飞蝗载脂蛋白对卵巢发育和脂质沉积的影响

doi:10.3864/j.issn.0578-1752.2024.04.007

摘要/Abstract

摘要：

【背景】脂质是生物体重要营养素之一，对于昆虫滞育、飞行、胚胎发育和能量调节至关重要。载脂蛋白（apolipophorin，apoLp）是昆虫脂蛋白颗粒的蛋白质部分，主要负责组织之间脂质转运。【目的】以世界性农业害虫飞蝗（Locusta migratoria）为研究对象，通过RNA干扰（RNA interference，RNAi）技术对其载脂蛋白生物学功能进行分析，探究载脂蛋白在飞蝗卵巢脂质运输中的作用，为害虫防治提供新的分子靶标。【方法】利用RNAi技术，分别对羽化后第1天（1 PAE，post adult eclosion）的成虫2个载脂蛋白基因（LmapoLp-II/I和LmapoLp-III）进行dsRNA注射，以dsGFP为对照，每头注射15 μg dsRNA;分别解剖羽化后第4、6、8天的卵巢进行观察;取羽化后第8天的卵巢，以EF1α作为内参基因，采用实时荧光定量PCR（RT-qPCR）方法检测沉默效率;利用脂质组学技术测定并分析对照和沉默LmapoLp-II/I后飞蝗卵巢脂代谢物差异，采用OPLS-DA模型的差异倍数（FC）、P值和VIP值相结合的方法筛选差异脂质;制备冰冻切片，利用Bodipy染色技术对沉默LmapoLp-II/I后飞蝗卵巢中性脂质的含量和分布进行观察;利用甘油三酯（triglyceride，TG）测定试剂盒，对沉默LmapoLp-II/I后飞蝗卵巢总甘油三酯含量进行测定。【结果】与对照组相比，分别注射dsLmapoLp-II/I和dsLmapoLp-III后，卵巢中靶基因的表达量均可被显著抑制，沉默效率分别为80.84%和92.89%;注射dsLmapoLp-II/I的飞蝗卵巢发育迟缓，而注射dsLmapoLp-III的飞蝗卵巢与对照组相同，均能够正常发育逐渐变大，颜色由白色逐渐变黄。沉默LmapoLp-II/I后，脂质组学分析共检测到1 166种上调代谢物和1 384种下调代谢物，其中20种甘油三酯显著下调;此外，总甘油三酯测定及Bodipy染色结果显示，卵巢总甘油三酯含量和中性脂质含量均较对照组显著降低。【结论】飞蝗LmapoLp-II/I是影响卵巢发育的主要载脂蛋白基因，其参与卵巢脂质的积累和运输。研究结果可为基于脂质运输基因为靶标的害虫防治提供理论依据。

关键词: 飞蝗, 载脂蛋白, RNA干扰, 卵巢, 脂质运输

Abstract:

【Background】Lipids are one of the important nutrients required by organisms, which play important physiological functions, including diapause, flight, embryonic development and energy regulation in insects. Apolipophorins (apoLps) are the main components of insect lipophorin particles, which are involved in the transport of lipids among different tissues.【Objective】The objective of this study is to investigate the function of apolipophorins (LmapoLp-II/I and LmapoLp-III) by RNA interference (RNAi) in ovarian lipid transport in Locusta migratoria, and to provide a new molecular target for pest control.【Method】RNAi was performed to silence two apolipophorin genes (LmapoLp-II/I and LmapoLp-III) at 1 day post adult eclosion (1 PAE), respectively. dsGFP was used as control, 15 μg dsRNA was injected into each insect. The ovaries at 4, 6 and 8 PAE were dissected and observed after dsRNA injection. The silencing efficiency of LmapoLps in the ovaries at 8 PAE was analyzed by reverse-transcription quantitative PCR (RT-qPCR), and EF1α was used as internal reference gene. Lipidomics technique was used to determine and analyze the differences of lipid metabolites in the ovaries between dsGFP- and dsLmapoLp-II/I-treated groups. The approach combining the fold change, the P value and the VIP value of the OPLS-DA model was adopted to screen for differential abundance of lipids. The contents of neutral lipids and total triglycerides were determined by Bodipy staining and triglyceride assay kit.【Result】After injection of dsLmapoLp-II/I and dsLmapoLp-III, the expression level of target genes could be significantly silenced by 80.84% and 92.89%, respectively. RNAi-mediated silencing of LmapoLp-II/I led to a retarded ovarian development, with significantly decreased neutral lipid content and triglyceride content, while the ovaries of L. migratoria injected with dsLmapoLp-III were the same as those in the control group, which could develop normally and gradually grow larger, with the color gradually changing from white to yellow. After LmapoLp-II/I silencing, a total of 1 166 up-regulated metabolites and 1 384 down-regulated metabolites were detected by lipidomics analysis, of which 20 triglycerides were significantly down-regulated.【Conclusion】LmapoLp-II/I is a major apolipophorin gene that affects ovarian development and is involved in the accumulation and transport of ovarian lipids. These research results not only enrich the research content of lipid metabolism in insects, but also help to find new targets for pest control.

Key words: Locusta migratoria, apolipophorin, RNA interference (RNAi), ovary, lipid transport

赵艺妍, 郭红芳, 刘卫敏, 赵小明, 张建珍. 飞蝗载脂蛋白对卵巢发育和脂质沉积的影响[J]. 中国农业科学, 2024, 57(4): 711-720.

ZHAO YiYan, GUO HongFang, LIU WeiMin, ZHAO XiaoMing, ZHANG JianZhen. Effects of Apolipophorin on Ovarian Development and Lipid Deposition in Locusta migratoria[J]. Scientia Agricultura Sinica, 2024, 57(4): 711-720.

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用途 Application	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	产物长度 Product size (bp)
dsRNA合成 dsRNA synthesis	dsLmapoLp-II/I-F	taatacgactcactatagggCTCCAAGCAGAAGGTCGTG	460
	dsLmapoLp-II/I-R	taatacgactcactatagggCAGGCTGAGATGAGAATGT	460
	dsLmapoLp-III-F	taatacgactcactatagggGCTGAATCACACCATCGTCA	420
	dsLmapoLp-III-R	taatacgactcactatagggTGGATGGAGTTCTGCAGGTT	420
	dsGFP-F	taatacgactcactatagggGTGGAGAGGGTGAAGG	571
	dsGFP-R	taatacgactcactatagggGGGCAGATTGTGTGGAC	571
RT-qPCR分析 RT-qPCR analysis	LmapoLp-II/I-F	AGCGATTTCATCCGGTGGC	103
	LmapoLp-II/I-R	GGTGTATGTCTGTCCCTTTT	103
	LmapoLp-III-F	ACGCTGCTCGCAGTCCTC	83
	LmapoLp-III-R	ACCGCCTCCGCGATGTT	83
	EF1α-F	AGCCCAGGAGATGGGTAAAG	155
	EF1α-R	CTCTGTGGCCTGGAGCATC	155