烟粉虱味觉受体基因BtabGR1和BtabGR2的克隆与表达模式分析

doi:10.3864/j.issn.0578-1752.2022.13.006

摘要/Abstract

摘要：

【目的】烟粉虱（Bemisia tabaci）为世界性分布的重要农林入侵害虫,寄主植物种类众多,但对不同寄主植物存在嗜性差异,而味觉受体（gustatory receptor,GR）基因在其取食选择等行为中发挥重要作用。本研究通过克隆烟粉虱味觉受体GR1和GR2基因,明确其在烟粉虱不同发育期和成虫不同组织中的表达特性,为基因功能的深入研究提供依据。【方法】从烟粉虱MED隐种成虫头部转录组中筛选获得味觉受体基因序列,利用巢式PCR技术克隆获得两个味觉受体基因的完整开放阅读框（open reading frame,ORF）序列,两个基因分别命名为BtabGR1和BtabGR2,运用生物信息学软件对其编码氨基酸序列特征、结构域等信息进行预测,基于邻接法构建BtabGR1、BtabGR2与其他半翅目昆虫味觉受体基因的系统进化树,并利用实时荧光定量PCR方法分析两个基因在烟粉虱不同发育期（卵、1—4龄若虫和雌、雄成虫）和雌、雄成虫不同组织（头、胸、腹、足）中的表达情况。【结果】克隆获得BtabGR1和BtabGR2基因序列（GenBank登录号：OL845904和OL845905）,完整开放阅读框为1 287和1 344 bp,分别编码428和447个氨基酸,预测蛋白分子量为 48.54和51.50 kD,理论等电点为8.85和8.74,具有4个和6个跨膜结构域。氨基酸序列比对和系统进化分析结果显示,BtabGR1 和BtabGR2与其他半翅目昆虫GR28b和GR43a-like基因亲缘关系较近。发育期表达谱分析表明,BtabGR1和BtabGR2在烟粉虱的不同发育历期均有表达,其中BtabGR1在成虫中表达量高于其他发育期,而BtabGR2在卵中的表达量最高;组织表达谱结果表明,BtabGR1和BtabGR2在烟粉虱雌、雄成虫不同组织中均有表达,且均在成虫头部高表达。【结论】BtabGR1和BtabGR2具有昆虫味觉受体基因的典型特征,二者均在烟粉虱成虫头部高表达,推测两基因在烟粉虱寄主植物适应过程中发挥重要作用,可作为烟粉虱新型防控措施的潜在靶标。

关键词: 烟粉虱, 味觉受体, 基因克隆, 序列分析, 表达谱

Abstract:

【Objective】Bemisia tabaci is an important agricultural and invasive pest worldwide. Although B. tabaci has many host plants, there are differences in the tropism of B. tabaci to these different hosts. The gustatory receptor genes (GRs) play an important role in its feeding selection and other behaviors. The objective of this study is to clone the two gustatory receptor genes (GR1 and GR2) of B. tabaci, and clarify their expression profiles at different developmental stages and in different adult tissues, so as to provide a theoretical basis for further functional study of the two gustatory receptor genes.【Method】The gustatory receptor genes were screened from the adult head transcriptome of B. tabaci. The open reading frame (ORF) of two gustatory receptor genes was cloned by nest PCR, and the two genes were named BtabGR1 and BtabGR2, respectively. Encoded amino acid sequence features and structure characteristics of BtabGR1 and BtabGR2 were analyzed by multiple bioinformatics methods. Phylogenetic tree between BtabGR1, BtabGR2 and other Hemiptera insects GRs was constructed using neighbor-joining method. The expression levels of the two genes at different developmental stages (egg, 1-4 instar nymphs, female and male adults), in different adult tissues (head, thorax, abdomen, leg) were detected by qRT-PCR.【Result】The cDNA sequences of BtabGR1 and BtabGR2 (GenBank accession number: OL845904 and OL845905) were obtained. The complete ORFs of these two genes are 1 287 and 1 344 bp in length, encoding 428 and 447 amino acids with the predicted molecular weight of 48.54 and 51.50 kD, the isoelectric point of 8.85 and 8.74, 4 and 6 transmembrane domains. Amino acid sequence alignment and phylogenetic analysis showed that BtabGR1 and BtabGR2 were closely related to the GR28b and GR43a-like genes of other Hemiptera insects. Developmental stages expression results showed that BtabGR1 and BtabGR2 were expressed at different developmental stages of B. tabaci. The expression level of BtabGR1 in female and male adults was higher than that in other developmental stages, while BtabGR2 was highly expressed in the egg. Tissue expression results showed that BtabGR1 and BtabGR2 were expressed in different tissues of adult B. tabaci, and the two genes were both highly expressed in the head of adults.【Conclusion】BtabGR1 and BtabGR2 have the typical characteristics of insect gustatory receptor genes, and both of them are highly expressed in the head of B. tabaci adults. It is speculated that these two genes play an important role in host plant adaptation of B. tabaci, and can be used as potential targets for novel control measures of B. tabaci.

Key words: Bemisia tabaci, gustatory receptor, gene cloning, sequence analysis, expression profile

渠成,王然,李峰奇,罗晨. 烟粉虱味觉受体基因BtabGR1和BtabGR2的克隆与表达模式分析[J]. 中国农业科学, 2022, 55(13): 2552-2561.

QU Cheng,WANG Ran,LI FengQi,LUO Chen. Cloning and Expression Profiling of Gustatory Receptor Genes BtabGR1 and BtabGR2 in Bemisia tabaci[J]. Scientia Agricultura Sinica, 2022, 55(13): 2552-2561.

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引物 Primer	引物序列 Primer sequence (5′-3′)	引物用途 Usage of primers
GR1-1441-F	CCTCTAGTTGTTCTTCTATCG	开放阅读框扩增 Amplification of ORF
GR1-1441-R	TGTCTGAATCATTTTTTATCC
GR1-ORF-F	ATGCAATTCCACTTGATTCCGACAT
GR1-ORF-R	TTATGAAGTTGAATTATGTGAAAAT
GR2-1495-F	AAAAAGGAAACATGAAAAAAA
GR2-1495-R	GAACTGTACGTATAAGAGCTG
GR2-ORF-F	ATGAAATTTAAAAACCGCACATTCT
GR2-ORF-R	TTAAGACACCATGTTTTGTTTTTCC
GR1-117-F	ACCTAAACAGTCCCAAGTGCT	实时荧光定量PCR qRT-PCR
GR1-117-R	CTTCGAACCCGTCACACGAA
GR2-181-F	AAAAGACAAGCTGGTCCCCG
GR1-181-R	GTCGGCTAAACTGTGGGTGT
EF1α-110-F	TAGCCTTGTGCCAATTTCCG
EF1α-110-R	CCTTCAGCATTACCGTCC
RPL29-144-F	TCGGAAAATTACCGTGAG
RPL29-144-R	GAACTTGTGATCTACTCCTCTCGTG