Expression of Lithocarols Biosynthesis Gene litI and Functional Analysis of Its Promoter

doi:10.13560/j.cnki.biotech.bull.1985.2023-1225

Abstract

Abstract:

【Objective】 Lithocarols are a novel class of polyisoprenyl diphenylketone compounds, which have a good antitumor activity. The lithocarols biosynthesis gene litI was cloned, expressed and purified, the promoter of the gene litI was functionally identified, which will lay a molecular foundation for the biosynthesis and transcriptional regulation of lithocarols. 【Method】 The biosynthetic gene litI was cloned and expressed in Escherichia coli, and the expressed protein LitI was preliminarily purified by nickel affinity chromatography column. The properties and structure of the protein were analyzed by bioinformatics method. The litI gene promoter fragment was amplified, the luciferase expression system was constructed to analyze the transcriptional activity of the promoter, and its functional components were predicted by the PlantCARE promoter analysis website. 【Result】 LitI protein is a hydrophilic protein, which has a relative molecular weight of 51 kD and a secondary structure including 51.32% α-helix, 8.99% extended chain, 3.95% β-angle, and 35.75% irregular curling. The gene litI promoter shows strong transcription activity, and it could initiate the transcription of ampicillin resistance gene in E. coli, and litI promoter contained TATA box and CAAT box. 【Conclusion】 LitI protein is obtained by heterologous expression, its properties and structure are analyzed, and the promoter fragment of litI gene with strong transcription activity is identified.

Key words: Phomopsis lithocarpus, polyisoprenyl benzophenones, biosynthetic gene, promoter, heterologous expression, bioinformatics

LI Meng-ran, YE Wei, LI Sai-ni, ZHANG Wei-yang, LI Jian-jun, ZHANG Wei-min. Expression of Lithocarols Biosynthesis Gene litI and Functional Analysis of Its Promoter[J]. Biotechnology Bulletin, 2024, 40(6): 310-318.

Figures/Tables 9

Fig. 1 Structures of compound 1-7

Fig. 2 PCR verification of gene litI A: Cloning of gene litI, M: DL5000 DNA marker; I: gene litI. B: PCR verification of bacterial liquid, M: DL5000 DNA marke; I1-5: positive colonies of pET-30a-litI

Fig. 3 Preliminary purification of protein LitI A: Optimization of protein LitI expression conditions. M: Blue Plus II protein marker（14-100 kD）; 1, 3, 5 and 6: supernatant for ①, ③, ② and ④; 2, 4, 7 and 8: sediment for ①, ③, ② and ④. B: Preliminary purification of protein LitI, M: protein marker; 1: binding buffer; 2: 10% imidazole eluent

Fig.4 Bioinformatics analysis of LitI protein A: Hydrophilicity/hydrophobicity analysis of protein LitI. B: Secondary structure analysis of protein LitI. C: Homology modeling for tertiary structure prediction of protein LitI

Fig. 5 Construction of pGL3-basic-promoter recombinant vector and analysis of its transcriptional activity A: Cloning of the litI promoter fragments; M: DL5000 DNA marker; I-1, I-2: promoter fragments amplified by PCR. B: PCR validation of colonies with pGL3-basic-litI promoter; M: DL5000 DNA marker; 1-5: PCR validation of colonies containing I-1 promoter; 6-10: PCR validation of colonies containing I-2 promoter. C: Luciferase RU values of the pGL3-basic-unloaded/pgpd/litI promoter vector in Trans5α cells

Fig.6 PCR verification of Trans5α colony and promoter vector map A: litI promoter fragment amplified with -pET28a-Amp homology arm primers； M1: Trans2K DNA marker. B: Colony PCR of Trans5α-pET28a-I-2； M2: DL5000 DNA marker; C: vector map of Trans5α-pET28a-I-2-AmpR

Fig.7 Growth of E. coli containing promoter fragments on ampicillin-resistant plates A: The plates of Trans5α-pEASY-T1-AmpR, Trans5α-pET28a-ACP-AmpR, and Trans5α-pET28a-I-2-AmpR, P: Trans5α-pEASY-T1-AmpR（Positive control）; N: Trans5α-pET28a-ACP-AmpR（Negative control）; I: Trans5α-pET28a-I-2-AmpR. B: The OD values of Trans5α-pET28a-ACP-AmpR, Trans5α-pEASY-T1-AmpR and Trans5α-pET28a-I-2-AmpR in LB medium at 0, 25, and 40 μg/mL AMP, respectively

Table 1 Sequence of litI promoter fragment I-2

启动子片段 Promoter fragment	启动子序列 Promoter sequence（5'-3'）
I-2	gcttgaatgcagcaagcaggcaactcgggcatcggagttgaccaatttggga caggcggaggtacatgtacccatgtagccatgcatacggccacctcccaag ataaatcacagctgatgatcagaccatattgctgagcatgttcgacaaacg ttttctgcgcagttagccgctatAAAAAtgttaatagaaggtaattcgatc gaaaccgtttgcaaataatattcagtgccacaagtcggtcttcaccggctc catacgattattcgcagatactgagacgacatactatctgttcattctcacctgaacatg

Fig. 8 Functional component analysis of litI promoter

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