was induced by IPTG.The survival rate of brown planthopper was significantly reduced by feeding artificial diet containing NlABCB-dsRNA.Therefore,the isolation and cloning of this gene will play an important role in insect resistance breeding of crops,especially in biological control of rice.