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

Abstract

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

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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References 48

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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

Response and Function of the Transcription Factor CncC in Cnaphalocrocis medinalis Infected with Baculovirus CnmeGV

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