白背飞虱酚氧化酶原PPO基因特性及其免疫应答

doi:10.3864/j.issn.0578-1752.2020.15.011

摘要/Abstract

摘要：

【目的】酚氧化酶（phenoloxidase,PO）是昆虫体内的免疫蛋白,在昆虫体内起重要的调节作用,主要以无活性的酚氧化酶原（prophenoloxidase,PPO）形式存在。本研究在转录组测序获得白背飞虱（Sogatella furcifera）3条PPO序列的基础上,通过分析白背飞虱体内不同SfPPO的发育和组织表达模式,并进一步抑制SfPPO的表达以及病原菌诱导,探讨SfPPO在白背飞虱体内的生物学功能。【方法】以白背飞虱3条SfPPO序列为研究对象,利用生物信息学方法分析其蛋白结构以及与其他昆虫之间的同源性。并以注射绿色荧光蛋白（green fluorescent protein,GFP）的dsRNA作为对照组,通过注射合成的dsSfPPO后采用实时荧光定量PCR（qRT-PCR）检测基因表达抑制效果;此外,为了探究SfPPO在白背飞虱生长发育和免疫应答方面的作用,利用qRT-PCR检测SfPPO在不同发育阶段及组织中的表达情况,以及注射大肠杆菌（Escherichia coli）和枯草芽孢杆菌（Bacillus subtilis）后3个SfPPO的诱导表达情况。【结果】SfPPO1、SfPPO2-1、SfPPO2-2的开放阅读框长度分别为2 079、999、2 070 bp,分别编码692、332、689个氨基酸,预测蛋白分子量分别为79.84、37.67、79.53 kD,等电点分别为6.43、9.56、6.20。生物信息学分析表明,白背飞虱的3个PPO蛋白具有较高的同源性,且与褐飞虱（Nilaparvata lugens）的亲缘关系最近;SfPPO1、SfPPO2-1、SfPPO2-2均在成虫第3天表达量最高,其中SfPPO2-1和SfPPO2-2的发育表达模式相似;SfPPO1和SfPPO2-1在表皮和脂肪体内表达量较高,而SfPPO2-2在翅和脂肪体内表达量较高,在头、足、中肠、马氏管中表达量相对较低;与注射dsGFP组相比,注射靶标基因的dsRNA后都能够显著沉默目的基因的表达,而且当SfPPO2-1表达被沉默后,SfPPO1出现显著高表达,说明不同的PPO之间可能具有补偿功能;大肠杆菌诱导后12 h,SfPPO2-1和SfPPO2-2表达量显著升高,SfPPO1表达量则在诱导24 h后显著升高;枯草芽胞杆菌诱导24 h后,SfPPO1、SfPPO2-1和SfPPO2-2表达量均显著升高。【结论】SfPPO1、SfPPO2-1、SfPPO2-2的表达存在组织和龄期特异性,且在不同菌诱导情况下存在免疫应答差异性。研究结果有助于更加全面、深入地探索白背飞虱酚氧化酶在调控昆虫发育及免疫中的潜在分子机理。

关键词: 白背飞虱, 酚氧化酶原, RNA干扰, 实时荧光定量PCR, 诱导表达, 免疫应答

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

张道伟,康奎,余亚娅,匡富萍,潘碧莹,陈静,唐斌. 白背飞虱酚氧化酶原PPO基因特性及其免疫应答[J]. 中国农业科学, 2020, 53(15): 3108-3119.

ZHANG DaoWei,KANG Kui,YU YaYa,KUANG FuPing,PAN BiYing,CHEN Jing,TANG Bin. Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera[J]. Scientia Agricultura Sinica, 2020, 53(15): 3108-3119.

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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′)	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