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飞蝗载脂蛋白受体基因LmLPR的表达及功能分析
Expression and functional analysis of the Locusta migratoria lipophorin receptor gene, LmLPR
赵艺妍;赵小明;杨 洋;马恩波;张建珍;刘卫敏
点击:1269次 下载:51次
DOI:10.7679/j.issn.2095-1353.2020.034
作者单位:山西大学应用生物学研究所,太原 030006;山西大学生命科学学院,太原 030006
中文关键词:飞蝗;载脂蛋白受体;LmLPR;表皮
英文关键词:Locusta migratoria; lipophorin receptor; LmLPR; integument
中文摘要:

【目的】 载脂蛋白受体(Lipophorin receptor,LPR)在昆虫体内脂类物质的转运中发挥着重要的作用。基于飞蝗转录组数据库获得飞蝗载脂蛋白受体基因LmLPR,分析其基因特性和表达特征,并通过RNA干扰(RNA interference,RNAi)技术对其生物学功能进行分析,探究LmLPR在飞蝗脂类物质转运中的作用。【方法】 基于课题组飞蝗转录组数据库,获得载脂蛋白受体基因,结合飞蝗基因组数据库,获得其全长开放阅读框(ORF)序列,克隆验证后对其编码蛋白序列特性进行分析;利用GENEDOC软件将该蛋白氨基酸序列与其他物种LPR氨基酸序列进行多序列比对,并使用MEGA 5.1软件中的Neighbor-Joining方法,将该序列与其它物种的同源序列进行聚类分析;通过RT-qPCR方法对其在5龄第2天飞蝗不同组织部位和不同发育天数表皮中的表达特性进行分析;通过RNAi技术及伊红染色方法对其生物学功能进行分析。【结果】 通过克隆和序列分析获得两种飞蝗LPR基因,即长型(LmLPR-L)和短型(LmLPR-S),序列特征分析发现LmLPR-L和LmLPR-S均有1个信号肽,4个表皮生长因子结构域,5个低密度脂蛋白受体YWTD结构域,以及1个跨膜域。不同于LmLPR-L,LmLPR-S在N端比LmLPR-L多一个低密度脂蛋白受体结构域(LmLPR-L含有6个,LmLPR-S含有7个),而在C端缺失38个氨基酸序列。系统进化树分析显示LmLPR-L和LmLPR-S与蜚蠊目德国小蠊Blattella germanica BgLPR-L和BgLPR-S聚为一支。RT-qPCR结果显示LmLPR-S在卵巢中高表达,在其他组织部位的表达量较低,而LmLPR-L在翅芽和卵巢中均高表达,在其他组织部位的表达量较低;时期表达显示LmLPR-SLmLPR-L在蜕皮后表达量均呈现先升高后降低的趋势,分别在第2天和第3天的表达量最高。在4龄期通过RNAi抑制LmLPR的表达后,发现飞蝗能够正常蜕皮,伊红染色结果显示表皮通透性没有显著改变。【结论】 飞蝗载脂蛋白受体基因LmLPR主要在翅芽和卵巢中高表达,其下调表达后不影响若虫的正常生长发育以及表皮的通透性。研究结果为深入探究载脂蛋白受体在昆虫表皮脂类物质转运过程中的作用机制提供了基础。


英文摘要:

[Objectives]  The Lipophorin receptor (LPR) plays an important role in lipid transport in insects. The lipophorin receptor gene of Locusta migratoria (LmLPR) was obtained from the transcriptome database, and its characteristics and expression was analyzed. RNA interference (RNAi) technology was used to explore the role of LmLPR in lipid transport. [Methods]  Based on a transcriptome database from our lab, LmLPR and its full-length Open Reading Frame (ORF) sequence were obtained and compared with the L. migratoria genomic database. Characteristics of the encoded protein sequence were analyzed after cloning and verification. The amino acid sequence of this protein was aligned with LPR amino acid sequences of other species using GENEDOC software. A phylogenetic tree of the relationship between LmLPR and homologous sequences from other species was constructed using the Neighbor-Joining method in MEGA 5.1 software. The expression pattern in different tissues of 2-day-old fifth-instar nymphs and different developmental stages were analyzed with RT-qPCR. The biological function of LmLPR was analyzed with RNAi technology and eosin staining. [Results]  Two types of L. migratoria LPR, namely a long type (LmLPR-L) and a short type (LmLPR-S), were obtained by cloning and sequence analysis. Sequence analysis revealed that both LmLPR-L and LmLPR-S have one signal peptide, four epidermal growth factor domains, five low-density lipoprotein receptor YWTD domains, and one transmembrane domain. Whereas LmLPR-L has 6 low-density lipoprotein receptor domains at the N-terminus, LmLPR-S has 7. A 38 amino acid sequence was detected at the C-terminus. The phylogenetic tree showed that LmLPR-L and LmLPR-S clustered together with the homologous BgLPR-L and BgLPR-S from Blattella germanica. The results of RT-qPCR showed that expression of LmLPR-S was high in ovarian tissue but low in other tested tissues. Expression of LmLPR-L was high in the wing pads and ovary but low in other tested tissues. The expression levels of LmLPR-S and LmLPR-L both first increased in the early stage of fifth instar, then decreased. Expression was highest on days 2 and 3 (N5D1 and N5D2), respectively. After silencing the expression of LmLPR by injecting dsLmLPR into 4th instar larvae, treated nymphs were able to complete the molt normally, as did the control group. The results of eosin staining found no evidence of significant change in cuticle permeability in both dsLmLPR and dsGFP-injected nymphs. [Conclusion]  The lipophorin receptor gene LmLPR is highly expressed in ovarian tissue, and down-regulating its expression did not affect the normal growth and development of nymphs, or the permeability of cuticle. These results provide a basis for further investigation of the role of the lipophorin receptor in the transport of insect cuticular lipids.

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