过表达veA基因对冠突散囊菌次级代谢的影响

doi:10.13304/j.nykjdb.2022.0170

摘要/Abstract

摘要：

冠突散囊菌是茯砖茶加工过程中的优势益生真菌，其产生的胞外酶和丰富的次生代谢产物影响成品茶的品质和口感。为探究veA对冠突散囊菌次级代谢的调控机制，使用无缝克隆及农杆菌转化技术构建了冠突散囊菌veA基因过表达突变株，使用高效液相色谱技术（high performance liquid chromatography，HPLC）和转录组测序初步解析过表达veA对冠突散囊菌次级代谢相关基因表达的影响。结果表明，与野生型冠突散囊菌相比，突变株发酵产物中多种化合物含量具有明显变化，物质吸收峰1的相对峰面积提高了4.09倍，且检测到3种新的物质吸收峰。转录组测序结果表明，与野生型相比，突变株的基因表达谱具有较大差异，共检测出737个显著差异表达基因（differentially expressed genes，DEGs），其中，282个基因显著上调表达，455个基因显著下调表达。DEGs主要富集在氨基酸代谢、蛋白酶和萜类化合物合成等通路。使用antiSMASH软件注释到545个次级代谢产物合成基因，10.64%的基因表达存在显著差异，其中，聚戊烯基合成酶基因、异戊二烯基二磷酸δ-异构酶基因和法尼基焦磷酸合成酶基因均显著上调表达。上述结果表明VeA能够激活一些次级代谢相关基因的表达，为冠突散囊菌的发酵应用和新颖次级代谢产物的挖掘提供了新的见解和方法。

关键词: 冠突散囊菌, veA基因, 过表达, 转录组, 次级代谢

Abstract:

Eurotium cristatus is a dominant fungus in the process of Fuzhuan tea， the extracellular enzymes and abundance of secondary metabolites affect the quality and taste of finished tea. To explore the regulation mechanism of veA on secondary metabolism of Eurotium cristatus， the veA overexpressed mutant was successfully constructed by seamless cloning and agrobacterium transformation technology， and the effect of overexpression of veA on E. cristatus was evaluated by high performance liquid chromatography （HPLC） and transcriptomic sequencing technology. The results showed that the contents of various compounds in the fermentation products of the mutant strain were significantly different from those of the wild type， 3 new compounds were detected and the absorption peak of compound 1 increased by 4.09 times. Also， there were significant differences in gene expression profiles between wild type and mutant strain. A total of 737 differentially expressed genes （DEGs） were detected， among which 282 genes were significantly up-regulated and 455 genes were significantly down-regulated. KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway showed that DEGs were mainly enriched in amino acid metabolism， proteasome and terpenoid backbone biosynthesis. AntiSMASH software annotated 545 genes related to secondary metabolite synthesis， of which 10.64% genes were significantly differentially expressed between wild type and mutant strain， including polypentenyl synthase gene， isoprene diphosphate δ-isomerase gene and farniki pyrophosphate synthase gene were significantly up-regulated. It suggested that overexpression of veA activated some secondary metabolite genes， which provided new insights and methods for further mining novel compounds of E. cristatus.

Key words: Eurotiumcristatus, veA, overexpression, transcriptome, secondary metabolism

中图分类号:

王云胜, 陈银翠, 程在, 张锦, 张传博. 过表达veA基因对冠突散囊菌次级代谢的影响[J]. 中国农业科技导报, 2023, 25(7): 77-86.

Yunsheng WANG, Yincui CHEN, Zai CHENG, Jin ZHANG, Chuanbo ZHANG. Effects of Overexpression of veA Gene on Secondary Metabolism of Eurotium cristatus[J]. Journal of Agricultural Science and Technology, 2023, 25(7): 77-86.

