Crops ›› 2022, Vol. 38 ›› Issue (1): 77-83.doi: 10.16035/j.issn.1001-7283.2022.01.011

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Cloning and Bioinformatics Analysis of FtC4H Gene from Tartary Buckwheat

Yin Guifang1(), Duan Ying2, Yang Xiaolin2, Cai Suyun2, Wang Yanqing1, Lu Wenjie1, Sun Daowang1, He Runli2(), Wang Lihua1()   

  1. 1Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences/ Yunnan Provincial Key Laboratory of Agricultural Biotechnology/Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Kunming 650205, Yunnan, China
    2College of Traditional Chinese Medicine and Food Engineering, Shanxi University of Chinese Medicine, Taiyuan 030619, Shanxi, China
  • Received:2021-01-18 Revised:2021-07-19 Online:2022-02-15 Published:2022-02-16
  • Contact: He Runli,Wang Lihua E-mail:1434651675@qq.com;herunli666@163.com;wanglihua70@hotmail.com

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Abstract:

In this paper, the key enzyme gene cinnamic acid-4-hydroxylase (FtC4H) in the benzene propanes metabolic pathway of tartary buckwheat was cloned, laying a foundation for further research on its function. The RNA of Yunqiao 1 and Xiaomiqiao husks at different developmental stages was extracted, and the FtC4H gene from tartary buckwheat was cloned by RT-PCR, and the characteristics of FtC4H protein were analyzed by bioinformatics. The phylogenetic tree of FtC4H protein was constructed and analyzed FtC4H gene expression. The results showed that the cloned gene sequence had complete cDNA open reading frame of 1299bp, encoding 432 amino acids. FtC4H was a hydrophilic and unstable basic protein with P450 superfamily conserved domain and without transmembrane domain; The secondary structure of FtC4H was complex; the prediction of tertiary structure showed a high similarity with 6vby.1.A sequence. The results of phylogenetic analysis showed that the FtC4H cloned in this study was different from other C4H genes of tartary buckwheat reported. qRT-PCR showed that the relative expression level of FtC4H in the flowers and leaves of Xiaomiqiao were significant higher than that of Yunqiao 1.

Key words: Tartary buckwheat, RT-PCR cloning, Cinnamic acid-4-hydroxylase, Bioinformatics analysis, qRT-PCR

Table 1

Primer sequences and their applications"

用途
Application		 引物名称
Primer name		 引物序列（5'-3'）
Primer sequence (5'-3')
RT-PCR FtC4H-F GTAATCTCAAAGCTCCGCGG
FtC4H-R ACACATAACAGCGACGATGAC
实时荧光定量PCR H3-F AAGAAGTCCCACAGATACCGC
qRT-PCR H3-R AGCCTCCTGAAGAGCTAGCAC
FtC4H-F GGTCAGTCGAGTGGGCATTA
FtC4H-R TTGGCGTCGTTGAGGTTC

Fig.1

The electrophoresis results of FtC4H fragments M: SM0331 DNA Marker; 1: Yunqiao 1; 2: Xiaomiqiao"

Fig.2

Nucleotide sequence and amino acid sequence of FtC4H"

Fig.3

Conservative structural domain analysis of FtC4H protein in tartary buckwheat"

Fig.4

Secondary structure prediction of FtC4H protein in tartary buckwheat Blue: alpha helix; red: extended chain; green: beta turn; purple: random curl"

Fig.5

Tertiary structure prediction of FtC4H protein in tartary buckwheat"

Fig.6

Multiple sequence alignment of FtC4H with C4H genes cloned from tartary buckwheat"

Fig.7

Multiple sequence alignment of FtC4H with C4H proteins cloned from tartary buckwheat The blue box is the proline-rich region; The red box is the heme binding domain; Substrate recognition sites (SRS) are highlighted in blue line; E-R-R triad is marked by green line"

Fig.8

Phylogenetic trees based on protein sequences of FtC4H and other plant sources C4H"

Fig.9

Relative expression of FtC4H gene in different organs of tartary buckwheat “*”and“**”indicate the significant difference at the level of 0.05 and 0.01, respectively"

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