Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes

doi:10.13560/j.cnki.biotech.bull.1985.2023-1074

Abstract

Abstract:

【Objective】This study aims to clone the sterol glycosyltransferase gene AoSGT1 from Asparagus officinalis, to assess its catalytic properties, and to elucidate its role in the biosynthesis of asparagus saponins and their metabolic regulation.【Method】We designed specific primers based on asparagus transcriptome data to amplify the complete open reading frame（ORF）of AoSGT1. The gene was sequenced and analyzed through bioinformatics, and its expression profile was investigated via quantitative real-time PCR（RT-qPCR）. We also constructed a pGEX-4T-3-AoSGT1 expression vector, expressed it in Escherichia coli BL21（DE3）, and induced the recombinant protein production.【Result】AoSGT1 has a length of 1 800 base pairs, encoding 599 amino acids, with a relative molecular weight of 66.72 kD. It is a hydrophilic protein with no transmembrane domains or signal peptides. The phylogenetic analysis revealed a close homology between AoSGT1 and Dioscorea zingiberensis Dz3GT2, indicating their membership in the UGT80B1 subfamily. Multiple sequence alignment revealed that the protein sequence contained conserved domains “PSBD Box” and “PSPG Box” of sterol glycosyltransferase, indicating its potential glycosylation activity at the 3β-OH position of steroidal compounds. RT-qPCR results showed that AoSGT1 was highly expressed in the roots, while expression in stems and flowers was relatively low. Additionally, SDS-PAGE results revealed that the target protein was efficiently expressed in a soluble form within E. coli, matching the predicted size.【Conclusion】The AoSGT1 gene was successfully cloned and demonstrated to exhibit tissue-specific expression in asparagus, suggesting its potential involvement in the biosynthesis of steroidal saponins in asparagus. Additionally, heterologous expression of the target protein in E. coli was successfully achieved.

Key words: Asparagus officinalis, steroidal saponins, sterol glycosyltransferase, gene cloning, bioinformatics analysis, RT-qPCR, prokaryotic expression, transcriptome

ZHONG Yun, LIN Chun, LIU Zheng-jie, DONG Chen-wen-hua, MAO Zi-chao, LI Xing-yu. Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes[J]. Biotechnology Bulletin, 2024, 40(4): 255-263.

Figures/Tables 8

Table 1 PCR primer sequences used in this study

引物Primer	引物序列Primer sequence（5'-3'）	用途Purpose
UAoSGT1	F：TCTTGATGCGATTACAGCGG R：GAGATGGCTACAAGGTGAAAGG	基因克隆 Gene clone
PGEX-AoSGT1	F：GTGGATCCCC GAATTCCATGAGGAGCGGAGATTTCGAAG R：GGCCGCTCGA GTCGACCTACAAGGTGAAAGGAAGACAACACC	原核表达 Prokaryotic expression
qRT-AoSGT1	F：ATCCTTTGGGTGGTGATCCG R：AGAGCTTCAGCAACATGCGT	实时荧光定量PCR RT-qPCR
EF1A	F：CTGGCCAGGGTGGTTCATGAT R：TAAGTCTGTTGAGATGCACC	内参基因 Reference gene

Fig. 1 Agarose gel electrophoresis of asparagus RNA and AOSGT1 gene amplification products A: Agarose gel electrophoresis of total RNA from various tissues of asparagus. B: Amplification of the AoSGT1 gene. M: DNA marker 2000. 1: Root. 2: Stem. 3: Flower. 4: AoSGT1

Fig. 2 Bioinformatics analysis of AoSGT1 A: NCBI conservative structural domain prediction. B: Transmembrane domain prediction. C: Signal peptide analysis

Fig. 3 Structural characteristics of asparagus AoSGT1 protein A: AoSGT1 secondary structure（Blue: α- Spiral; green: β- Corner; orange: irregular curl; purple: no secondary structure）. B: AoSGT1 3D structure prediction（Blue: helix; pink: sheet; orange: loop）

Fig. 4 Phylogenetic tree of asparagus AoSGT1 and SGTs from other plants

Fig. 5 Multiple sequence alignment analysis of asparagus AoSGT1 Black box area: PSBD Box structural domain; blue box area: PSPG Box structure domain

Fig. 6 Relative expression levels of AoSGT1 in different tissue parts of asparagus

Fig. 7 SDS-PAGE gel electrophoresis of prokaryotic expression of AoSGT1 M: Protein marker. 1: Empty supernatant. 2: Empty sediment. 3: AoSGT1 supernatant. 4: AoSGT1 sediment

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