Phomopsis tersa FS441聚酮杂萜类化合物生物合成基因启动子的鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-0347

摘要/Abstract

摘要：

【目的】Phomeroids为分离自海洋真菌Phomopsis tersa FS441的聚酮杂萜类新骨架化合物，具有良好的抗肿瘤活性。为便于后期对phomeroids生物合成关键基因萜类环化酶编码基因ctg1509和P450单加氧酶编码基因ctg1511进行转录调控，对其启动子功能进行鉴定。【方法】对海洋真菌P. tersa FS441中ctg1509、ctg1511基因启动子p1509、p1511进行克隆，用荧光素酶报告基因载体分析启动子的转录活性，并进一步采用点板实验和生长曲线鉴定其在大肠杆菌中启动氨苄青霉素抗性基因转录的功能，采用PlantCARE数据库分析启动子的调控元件。【结果】p1509的启动子转录活性最强，其活性远强于阳性对照PgpdA启动子，p1509和p1511能在大肠杆菌中启动氨苄青霉素抗性基因的转录。p1509、p1511启动子区域除了含有CAAT-box、TATA-box核心元件，还有参与低温响应、光响应等顺式调控元件。【结论】验证了启动子p1509和p1511的功能。发掘了一个转录活性强于阳性对照的新型强启动子p1509，该启动子含有丰富的调控元件。

关键词: 生物合成基因启动子, 转录活性, Phomopsis tersa, 聚酮杂萜, 元件分析

Abstract:

【Objective】 Phomeroids are novel skeleton polyketide meroterpenoids isolated from the marine fungus Phomopsis tersa FS441, demonstrating promising anti-tumor activity. To facilitate the transcriptional regulation of the crucial genes for the biosynthesis of phomeroids, including the terpene cyclase gene ctg1509 and the P450 monooxygenase gene ctg1511, the functions for the promoters of these two genes were identified in this study.【Method】 This study cloned the promoter p1509 and p1511 of ctg1509 and ctg1511 genes in marine fungus P. tersa FS441. The transcriptional activities of the p1509 and p1511 promoters were validated using luciferase reporter gene vectors, the plate experiment and the growth curve were employed to identify the function of the two promoters. Moreover, PlantCARE database was used to analyze the regulatory elements of the promoter. 【Result】 The promoter of p1509 has the strongest transcriptional activity, which is stronger than the positive promoter PgpdA. p1509 and p1511 promoters can initiate the transcription of ampicillin resistant gene AmpR in Escherichia coli. p1509 and p1511 promoters’ regions not only contain core components such as CAAT box and TATA box, but also include cis-acting regulatory elements involved in low temperature response and light response. 【Conclusion】 This study confirmed the function of p1509 and p1511 promoters, and excavated a novel strong promoter, p1509, which had stronger transcriptional activity than the positive control and contained abundant regulatory elements.

Key words: promoters for biosynthetic genes, transcriptional activity, Phomopsis tersa, polyketide meroterpenoids, element analysis

刘玉萍, 张维阳, 章卫民, 叶伟, 李冬利. Phomopsis tersa FS441聚酮杂萜类化合物生物合成基因启动子的鉴定[J]. 生物技术通报, 2024, 40(12): 248-255.

LIU Yu-ping, ZHANG Wei-yang, ZHANG Wei-min, YE Wei, LI Dong-li. Identification of the Promoter for the Biosynthesis Gene of Polyketide Meroterpenoids in Phomopsis tersa FS441[J]. Biotechnology Bulletin, 2024, 40(12): 248-255.

图/表 7

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启动子Promoter	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
p1509	CCGCTCGAGCGTTTATAATGAGAATACATAGCG	CCCAAGCTTCCGAACGCGTAGTAAACG
p1511	ACGCGTGCTAGCCCGGGCAACACGTAGGAAGAAGAGGGTC	AACAGTACCGGAATGCCATGACAATTTAAACTOTGTTAAGGTAC

启动子Promoter	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
p1509	CCGCTCGAGCGTTTATAATGAGAATACATAGCG	CCCAAGCTTCCGAACGCGTAGTAAACG
p1511	ACGCGTGCTAGCCCGGGCAACACGTAGGAAGAAGAGGGTC	AACAGTACCGGAATGCCATGACAATTTAAACTOTGTTAAGGTAC

启动子 Promoter	分值 Score	核心序列 Core sequence
p1509	0.97	TTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATG
p1511	0.92	AATATTTTGTTAAAATTCGCGTTTAAATTTTTGTTAAATCAGCTCATTTT

启动子 Promoter	分值 Score	核心序列 Core sequence
p1509	0.97	TTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATG
p1511	0.92	AATATTTTGTTAAAATTCGCGTTTAAATTTTTGTTAAATCAGCTCATTTT

启动子Promoter	元件名称Component name	元件功能Component functions
p1509/p1511	ABRE	参与脱落酸反应性的顺式作用元件Cis-acting elements involved in abscisic acid reactivity
	MBS	参与干旱诱导的MYB结合位点MYB binding sites involved in drought induced stress
	TGACG-motif	参与MeJA反应的顺式调控元件Cis-acting regulatory elements involved in MeJA reaction
	TATA-box	转录开始的核心启动子元件Core promoter elements for transcription initiation
	CAAT-box	启动子和增强子区域中顺式作用元件Cis-acting elements in promoter and enhancer regions
	CGCTA-motif	参与MeJA反应的顺式调控元件Cis-regulating elements involved in MeJA reaction
p1509	G-Box	参与光反应的顺式调节元件Cis-regulating elements involved in light reactions
	GATA-motif	光反应的顺式调节元件Cis-regulating element for light reaction
	GCN4-motif	参与胚乳表达调控元件Elements involved in regulating endosperm expression
	P-Box	赤霉素反应元件Gibberellin reaction element
	TATC-Box	参与赤霉素反应的顺式作用元件Cis-acting elements involved in gibberellin reaction
	TGA-element	激活反应元件Activate reaction elements
p1511	LTR	低温顺式响应元件Low temperature cis-responsive element
	I-Box	光响应元件Photoresponsive element
	ARE	厌氧诱导顺式作用元件Anaerobic induced cis acting element
	TCT-motif	光响应元件Photoresponsive element
	GT1-motif	光响应元件Photoresponsive element