Functional Verification of GhP450-94C1 that a Verticillium wilt Resistant Gene in Gossypium hirsutum L.

doi:10.7668/hbnxb.20192979

Abstract

Abstract:

To explore the biological function of cotton cytochrome P450 gene GhP450-94C1 in cotton Verticillium wilt response, and lay a foundation for cotton Verticillium wilt resistance gene mining and disease resistance breeding. A cytochrome P450 gene, GhP450-94C1, was cloned through transcriptome screening. The physicochemical properties of the gene were analyzed by bioinformatics methods. The expression pattern of GhP450-94C1 under Verticillium wilt induction was analyzed by Real-time quantitative polymerase chain reaction (qRT-PCR). Virus-induced gene silencing (VIGS) technology was used to preliminarily explore its biological function in cotton resistance to Verticillium wilt. The main results were as follows: upland cotton cytochrome P450 gene, GhP450-94C1, was obtained by cloning. The open reading frame (ORF) was 1 503 bp, encoding an acidic, hydrophilic and unstable transmembrane protein with 500 amino acids. The molecular formula was C₂₅₉₇H₄₀₂₅N₆₉₁O₇₂₅S₂₂ with a molecular weight of 57.23 ku, which was located in the endoplasmic reticulum membrane and contained a P450 domain. There was 86.45% probability of signal peptide; the secondary structure prediction showed that the protein contained 24 α-helixes and 8 β-sheets. This gene responds to Verticillium wilt infection, and after inhibiting its expression, the sensitivity of plants to Verticillium wilt is enhanced. GhP450-94C1 is a positive regulator of cotton resistance to Verticillium wilt.

Key words: Cotton Verticillium wilt, GhP450-94C1, Gene cloning, Virus-induced gene silencing

HU Ziyao, LI Xiuqing, DAI Peihong, LEI Jianfeng, LIU Jianfei, ZHAO Yi, DENG Jiahui, LIU Chao, LIU Xiaodong, LI Yue. Functional Verification of GhP450-94C1 that a Verticillium wilt Resistant Gene in Gossypium hirsutum L.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 72-81. doi: 10.7668/hbnxb.20192979.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192979

http://www.hbnxb.net/EN/Y2022/V37/I6/72

Figures/Tables 10

Tab.1 Bioinformatics analysis tools and corresponding functions

分析工具 Analysis tools	功能 Function	网址 URL
ExPASy-ProtScale	蛋白理化性质分析	http://web.expasy.org/protparam/
SignalP	信号肽预测	http://www.cbs.dtu.dk/services/SignalP
TMHMM	跨膜结构域预测	http://www.cbs.dtu.dk/services/TMHMM/
LocTree	亚细胞定位分析	https://www.rostlab.org/services/loctree2/
SMART	蛋白结构域预测	http://smart.embl-heidelberg.de/
PSIPRED	蛋白二级结构预测	http://bioinf.cs.ucl.ac.uk/psipred/
SWISS-MODEL	蛋白三级结构预测	https://swissmodel.expasy.org/

Tab.2 Primer and application

引物名称 Primer name	正向引物(5'—3') Forward primer(5'—3')	反向引物(5'—3') Reverse primer(5'—3')	用途 Application
GhP450-94C1	ATGGATTCACAACTTTCCTTG	TTAAGCTTCCCTTTCTTGGAC	基因克隆
q-GhP450-94C1	GTCACCTACCATCCATATGC	CACCAATGCTATTTCCTTCCC	qRT-PCR
q-GhUBQ7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG	qRT-PCR
q-GhCLA1	GCCCTTTGTGCATCTTC	CTCTAGGGGCATTGAAG	qRT-PCR
CM-GhP450-94C1	GAATTCGATTTCGATAATCTTTGTGATTGG	GGTACCATTCGAGGCAACCAGGGTC	载体构建

Fig.1 Cloning of GhP450-94C1 gene M.Trans 2000 PlusⅡ DNA Marker.The same as Fig.6.

Fig.2 The prediction protein domain of GhP450-94C1

Fig.3 The prediction signal peptide of GhP450-94C1 protein

Fig.4 The prediction secondary and tertiary structure of GhP450-94C1 protein A.The prediction secondary structure of GhP450-94C1 protein;B.The prediction tertiary structure of GhP450-94C1 protein.

Fig.5 Analysis of the expression patterns of GhP450-94C1 under the treatment of Verticillium wilt The same lowercase letter indicates no significant difference(P>0.05),and different lowercase letters indicate significant difference(P<0.05).The same as Fig.7—8.

Fig.6 Construction of VIGS vector of GhP450-94C1 A.Construction of VIGS vector of GhP450-94C1;B.PCR amplification of target fragment of GhP450-94C1;C.Identification of TRV∶GhP450-94C1 vectors by restriction enzyme digestion.

Fig.7 Silencing efficiency of GhP450-94C1 gene A.The phenotype of silenced GhCLA1;B,C.Silencing efficiency of GhCLA1 and GhP450-94C1.

Fig.8 Disease resistance identification of GhP450-94C1 gene silencing plants A.Phenotype analysis of plants after GhP450-94C1 gene silencing;B.Disease index statistics;C.Stem cutting phenotype;D.Restoration culture of pathogenic bacteria.

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