二化螟P糖蛋白基因的克隆分析及对杀虫剂的诱导响应

doi:10.3864/j.issn.0578-1752.2021.19.008

Abstract

Abstract:

【Objective】The CsPgp was cloned from Chilo suppressalis, and the molecular characteristics and expression profiles of CsPgp were analyzed. Transcriptional responses as well as the potential transcriptional regulation mechanism of CsPgp to two common used insecticides (chlorantraniliprole and abamectin) were also studied.【Method】The full length of CsPgp was cloned from C. suppressalis using the gene cloning technology. The molecular characteristics and the transcription factor binding sites in 5′ transcriptional regulatory region of CsPgp were analyzed employing the bioinformatics technologies. Expression profiles of CsPgp in different stages and tissues of C. suppressalis, and the transcriptional responses of CsPgp to different doses of chlorantraniliprole and abamectin treatment were determined using the real-time quantitative PCR.【Result】The full length of CsPgp cDNA is 4 584 bp and consists of 23 exons. The encoding protein has 1 259 amino acids containing two transmembrane regions and two nucleotide binding domains and the typical structural features of ABC transporter family such as the Walker A, Walker B and D, H, P, Q-Loop which have important function in substrate transfer. CsPgp was mainly expressed in larval stage of C. suppressalis, especially in the 3rd and 4th instar larvae, while CsPgp showed low expression levels in the pupal and adult stages. Analysis of the tissue expressions showed that CsPgp was predominately expressed in the foregut and midgut, and had very low expression levels in other tissues including hindgut, fat body and Malpighian tubule. No significant change of CsPgp expression was found in the 3rd instar larvae of C. suppressalis after treated with LC₃₀ and LC₇₀ of chlorantraniliprole for 12 and 24 h, respectively, when compared with the control groups. However, the expressions of CsPgp were significantly up-regulated in larvae after treated with LC₃₀ of chlorantraniliprole for 36 h, while the expressions of CsPgp were significantly down-regulated in larvae after treated with LC₇₀ of chlorantraniliprole for 36 h. In the 0.05 mg·L^-1 of abamectin treatment, CsPgp was remarkably down-regulated at 12 h post-treatment, while the expressions of CsPgp were not significantly changed at 24 and 36 h post-treatment, respectively. However, CsPgp was significantly induced in larvae after treated with 0.15 mg·L^-1 of abamectin for 24 and 36 h, respectively. Sequence analysis of the 5′ transcriptional regulatory region of CsPgp showed that multiple transcription factor binding sites were predicted in the 5′ transcriptional regulatory region of CsPgp, including five potential CncC binding sites.【Conclusion】CsPgp was highly expressed in the midgut of C. suppressalis and could be induced by chlorantraniliprole and abamectin, which indicated that CsPgp might involve in the detoxification metabolism of chlorantraniliprole and abamectin in C. suppressalis. Multiple CncC binding sites were found in the 5′ transcriptional regulatory region of CsPgp which might have important regulatory effects on the expression of CsPgp. It was speculated that CsPgp might be regulated by transcription factor CncC and participated in the detoxification metabolism of chlorantraniliprole or abamectin when C. suppressalis was exposed to chlorantraniliprole or abamectin.

Key words: Chilo suppressalis, P-glycoprotein, molecular characteristic, insecticide induction, transcriptional regulation

MENG XiangKun,WU ZhaoLu,YANG XueMei,GUAN DaoJie,WANG JianJun. Cloning and Analysis of P-glycoprotein Gene and Its Transcriptional Response to Insecticide in Chilo suppressalis[J].Scientia Agricultura Sinica, 2021, 54(19): 4121-4131.

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引物名称Primer name	序列Sequence (5′ to 3′)	作用Function
CsPgp F	CAGCTTTGAATCAGGACTTTGC	CsPgp基因片段扩增 Gene fragment amplification of CsPgp
CsPgp R	ATCCACTCCGATTGAGGTTGTA	CsPgp基因片段扩增 Gene fragment amplification of CsPgp
CsPgp 5′R1	CCATCCCTTCACAAGTGCCATTAT	CsPgp 5′端片段扩增 5′ end fragment amplification of CsPgp
CsPgp 5′R2	CGAACGCCGATTGGTAGAAAAC	CsPgp 5′端片段扩增 5′ end fragment amplification of CsPgp
CsPgp 3′F1	GCGTTGGATACGGAAAGCGAAAA	CsPgp 3′端片段扩增 3′ end fragment amplification of CsPgp
CsPgp 3′F2	AGTGCGGGACGCCGACCTTATA	CsPgp 3′端片段扩增 3′ end fragment amplification of CsPgp
DLCsPgp F	GCAGGATGCACCACTCCTATCA	CsPgp定量分析 Quantitative analysis of CsPgp
DLCsPgp R	TGGCCGCTCCGACTATACAGTTA
EF-1α F	TGAACCCCCATACAGCGAATCC
EF-1α R	TCTCCGTGCCAACCAGAAATAGG
CsPgp 5′F	GCACTATTACACTTTTTAATGAG	CsPgp 5′侧翼区DNA序列克隆 5′ lateral DNA sequence cloning of CsPgp
CsPgp 5′R	GCGTTTCCGTTCAAATCATAAG

Cloning and Analysis of P-glycoprotein Gene and Its Transcriptional Response to Insecticide in Chilo suppressalis

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