稻纵卷叶螟转录因子CncC对杆状病毒CnmeGV感染的响应及功能

doi:10.3864/j.issn.0578-1752.2023.13.005

摘要/Abstract

摘要：

【目的】研究稻纵卷叶螟（Cnaphalocrocis medinalis）转录因子CncC（Cap‘n’Collar Isoform C）及其调控的抗氧化酶基因在抗稻纵卷叶螟颗粒体病毒（Cnaphalocrocis medinalis granulovirus，CnmeGV）感染中的作用，进一步丰富昆虫抗杆状病毒感染的免疫调控机制研究。【方法】使用RT-PCR克隆得到稻纵卷叶螟CncC（CmCncC）cDNA全长序列并分析其蛋白结构；以10⁶ OB/mL浓度的CnmeGV或同浓度的CnmeGV和1 mmol·L^-1 CncC抑制剂——ML385混合液饲喂稻纵卷叶螟3龄幼虫，感染后6—48 h取样，分析ML385对CnmeGV诱导的稻纵卷叶螟丙二醛（MDA）含量变化的影响，同时采用RT-qPCR分析病毒DNA复制水平、稻纵卷叶螟CmCncC、谷胱甘肽过氧化物酶-3（glutathione peroxidase-3，GPx-3）、锰超氧化物歧化酶（manganese superoxide dismutase，Mn-SOD）和硫氧还蛋白过氧化物酶（thioredoxin peroxidase，TPx）等基因的表达水平，并每隔24 h统计幼虫死亡数，绘制10 d内幼虫的Kaplan-Meier生存曲线。使用1 mmol·L^-1 ML385或1 mmol·L^-1 ML385和200 μg·mL^-1抗氧化剂N-乙酰-L-半胱氨酸（NAC）混合液饲喂感染的幼虫，使用RT-qPCR分析不同时间病毒DNA复制水平。【结果】CmCncC cDNA全长为3 404 bp，包括321 bp的5′-UTR，847 bp的3′-UTR和2 211 bp的ORF，编码一个长度为736 aa的蛋白，预测蛋白质分子质量为75.8 kDa。稻纵卷叶螟感染CnmeGV后丙二醛含量在感染的前12 h内显著上调，随后在24 h恢复到正常水平，而在48 h低于对照组。病毒感染导致CmCncC在12、24和48 h显著上调表达，分别为对照组的1.56、2.16和2.63倍，NAC处理显著降低了CnmeGV诱导的CmCncC表达上调的倍数，分别降低至对照组的48.12%、59.83%和56.32%。ML385处理导致病毒基因拷贝数在12和24 h分别增至对照组的1.79和1.76倍，同时导致稻纵卷叶螟感染CnmeGV 10 d后的死亡率显著升高，从单CnmeGV处理组的73.33%升至94.14%，而NAC处理能够减轻由于CncC抑制导致的病毒基因拷贝数的上调。CnmeGV感染分别导致GPx-3表达量在感染后48 h上调至对照组的3.68倍，Mn-SOD表达量在12、24和48 h分别上调至对照组的1.73、2.62和2.77倍，TPx表达量在12和24 h分别上调至对照组的1.76和2.10倍，但48 h恢复至对照组水平。使用NAC或CncC抑制剂处理感病幼虫发现，GPx-3、Mn-SOD和TPx对CnmeGV感染的响应被显著削弱。【结论】CmCncC介导了CnmeGV感染诱导的GPx-3、Mn-SOD和TPx上调表达，降低CnmeGV感染导致的氧化应激，从而限制病毒在其体内的复制并降低氧化损伤。

关键词: 稻纵卷叶螟, 稻纵卷叶螟颗粒体病毒, CncC转录因子, 氧化应激, 抗氧化基因, 杆状病毒

Abstract:

