埃及伊蚊丝氨酸蛋白酶抑制蛋白基因家族的筛选和表达谱分析

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (6): 527-535.

埃及伊蚊丝氨酸蛋白酶抑制蛋白基因家族的筛选和表达谱分析

程金芝^1,², 刘鉴^1,², 翟慧^1,², 吕清巧^1,², 颜凤¹, 商正玲³, 吴家红^1,^2,^*

1 贵州医科大学,基础医学院寄生虫学教研室,现代病原生物学实验室
2 贵州医科大学,微生物与生化药学工程中心
3贵州医科大学,基础医学院免疫学教研室,贵阳,550025

收稿日期:2017-04-25 出版日期:2017-12-30 发布日期:2018-01-10
通讯作者: 吴家红
基金资助:
国家自然科学基金（No. 81260260,No. 81060318）;贵州省优秀青年科技人才培养对象专项资金（黔科合人字（2013）16号）;贵州省科技合作计划项目（黔科合LH字[2015]7355）;贵州省基础科技平台项目[黔科平台（2012）4006号]

Identification and expression of the Aedes aegypti serpin gene family

Jin-zhi CHENG^1,², Jian LIU^1,², Hui ZHAI^1,², Qing-qiao LV^1,², Feng YAN¹, Zheng-ling SHANG³, Jia-hong WU^1,^2,^*

1 Department of Parasitology, Laboratory for Modern Pathogen Biology, Basic Medical College
2 Microbial and Biochemical Pharmaceutical Engineering Center
3 Department of Immunology, Basic Medical College, Guizhou Medical University, Guiyang 550025, China

Received:2017-04-25 Online:2017-12-30 Published:2018-01-10
Contact: Jia-hong WU
Supported by:
Supported by the National Natural Science Foundation of China(No. 81260260, No. 81060318);Special Funds of Developing Guizhou Province Outstanding Youth in Technology and Science[No.（2013）16];Science and Technology Cooperation Program of Guizhou Province[No.[2015]7355];Project of Basic Science and Technology Platform in Guizhou Province (No. 2012[4006])

摘要/Abstract

摘要：

目的筛选并鉴定埃及伊蚊（Aedes aegypti）全基因组中的丝氨酸蛋白酶抑制蛋白（serine protease inhibitor, serpin）基因,分析其基因结构以及在不同发育时期和不同组织中的表达谱。方法运用生物信息学方法在埃及伊蚊全基因组数据库中筛选并鉴定埃及伊蚊serpin基因家族,利用Vector NTI11.0软件进行同源性比对、保守性分析,采用MEGA7.0软件构建系统进化树。实时荧光定量PCR（qRT-PCR）检测埃及伊蚊不同发育时期（卵、Ⅰ~Ⅳ龄幼虫、蛹、雄蚊和未吸血雌蚊）,以及未吸血雌蚊不同组织（唾液腺、中肠、脂肪体和卵巢）的serpin基因表达变化。结果从埃及伊蚊全基因组中筛选出26条serpin基因序列。生物信息学分析结果显示,19条具有信号肽,16条推测具有抑制酶活性。进化分析表明,埃及伊蚊serpin基因家族被分为A、B和C分支,B分支分为4组,每分支均具有同源性,且不同成员间在进化上相对保守。qRT-PCR结果显示,AaSRPN25在雌蚊中特异性表达,相对表达量为0.074 ± 0.015（P < 0.01）;AaSRPN19~22在雄蚊中丰富表达;AaSRPN23在Ⅰ龄幼虫时期特异性高表达,而且在唾液腺中也特异性高表达,相对表达量分别为22.9 ± 5.28和4.24 ± 1.39（P < 0.01）;AaSRPN25在唾液腺中特异性高表达,相对表达量为74.44 ± 25.76（P < 0.01）,AaSRPN6、AaSRPN14在中肠中的表达量最高,相对表达量分别为1.80 ± 0.33、0.703 ± 0.501;AaSRPN17、AaSRPN20在脂肪体中的表达量最高,相对表达量分别为0.34 ± 0.09、0.15 ± 0.03。结论从埃及伊蚊全基因组中筛选出26条serpin基因序列,其中19条具有信号肽,16条推测具有抑制酶活性的作用。

关键词: 埃及伊蚊, 丝氨酸蛋白酶抑制剂, 实时荧光定量PCR, 时空表达谱

Abstract:

