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

摘要/Abstract

摘要：

【目的】克隆红铃虫（Pectinophora gossypiella）性信息素合成激活肽（pheromone biosynthesis activating neuropeptide,PBAN）基因,分析其序列特征,阐明该基因在红铃虫不同发育阶段中的表达规律,以及与交配行为、棉花挥发物间的调控关系,为进一步揭示红铃虫性信息素合成释放机制提供科学依据。【方法】利用RACE技术克隆红铃虫性信息素合成激活肽基因PgosPBAN的全长cDNA序列,应用DNAMAN 6.0对其进行序列分析,利用Protparam、Chou & Fasman等在线分析软件进行蛋白二级结构预测和生物信息学分析;利用实时荧光定量PCR（qRT-PCR）技术检测PgosPBAN在红铃虫不同发育阶段的表达规律,分析交配行为和棉花挥发物对PgosPBAN表达水平的影响。【结果】获得了PgosPBAN全长cDNA序列,GenBank登录号为KY987647,该基因cDNA全长1 461 bp,其开放阅读框（ORF）为618 bp,编码205个氨基酸,5′端非编码区长121 bp,3′端非编码区长722 bp;PgosPBAN编码的氨基酸序列包含了滞育激素、α神经肽、β神经肽、PBAN和γ神经肽5种神经肽,N端还含有一个23氨基酸的信号肽;预测该蛋白分子量为2.41 kD,等电点为9.25;同源性及系统进化树分析发现,PgosPBAN与15种鳞翅目昆虫的PBAN位于同一分支,其中与PgosPBAN进化关系最近的是二化螟（Chilo suppressalis）的PBAN（GenBank登录号：ALM30314.1）,表明这两个基因可能有共同的祖先基因;PgosPBAN在红铃虫不同发育阶段存在明显的表达特异性,以成虫期的表达量较高,幼虫期次之,蛹期最低;PgosPBAN在红铃虫雌、雄成虫体内均有表达,且1—5 d内雄性成虫PgosPBAN表达量显著高于雌性成虫;交配后1—3 d的红铃虫体内PgosPBAN表达水平显著高于处女蛾;暴露于棉花挥发物1—5 d雄成虫PgosPBAN表达水平未受到显著影响,而1、8 d雌成虫及8 d雄成虫PgosPBAN表达水平均显著低于对照。【结论】明确了PgosPBAN的核苷酸、氨基酸序列特征,分析了该蛋白的二级结构特征。根据PgosPBAN在红铃虫不同发育阶段的表达规律以及与交配行为、棉花挥发物的调控关系,推测该基因不仅参与了红铃虫雌性信息素的合成释放,在调控雄性信息素以及调节生长发育等方面可能也扮演着重要角色。

关键词: 红铃虫, 性信息素合成激活肽, 表达分析, 交配行为, 棉花挥发物

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

许冬,王玲,丛胜波,王金涛,李文静,万鹏. 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[J]. 中国农业科学, 2018, 51(23): 4449-4458.

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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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