甜菜夜蛾双重氧化酶基因SeDuox的克隆、表达及功能分析

doi:10.3864/j.issn.0578-1752.2021.18.008

摘要/Abstract

摘要：

【目的】昆虫双重氧化酶（dual oxidase,Duox）介导产生活性氧（reactive oxygen species,ROS）是调节昆虫肠道微生物动态平衡的重要免疫机制。本研究通过分析甜菜夜蛾（Spodoptera exigua）双重氧化酶基因（SeDuox）的序列特征与表达模式,RNAi技术探究SeDuox基因沉默对甜菜夜蛾肠道细菌载量的影响,分析SeDuox对苏云金杆菌（Bacillus thuringiensis,Bt）的免疫响应,从而明确SeDuox在昆虫肠道免疫调控中的作用,为甜菜夜蛾的综合防控提供新的思路和作用靶标。【方法】利用反转录PCR（RT-PCR）技术克隆SeDuox,序列特征进行生物信息学分析,采用ClustalX和MEGA_X软件构建系统发育树;克隆SeDuox膜外91—1 767 bp编码基因SeDuox-OM,利用Bac to Bac表达系统构建Bacmid-SeDuox-OM,脂质体转染法转染昆虫细胞Sf9,表达SeDuox-OM膜外蛋白;采用实时荧光定量PCR（RT-qPCR）方法分析甜菜夜蛾不同组织（围食膜、血淋巴、脂肪体、马氏管、中肠和表皮）和不同发育时期（卵、1—5龄幼虫、雌蛹、雄蛹）SeDuox的表达水平;利用RNAi技术进行功能分析,向4龄幼虫注射SeDuox的dsRNA,以注射绿色荧光蛋白（green fluorescent protein,GFP）的dsRNA作为对照,注射48 h和72 h后检测基因沉默效果及肠道细菌载量的变化;向甜菜夜蛾幼虫饲喂Bt,RT-qPCR方法分析SeDuox及相关免疫基因的诱导表达情况。【结果】SeDuox序列全长为4 497 bp,编码1 498个氨基酸,预测蛋白质的相对分子量为171.62 kD。SMART分析结构域发现SeDuox含有过氧化物酶结构域（peroxidase）、烟酰胺腺嘌呤二核苷酸结构域（NAD）、N-端钙离子结合域（EFH）、铁还原酶结构域（Ferric）和黄素腺嘌呤二核苷酸结构域（FAD）,与斜纹夜蛾（Spodoptera litura）、草地贪夜蛾（Spodoptera frugiperda）等昆虫的Duox结构域类似。TMHMM分析显示有7个跨膜区,跨膜片段SeDuox-OM 在昆虫细胞中成功表达约80 kD的重组蛋白;RT-qPCR分析结果显示SeDuox在甜菜夜蛾4龄幼虫不同组织中均有表达,其中围食膜和中肠表达量较高;在不同发育时期均有表达,但在卵期表达量最高。与注射dsGFP相比,注射 dsSeDuox 的甜菜夜蛾幼虫在48 h和72 h时,SeDuox表达量分别下调了62.08%和74.94%,肠道微生物载量显著升高。甜菜夜蛾幼虫取食Bt GS36 48 h时,SeDuox表达量显著增加,随着侵染时间的延长,其表达量呈现出先升高后降低再趋于稳定的特点。【结论】甜菜夜蛾SeDuox具有保守的结构域,在中肠和围食膜中高表达,SeDuox在宿主肠道免疫调控中起重要作用,可能与抗菌肽基因协同作用抵御Bt的侵染。

关键词: 双重氧化酶, 甜菜夜蛾, 基因表达, RNAi, 免疫调控, 苏云金杆菌

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)

付超然, 李亚子, 吴涵, 赵丹, 郭巍, 郭晓昌. 甜菜夜蛾双重氧化酶基因SeDuox的克隆、表达及功能分析[J]. 中国农业科学, 2021, 54(18): 3881-3891.

FU ChaoRan, LI YaZi, WU Han, ZHAO Dan, GUO Wei, GUO XiaoChang. Cloning, Expression and Functional Analysis of SeDuox from Spodoptera exigua[J]. Scientia Agricultura Sinica, 2021, 54(18): 3881-3891.

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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