【Objective】The objective of this study is to research the role of transcription factor CncC (Cap ‘n’ Collar Isoform C) of Cnaphalocrocis medinalis and its regulation of antioxidant enzyme genes in the infection of Cnaphalocrocis medinalis granulovirus (CnmeGV), and to further enrich the study of immune regulation mechanism of insects against baculovirus.【Method】The full-length cDNA sequence of CncC in C. medinalis (CmCncC) was cloned by RT-PCR, and its protein structure was analyzed. Third instar larvae of C. medinalis were fed with 10⁶ OB/mL concentration of CnmeGV or a mixture of the same concentration of CnmeGV and 1 mmol·L^-1 CncC inhibitor ML385. Samples were taken 6-48 h after infection to analyze the effect of ML385 on the change of malondialdehyde (MDA) content induced by CnmeGV, and viral DNA replication levels, the expression levels of CmCncC, glutathione peroxidase-3 (GPx-3), manganese superoxide dismutase (Mn-SOD), and thioredoxin peroxidase (TPx) were analyzed by RT-qPCR. The number of larval deaths was counted every 24 hours, and the Kaplan-Meier survival analysis of the larvae within 10 d was conducted. Infected larvae were fed with 1 mmol·L^-1 ML385 or a mixture of 1 mmol·L^-1 ML385 and 200 μg·mL^-1 antioxidant N-acetyl-L-cysteine (NAC), and viral DNA replication levels at different times were analyzed using RT-qPCR.【Result】The full length cDNA of CmCncC was 3 404 bp, including a 321 bp 5′-UTR, an 847 bp 3′-UTR, and a 2 211 bp ORF that encoded a protein of 736 amino acids. The predicted molecular weight of the protein was 75.8 kDa. The MDA content in C. medinalis significantly increased within the first 12 h of CnmeGV infection and returned to normal levels at 24 h, but was lower than the control group at 48 h. The expression of CmCncC was significantly up-regulated at 12, 24, and 48 h after viral infection, by 1.56, 2.16, and 2.63 folds of the control group, respectively. NAC treatment significantly reduced the fold increase of CmCncC expression induced by CnmeGV, to 48.12%, 59.83%, and 56.32% of the control group, respectively. Treatment with the ML385 caused an increase in viral gene copy number to 1.79 and 1.76 folds of the control group at 12 and 24 h, respectively, and significantly increased the mortality rate of C. medinalis infected with CnmeGV 10 d after treatment, from 73.33% to 94.14%. NAC treatment partially alleviated the up-regulation of viral gene copy number caused by CncC inhibition. CnmeGV infection led to a 3.68-fold increase in GPx-3 expression level at 48 h after infection, and 1.73-, 2.62-, and 2.77-folds increase in Mn-SOD expression level at 12, 24, and 48 h after infection, respectively. The expression level of TPx increased by 1.76 and 2.10 folds at 12 and 24 h after infection, respectively, but returned to the control group level at 48 h, and treatment with NAC or the CncC inhibitor significantly weakened the responses of GPx-3, Mn-SOD, and TPx to CnmeGV infection.【Conclusion】CmCncC mediates the CnmeGV infection-induced up-regulation of GPx-3, Mn-SOD and TPx mRNA levels and reduces oxidative stress caused by CnmeGV infection, thereby limiting virus replication, and reducing oxidative damage.

Key words: Cnaphalocrocis medinalis, Cnaphalocrocis medinalis granulovirus (CnmeGV), CncC transcription factor, oxidative stress, antioxidant gene, baculovirus

张楠, 韩光杰, 刘琴, 李传明, 祁建杭, 陆玉荣, 夏杨, 徐健. 稻纵卷叶螟转录因子CncC对杆状病毒CnmeGV感染的响应及功能[J]. 中国农业科学, 2023, 56(13): 2491-2503.

ZHANG Nan, HAN GuangJie, LIU Qin, LI ChuanMing, QI JianHang, LU YuRong, XIA Yang, XU Jian. Response and Function of the Transcription Factor CncC in Cnaphalocrocis medinalis Infected with Baculovirus CnmeGV[J]. Scientia Agricultura Sinica, 2023, 56(13): 2491-2503.

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引物名称 Primer name	序列 Sequence (5′ to 3′)	作用 Function
CncC1F	TTCCGAGGTTGCTCGAAGAC	CmCncC cDNA中间片段扩增 Amplification of intermediate fragments of CmCncC cDNA
CncC1R	GTGGTGGTTCGTCGGGTAG
CncC2F	TGTCACTTTTCAAACCACACCA
CncC2R	TGGTGTGGTTTGAAAAGTGACA
CncC5RACE1R	TGCACCGGGTCCTCGAGGGAGAAGCC	CmCncC cDNA 5′端第一轮扩增 The first round PCR of CmCncC cDNA 5′ end
CncC5RACE2R	CGTGGCCTGAGGACCCTCCATCTGCACC	CmCncC cDNA 5′端第二轮扩增 The second round PCR of CmCncC cDNA 5′ end
CncC3RACE1F	CGCATCAGCCGCACGCGCATCCGCAT	CmCncC cDNA 3′端第一轮扩增 The first round PCR of CmCncC cDNA 3′ end
CncC3RACE2F	CGCATCCGCATCCGCACACCCATCCG	CmCncC cDNA 3′端第二轮扩增The second round PCR of CmCncC cDNA 3′ end
CncCqF	CAGAACTGTCGCAAACGCAA	CmCncC mRNA定量分析 Quantitative analysis of CmCncC mRNA
CncCqR	TGGAACACGTGTCTGTAGAGC	CmCncC mRNA定量分析 Quantitative analysis of CmCncC mRNA
GPxqF	ATCCGTGTCTCCAACCACAC	GPx-3 mRNA定量分析 Quantitative analysis of GPx-3 mRNA
GPxqR	GCCTGGCAGTACACAGAACT	GPx-3 mRNA定量分析 Quantitative analysis of GPx-3 mRNA
Mn-SODqF	GGTGACGTTCAGGTTGTTCAC	Mn-SOD mRNA定量分析 Quantitative analysis of Mn-SOD mRNA
Mn-SODqR	CCCAGAATCTGTCCCACTCCC	Mn-SOD mRNA定量分析 Quantitative analysis of Mn-SOD mRNA
TPxqF	AGGCTTTCGATCCCTTCTGC	TPx mRNA定量分析 Quantitative analysis of TPx mRNA
TPxqR	GGTGGGGGTCACTGATGAAG	TPx mRNA定量分析 Quantitative analysis of TPx mRNA
β-actinF	ATGGTCGGCATGGGACAG	内参基因 Internal reference gene
β-actinR	GAGTTCATTGTAGAAGGTGT	内参基因 Internal reference gene
granulin qF	GACCCGACAACATTGCACAC	granulin的定量分析 Quantitative analysis of granulin
granulin qR	ACGTACACGAGAGGACGGTA	granulin的定量分析 Quantitative analysis of granulin