
棉蚜AgosOBP5基因克隆、表达纯化及 结合特性研究
Cloning, expression, purification and binding characteristics of the AgosOBP5 gene in Aphis gossypii
王铁矿1** 李峰奇2 刘思雨1 李增凤3 李秋荣1***
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DOI:10.7679/j.issn.2095-1353.2025.012
作者单位:1. 青海大学农林科学院,青海省农业有害生物综合治理重点实验室,农业农村部西宁作物有害生物科学观测实验站,西宁 810016; 2. 贵州大学绿色农药全国重点实验室,精细化工研究开发中心,贵阳 550025;3. 都兰林晟防沙治沙有限责任公司,都兰 816100
中文关键词:棉蚜;气味结合蛋白;基因克隆;表达纯化;荧光竞争结合
英文关键词: Aphis gossypii; odorant-binding proteins; gene cloning; expression and purification; fluorescence competitive binding
中文摘要:
【目的】 棉蚜Aphis gossypii是枸杞Lycium chinense、棉花Gossypium herbaceum等作物上的一种成灾性害虫。明确气味结合蛋白与气味分子的结合特征及其在棉蚜嗅觉识别过程中发挥的作用,可为今后利用化学挥发物制作诱芯防治棉蚜提供理论依据。【方法】 基于转录组数据对棉蚜气味结合蛋白(OBPs)AgosOBP5基因进行全长克隆并开展生物信息学分析;构建原核表达载体并用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测;利用荧光竞争结合实验研究AgosOBP5重组蛋白与水杨酸甲酯、苯乙醇、苄醇、壬醛、1-辛烯-3-醇、棕榈酸这6种植物源气味分子的结合特征。【结果】 棉蚜AgosOBP5基因全长为675 bp,编码224个氨基酸,含有12个保守半胱氨酸,预测理论相对分子量为54.50 kD。序列比对和系统进化树分析表明,棉蚜AgosOBP5氨基酸序列与大豆蚜Aphis glycines AglyOBP5同源性最高。经异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导和SDS-PAGE检测,证明了AgosOBP5重组蛋白是可溶性蛋白,能够在上清液中表达。6种气味分子均可将AgosOBP5与N-苯基-1-萘胺(1-NPN)荧光探针体系的荧光强度值降到50%以下,不同的是水杨酸甲酯、2-苯乙醇、苄醇、壬醛、1-辛烯-3-醇这5种化合物从初始滴加,荧光值即开始下降,随后一直保持不断下降的趋势,而棕榈酸在浓度低于96 μmol·L-1时荧光值呈升高趋势,随自身浓度不断升高后转为下降趋势。【结论】 本研究明确了棉蚜AgosOBP5基因的核苷酸及其编码氨基酸序列,根据AgosOBP5与植物源挥发物水杨酸甲酯、2-苯乙醇、苄醇、壬醛、1-辛烯-3-醇的良好结合能力,推断AgosOBP5蛋白可能在棉蚜的嗅觉信息识别中起着重要的作用。
英文摘要:
[Aim] Aphis gossypii is a serious pest of the
Chinese wolfberry, cotton and other crops. The aim of this study was to
identify the binding characteristics of odorant-binding proteins with odor
molecules and establish their role in the olfactory recognition process in A. gossypii. The findings will establish
a theoretical basis for using chemical volatiles in developing lures aimed at
the future prevention and control of A.
gossypii. [Methods] Using transcriptome data, a full-length
sequence of the odorant-binding proteins (OBPs) AgosOBP5 gene was
obtained and bioinformatics analysis was performed. The prokaryotic expression
of AgosOBP5 was constructed and detected by Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Fluorescence competitive
binding experiment was used to study the binding characteristics of the
AgosOBP5 recombinant protein with six different odor molecules derived from
plants. [Results] The total length of the A. gossypii gene AgosOBP5 was 675 bp, encoding 224 amino acids,
including 12 conserved cysteines. The predicted theoretical relative molecular
weight was 54.50 kD. Sequence alignment and phylogenetic tree analysis show
that the amino acid sequence of AgosOBP5
in A. gossypii has the highest
homology with the AglyOBP5 in soybean aphid A. glycines. By Isopropyl β-D-thiogalactoside
(IPTG) induction and SDS-PAGE detection, it demonstrates that the recombinant
protein AgosOBP5 is soluble and can be expressed in the supernatant. The test
results show that these six odor molecules can reduce the fluorescence
intensity value of the AgosOBP5 and N-phenyl-1-naphthylamine (1-NPN)
fluorescent probe system to less than 50%. Starting from the initial dropwise
addition of the five compounds including methyl salicylate, 2-phenylethanol,
benzyl alcohol, nonanal, and 1-octen-3-ol, the fluorescence value began to
decrease and continued to maintain a downward trend. However, palmitic acid
initially showed an increasing trend at the concentrations below 96 μmol·L-1, this
trend reversed later and showed a continuous decrease at other higher
concentrations. [Conclusion] This study identified the nucleotide and
amino acid sequences of protein encoded by AgosOBP5 in A. gossypii. The protein AgosOBP5 has a
higher binding affinity with methyl salicylate, 2-phenylethanol, benzyl
alcohol, nonanal, and 1-octen-3-ol the five kinds of green leaf volatiles. It
is inferred that AgosOBP5 protein may play an important role in the ordor information
recognition of A. gossypii.