Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (15): 3108-3119.doi: 10.3864/j.issn.0578-1752.2020.15.011

• PLANT PROTECTION • Previous Articles     Next Articles

Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera

ZHANG DaoWei1(),KANG Kui1,YU YaYa2,KUANG FuPing1,PAN BiYing3,CHEN Jing2,TANG Bin1,3()   

  1. 1College of Biology and Agriculture, Zunyi Normal University/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, Zunyi 563006, Guizhou
    2School of Basic Medical Science, Zunyi Medical University, Zunyi 563006, Guizhou
    3College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
  • Received:2020-01-16 Accepted:2020-03-13 Online:2020-08-01 Published:2020-08-06
  • Contact: Bin TANG E-mail:zhangdw1000@163.com;tbzm611@163.com

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Abstract:

【Objective】Phenoloxidase (PO) is an immune protein and plays an important regulatory role in insects. It exists as the form of inactive prophenoloxidase (PPO). In this study, based on the three PPO sequences of Sogatella furcifera by transcriptome sequencing, the biological functions of SfPPOs were further explored by analyzing the developmental and tissue expression patterns of different SfPPOs, suppressing the expression of SfPPOs and inducing by pathogenic bacteria.【Method】Taking three SfPPO sequences as research objects, the protein structure and homology with other insects were analyzed by bioinformatics method. In addition, dsRNA injected with green fluorescent protein (GFP) was used as a control group, and the inhibitory effect of target gene was detected by quantitative real-time PCR (qRT-PCR) after injecting with dsSfPPO. In order to investigate the role of SfPPO in the growth and immune response of S. furcifera, qRT-PCR was used to detect the expression of SfPPO at different developmental stages and in different tissues, as well as the induced expression of three SfPPOs after injecting with Escherichia coli and Bacillus subtilis.【Result】The open reading frames (ORF) of SfPPO1, SfPPO2-1, and SfPPO2-2 are 2 079, 999, and 2 070 bp in length, respectively, which encoding 692, 332, and 689 amino acids, respectively. The predicted protein molecular weights are 79.84, 37.67, and 79.53 kD, and the isoelectric points are 6.43, 9.56, and 6.20, respectively. Bioinformatics analysis showed that the three PPO proteins of S. furcifera had high homology and were closely related to Nilaparvata lugens. SfPPO1, SfPPO2-1 and SfPPO2-2 were all highly expressed on the 3rd day of adult, and the developmental expression patterns of SfPPO2-1 and SfPPO2-2 were similar. The tissue expression results showed that SfPPO1 and SfPPO2-1 were highly expressed in the epidermis and fat body, while SfPPO2-2 was highly expressed in the wing and fat body, and the expressions of all the three genes were relatively low in head, foot, midgut and Malpighian tubule. Compared with the dsGFP group, injecting with the dsRNA of target gene could significantly silence the expression of target gene, and when the SfPPO2-1 expression was silenced, SfPPO1 appeared to be significantly overexpressed, indicating that there might be compensatory function among different PPOs. The expression levels of SfPPO2-1 and SfPPO2-2 were significantly increased after 12 h of induction with E. coli, while the expression of SfPPO1 was significantly increased after 24 h of induction. In addition, the expression levels of SfPPO1, SfPPO2-1 and SfPPO2-2 were all significantly increased after 24 h of induction with B. subtilis.【Conclusion】The expression of SfPPO1, SfPPO2-1 and SfPPO2-2 has tissue and development specificity, and there are differences in immune response under the induction of different pathogenic bacteria. The results are helpful to explore the potential molecular mechanism of phenoloxidase in insect development and immunity.

Key words: Sogatella furcifera, prophenoloxidase (PPO), RNA interference (RNAi), quantitative real-time PCR (qRT-PCR), inducible expression, immune response

Table 1

Primers used for qRT-PCR and primers of dsPPOs and dsGFP"

引物名称
Primer name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)		 产物长度
Length (bp)
qSf18s CGCTACTACCGATTGAA GGAAACCTTGTTACGACTT 165
qSfPPO1 GACTCTGCCTCCCTACACC AGCGAGCGAACACGTTGC 176
dsSfPPO1 TGGGCATCAACCTACATCA TGCTAACAGCCTCGTACAAAC 360
dsSfPPO1-T7 T7-TGGGCATCAACCTACATCA T7-TGCTAACAGCCTCGTACAAAC 410
qSfPPO2-1 AGAACTCACCTCGCAACCA CTGAAGATGCTGCACCCTAG 116
dsSfPPO2-1 AGCATCTTCAGCATCAGCCTTTC T7-GGCATTCCCTCGGCGTTT 361
dsSfPPO2-1-T7 T7-AGCATCTTCAGCATCAGCCTTTC GGCATTCCCTCGGCGTTT 411
qSfPPO2-2 TGCTCGCATCACTCATCT ACAAATCTGTCCACCTCAA 177
dsSfPPO2-2 GGCATCAACCTACACCAT CCCTGCGTATTTATCACC 383
dsSfPPO2-2-T7 T7-GGCATCAACCTACACCAT T7-CCCTGCGTATTTATCACC 433
dsSfGFP AAGGGCGAGGAGCTGTTCACCG CAGCAGGACCATGTGATCGCGC
dsSfGFP-T7 T7-AAGGGCGAGGAGCTGTTCACCG T7-CAGCAGGACCATGTGATCGCGC

Fig. 1

Nucleotide and amino acid sequences of SfPPO Initiation and termination codons are indicated in bold and underlined. The nucleotide sequences reported in this study have been submitted to GenBank (Accession numbers: MN895440, MN895441, and MN895442)。A: SfPPO1; B: SfPPO2-1; C: SfPPO2-2"

Fig. 2

Phylogenetic tree of SfPPOs and PPO proteins from other insect species based on the amino acid sequence (neighbor-joining method)"

Fig. 3

Relative expression of SfPPOs at different developmental stages of S. furcifera Different lowercases on the bars indicate significant differences (P<0.05)。 The same as Fig. 4"

Fig. 4

Relative expression of SfPPOs in different tissues of S. furcifera"

Fig. 5

Relative expression of SfPPOs after injection for 48 h and 72 h"

Fig. 6

Response of SfPPOs after bacteria induction"

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