红海榄肉桂酸-4-羟基化酶基因的克隆与表达分析

doi:10.11978/2021184

Abstract

Abstract:

Cinnamate-4-hydroxylase is a key enzyme in lignin synthesis which plays an important role in plant tolerance to heavy metal stress. In this study, a cinnamate-4-hydroxylase gene named RsC4H was cloned from the leaves of Rhizophora stylosa by homologous cloning and rapid amplification of cDNA ends (RACE). Bioinformatics analysis showed that the full length of RsC4H gene cDNA was 2006bp, the length of open reading frame was 1572bp, and encoded 523 amino acids. The relative molecular weight of the encoded protein is 60.18kD. It is a hydrophilic unstable protein with secondary structure alpha-helix (47.42%) and random coil (32.70%), this protein contains a transmembrane structure at the N-terminal and C-terminal, respectively, and does not contain signal peptides. It is mainly distributed on the membrane structure or endoplasmic reticulum to function, and belongs to P450 superfamily. RsC4H protein has high similarity with C4H protein of other plants. The phylogenetic tree results showed that it was closer to Cucurbita maxima (xp_023007159.1) and Cucurbita moschata (xp_022947682.1). qRT-PCR results showed that Rhizophora stylosa could quickly respond to heavy metal stress, improve the expression of RsC4H gene, promote lignin production, increase cell wall thickness and prevent metal ions from entering cells. The results enrich the gene pool of mangrove plants resistant to heavy metal stress, and lay a foundation for revealing the mechanism of heavy metal stress tolerance of mangrove at the molecular level.

Key words: Rhizophora stylosa, cinnamate-4-hydroxylase, gene cloning, bioinformatics analysis

CLC Number:

Q943.2

XIE Yong, WANG Youshao, ZHANG Weishi. Cloning and expression analysis of Cinnamate-4-hydroxylase gene from Rhizophora stylosa[J].Journal of Tropical Oceanography, 2022, 41(6): 20-27.

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References 20

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引物名称	引物序列(5′ to 3′)
C4H-F(中间片段)	AAGGGCCAGGACATGGTNTTYAC
C4H-R(中间片段)	CTCGTTGATCTCNCCYTTYTGYTG
C4H-GSP-5′	TCACGCTCTTGGTGCTTC
C4H-NGSP-5′	CAAAGCTCTGTGCCAATC
C4H-GSP-3′ (RACE)	GCAGTACCGATATGGTTGGG
C4H-NGSP-3′ (RACE)	GGGATTGTGCTCAGAAGAAG
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
UPM short	CTAATACGACTCACTATAGGGC
18S rRNA-F(qRT-PCR)	ATTGGAGGGCAAGTCTGGTG
18S rRNA-R(qRT-PCR)	GATCGACCCATCCCAAGGTC
C4H-F(qRT-PCR)	TTGATAGCAGGTTTGAGAGTGAGG
C4H-R(qRT-PCR)	CCTCTCAAGAATGGTCTCAAAATG

预测项目	软件版本/网址
同源比对	Nucleotide BLAST
开放阅读框查找	http://www.bio-soft.net/sms/index.html
理化性质预测	https://web.expasy.org/protparam/
保守结构域查找	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
信号肽分析	http://www.cbs.dtu.dk/services/SignalP-4.0/
跨膜结构查询	http://www.cbs.dtu.dk/services/TMHMM/
二级结构	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
三维模型	https://swissmodel.expasy.org/
亚细胞定位	https://wolfpsort.hgc.jp/
系统发育树构建	MEGA6

Cloning and expression analysis of Cinnamate-4-hydroxylase gene from Rhizophora stylosa

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