Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (23): 5021-5031.doi: 10.3864/j.issn.0578-1752.2021.23.008

• PLANT PROTECTION • Previous Articles     Next Articles

Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Its 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
  • Received:2021-06-02 Accepted:2021-07-02 Online:2021-12-01 Published:2021-12-06
  • Contact: Bin TANG E-mail:gexinzhu2019@163.com;tbzm611@hznu.edu.cn

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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)

Table 1

Primers used for dsRNA synthesis and real-time fluorescent quantitative PCR (qRT-PCR)"

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

Table 2

Basic physicochemical properties of HaPYK sequences"

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

Fig. 1

Prediction of conserved domain of HaPYK amino acid sequences"

Fig. 2

Prediction of the secondary structure of HaPYKs"

Fig. 3

Three-dimensional structure prediction of HaPYK proteins"

Fig. 4

Phylogenetic tree of HaPYK and pyruvate kinases from other insect species based on amino acid sequence"

Fig. 5

Relative mRNA expression levels of HaPYKs after RNAi Data were presented as mean±SE. * indicated significant differences (P<0.05), **indicated extremely significant differences (P<0.01). The same as below"

Fig. 6

Changes in the expression levels of genes related to the trehalose metabolism pathway of H. axyridis after RNAi"

Fig. 7

Changes in trehalase activities of H. axyridis after RNAi"

Fig. 8

Changes in carbohydrate content of H. axyridis after RNAi"

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