淡色库蚊与骚扰库蚊触角嗅觉基因表达分析

doi:10.12140/j.issn.1000-7423.2019.04.015

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (4): 453-457.doi: 10.12140/j.issn.1000-7423.2019.04.015

淡色库蚊与骚扰库蚊触角嗅觉基因表达分析

周洁^1,²(), 李春晓², 兰策介², 高剑², 刘钦梅³, 孙爱娟⁴, 赵彤言^1,^2,^*()

1 安徽医科大学,合肥 230000
2 军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室,媒介生物危害和自然疫源性疾病北京市重点实验室,北京 100071
3 浙江省疾病预防控制中心,杭州 310051
4 苏州市疾病预防控制中心,苏州 215004

收稿日期:2019-04-26 出版日期:2019-08-30 发布日期:2019-09-05
通讯作者: 赵彤言
作者简介:
作者简介：周洁（1993-）,女,硕士研究生,从事媒介生物学研究。E-mail: 540767140@qq.com
基金资助:
国家自然科学基金（No. 81371847）;国家科技重大专项（No. 2017ZX10303404）

Comparative analysis of antennal olfactory gene expression between Culex pipiens pallens and Culex pipiens molestus

Jie ZHOU^1,²(), Chun-xiao LI², Ce-jie LAN², Jian GAO², Qin-mei LIU³, Ai-juan SUN⁴, Tong-yan ZHAO^1,^2,^*()

1 Anhui Medical University, Hefei 230032, China
2 Beijing Key Laboratory of Vector Borne and Natural Focus Infectious Diseases, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071,China
3 Zhejiang Provincial Centre for Disease Control and Prevention, Hangzhou 310051, China
4 Suzhou Municipal Centre for Disease Control and Prevention, Suzhou 215004, China

Received:2019-04-26 Online:2019-08-30 Published:2019-09-05
Contact: Tong-yan ZHAO
Supported by:
Supported by National Natural Science Foundation of China（No. 81371847）, National Science and Technology Major Project（No. 2017ZX10303404）

摘要/Abstract

摘要：

目的了解淡色库蚊与骚扰库蚊触角中差异性表达的嗅觉基因,为揭示二者遗传分化特征提供参考依据。方法分别设置淡色库蚊吸血前、后,骚扰库蚊吸血前、后等4个实验组。每组共设4个生物学重复样品,每个样品取20只蚊虫触角。采用TRIzol法提取各组样品总RNA,采用NEBNext Ultra^TM RNA^® 文库构建试剂盒构建文库。对转录组基因进行测序,通过FPKM值差异性分析,筛选淡色库蚊与骚扰库蚊分别在吸血前、后表达差异显著的嗅觉基因,并进行实时荧光定量PCR（qRT-PCR）验证。结果淡色库蚊吸血前、后触角转录组共注释了50个气味结合蛋白（OBP）和31个气味受体（OR）基因,骚扰库蚊吸血前、后触角转录组共注释了36个OBP和12个OR。淡色库蚊吸血后较吸血前显著上调的OBP有4个,无显著下调的OBP。骚扰库蚊吸血后较吸血前显著上调和下调的OBP分别有3个。淡色库蚊吸血后较吸血前无显著上调的OR,显著下调的OR有3个。骚扰库蚊吸血后较吸血前无显著上调的OR,显著下调的OR有1个。qRT-PCR验证结果显示,淡色库蚊吸血后OBP17显著上调;骚扰库蚊吸血后OBP31、OBP44表达量显著上调,其中OBP44与测序结果一致,OBP31与测序结果相反。吸血前,骚扰库蚊OBP17显著高于淡色库蚊;吸血后,骚扰库蚊OBP31、OBP44显著高于淡色库蚊,OBP99a显著低于淡色库蚊。骚扰库蚊吸血后OR31、OR65、CPIJ001069表达量均显著上调,其中OR65和CPIJ001069表达量显著高于淡色库蚊(P < 0.05)。淡色库蚊吸血后无显著变化的OR基因。结论淡色库蚊与骚扰库蚊的嗅觉基因OBP和OR表现出了进化分化,OBP17、OBP44可能参与调控骚扰库蚊吸血后行为。OR31、OR65、CPIJ001069可能参与调控骚扰库蚊吸血后行为。

关键词: 淡色库蚊, 骚扰库蚊, 高通量分析技术, 嗅觉基因

Abstract:

