中国农业科学

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异色瓢虫丙酮酸激酶基因序列分析与调控海藻糖代谢功能

葛欣竺,史宇星,王莎莎,刘智慧,蔡文杰,周敏,王世贵,唐斌   

  1. 杭州师范大学生命与环境科学学院,杭州311121
  • 出版日期:2021-07-19 发布日期:2021-07-19

Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Regulation of Trehalose Metabolism

GE XinZhu, SHI YuXing, WANG ShaSha, LIU ZhiHui, CAI WenJie, ZHOU Min, WANG ShiGui, TANG Bin   

  1. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121
  • Published:2021-07-19 Online:2021-07-19

摘要: 【目的】丙酮酸激酶(PYK)是一种调节性糖酵解酶,负责调节糖酵解和糖异生之间的平衡,所以具有重要的生理作用。本研究旨在通过分析异色瓢虫(Harmonia axyridis3条HaPYK的序列结构,并结合RNA干扰技术,探讨HaPYK在瓢虫体内的生物学功能。【方法】基于从异色瓢虫全基因组中获得的3条HaPYK序列(被分别命名为HaPYK1-1HaPYK1-2HaPYK2),通过生物信息学方法分析其理化性质和序列结构,以及与其他昆虫之间的同源性。然后采用显微注射的方法将体外合成的dsRNA导入羽化第1天成虫体内,在注射后48和72 h分别收集试验材料,提取虫体总RNA后进行反转录,随后采用实时荧光定量PCR技术测定3个HaPYK的表达水平以评估RNA干扰效果,并检测与海藻糖代谢相关基因的表达水平,最后检测葡萄糖、糖原、海藻糖含量以及两种海藻糖酶的活性。【结果】HaPYK1-1HaPYK1-2HaPYK2的开放阅读框分别为1 350 、1 569 、1 656 bp;蛋白大小分别为449、522、551 aa,理论等电点分别为5.04、5.02、5.01。保守结构域预测得到,三条HaPYK均属于Pyruvate_kinase超家族。HaPYK二级结构主要是α-螺旋和无规则卷曲,另外也含有延伸链和β-转角。三条序列的寡聚物类型均为同源四聚体。系统进化树显示,HaPYK与同为鞘翅目的昆虫具有较近的亲缘关系,如马铃薯甲虫(Leptinotarsa decemlineata)和赤拟谷盗(Tribolium castaneum)。另外,注射dsHaPYK1-1后,HaPYK1-2HaTRE1-4的表达量显著下调,而HaPYK2HaTRE1-1HaTRE1-2HaTRE2-likeHaTPS的表达量显著上调;注射dsHaPYK1-2后,HaTRE1-1、HaTRE1-2HaTRE1-3HaTRE1-5HaTRE2HaTPS的表达量显著增加;注射dsHaPYK2后,仅HaTRE1-148 h表现为显著上调,HaTRE1-3HaTRE1-4HaTRE1-5HaTRE2HaTRE2-like的表达量均下调。与对照组相比,在dsHaPYK1-2组中48 h时TRE1活性显著降低,海藻糖和糖原含量显著增加;在dsHaPYK1-1dsHaPYK248 h时葡萄糖含量减少,72 h时葡萄糖、海藻糖含量增加及TRE2的酶活显著提高;在dsHaPYK248 h时,海藻糖显著减少,而糖原含量显著增加。【结论】异色瓢虫体内存在3个HaPYK,通过导入外源dsHaPYK能成功干扰靶标基因的表达水平,且抑制表达后海藻糖代谢会受到影响,但3个HaPYK的调控机制不尽相同。

关键词: 异色瓢虫, 生物信息学, 丙酮酸激酶, 海藻糖代谢, RNA干扰

Abstract: 【ObjectivePyruvate kinase (PYK) is a glycolytic enzyme that regulates the balance between glycolysis and gluconeogenesis, so it has an important physiological role in insects. This study aims to explore the biological functions of HaPYK in Harmonia axyridis by analyzing the sequence structure of the three HaPYKs and RNA interference technology. MethodBased on the three HaPYK sequences obtained from the whole genome of H. axyridis (named HaPYK1-1, HaPYK1-2, HaPYK2, respectively), we analyzed its physicochemical properties, sequence structure and homology with other insects through bioinformatics knowledge. Then, the dsRNA synthesized in vitro was injected into the adult on the first day of emergence by microinjection. The experimental materials were collected 48 and 72 h after injection, and the total RNA extraction and reverse transcription experiments were performed. In order to evaluate the inhibitory effect, we used real-time fluorescent quantitative PCR technology to determine the expression levels of three HaPYKs. Finally, the expression levels of genes related to trehalose metabolism, the content of glucose, glycogen, trehalose, and the activities of two trehalase enzymes were detected. 【Result】The experimental data showed that the open reading frames of HaPYK1-1, HaPYK1-2 and HaPYK2 were 1 350, 1 569 and 1 656 bp, respectively. The protein sizes were 449, 522 and 551 aa, and the theoretical isoelectric points were 5.04, 5.02 and 5.01, respectively. The conserved domains predicted that the three HaPYKs belonged to the pyruvate_kinase superfamily. The secondary structure of HaPYK was mainly α-helix and random coil, but also contained extended chain and β-corner. The oligomer types of the three sequences were all homologous tetramer. The phylogenetic tree showed that HaPYK was closely related to the same coleoptera insects, such as Leptinotarsa decemlineata and Tribolium castaneum. In addition, after injection of dsHaPYK1-1, the expression levels of HaPYK1-2 and HaTRE1-4 were significantly down-regulated, while the expression levels of HaPYK2, HaTRE1-1, HaTRE1-2, HaTRE2-like, and HaTPS were significantly up-regulated; after injection of dsHaPYK1-2, the expression of HaTRE1-1, HaTRE1-2, HaTRE1-3, HaTRE1-5, HaTRE2, HaTPS increased significantly; after injection of dsHaPYK2, only HaTRE1-1 was significantly up-regulated at 48 h, and the expression levels of HaTRE1-3, HaTRE1-4, HaTRE1-5, HaTRE2, HaTRE2-like were down-regulated. Compared with the control group, in the dsHaPYK1-2 group, the activity of TRE1 reduced significantly at 48 h, and the content of trehalose and glycogen was increased significantly; in dsHaPYK1-1 or dsHaPYK2, the glucose content decreased at 48 h, the glucose and trehalose content increased at 72 h, and the enzyme activity of TRE2 was increased significantly; at 48 h in dsHaPYK2 group, trehalose significantly reduced, but a significant increase in glycogen content.ConclusionThere are three HaPYKs in H. axyridis. By injecting exogenous dsHaPYK, the expression level of target genes can be successfully reduced. Suppressing their expression levels could affect the metabolism of trehalose, and the regulatory mechanisms of the three HaPYKs are not the same. The above results provide theoretical basis and help for further research on the energy metabolism of H. axyridis and other biological functions of HaPYK in H. axyridis.

Key words: Harmonia axyridisbioinformaticspyruvate kinase (PYK), trehalose metabolism; RNA interference (RNAi)