Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (18): 3881-3891.doi: 10.3864/j.issn.0578-1752.2021.18.008

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Expression and Functional Analysis of SeDuox from Spodoptera exigua

FU ChaoRan1(),LI YaZi1,WU Han1,ZHAO Dan1(),GUO Wei1,2(),GUO XiaoChang1   

  1. 1College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
    2Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2021-01-04 Accepted:2021-02-07 Online:2021-09-16 Published:2021-09-26
  • Contact: Dan ZHAO,Wei GUO E-mail:1354598764@qq.com;zhaodan@hebau.edu.cn;guowei05@caas.cn

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Abstract:

【Objective】The production of reactive oxygen species (ROS) mediated by dual oxidase (Duox) is an important immune mechanism to regulate the dynamic balance of insect intestinal microorganisms. The objective of this study is to analyze the sequence characteristics and expression patterns of SeDuox from Spodoptera exigua, explore the effect of SeDuox silencing on the intestinal bacterial load of host by RNAi and analyze the immune response of SeDuox to Bacillus thuringiensis (Bt), so as to clarify the role of SeDuox in the intestinal immune regulation of insects, and provide new ideas and targets for the comprehensive control of S. exigua.【Method】The SeDuox was cloned by reverse transcription PCR (RT-PCR), and the sequence characteristics were analyzed by bioinformatics. The phylogenetic tree was constructed by ClustalX and MEGA_X software. The extramembrane fragment of SeDuox (91-1 767 bp) named SeDuox-OM was cloned by PCR. The Bacmid-SeDuox-OM was constructed using the Bac to Bac expression system and transfected into the Sf9 insect cells by liposome transfection to express SeDuox-OM. Real-time quantitative PCR (RT-qPCR) was used to analyze the expression level of SeDuox in different tissues (peritrophic membrane, hemolymph, fat body, Malpighian tubules, midgut, epidermis) and developmental stages (egg, 1st- to 5th-instar larvae, female pupa, male pupa). The 4th instar larvae were injected with dsSeDuox using RNAi technology for functional analysis, with dsGFP as control group. The efficiency of gene silence and intestinal bacterial load changes were detected at 48 h and 72 h after injection. In addition, Bt was fed to S. exigua larvae to detect relative expression of SeDuox and related immune genes by RT-qPCR.【Result】The length of open reading frame (ORF) of SeDuox is 4 497 bp, encoding 1 498 amino acids, and the deduced molecular weight of protein is 171.62 kD. SMART analysis showed that SeDuox domain is similar to that of Spodoptera litura, Spodoptera frugiperda, including peroxidase domain, nicotinamide adenine dinucleotide binding domain (NAD), N-terminal calcium binding domain (EFH), iron reductase binding domain (Ferric) and flavin adenie dinucleotide binding domain (FAD). TMHMM analysis showed that there are seven transmembrane regions. The transmembrane fragment named SeDuox-OM was successfully expressed about 80 kD recombinant protein in insect cells. The results of RT-qPCR showed that SeDuox was expressed in different tissues of the 4th instar larvae of S. exigua, and the expression level was higher in peritrophic membrane and midgut. It was expressed in each developmental stage, and highly expressed at the egg stage. Compared with dsGFP injection, dsSeDuox injection significantly reduced the expression of SeDuox, which was reduced by 62.08% and 74.94% at 48 h and 72 h, respectively, and the load of intestinal microbial in S. exigua larvae was significantly increased. Compared with the control group, the expression level of SeDuox was significantly increased at 48 h after being fed on Bt GS36. With the extension of infection time, the expression level of SeDuox was increased at first, then decreased and turned to stabilize finally.【Conclusion】SeDuox contains conserved domains, which is highly expressed in the midgut and perinogphic matrix. SeDuox plays an important role in the regulation of host intestinal immunity, it probably has synergistic effect with antimicrobial peptide gene against Bt infection.