Objective To analyze the difference in antennal olfactory gene expression between Culex pipiens pallens and Cx. pipiens molistus to provide a reference for revealing the characteristics of their genetic differentiation. Methods Total RNAs were extracted from antennas of Cx. pipiens pallens and Cx. pipiens molistus before and after blood feeding, and the RNA libraries were constructed using NEBNext Ultra^TM RNA ^® RNA Library Prep Kit. The transcriptome were obtained by RNAseq and their difference in different species of Cx. was analyzed by FPKM. Those olfactory genes with significant difference before and after blood feeding were identified by qRT-PCR. Results RNA-Seq results identified 50 odorant binding protein (OBP) genes and 31 odor receptor(OR) genes expressed in Cx. pipiens pallens, 36 OBPs and 12 ORs in Cx. pipiens molestus. The regulation trend of OBP44 was, in consistence, the same with the RNAseq results while OBP31 was contrary to that. The expression of OBP17 was significantly higher in Cx. pipiens molestus than in Cx. pipiens pallens before blood-sucking, and the expressions of OBP31 and OBP44 displayed the same trend after blood-sucking while the expression of OBP99a was significantly lower than Cx. pipiens pallens(P < 0.05). Expression of OR31, OR65 and CPIJ001069 were significantly up-regulated in Cx. pipiens molestus post blood feeding, but not in Cx. pipiens pallens, consistent with RNAseq results. Expression of OR65 and CPIJ001069 was significantly higher in Cx. pipiens molestus than that in Cx. pipiens pallens post blood feeding(P < 0.05). There was no significant change of OR gene expression in Cx. pipens pallents post blood feeding. Conclusion OBPs and ORs of Cx. pipiens pallens and Cx. pipiens molestus showed genetic differentiation. OBP17 and OBP44 is possibly involved in the regulation of behavior post blood feeding of Cx. pipiens molestus. OR31, OR65, and CPIJ001069 could be involved in regulating the behavior of Cx. pipiens molestus post blood feeding.

Key words: Culex pipiens pallens, Culex pipiens molestus, High-throughput analysis, Olfactory gene

中图分类号:

R384.1

周洁, 李春晓, 兰策介, 高剑, 刘钦梅, 孙爱娟, 赵彤言. 淡色库蚊与骚扰库蚊触角嗅觉基因表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 453-457.

Jie ZHOU, Chun-xiao LI, Ce-jie LAN, Jian GAO, Qin-mei LIU, Ai-juan SUN, Tong-yan ZHAO. Comparative analysis of antennal olfactory gene expression between Culex pipiens pallens and Culex pipiens molestus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(4): 453-457.

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参考文献 18

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基因名称 Gene name	引物（5′→3′） Primer sequences( 5′→3′)	产物长度/bp Product length/bp
RPL8	R: GTATCGACAAGCCCATCCTGA F: ACCTGGCGGCGTTCCT	176
OR58	R: ACTGTTCTGCTCGGAGTCTCTGTC F: TTCGGCGTCGTTGAAGTTAGCG	160
OR65	R: ACGCTCTCATGGATCGCAGGATA F: CCAGAACTGTTGGTACGAGGCAA	85
OR31	R: GTCCTGTTCTGGCTGTCGTTGAT F: GAATGTTCGTACCACGGCATATCG	119
CPIJ001069	R: AGGATTCACGCAGGTCGTCACA F: ATCGGAGGCATCTTCGTCTTGGA	108
CPIJ019566	R: AACGCTTGCTGCCGAACTTCC F: TGCTGCTGCTTCTCGGTCCAA	110
CPIJ013183	R: TCGCTCTCGTCGCTGGTGTATT F: GCTCGTGTTGATGTGCTTGATTCG	204
OBP23	R: CAGGAGAAGGCGGAACGGTT F: CAGAAACAGCGAACATCCAGCAAA	157
OBP31	R: AGTTGCGTGATGCAGAAGATTGGA F: TCGTTAGTCGTGCCTTCACATTGG	143
OBP44	R: GAGTTCACATGGACGCCACCTG F: ACAATACGCATCATCCGCCAAGG	131
OBP99a	R: GGACAAGAACACCGACGACAACT F: TGTTAGAGCCAGCGTTTCCTTCAA	184
OBP17	R: GGTGCTTCGTGCAGTGCTTCT F: GGTCAAACTCGGCGGACATCTTT	102
CPIJ016952	R: CGTATGCTCAGGTTCAGCTTCTCC F: CGTAGGCAACTTCGCACTTGTCTT	114

淡色库蚊与骚扰库蚊触角嗅觉基因表达分析

Comparative analysis of antennal olfactory gene expression between Culex pipiens pallens and Culex pipiens molestus

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