红铃虫性信息素合成激活肽基因克隆、序列特征及在不同发育阶段的表达分析

doi:10.3864/j.issn.0578-1752.2018.23.005

Abstract

Abstract:

【Objective】The objective of this study is to clone the pheromone biosynthesis activating neuropeptide (PBAN) gene of pink bollworm (Pectinophora gossypiella), analyze its sequence characteristics, clarify its expression patterns in different developmental stages as well as correlation with mating behavior and cotton volatiles, which will provide a scientific basis for further revealing the biosynthesis and release mechanisms of sex pheromone in P. gossypiella.【Method】The full cDNA sequence of PgosPBAN was cloned by RACE technique. Gene splicing and amino acid sequence were analyzed by the software of DNAMAN 6.0, protein secondary structure prediction of PgosPBAN and bioinformatics information were predicted using Protparam and Chou & Fasman. The expression patterns of PgosPBAN in different developmental stages of P. gossypiella were detected and the effects of mating behavior and cotton volatiles on the expression level of PgosPBAN were analyzed using real-time quantitative PCR (qRT-PCR). 【Result】The full cDNA sequence of PgosPBAN (GenBank accession number: KY987647) is obtained, and the total length of cDNA is 1 461 bp. The open reading frame (ORF) is 618 bp, encoding 205 amino acid residues. The length of 5′-untranslated region (5′ UTR) and 3′-untranslated region (3′ UTR) is 121 and 722 bp, respectively. The amino acid sequence encoded by PgosPBAN contains five peptides, including diapause hormone homolog, α-SGNP, β-SGNP, PBAN and γ-SGNP, and a signal peptide of 23 amino acid residues at the N terminus. The predicted molecular mass and isoelectric point are 2.41 kD and 9.25, respectively. Homology and phylogenetic tree analysis showed that PgosPBAN and PBAN of 15 Lepidoptera insects were located in the same branch, and PgosPBAN had the closest relationship with Chilo suppressalis PBAN (GenBank accession number: ALM30314.1), suggesting that the two genes likely developed from a common ancestral gene. The expression of PgosPBAN was specific in different developmental stages, which was higher in adult stage, second in larval stage, and the lowest in pupal stage. PgosPBAN was expressed in both female and male adults, and the expression of PgosPBAN in male adults was significantly higher than that in female adults from the 1st-day to 5th-day old. The expression level of PgosPBAN in P. gossypiella at 1-3 days after mating was significantly higher than that in virgin moth. After exposed to cotton volatiles for 1-5 days, the expression level of PgosPBAN in male adults had no significant difference compared with the control, but the expression level of PgosPBAN at 1st, 8th day of female adults and 8th day of male adults was significantly lower than that in control. 【Conclusion】The sequence characteristics of nucleotides and amino acids of PgosPBAN were clarified, and the secondary structure characteristics of the protein were analyzed. According to the expression of PgosPBAN in different developmental stages of P. gossypiella and its relationship with mating behavior and the regulation of cotton volatiles, it is speculated that this gene is not only involved in the synthesis and release of female pheromone in P. gossypiella, it may also play an important role in regulating male pheromones and regulating growth and development.

Key words: pink bollworm (Pectinophora gossypiella), pheromone biosynthesis activating neuropeptide (PBAN), expression analysis, mating behavior, cotton volatiles

XU Dong,WANG Ling,CONG ShengBo,WANG JinTao,LI WenJing,WAN Peng. Cloning, Sequence Analysis and Expression of Pheromone Biosynthesis Activating Neuropeptide (PBAN) Gene in Different Development Stages of Pectinophora gossypiella[J].Scientia Agricultura Sinica, 2018, 51(23): 4449-4458.

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References 40

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Primer use
PgosPBAN-F	CTBTGGTTCGGYCCYMGACTMGG	cDNA克隆 cDNA cloning
PgosPBAN-R	CATSGTBGKSCCBAGCCTKGGBGAGAAGT	cDNA克隆 cDNA cloning
3′ PgosPBAN-Outer	CAAACCTACCTCCGTCTTCTT	3′端cDNA扩增 3′-cDNA end amplification
3′ PgosPBAN-Inner	CGCCCTAAAATACTACTACGAC	3′端cDNA扩增 3′-cDNA end amplification
5′ PgosPBAN-Outer	GTCAGCTAACCTCCTTCCGAG	5′端cDNA扩增 5′-cDNA end amplification
5′ PgosPBAN-Inner	CACTCTCGTATCGCTTCCTCT	5′端cDNA扩增 5′-cDNA end amplification
PgosPBAN-O-F	GCAACAGTAAATCGTAGAAACA	开放阅读框扩增 ORF amplification
PgosPBAN-O-R	GATGAGCATAAACCAGCCAAT	开放阅读框扩增 ORF amplification
PgosPBAN-Q-F	GGCAAGCGTTCTTTCCATCC	实时荧光定量PCR qRT-PCR
PgosPBAN-Q-R	GCCTCGTCATCAGCCTGTG	实时荧光定量PCR qRT-PCR
ACS1	CACCGTGCCCATCTATGAAGG
ACR1	GACGATTTCCCTCTCAGCGGT

Cloning, Sequence Analysis and Expression of Pheromone Biosynthesis Activating Neuropeptide (PBAN) Gene in Different Development Stages of Pectinophora gossypiella

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