本氏烟半胱氨酸蛋白酶基因家族特征及其在TMV侵染中的功能

doi:10.3864/j.issn.0578-1752.2022.21.008

Abstract

Abstract:

【Objective】The objective of this study is to identify the effects of Nicotiana benthamiana cysteine proteinase (CP) induced by the antiviral compound chloroinconazide (CHI) on the infection of tobacco mosaic virus (TMV) and its gene family characteristics, and to provide a theoretical basis for understanding the antiviral molecular mechanism and chemical regulation of Solanaceae crops.【Method】Genome-wide analysis of NbCP genes was identified from Sol Genomics Network and the evolutionary relationships, motifs and promoter cis-acting elements of NbCP gene family were analyzed by bioinformatics. According to the results of bioinformatics analysis, the expression of NbCP genes after TMV infection was analyzed by real-time fluorescence quantitative PCR (qRT-PCR), so as to screen out potential disease-resistant protein. The effect of this key protein on TMV-GFP infection was verified by gene silencing mediated by tobacco rattle virus (TRV) and gene overexpression mediated by potato virus X (PVX). qRT-PCR and biochemical methods were combined to determine the antiviral mechanism of this key protein.【Result】There were 24 NbCP genes in N. benthamiana, which were named NbCP1-NbCP24 according to their chromosome location. The phylogenetic analysis showed that the NbCPs were divided into five subfamilies, of which Group V contained seven NbCPs, while Group I contained only two NbCPs. Motif analyses showed that NbCPs of different branches had similar motif distribution. Promoter cis-acting elements analysis showed that NbCP family genes were all regulated by light, and several NbCP family gene promoters contained hormone response elements such as methyl jasmonate (MeJA), methyl salicylate (MeSA), abscisic acid (ABA), gibberellin (GA) and auxin (IAA). Combined with bioinformatics analysis, five key potential disease-resistant related NbCP genes (NbCP8, NbCP12, NbCP13, NbCP18, and NbCP22) were screened from the above subfamilies, and the expression of these NbCP genes under TMV infection at the 5th day was analyzed by qRT-PCR, and it was found that the expression difference of NbCP8 was the greatest, which increased 2.6 times. The expression of NbCP8 was highest in leaves, followed by stems and roots, and lowest in flowers. TRV-mediated NbCP8 silencing significantly increased TMV-GFP infection, while PVX-mediated NbCP8 overexpression significantly inhibited TMV-GFP infection, suggesting that NbCP8 acts as a positive regulatory factor in plant to inhibit virus infection. Furthermore, silencing NbCP8 significantly inhibited the expression of salicylic acid (SA) signaling pathway related gene PR1 and jasmonic acid (JA) signaling pathway related gene MYC2, while the expressions of NPR1, COI1, and ABA related genes ABA1, NCED1 were not significantly affected. The overexpression of NbCP8 enhanced the expression of PR1 and ABA1, but had no significant effect on expression of NPR1, MYC2, COI1 and NCED1. 【Conclusion】N. benthamiana NbCPs are involved in plant stress defense, while NbCP8 plays a key role in TMV defense, which can induce SA signaling pathway to participate in the defense. The research results will provide evidence for the molecular mechanism of antiviral in Solanaceae crops.

Key words: Nicotiana benthamiana, genome-wide, cysteine proteinase (CP), NbCP8, tobacco mosaic virus (TMV)

PEI YueHong,LI FengWei,LIU WeiNa,WEN YuXia,ZHU Xin,TIAN ShaoRui,FAN GuangJin,MA XiaoZhou,SUN XianChao. Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection[J].Scientia Agricultura Sinica, 2022, 55(21): 4196-4210.

Figures/Tables 10

Table 1

Table 2

Identification and characterization of NbCP gene family"

