Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (19): 4169-4178.doi: 10.3864/j.issn.0578-1752.2021.19.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Identification of γ-Glutamyl Transpeptidase AcGGT Gene from Onion (Allium cepa)

XU HuanHuan1,2(),LI Yi1,GAO Wei1,WANG YongQin2(),LIU LeCheng1()   

  1. 1College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, Hubei
    2Vegetable Research Center, Beijing Academy of Agriculture and Forestry/Key Laboratory of Biology and Genetic Improvement of Horticultural (North China), Ministry of Agriculture and Rural Affairs, P.R. China/Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing 100097
  • Received:2020-11-12 Accepted:2021-01-08 Online:2021-10-01 Published:2021-10-12
  • Contact: YongQin WANG,LeCheng LIU E-mail:sr19951010@126.com;wangyqly@163.com;516119@yangtzeu.edu.cn

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Abstract:

【Objective】Alliin metabolized by Allium plants had important pharmaceutical value. γ-glutamyl transpeptidase was a key enzyme in the deglutamylation step of the process of alliin synthesis. Studying the function of γ-glutamyl transpeptidase gene in onion can reveal the role of γ-glutamyl transpeptidase in alliin synthesis pathway, providing theoretical basis for alliin synthesis in vitro and laying a foundation for further study on alliin synthesis mechanism. 【Method】Using onion as material, the primers were designed according to onion RNA-seq database, and the gene, γ-glutamyl transpeptidase, was cloned from onion by RT-PCR and analyzed by bioinformatics. The CAMV 35S-AcGGT-GFP vector was used to bombard onion inner epidermis cells with gold powder plasmid microcarrier by particle bombardment technology, and the subcellular localization of AcGGT was determined by fusion green fluorescent expression protein. The Saccharomyces cerevisiae expression vector with AcGGT was constructed, transforming and inducing the expression of AcGGT, and using the method of transforming glutamyl-p-nitroaniline to p-nitroaniline by γ-glutamyl transpeptidase to determine the glutamyl transpeptidase activity of the total protein of Saccharomyces cerevisiae transferred into AcGGT. Real time quantitative PCR was used to analyze the differential expression pattern of the gene in onion tissues. The activity of endogenous transpeptidase in onion tissues was determined by the method of γ-glutamyl transpeptidase catalyzing the production of p-nitroaniline from p-nitroaniline. 【Result】AcGGT was cloned and its length was 1 869 bp. Bioinformatics analysis showed that AcGGT encoded 622 amino acids, protein domain prediction showed that it had glutamyl transpeptidase domain, secondary structure was mainly α - helix, transmembrane region analysis suggested that GGT protein had transmembrane region, amino acid multiple alignment results showed that GGT in plants had certain conservation, evolutionary analysis showed that AcGGT was related to garlic AsGGT2, and the relationship is closest. The fluorescence signal of CaMV 35S-AcGGT-GFP fusion protein was located in the vacuole, indicating that the protein encoded by CaMV 35S-AcGGT-GFP was located in the vacuole. The results of glutamyl transpeptidase activity assay showed that the glutamyl transpeptidase activity of yeast transformed with AcGGT was significantly higher than the control, indicating that the protein encoded by AcGGT had transpeptidase activity. The results of differential expression analysis of AcGGT showed that the expression of AcGGT was mainly in leaf sheath, bulb and leaf sheath followed by. The activity of glutamyl transpeptidase in different tissues, the highest activity in leaf, followed by leaf sheath. Correlation analysis showed that there was no significant correlation between the activity of transglutaminase and the expression of AcGGT. 【Conclusion】The enzymatic reaction data of exogenous AcGGT expression were obtained. The deglutination of alliin synthesis pathway preceded S-oxygenation; there was no significant correlation between AcGGT expression and endogenous transglutaminase activity in onion, suggesting that there may be multiple transglutaminase genes in onion.

Key words: onion, γ-glutamyl transpeptidase, bioinformatics, subcellular localization, eukaryotic expression, expression pattern

Table 1

Primers used in this study"

名称 Name 引物序列 Primer sequence (5′-3′) 引物用途 Use of primer
GGT-F ATGGAACCGGCGCATGATGA 基因克隆
Gene cloning
GGT-R TCACACATGCAGGACTTCCATCT
PYBA1332-GGT-F GGAGAGGACAATTGGAGCTCATGGAACCGGCGCATGATGA 亚细胞定位
Subcellular Localization
PYBA1332-GGT-R CCCTTGCTCACCATGGTACCCACACATGCAGGACTTCCATCT
pYES2-GGT-F GGAATATTAAGCTTGGTACCATGGAACCGGCGCATGATG 真核表达
Prokaryotic Expression
pYES2-GGT-R CCTCTAGATGCATGCTCGAGTCACACATGCAGGACTTCC
AcTUB-F TCAGTCCAGTAGGAGGAATGTCGC 实时荧光定量PCR
qRT-PCR
AcTUB-R CTGTCTTCAGAGGCAAGATGAGCAC
qPCR-GGT-F AGTTGAAGTCACAGAGGCAC 实时荧光定量PCR
qRT-PCR
qPCR-GGT-R CAAGCCTTTCACAGCATCAAG

Fig. 1

PCR amplification of onion AcGGT M：DL5000 DNA Marker;1—3：AcGGT"

Fig. 2

Coding region sequence of AcGGT"

Fig. 3

Sequence alignment of the representative γ-glutamyl transpeptidases sequences from the indicated species Black line: Predicted GGT signal peptide; *: Predicted N-linked glycosylation site; Arrow: Conservative protease cleavage site between large subunit and small subunit"

Fig. 4

Prediction of AcGGT conserved domain and AcGGT secondary structure A: Prediction of the conserved domain of γ-glutamyl transpeptidase; B: Prediction of the secondary structure of γ-glutamyl transpeptidase"

Fig. 5

Phylogenetic tree of AcGGT protein sequences from onion and other species"

Fig. 6

Phylogenetic tree of GGT protein sequences from onion and other species *：Allium cepa AcGGT"

Fig. 7

Subcellular localization of 35S-AcGGT-GFP in onion epidermal cell"

Fig. 8

Activity determination of AcGGT protein heterologously expressed in yeast **: Indicate the significant difference (P<0.01) compared with control"

Fig. 9

Difference of AcGGT expression and activity of transglutaminase in onion R: Root; B: Bulb; S: Sheath; L: Leaf"

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