Crops ›› 2022, Vol. 38 ›› Issue (3): 73-79.doi: 10.16035/j.issn.1001-7283.2022.03.010

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Molecular Cloning and Bioinformatics Analyzing of Laccase in Fagopyrum tataricum

Yang Xiaolin1(), Duan Ying1, Cai Suyun1, He Runli1(), Yin Guifang2, Wang Yanqing2, Lu Wenjie2, Sun Daowang2, Wang Lihua2()   

  1. 1College of Traditional Chinese Medicine and Food Engineering, Shanxi University of Traditional Chinese Medicine, Taiyuan 030619, Shanxi, China
    2Biotechnology 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 650221, Yunnan, China
  • Received:2021-07-07 Revised:2021-11-08 Online:2022-06-15 Published:2022-06-20
  • Contact: He Runli,Wang Lihua E-mail:15733605992@163.com;herunli666@163.com;wanglihua70@hotmail.com

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Abstract:

In this experiment, to study the gene function of laccase protein in the development of tartary buckwheat, two laccase genes from tartary buckwheat were cloned using RT-PCR technology. Bioinformatics techniques were used to predict the domain, secondary and tertiary protein structure, and to perform the protein homology alignment and phylogenetic evolutionary tree analysis. The results showed that the open reading frame of the FtLAC-1 gene sequence was 1695bp, coding for 564 amino acids, and the open reading frame of the FtLAC-2 gene sequence was 1707bp, coding for 568 amino acids. The predicted molecular weights of FtLAC-1 and FtLAC-2 proteins were 61.41 and 62.47kDa and their theoretical isoelectric points were 9.45 and 9.41, respectively. They were hydrophilic proteins. The sequence similarity of the two genes was low and the amino acid sequence homology was not high. qRT-PCR analysis of its differential expression in different tissues and organs of tartary buckwheat. This study provided a theoretical basis for further exploring the functions of the FtLAC-1 and FtLAC-2 genes in thin buckwheat husk formation.

Key words: Fagopyrum tataricum, Laccase, Gene cloning, Bioinformatics, Expression analysis

Table 1

Sequences of primers and their usage"

引物名称
Primer name		 引物序列（5'-3'）
Primer sequence（5'-3'）		 用途
Usage
LAC-1-1F AGTCACTCCACTACAGCAACACACG 基因克隆
LAC-1-1468R CGGATGGCTTTCAGGTGCTA
LAC-1-1285F CAACATAAGTGGGGTATTCACGGAT
LAC-1-2131R TTCAGAGAAGGATTTCAAACAACAA
LAC-2-47F TTCAAACCACACACAAAACCCTA
LAC-2-1437R CACCACCTTCGTACCGTTCATC
LAC-2-1067F CACTCCCAGCCATCAACGAC
LAC-2-2101R CGGAGCAAGAGGGAAGAAACA
LAC-1-F GGAAGGGGTCCTAACGAGTCT 实时荧光
定量PCR
LAC-1-R CCTATTGTCTCCTTTGCTGGTT
LAC-2-F TGGTGGAACAACGATACGG
LAC-2-R CGGGAAAGCCATTGAAAGT
H3-F AAGAAGTCCCACAGATACCGC
H3-R AGCCTCCTGAAGAGCTAGCAC

Fig.1

Amplification of the LAC-1 and LAC-2 genes from tartary buckwheat M: SM0331 DNA marker; 1, 2: H-LAC-1; 3, 4: B-LAC-1; 5, 6: H-LAC-2; 7, 8: B-LAC-2; H: Yunqiao 1 (thick shell); B: Millet buckwheat (thin shell)"

Fig.2

Conserved domains of gene-encoded proteins of FtLAC-1 and FtLAC-2"

Fig.3

Analysis of hydrophobicity of laccase proteins"

Fig.4

The prediction of tertiary structural model of laccase proteins"

Fig.5

Amino acid sequence alignment of different plant laccase proteins"

Fig.6

Phylogenetic tree analysis of laccase proteins from different species"

Fig.7

Relative expression of FtLAC-1 and FtLAC-2 genes in various organs “*”means the difference is significant at 0.05 level,“**”means the difference is extremely significant at 0.01 level"

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