名称 Name	基因定位 Gene locus ID	蛋白质编码区长度 CDS (bp)	分子量 MW (Da)	等电点 pI	分子式 Formula
NbCP1	Niben101Scf00712g02010.1	1068	39921.29	5.74	C₁₇₉₀H₂₇₅₆N₄₆₈O₅₃₈S₁₅
NbCP2	Niben101Scf00973g01002.1	1113	40808.02	5.95	C₁₈₀₁H₂₇₈₆N₄₉₈O₅₅₁S₁₈
NbCP3	Niben101Scf01369g00024.1	1068	39907.19	5.64	C₁₇₈₂H₂₇₄₆N₄₇₀O₅₄₀S₁₆
NbCP4	Niben101Scf01631g00002.1	999	36623.07	5.29	C₁₆₁₄H₂₄₈₆N₄₂₈O₅₁₂S₁₇
NbCP5	Niben101Scf02159g01012.1	1086	40558.46	5.78	C₁₈₀₀H₂₇₅₅N₄₈₉O₅₅₄S₁₄
NbCP6	Niben101Scf02336g00010.1	1311	48758.34	5.71	C₂₁₇₅H₃₃₄₉N₅₇₃O₆₅₆S₂₃
NbCP7	Niben101Scf02336g00012.1	1482	55407.91	5.82	C₂₄₆₀H₃₇₇₁N₆₆₁O₇₃₆S₃₂
NbCP8	Niben101Scf02763g05012.1	1059	38333.52	7.00	C₁₇₁₂H₂₆₅₈N₄₆₄O₅₁₁S₁₃
NbCP9	Niben101Scf02832g01014.1	1074	38845.02	5.09	C₁₇₀₅H₂₆₇₉N₄₇₃O₅₂₇S₁₉
NbCP10	Niben101Scf02853g06021.1	1086	40508.59	6.20	C₁₇₉₈H₂₇₆₅N₄₉₁O₅₄₈S₁₅
NbCP11	Niben101Scf02976g00007.1	1086	39876.46	6.00	C₁₇₈₁H₂₇₃₃N₄₈₁O₅₂₀S₂₁
NbCP12	Niben101Scf03867g02046.1	1092	40222.77	5.67	C₁₈₀₁H₂₇₃₉N₄₇₉O₅₂₆S₂₂
NbCP13	Niben101Scf04209g00004.1	1023	37952.64	6.67	C₁₆₇₄H₂₅₆₆N₄₆₀O₅₁₅S₁₈
NbCP14	Niben101Scf04653g01012.1	1059	39469.28	6.23	C₁₇₄₇H₂₆₉₁N₄₈₃O₅₃₇S₁₃
NbCP15	Niben101Scf04934g02006.1	1035	38571.66	8.89	C₁₇₁₁H₂₆₂₈N₄₆₈O₅₁₃S₁₉
NbCP16	Niben101Scf05035g01005.1	2052	74678.53	5.18	C₃₂₆₁H₄₉₇₃N₈₉₁O₁₀₃₂S₄₇
NbCP17	Niben101Scf05047g04012.1	987	36843.67	5.72	C₁₆₄₉H₂₅₂₂N₄₃₄O₄₉₆S₁₅
NbCP18	Niben101Scf05135g02002.1	1125	42280.54	6.73	C₁₈₆₁H₂₈₈₃N₅₂₃O₅₇₂S₁₇
NbCP19	Niben101Scf05469g01014.1	336	11947.10	4.37	C₅₀₅H₇₇₈N₁₄₀O₁₇₈S₉
NbCP20	Niben101Scf05469g01024.1	1623	59602.30	4.45	C₂₆₃₈H₃₉₆₄N₆₈₂O₈₄₀S₂₉
NbCP21	Niben101Scf08921g02020.1	1584	59286.05	8.87	C₂₆₃₂H₄₀₆₉N₇₂₉O₇₈₉S₂₃
NbCP22	Niben101Scf10490g00007.1	990	36122.34	4.81	C₁₅₉₇H₂₄₄₆N₄₂₀O₅₀₉S₁₄
NbCP23	Niben101Scf11030g00003.1	720	26115.98	7.95	C₁₁₄₆H₁₇₈₆N₃₁₀O₃₄₄S₂₂
NbCP24	Niben101Scf15022g00015.1	1113	40804.10	6.01	C₁₇₉₇H₂₇₉₀N₅₀₂O₅₄₈S₁₉

Table 2

Fig. 1

Fig. 2

Fig. 3

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Fig. 8

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引物名称 Primers name		引物序列F Primer sequence F (5′-3′)	引物序列R Primer sequence R (5′-3′)
PCR	NbCP8	ATGTCTCGTTTCTCACTTCTATTGGC	TCCTACCCTGTCGTTGCCTGA
qRT-PCR	CP8	TCGCTTTGCTCACAGGTATG	TGGTGGCTGAACAGTTTTGA
	CP12	GAAAGCTGCACTGAACCCTC	GTGCTACAGTTAGGCCAAGC
	CP22	CGGCAGTGAGAGAGCATTTC	CCACCCTTCCTCATAAGCCA
	CP18	CCCTTATCGCGGTGTTGAAG	GATGCTTCAATGGCTACGCA
	CP13	TAAGAACCAAGGGCAATGCG	GAGACCTCCTTGACAACCCA
	MYC2	CGGTTCTTCTTCCGTCTTCTT	ACGCTGTTGAAGGGTTTCT
	TMV MP	TTAGGTTCCCTGACGGTGAC	ACCGTTGCGTCGTCTACTCT
	ABA1	GCCGTTGTTTGTTCATCTC	ACTGTATCACCTTGCCTCC
	NCED1	ACCCGACTCGATTTTCAATG	AACTTTTGGCCATGGTTCTG
	COI1	CAGCAGCCCATTGTTTCTTAC	TACTGGCCAAGTACTTCCAATC
	PR1	CGTGAAGATGTGGGTCAATG	CCATACGGACGTTGTCCTCT
	NPR1	TGTTGCTGCCATGAGAAAAG	TAACCGATCCTTTGGAGCAG
	Actin	CGGTGCCCACTTTCCGATCT	TCCTCACCGTCAGCCATTTT
TRV	TRV:NbCP8	CGCGGATCCGGCTGCAATGGTGGGCT	CCGCTCGAGTACATCCATGGGAGTGTTGC
PVX	PVX:NbCP8	ACGCGTCGACATGTCTCGTTTCTCACTTCTATTGGC	CCATCGATCATGTCTCGTTTCTCACTTCTATTGGC

Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection

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