Lithocarols类化合物生物合成基因litI的表达及其启动子功能分析

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

摘要/Abstract

摘要：

【目的】 Lithocarols为新型的多异戊烯基二苯甲酮类化合物，具有良好的抗肿瘤活性。对lithocarols生物合成基因litI进行克隆和表达纯化，并对该基因的启动子进行功能鉴定，为lithocarols的生物合成及转录调控奠定分子生物学基础。【方法】 将litI基因扩增后进行原核表达，采用镍亲和层析初步纯化目的蛋白LitI，利用生物信息学方法分析该蛋白的性质和结构。同时扩增litI基因启动子片段，构建荧光素酶表达系统，分析启动子的转录活性，利用PlantCARE启动子分析网站对litI基因启动子的功能组件进行预测。【结果】 LitI蛋白为亲水性蛋白，其相对分子量为51 kD，二级结构包括51.32%的α-螺旋、8.99%的延伸链、3.95%的β-转角以及35.75%无规则卷曲。litI基因启动子具有较强的转录活性且在大肠杆菌中具有启动氨苄青霉素抗性基因表达的功能，其功能组件包含TATA box和CAAT box。【结论】 通过异源表达获得了LitI蛋白，分析了其性质和结构，并鉴定了具有较强转录活性的litI基因启动子片段。

关键词: Phomopsis lithocarpus, 多异戊烯基二苯甲酮类化合物, 生物合成基因, 启动子, 异源表达, 生物信息学

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

李梦然, 叶伟, 李赛妮, 张维阳, 李建军, 章卫民. Lithocarols类化合物生物合成基因litI的表达及其启动子功能分析[J]. 生物技术通报, 2024, 40(6): 310-318.

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.

图/表 9

图1 化合物结构1-7

Fig. 1 Structures of compound 1-7

图2 基因litI的PCR验证 A：基因litI的克隆，M：DL5000 DNA marker；I：基因litI。B：菌液PCR验证，M：DL5000 DNA marker；I1-5：pET-30a-litI阳性单克隆

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

图3 蛋白LitI的初步纯化 A：蛋白LitI表达条件的优化，M：Blue Plus II protein marker（14-100 kD）；1、3、5、6：条件①、③、②、④的上清；2、4、7、8：条件①、③、②、④的沉淀。B：蛋白LitI的初步纯化，M： protein marker（14-100 kD）；1：穿过液；2：10%咪唑洗脱液

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

图4 LitI蛋白的生物信息学分析 A：蛋白LitI的亲水性/疏水性分析；B：LitI蛋白的二级结构分析；C：LitI蛋白三级结构预测同源建模

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

图5 pGL3-basic-启动子重组载体的构建及其转录活性分析 A：litI启动子片段的克隆，M：DL5000 DNA marker；I-1，I-2：PCR扩增得到的启动子片段；B：含 pGL3-basic-litI启动子的菌落PCR 验证，M：DL5000 DNA marker；1-5：含I-1启动子的菌落PCR验证；6-10：含I-2启动子的菌落PCR验证；C：pGL3-basic-空载/pgpd/litI 启动子载体的荧光强度分析

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

图6 Trans5α 菌落PCR验证及启动子载体图谱 A：含-pET28a-Amp同源臂引物扩增的litI启动子片段，M1：Trans2K DNA marker；B：Trans5α-pET28a-I-2菌落PCR，M2：DL5000 DNA Marker；C：Trans5α-pET28a-I-2-AmpR载体图谱

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

图7 含启动子片段的大肠杆菌在Amp抗性平板上的生长情况 A：Trans5α-pEASY-T1-AmpR、Trans5α-pET28a-ACP-AmpR、Trans5α-pET28a-I-2-AmpR点板结果，P：Trans5α-pEASY-T1-AmpR（阳性对照）；N：Trans5α-pET28a-ACP-AmpR（阴性对照）；I：Trans5α-pET28a-I-2-AmpR；B：Trans5α-pET28a-ACP-AmpR、Trans5α-pEASY-T1-AmpR、Trans5α-pET28a-I-2-AmpR分别在0、25、40 μg /mL AMP浓度的LB培养基中的OD值

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

表1 litI启动子片段I-2的序列

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

图8 litI启动子功能元件分析

Fig. 8 Functional component analysis of litI promoter

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