Key words: dual oxidase (Duox), Spodoptera exigua, gene expression, RNAi, immune regulation, Bacillus thuringiensis (Bt)

Table 1

Primer information"

引物名称
Primer name		 引物序列
Primer sequence
SeDuox-F GGTTCAACAATAGAGCTTACCC
SeDuox-R TGACTTGCTCGCATGCGGACAT
SeDuox-OM-F CGGATCCGCCAGAAGTGTACTACGAGAAGC
SeDuox-OM-R CGGAATTCGTTGCCCTCGAAGTAGTCATAG
SeActin-F CTACCTCACGCCATTCTC
SeActin-R AACCTGAGTCTTTGTGTACCTCC
SeqPCR-F CGTATACTAATGGACCAGGTTTTCG
SeqPCR-R AACCTGAGTCTTTGTGTACCTCC
dsSeDuox-T7-F TAATACGACTCACTATAGGCCGCCATACTACTGA
GAGAC
dsSeDuox-T7-R TAATACGACTCACTATAGGGATGACCCGTGGATT
CTAAC
dsSeDuox-F CCGCCATACTACTGAGAGAC
dsSeDuox-R GATGACCCGTGGATTCTAAC
dsGFP-T7-F TAATACGACTCACTATAGGCCACAAGTTCAGCGT
GT
dsGFP-T7-R TAATACGACTCACTATAGGAGTTCACCTTGATGC
CG
dsGFP-F CCACAAGTTCAGCGTGT
dsGFP-R AGTTCACCTTGATGCCG
16S-F TCCTACGGGAGGCAGCAGT
16S-R GGACTACCAGGGTATCTAATCCTGTT
Attacin-F GGACTGACCGTGATGAAGG
Attacin-R CGTTGTGGTTGTCGTTGTG
Definsin-F GACTTGGACGCGAAATTGA
Definsin-R TGGTAGACAATGCTCCTGG
PGRP-F CACTCGAACTTGCAATACGG
PGRP-R ACCAATCACGAACGAGGACC
Es-F CCCTTATTGTTAGTTGCCATCATT
Es-R ACTCGTTGTACTTCCCATTGT
Bc-F GCCCTGGTATGTATATTGGATCTAC
Bc-R GGTCATAATAACTTCTACAGCAGGA
下划线分别表示酶切位点和T7 RNA聚合酶启动子
Restriction enzyme cutting site and T7 RNA polymerase promoter are underlined

Fig. 1

Analysis of amino acid sequence of SeDuox The signal peptide sequence of SeDuox protein is marked with black solid line, the transmembrane region is labeled with rectangle, the peroxidase domain is labeled with green wavy line, the N-terminal calcium binding domain (EFH) is labeled with short dashed line, the iron reductase binding domain (Ferric) is labeled with double dashed line, the flavin adenie dinucleotide binding domain (FAD) is labeled with double solid line, the nicotinamide adenine dinucleotide binding domain (NAD) is labeled with long dashed line, the O-glycosylation site is marked with the red arrow, and the N-glycosylation site is marked with the black arrow"

Fig. 2

The prediction of transmembrane domain and the domain analysis of the SeDuox"

Fig. 3

Phylogenetic tree of Duox amino acid sequence of S. exigua and other insects"

Fig. 4

PCR amplification of SeDuox and SeDuox-OM"

Fig. 5

Expression of recombinant protein SeDuox-OM in Sf9 cells"

Fig. 6

mRNA expression of SeDuox in different tissues and developmental stages of S. exigua"

Fig. 7

Electrophoresis detection of dsSeDuox and dsGFP of S. exigua"

Fig. 8

Silencing efficiency of SeDuox induced by RNAi Data are representative of three independent experiments (mean±SE), *P<0.05, **P<0.01with a Student’s t-test. The same as Fig. 9"

Fig. 9

Effects of SeDuox gene silencing on intestinal flora"

Fig. 10

Effects of Bt GS36 on the expression of SeDuox and antimicrobial peptides of S. exigua"

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