Objective To identify serpin genes from the whole genome of Aedes aegypti, and analyze the structure of the gene and its expression at various developmental stages and in different tissues. Methods The serpin gene family was identified in Ae. aegypti genome database. Sequences were aligned for homology analysis and conservation analysis using Vector NTI11.0 softwares. The phylogenetic tree was constructed using MEGA7.0 software. qRT-PCR was performed to examine serpin expression at different developmental stages (eggs, larva of Ⅰ-Ⅳ stages, pupa, male mosquito and blood-unfed female mosquito) and in different tissues(salivary gland, midgut, fat body and ovary) of blood-unfed female mosquitos. Results Twenty-six gene sequences of serpin were identified from Ae. aegypti genome. Bioinformatics analysis revealed that 19 gene sequences harbored a signal peptide, and 16 were predicted to be inhibitory on enzymes. Phylogenetic analysis demonstrated that the serpin gene family of Ae. aegypti was divided into A, B and C clusters, in which cluster B was split into 4 groups. Each cluster showed homology, and members of each cluster were conservative. Results of qRT-PCR showed that AaSRPN25 was expressed specifically in female mosquitos (0.074 ± 0.015, P < 0.01), while AaSRPN19, AaSRPN20, AaSRPN21 and AaSRPN22 were enriched in male mosquitos. AaSRPN23 had a high expression level not only in the stage-Ⅰlarvae(22.9 ± 5.28) but also in the salivary gland (4.24 ± 1.39)(P < 0.01); AaSRPN25 were highly expressed specifically in the salivary gland (74.44 ± 25.67, P < 0.01); AaSRPN6 and AaSRPN14 had the highest expression in the mid gut(1.80 ± 0.33 and 0.703 ± 0.501, respectively); AaSRPN17 and AaSRPN20 had the highest expression in the fat body (0.34 ± 0.09 and 0.15 ± 0.03, respectively). Conclusion 26 Serpin sequences of Ae. aegypti have been identified in the whole genome, in which 19 sequences owned the signal peptide, and 16 sequences were speculated to have an inhibiting enzyme effect.

Key words: Aedes aegypti, Serpin, qRT-PCR, Expression profile

中图分类号:

R384.113

程金芝, 刘鉴, 翟慧, 吕清巧, 颜凤, 商正玲, 吴家红. 埃及伊蚊丝氨酸蛋白酶抑制蛋白基因家族的筛选和表达谱分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 527-535.

Jin-zhi CHENG, Jian LIU, Hui ZHAI, Qing-qiao LV, Feng YAN, Zheng-ling SHANG, Jia-hong WU. Identification and expression of the Aedes aegypti serpin gene family[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 527-535.

