中国农业科学 ›› 2022, Vol. 55 ›› Issue (13): 2552-2561.doi: 10.3864/j.issn.0578-1752.2022.13.006
收稿日期:
2021-12-17
接受日期:
2022-01-18
出版日期:
2022-07-01
发布日期:
2022-07-08
联系方式:
渠成,E-mail: quch1990@sina.com。
基金资助:
QU Cheng(),WANG Ran,LI FengQi,LUO Chen(
)
Received:
2021-12-17
Accepted:
2022-01-18
Published:
2022-07-01
Online:
2022-07-08
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。
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.
表1
引物序列"
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 |
图4
BtabGR1、BtabGR2与其他半翅目昆虫味觉受体氨基酸序列比对 SflaGR28b:甘蔗黄蚜Sipha flava (XP_025417386.1);AgosGR28b:棉蚜Aphis gossypii (XP_027840408.1);ApisGR28b、ApisGR43a-like:豌豆蚜Acyrthosiphon pisum (XP_008178128.1, XP_003244120.4);MperGR43a-like:桃蚜Myzus persicae (XP_022165389.1);MsacGR43a-like:高粱蚜Melanaphis sacchari (XP_025195577.1);Consensus:共有序列Consensus sequence。黑色区域代表相似度100%,粉红色区域代表相似度大于75%,浅蓝色区域代表相似度大于50% Black areas indicate 100% similarity, pink areas indicate more than 75% similarity, blue areas indicate more than 50% similarity"
图5
基于氨基酸序列构建的BtabGR1、BtabGR2与其他半翅目昆虫味觉受体的系统进化树 GR蛋白来源物种及GenBank登录号Origin species of GRs and their GenBank accession numbers。BtabGR1、BtabGR2:烟粉虱Bemisia tabaci (OL845904, OL845905);ApisGR43a-like、ApisGR28b、ApisGR64f-like:豌豆蚜Acyrthosiphon pisum (XP_003244120.4, XP_008178128.1, XP_016660313.2); AcraGR43a-like、AcraGR64e-like、AcraGR64f-like、AcraGR28b:豆蚜Aphis craccivora (KAF0773920.1, KAF0765871.1, KAF0765661.1, KAF0749339.1);AgosGR28b、AgosGR43a-like、AgosGR64f-like、AgosGR64e-like:棉蚜Aphis gossypii (XP_027840408.1, XP_027840395.1, XP_027847490.1, XP_027847461.1);DcitGR64f-like:柑橘木虱Diaphorina citri (XP_017300309.2);DnoxGR64f-like:麦双尾蚜Diuraphis noxia (XP_015369418.1); MperGR43a-like、MperGR64f-like:桃蚜Myzus persicae (XP_022165389.1, XP_016660313.2);MsacGR43a-like、MsacGR64f-like、MsacGR28b:高粱蚜Melanaphis sacchari (XP_025195577.1, XP_025204513.1, XP_025202779.1);NlugGR28b、NlugGR64f-like:褐飞虱Nilaparvata lugens (XP_039287957.1, XP_022186608.2);RmaiGR43a-like、RmaiGR64f-like:玉米蚜Rhopalosiphum maidis (XP_026807230.1, XP_026822156.1);SflaGR28b:甘蔗黄蚜Sipha flava (XP_025417386.1)"
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