白纹伊蚊胰岛素样肽基因的鉴定和时空表达分析

doi:10.11853/j.issn.1003.8280.2023.02.010

摘要/Abstract

摘要： 目的筛选并鉴定白纹伊蚊全基因组中的胰岛素样肽（insulin-like peptides，ILP）基因，分析其基因结构以及在不同发育时期和不同组织的表达谱。方法基于埃及伊蚊ILPs家族成员序列，在Vectorbase网站上的白纹伊蚊全基因组数据库做同源搜索，SignalP 6.0 进行信号肽预测，MEGA 11.0软件包对ILPs基因结构特点进行分析，最大似然法构建系统进化树。实时荧光定量PCR（qRT-PCR）检测不同发育时期（卵、4龄幼虫、蛹、成蚊），以及吸血前后雌蚊不同组织（头部、脂肪体、中肠、胸部、卵巢）的白纹伊蚊胰岛素样肽（insulin-like peptides of Aedes albopictus，AalbILPs）基因表达变化。采用SPSS 20.0软件做数据统计分析，Tukey’s HSD检验和t检验分析不同发育时期和吸血前后不同组织中AalbILPs基因的表达差异。结果在白纹伊蚊全基因组中获得7条ILPs开放阅读框序列。7条AalbILPs序列均具有胰岛素超家族的保守特征，由连续的信号肽、B、C和A链组成前肽。其中AalbILP6具有截短的C链和羧基末端延伸，类似脊椎动物的胰岛素生长因子。系统进化树显示，AalbILP1、2、3和5在蚊虫中最为保守，AalbILP6其次，而AalbILP4、7为伊蚊所特有。qRT-PCR结果显示，AalbILP1、2、3和5在不同发育阶段均有表达。AalbILP5在雄蚊中相对表达量为1.358 3±0.576 9，与其他发育阶段和雌蚊相比最高（q_卵=6.572，q_幼虫=5.771，q_蛹=5.409，q_雌蚊=3.115，均P＜0.05）。AalbILP3和4在成蚊头部特异性表达，AalbILP6主要在雌蚊卵巢表达；AalbILP7为假基因，未转录。与未吸血蚊虫相比，吸血诱导AalbILP3和4在雌蚊头部的表达分别上调2.60和1.68倍（t_AalbILP3-_头部=9.596，P_AalbILP3-_头部＜0.001；t_AalbILP4-_头部=4.524，P_AalbILP4-_头部=0.001）；AalbILP1在头部、脂肪体以及中肠的表达分别上调10.33、6.07和3.79倍（t_头部=4.255，P_头部=0.001；t_脂肪体=4.305，P_脂肪体=0.001；t_中肠=10.480，P_中肠＜0.001），胸部和卵巢的表达分别下调4.24和2.17倍（t_胸部=7.922，P_胸部＜0.001；t_卵巢=3.752，P_卵巢=0.003）；AalbILP6在雌蚊中肠和卵巢的相对表达量分别上调11.91和2.16倍（t_中肠=5.799，P_中肠＜0.001；t_卵巢=9.074，P_卵巢＜0.001）。结论在白纹伊蚊全基因组中共获得6条ILPs序列，并成功构建其时空表达谱。

关键词: 白纹伊蚊, 胰岛素样肽, 时空表达, 血餐

Abstract: Objective To identify the insulin-like peptide (ILP) genes from the whole genome of Aedes albopictus, and to analyze the gene structure and its expression profiles at various developmental stages and in different tissues. Methods The ILP gene family was identified by homology comparison in the whole genome database of Ae. albopictus on the Vectorbase website. Signal peptide prediction was performed by SignalP 6.0 software. The structural features of ILP genes were analyzed by MEGA 11.0 software. The phylogenetic tree was constructed by the maximum likelihood method. Quantitative real-time PCR (qRT-PCR) was used to detect the changes in ILP gene expression at different mosquito developmental stages (eggs, fourth instar larvae, pupae, and adults) and in different tissues (head, fat body, midgut, thorax, and ovary) of female mosquitoes before and after blood feeding. SPSS 20.0 software was used for statistical analysis. The Tukey’s HSD test was used to analyze the expression of AalbILPs at different developmental stages, and the t-test was used to compare the expression of AalbILPs in different tissues before and after blood feeding. Results Seven AalbILP open reading frame sequences were identified from the whole genome of Ae. albopictus. The seven AalbILP sequences had the conserved characteristics of the insulin superfamily, and the propeptide consisted of continuous signal peptides, B, C, and A chains; AalbILP6 had a truncated C chain and carboxy-terminal extension, similar to the insulin growth factor in vertebrates. The clustering evolutionary tree demonstrated that AalbILP1, 2, 3, and 5 were most conservative among mosquitoes, followed by AalbILP6, and both AalbILP4 and 7 were unique to Aedes. The results of qRT-PCR showed that AalbILP1, 2, 3, and 5 were expressed at different developmental stages. Compared with other developmental stages, AalbILP5 was the highest expressed in male adult mosquitoes (1.358 3±0.576 9; q_egg=6.572, q_larva=5.771, q_pupa=5.409, q_female=3.115, all P<0.05). AalbILP 3 and 4 were specifically expressed in the head of the whole mosquito, and AalbILP6 was principal expressed in the ovaries of female adult mosquitoes. AalbILP7 is a pseudogene and is not transcribed. Compared to the non-blood mosquitoes, the expression level of AalbILP3 and AalbILP4 in the head of female mosquitoes were significantly upregulated by 2.60 and 1.68 times (t_{AalbILP3- head}=9.596, P_{AalbILP3- head}<0.001; t_{AalbILP4-head}=4.524, P_{AalbILP4-head}=0.001); The expression of AalbILP1 in the head, fat body and midgut was up-regulated by 10.33, 6.07, and 3.79 times（t_head=4.255, P_head=0.001; t_{fat body}=4.305, P_{fat body}=0.001; t_migut=10.480, P_migut<0.001）, but the expression of throax and ovary decreased 4.24 and 2.17 times （t_throax=7.922, P_throax<0.001; t_ovary=3.752, P_ovary=0.003）; The expression of AalbILP6 in midgut and ovary was up-regulated 11.91 and 2.16 times (t_migut=5.799, P_migut<0.001; t_ovary=9.074, P_ovary<0.001). Conclusions Six ILP sequences have been identified in the whole genome of Ae. albopictus and the spatiotemporal expression profiles of ILPs have been constructed.

Key words: Aedes albopictus, Insulin-like peptide, Spatiotemporal expression, Blood meal

中图分类号:

R384.1

肖秋秋, 李威仪, 何珊, 程金芝, 彭哲慧, 吴家红. 白纹伊蚊胰岛素样肽基因的鉴定和时空表达分析[J]. 中国媒介生物学及控制杂志, 2023, 34(2): 204-211.

XIAO Qiu-qiu, LI Wei-yi, HE Shan, CHENG Jin-zhi, PENG Zhe-hui, WU Jia-hong. Identification and spatiotemporal expression of the insulin-like peptide genes in Aedes albopictus[J]. Chinese Journal of Vector Biology and Control, 2023, 34(2): 204-211.

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