图/表 9

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引物 Primer	序列 Sequence （5’-3’）
veAF	AGACATCACCATGGTAGATCTATGGCGACGCGAATGCCC
veAR	GGGGAAATTCGAGCTGGTCACCTTAACTAGAAAAAGCCGGCGG
GAPDHF	CTGCCGTATCGAGAAGGGTG
GAPDHR	GATGAAGTTGGGGTTGAGGG
QveAF	CGACATCACCTTTGCCTAC
QveAR	TCGGGAAAGATGAAGTAGC

引物 Primer	序列 Sequence （5’-3’）
veAF	AGACATCACCATGGTAGATCTATGGCGACGCGAATGCCC
veAR	GGGGAAATTCGAGCTGGTCACCTTAACTAGAAAAAGCCGGCGG
GAPDHF	CTGCCGTATCGAGAAGGGTG
GAPDHR	GATGAAGTTGGGGTTGAGGG
QveAF	CGACATCACCTTTGCCTAC
QveAR	TCGGGAAAGATGAAGTAGC

样品 Sample	原始数据 Raw reads	质控数据 Clean reads	错配率 Error rate/%	Q20/%	Q30/%
OE：：veA1_1	53 215 236	52 880 844	0.023 9	98.44	95.28
OE：：veA1_2	58 759 294	58 355 148	0.024 0	98.4	95.21
OE：：veA1_3	56 979 242	56 535 564	0.024 1	98.36	95.11
WT1	52 014 002	51 659 080	0.024 0	98.41	95.24
WT2	57 594 222	57 246 210	0.023 9	98.43	95.26
WT3	58 672 768	58 282 756	0.023 9	98.42	95.25

样品 Sample	原始数据 Raw reads	质控数据 Clean reads	错配率 Error rate/%	Q20/%	Q30/%
OE：：veA1_1	53 215 236	52 880 844	0.023 9	98.44	95.28
OE：：veA1_2	58 759 294	58 355 148	0.024 0	98.4	95.21
OE：：veA1_3	56 979 242	56 535 564	0.024 1	98.36	95.11
WT1	52 014 002	51 659 080	0.024 0	98.41	95.24
WT2	57 594 222	57 246 210	0.023 9	98.43	95.26
WT3	58 672 768	58 282 756	0.023 9	98.42	95.25

基因编号 Gene ID	功能注释 Function annotation	变化倍数 Fold change	调控 Regulate
SI65_05759	氧化还原酶 Flavoprotein-ubiquinone oxidoreductase	1.01^ns	上调Up
SI65_05760	反转录酶 Reverse transcriptase	0.61^ns	下调Down
SI65_05761	TATA框结合蛋白 TATA-box-binding protein	0.72^ns	下调Down
SI65_05762	L-甲基哌啶氧化酶 L-pipecolate oxidase	1.50^*	上调Up
SI65_05763	甲基转移酶 Methyltransferase	1.05^ns	上调Up
SI65_05764	假设蛋白 Hypothetical protein	1.45^ns	上调Up
SI65_05765	C6指域转录因子 C6 finger domain transcription factor	1.25^ns	上调Up
SI65_05766	NADH-生化色素b5还原酶 NADH-cytochrome b5 reductase	1.27^ns	上调 Up
SI65_05767	甲基转移酶 Methyltransferase	1.72^*	上调Up
SI65_05768	羧基酸合成酶 Carboxylic acid synthase	2.02^*	上调Up
SI65_05769	金属化合物-β-内酰胺酶 Metallo-beta-lactamase	2.31^*	上调Up
SI65_05770	细胞色素P450单加氧酶 Cytochrome P450 monooxygenase	1.92^ns	上调Up
SI65_05771	细胞色素P450单加氧酶 Cytochrome P450 monooxygenase	2.47^*	上调 Up
SI65_05772	氧化还原酶 Oxidoreductase	2.67^*	Up 上调
SI65_05773	乙酰基转移酶 Acetyltransferase	2.74^*	Up 上调
SI65_05774	假定蛋白 Hypothetical protein	NA	NA
SI65_05775	假定蛋白 Hypothetical protein	0.55^*	Down 下调
SI65_05776	转录因子 Transcription factor	0.64^*	Down 下调