家蚕RNAi效率相关核酸酶基因对RNAi效率的影响

doi:10.16380/j.kcxb.2023.03.004

昆虫学报 ›› 2023, Vol. 66 ›› Issue (3): 303-311.doi: 10.16380/j.kcxb.2023.03.004

家蚕RNAi效率相关核酸酶基因对RNAi效率的影响

陈咏琪¹, 尹延萍¹, 冯嘉伟¹, 白新宇¹, 李庆荣², 钟仰进¹, 杨婉莹^1,*

(1. 华南农业大学动物科学学院, 广东省农业动物基因组学与分子育种重点实验室, 广州 510642; 2. 广东省农业科学院蚕业与农产品加工研究所, 广州 510610)

出版日期:2023-03-20 发布日期:2023-04-23

Effects of RNAi efficiency-related nuclease gene on RNAi efficiency in Bombyx mori

CHEN Yong-Qi¹, YIN Yan-Ping¹, FENG Jia-Wei¹, BAI Xin-Yu¹, LI Qing-Rong², ZHONG Yang-Jin¹, YANG Wan-Ying^1,*

(1. Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science of South China Agricultural University, Guangzhou 510642, China; 2. Institute of Sericulture and Processing of Agricultural Products, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China)

Online:2023-03-20 Published:2023-04-23

摘要/Abstract

摘要： 【目的】鳞翅目(Lepidoptera)昆虫消化系统存在的核酸酶是导致RNAi低效的主要原因之一，本研究旨在探索家蚕Bombyx mori RNAi效率相关核酸酶(RNAi efficiency-related nuclease, REase)BmREase对家蚕RNAi低效的影响。【方法】利用RT-PCR对家蚕BmREase cDNA全长序列进行同源克隆和生物信息学分析，并采用最大似然法进行系统发育分析；利用qRT-PCR检测BmREase在游走期家蚕不同组织(头、表皮、脂肪体、中肠、气管、马氏管和丝腺)中的特异性表达。通过向游走期家蚕注射BmREase, 家蚕蜕皮激素受体(ecdysone receptor, EcR)基因BmEcR, 超气门蛋白(ultraspiracle, USP)基因BmUSP和细胞因子sp-tzle1(BmSpz1)基因BmSpz1的dsRNA进行RNAi，分析干扰BmREase表达后是否可以提高靶基因dsRNAs的干扰效率。【结果】RT-PCR扩增得到家蚕BmREase(GenBank登录号: XM_021350017.2)全长cDNA序列，其ORF长2 241 bp，编码746个氨基酸残基；生物信息学分析发现，BmREase与人核酸外切酶I 3qe9.1具有极其相似的结构，其Thr7, His33, Ala37, Arg93, Lys97, Tyr159, Asp160, Ser161和Asn174组成的活性结构域能够与核苷酸序列结合，暗示BmREase具有核酸酶活性。qRT-PCR结果显示，BmREase在家蚕游走期的中肠和马氏管中高表达，说明BmREase主要在家蚕消化系统中表达。在家蚕游走期注射dsRNA，BmREase的表达量比空白对照组的高，RNAi干扰BmREase提高了dsBmEcR, dsBmUSP和dsBmSpz1的干扰效率。【结论】家蚕体内存在与人类核酸外切酶相似功能的酶BmREase，其存在可能影响dsRNA在家蚕体内的干扰效果。本研究为利用RNAi进行家蚕基因功能的研究以及进一步开发RNAi防治害虫具有指导性意义。

关键词: 家蚕, RNA干扰, dsRNA, RNAi效率相关核酸酶, BmREase, dsRNA外切酶

Abstract: 【Aim】 The nucleases in the digestive system of Lepidoptera are one of the main reasons for the low efficiency of RNAi. This study aims to explore the effects of RNAi efficiency-related nuclease (REase) of Bombyx mori BmREase on the low efficiency of RNAi in B. mori. 【Methods】 The full-length cDNA sequence of BmREase was homologously cloned from B. mori by RT-PCR and bioinformatically analyzed, and phylogenetic analysis was carried out by maximum likelihood method. qRT-PCR was used to detect the specific expression of BmREase in different tissues (head, cuticle, fat body, midgut, trachea, Malpighian tubules and silk gland) of B. mori at the wandering stage. Furthermore, by injecting the dsRNAs of BmREase, ecdysone receptor (EcR) gene BmEcR, ultraspiracle (USP) gene BmUSP and cytokine sp-tzle1 (Spz1) gene BmSpz1 of B. mori into the wandering B. mori for RNAi, we analyzed whether interferring the expression of BmREase can improve the interference efficiency of dsRNAs of target genes.【Results】The full-length cDNA sequence of BmREase (GenBank accession no.: XM_021350017.2) of B. mori was obtained by RT-PCR amplification. The open reading frame of BmREase is 2 241 bp in length, encoding 746 amino acid residues. Bioinformatics analysis showed that BmREase has a very similar structure to human exonuclease I 3qe9.1, and the active domain composed of Thr7, His33, Ala37, Arg93, Lys97, Tyr159, Asp160, Ser161 and Asn174 can bind to the nucleic acid sequence, indicating that BmREase has nuclease activity. qRT-PCR results showed that BmREase was highly expressed in the wandering midgut and Malpighian tubules of B. mori, indicating that the nuclease mainly exists in the digestive system of B. mori. The expression level of BmREase in the wandering B. mori injected with dsRNA was higher than that of the blank control group. When BmREase was interfered by RNAi, the interference efficiency of dsBmEcR, dsBmUSP and dsBmSpz1 were increased.【Conclusion】 BmREase, which has a similar function to human exonuclease, may affect the interference efficiency of dsRNA in B. mori. This study is instructive for using RNAi to study the gene function of B. mori and to further develop RNAi for pest control.

Key words: Bombyx mori, RNA interference, dsRNA, RNAi efficiency-related nuclease; BmREase, dsRNA exonuclease

陈咏琪, 尹延萍, 冯嘉伟, 白新宇, 李庆荣, 钟仰进, 杨婉莹. 家蚕RNAi效率相关核酸酶基因对RNAi效率的影响[J]. 昆虫学报, 2023, 66(3): 303-311.

CHEN Yong-Qi, YIN Yan-Ping, FENG Jia-Wei, BAI Xin-Yu, LI Qing-Rong, ZHONG Yang-Jin, YANG Wan-Ying. Effects of RNAi efficiency-related nuclease gene on RNAi efficiency in Bombyx mori[J]. Acta Entomologica Sinica, 2023, 66(3): 303-311.

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家蚕RNAi效率相关核酸酶基因对RNAi效率的影响

Effects of RNAi efficiency-related nuclease gene on RNAi efficiency in Bombyx mori

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