图/表 5

表1

表2

埃及伊蚊serpin基因家族的序列结构特征

基因名称Gene name	Vectorbase 登录号 VectorBase No.	基因登录号GenBank No.	目标蛋白酶Target proteinase	基因长度Gene length /bp	信号肽 Signal peptide	编码蛋白长度 Length of aa	serpin位置（aa） serpin domain（aa）	内含子数目Number of intron	定位Location
AaSRPN1	AAEL005670	XM_001651183	胰蛋白酶Trypsin	1 733	1~27	425	55~418	8	SuperContig supercont1.169: 334 419-358 419 reverse strand
AaSRPN2	AAEL005673	XM_001647962	胰蛋白酶Trypsin	1 344	1~17	447	69~445	8	SuperContig supercont1.1000: 73 345-93 470 forward strand
AaSRPN3	AAEL005665	XM_001651182	未知Unknown	1 631	1~24	412	52~410	8	SuperContig supercont1.169: 311 490-319 677 reverse strand
AaSRPN4A	AAEL013936	XM_001664084	胰凝乳蛋白酶Chymotrypsin	1 962	1~20	653	46~651	4	SuperContig supercont1.963: 225 626-270 242 reverse strand
AaSRPN4B		XM_001664083		1 836	1~20	611	46~609	4
AaSRPN4C		XM_001664081		1 602	1~20	533	46~531	4
AaSRPN4D		XM_001664082		1 611	1~20	536	46~534	4
AaSRPN5A	AAEL014141	XM_001648042	未知Unknown	1 764	1~27	465	46~463	4	SuperContig supercont1.1016: 284 132-291 292 forward strand
AaSRPN5B		XM_001648043		1 834	1~27	465	46~463	4
AaSRPN6	AAEL010769	XM_001654824	未知Unknown	987		328	1~326	3	SuperContig supercont1.507: 776-1 792 forward strand
AaSRPN7	AAEL002699	XM_001662132	胰蛋白酶Trypsin	1 146	1~18	381	33~380	3	SuperContig supercont1.65: 1 122 350-1 123 562 reverse strand
AaSRPN8	AAEL011777	XM_001661855	胰蛋白酶Trypsin	1 149		382	8~397	5	SuperContig supercont1.612: 384 333-397 593 forward strand
AaSRPN9	AAEL008364	XM_001653169	未知Unknown	2 302	1~21	423	53~422	5	SuperContig supercont1.320: 451 734-481 311 reverse strand
AaSRPN10A	AAEL007765	XM_001658592	胰蛋白酶Trypsin	1 161		386	18~377	5	SuperContig supercont1.279: 1 261 428-1 283 547 forward strand
AaSRPN10B		XM_001658594		1 122		373	18~373	5
AaSRPN10C		XM_001658591		1 131		376	18~375	5
AaSRPN10D		XM_001659593		1 140		379	18~376	5
AaSRPN11	AAEL003686	XM_001657054	胰凝乳蛋白酶Chymotrypsin	1 497	1~18	498	126~495	5	SuperContig supercont1.94: 409 162-424 050 forward strand
AaSRPN12	AAEL003653	XM_001657051 XM_001657052	未知Unknown	1 521		506	175~501	7	SuperContig supercont1.94: 346 859-356 370 forward strand
AaSRPN14	AAEL002731	XM_001662138	胰蛋白酶Trypsin	1 203	1~20	400	35~394	3	SuperContig supercont1.65: 1 233 407-1 234 680 reverse strand
AaSRPN16	AAEL014138	XM_001648088	未知Unknown	1 811	1~23	495	96~493		SuperContig supercont1.1016: 91 775-93 585 reverse strand
AaSRPN17	AAEL003697	XM_001657053	胰凝乳蛋白酶Chymotrypsin	1 023		340	2~338	3	SuperContig supercont1.94: 395 963-397 043 forward strand
AaSRPN19	AAEL006137	XM_001657462	Unknown	1 308		435	10~433	3	SuperContig supercont1.191: 446 239-451 917 forward strand
AaSRPN20	AAEL002720	XM_001662133	胰蛋白酶Trypsin	1 164	1~17	387	31~385	4	SuperContig supercont1.65: 1 149 985-1 171 765 forward strand
AaSRPN21	AAEL002730	XM_001662139	胰蛋白酶Trypsin	1 149	1~15	382	32~380	2	SuperContig supercont1.65: 1 247 238-1 248 443 forward strand
AaSRPN22	AAEL002715	XM_001662140	胰蛋白酶Trypsin	1 134		377	22~369	4	SuperContig supercont1.65: 1 277 021-1 287 800 forward strand
AaSRPN23	AAEL002704	XM_001662136 XM_001662137	胰蛋白酶Trypsin	1 254	1~19	417	39~395	4	SuperContig supercont1.65: 1 197 986-1 199 365 reverse strand
AaSRPN24	AAEL017249		未知Unknown	1 095	1~19	364	28~362	3	SuperContig supercont1.359: 63 896-65 053 reverse strand
AaSRPN25	AAEL007420	XM_001658312	未知Unknown	1 245	1~19	414	37~393	3	SuperContig supercont1.255: 1 148 690-1 150 060 forward strand
AaSRPN26	AAEL003182	XM_001656462	弹性蛋白酶Elastase	1 257	1~18	418	51~394	3	SuperContig supercont1.81: 289 404-290 790 reverse strand
AaSRPN27	AAEL014079	XM_001647961	胰蛋白酶Trypsin	1 344	1~17	447	78~455	8	SuperContig supercont1.169: 286 950-289 171 reverse strand
AaSRPN28	AAEL014078	XM_001651181	胰蛋白酶Trypsin	1 230	1~17	409	40~407	8	SuperContig supercont1.1000: 130 402-134 622 forward strand
AaSRPN29	AAEL014980	XM_001650108	未知Unknown	1 257	1~18	418	52~410	3	SuperContig supercont1.1378: 18-5 781 reverse strand

表2

图1

图2

图3

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