异色瓢虫丙酮酸激酶基因序列分析及其调控海藻糖代谢功能

doi:10.3864/j.issn.0578-1752.2021.23.008

摘要/Abstract

摘要：

【目的】丙酮酸激酶（pyruvate kinase,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,具有重要的生理作用。本研究通过分析异色瓢虫（Harmonia axyridis）3条HaPYK的序列结构,并结合RNA干扰（RNAi）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HaPYK1-1、HaPYK1-2、HaPYK2）,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（qRT-PCR）技术测定3个HaPYK的表达水平以评估RNAi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】HaPYK1-1、HaPYK1-2、HaPYK2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。保守结构域预测表明3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。3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）。注射dsHaPYK1-1后,HaPYK1-2和HaTRE1-4的表达量显著下调,而HaPYK2、HaTRE1-1、HaTRE1-2、HaTRE2-like、HaTPS的表达量显著上调;注射dsHaPYK1-2后,HaTRE1-1、HaTRE1-2、HaTRE1-3、HaTRE1-5、HaTRE2、HaTPS的表达量显著上调;注射dsHaPYK2后,仅HaTRE1-1表达量在48 h表现为显著上调,HaTRE1-3、HaTRE1-4、HaTRE1-5、HaTRE2、HaTRE2-like的表达量均下调。与对照组相比,在dsHaPYK1-2组中48 h时可溶性海藻糖酶（TRE1）活性显著降低,海藻糖和糖原含量显著增加;在dsHaPYK1-1或dsHaPYK2组中48 h时葡萄糖含量减少,72 h时葡萄糖、海藻糖含量增加,膜结合型海藻糖酶（TRE2）活性显著提高;在dsHaPYK2组48 h时,海藻糖含量显著减少,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:

【Objective】Pyruvate kinase (PYK) is a regulatory 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.【Method】Based on the three HaPYK sequences (named HaPYK1-1, HaPYK1-2, HaPYK2, respectively) obtained from the whole genome of H. axyridis, the physicochemical properties, sequence structure and homology with other insects through bioinformatics knowledge were analyzed. Then, the dsRNA synthesized in vitro was injected into the adult on the 1st 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, real-time fluorescent quantitative PCR (qRT-PCR) technology was used 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 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. 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 levels of HaTRE1-1, HaTRE1-2, HaTRE1-3, HaTRE1-5, HaTRE2, and 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 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 increased significantly; at 48 h in dsHaPYK2 group, the trehalose content significantly reduced, while glycogen content significantly increased.【Conclusion】There are three HaPYKs in H. axyridis. By injecting exogenous dsHaPYK, the expression level of target genes can be successfully reduced. Suppressing their expression levels can affect the metabolism of trehalose, and the regulatory mechanisms of the three HaPYKs are not the same.

Key words: Harmonia axyridis, bioinformatics, pyruvate kinase (PYK), trehalose metabolism, RNA interference (RNAi)

葛欣竺,史宇星,王莎莎,刘智慧,蔡文杰,周敏,王世贵,唐斌. 异色瓢虫丙酮酸激酶基因序列分析及其调控海藻糖代谢功能[J]. 中国农业科学, 2021, 54(23): 5021-5031.

GE XinZhu,SHI YuXing,WANG ShaSha,LIU ZhiHui,CAI WenJie,ZHOU Min,WANG ShiGui,TANG Bin. Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Its Regulation of Trehalose Metabolism[J]. Scientia Agricultura Sinica, 2021, 54(23): 5021-5031.

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引物名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Application
dsHaPYK1-1	ACATGGTCTTCGCCTCCTTCA	TGCAGCCAGACACTTAGCAGA	合成dsRNA dsRNA synthesis
dsHaPYK1-2	ACAAGGGACGCTGACACTGT	TCAAGTGTGCAGATCGTCCAGT
dsHaPYK2	ATAGTAACGTCGCGGTCACT	GATCCTCCTTTGATGCGCTG
dsGFP	AAGGGCGAGGAGCTGTTCACCG	CAGCAGGACCATGTGATCGCGC
dsHaPYK1-1-T7	T7-ACATGGTCTTCGCCTCCTTCA	T7-TGCAGCCAGACACTTAGCAGA
dsHaPYK1-2-T7	T7-ACAAGGGACGCTGACACTGT	T7-TCAAGTGTGCAGATCGTCCAGT
dsHaPYK2-T7	T7-ATAGTAACGTCGCGGTCACT	T7-ATAGTAACGTCGCGGTCACT
dsGFP-T7	T7-AAGGGCGAGGAGCTGTTCACCG	T7-CAGCAGGACCATGTGATCGCGC
qHaPYK1-1	AGTTGACCACAGACAAGGCT	AGACAGCAGGTAGATCGACG	qRT-PCR
qHaPYK1-2	CTGGACGATCTGCACACTTG	CACGAGCGTTCACATCTTGT
qHaPYK2	GCAAACTTGTTCAGCCAGGA	GCAGCCTGATGTTTCTCTCG
qHaTRE1-1	CTTCGCCAGTCAAATCGTCA	CCGTTTGGGACATTCCAGAT
qHaTRE1-2	TGACAACTTCCAACCTGGTAATG	TTCCTTCGAGACATCTGGCTTA
qHaTRE1-3	ACAGTCCCTCAGAATCTATCGTC	GGAGCCAAGTCTCAAGCTCATC
qHaTRE1-4	TTACTGCCAGTTTGATGACCAT	CATTTCGCTAATCAGAAGACCCT
qHaTRE1-5	TGATGATGAGGTACGACGAGA	GTAGCAAGGACCTAACAAACTG
qHaTRE2-like	TTCCAGGTGGGAGATTCAGG	GGGATCAATGTAGGAGGCTGTG
qHaTRE2	CAATCAGGGTGCTGTAATGTCG	CGTAGTTGGCTCATTCGTTTCC
qHaTPS	GACCCTGACGAAGCCATACC	AAAGTTCCATTACACGCAC
qrp49	GCGATCGCTATGGAAAACTC	TACGATTTTGCATCAACAGT

基因 Gene	登录号 Accession number	开放阅读框长度 ORF length (bp)	分子量 Molecular weight (Da)	理论等电点Theoretical pI	大小 Size (aa)	原子总数 Total number of atoms	脂肪系数 Aliphatic index	不稳定系数 Instability index
HaPYK1-1	MZ327965	1350	110346.47	5.04	449	14206	30.52	36.04
HaPYK1-2	MZ327966	1569	127708.83	5.02	522	16470	32.63	41.01
HaPYK2	MZ327967	1656	134975.87	5.01	551	17416	32.55	38.92