Q型烟粉虱扬州种群cyp6cm1基因内含子抗性相关多态性检测及其5′侧翼序列克隆

Detection of single-nucleotide polymorphisms(SNPs) associated with resistance to neonicotinoid insecticides in the intron of cyp6cm1 gene and cloning of its 5′ flanking sequence in a Q-biotype Bemisia tabaci population from Yangzhou

【目的】烟粉虱Bemisia tabaci（Gennadius）已对包括有机磷类、拟除虫菊酯类、新烟碱类和昆虫生长调节剂等多种杀虫剂产生了不同程度的抗药性,其中尤以对新烟碱类杀虫剂的抗性问题最为突出。本研究旨在克隆Q型烟粉虱扬州种群细胞色素P450基因cyp6cm1片段序列及其5′侧翼序列。【方法】分别应用PCR和基因组步移技术克隆cyp6cm1基因片段序列及其5′侧翼序列。【结果】cyp6cm1基因片段序列包括63 bp的外显子片段和826~829 bp的内含子片段。多重序列比对发现,在内含子第195、230和242等3个碱基处存在与新烟碱类杀虫剂抗性相关的单核苷酸多态性（SNPs）。进一步利用基因组步移技术获得了长度为962 bp的cyp6cm1基因5′侧翼序列,利用NNPP在线分析软件预测转录起始位点为位于起始密码子上游57 bp处的碱基A;Con Site软件分析发现,cyp6cm1基因5′侧翼序列具有XRE-Ah R、CREB、Oct-1和Broad-complex-4等多种转录因子结合位点。

[Objectives] The whitefly, Bemisia tabaci, has developed resistance to a wide range of insecticides, including organophosphates, pyrethroids, insect growth regulators, and especially neonicotinoids. To understand the molecular mechanisms underlying neonicotinoid resistance, the genomic DNA fragment of the cytochrome P450 gene cyp6cml and its 5＇ flanking sequence were cloned in Q-biotype B. tabaci from Yangzhou. [Methods] PCR and genome walking were used to isolate the genomic DNA fragment of the cyp6cml gene and its flanking sequence. [Resultsl The genomic fragment of the cyp6cml gene contained the last 63 bp of the first exon of the cyp6cml gene together with the first 826-829 bp of the adjacent intron. Three single-nucleotide polymorphisms （SNPs） associated with resistance to neonicotinoid insecticides were identified by multiple sequences alignment, thesewere located at positions 195, 230 and 242 of the intron, respectively. In addition, the 962 bp 5＇ flanking sequence of cyp6cml gene of Q-biotype B. tabaci was cloned by genome walking. NNPP online analysis software predicted the transcription initiation site as the nucleotide A, which is located 57 bp upstream of the initiation codon. ConSite software predicted several transcription factor binding sites in this region, including XRE-AhR, CREB, Oct-1 and Broad-complex-4.