开花期番茄circRNA的测序分析及功能分析

doi:10.3864/j.issn.0578-1752.2023.21.012

摘要/Abstract

摘要：

【背景】开花期作为植物生长发育过程中至关重要的时期之一，直接影响植物果实成熟和种子发育。circRNA是一类普遍存在于真核细胞的共价闭环RNA分子，在番茄的发育过程和应激反应中起重要调节作用。但目前番茄的circRNA研究主要集中在果实和叶片中，缺少对番茄花期circRNA较为系统的研究。【目的】鉴定和分析开花期番茄circRNA，对番茄中miRNA、circRNA的功能研究有重要意义，也为番茄生长、发育和胁迫响应机制研究奠定基础。【方法】选择开花期番茄植株的花、根、叶3个组织样品进行circRNA测序，每个样品3个重复，鉴定circRNA并对其基本特性进行分析；筛选组织特异性的circRNA检测成环能力，并对鉴定的circRNA宿主基因进行GO分析和KEGG分析，通过生物信息学方法预测分析circRNA的作用方式和作用位点，构建番茄响应生长发育的潜在circRNA-miRNA-mRNA互作调控网络。【结果】利用高通量测序方法，共获得532个circRNA，其中83%为外显子类型，且circRNA在花期番茄各染色体中的分布不均匀，其中1号染色体产生的circRNA最多，5号染色体最少。对开花期番茄花、叶、根3个组织的circRNA差异表达分析表明，花与叶中差异表达的circRNA有79个、花与根中差异表达的circRNA 133个、叶与根中差异表达的circRNA 132个。其中花、叶、根3个组织中均有显著差异表达的circRNA 14个。从14个差异表达circRNA中随机选择8个circRNA进行成环能力检测，结果表明这8个circRNA均具有成环能力。GO分析和KEGG分析表明开花期番茄中的circRNA主要与核酸、蛋白及其他小分子物质结合以及各类生物大分子的合成和代谢相关。最后构建了14个circRNA、10个miRNA和136个mRNA组成的番茄circRNA-miRNA-mRNA互作调控网络。【结论】开花期番茄circRNA具有明显的组织特异性，共鉴定到342个组织特异性的circRNA，其中均显著特异性表达的14个，成功鉴定成环8个，构建了开花期番茄特异性的circRNA-miRNA-mRNA互作调控网络，为后续开花期circRNA的研究奠定了基础。

关键词: circRNA, 番茄, 开花期, miRNA, ceRNA

Abstract:

【Background】As one of the most important periods in plant growth and development, the flowering period directly affects fruit ripening and seed development. circRNAs are a class of covalent closed-loop RNA molecules that are ubiquitous in eukaryotic cells and play an important role in the regulation of tomato development and stress response. However, the current circRNA studies on tomato mainly focus on fruit and leaves, and there is a lack of systematic studies on tomato circRNA at flowering stage.【Objective】Identification and analysis of circRNs in flowering tomato could be of great significance for the functional study of miRNA and circRNA in tomato, and also layed a foundation for the study of tomato growth, development and stress response mechanism.【Method】circRNA sequencing was performed on 3 tissue samples of flowers, roots and leaves of flowering tomato plants, with 3 replicates for each sample. circRNAs were identified and their basic characteristics were analyzed. The cycle-forming ability of tissue-specific circRNAs was screened, and the host genes of identified circRNAs were analyzed by GO analysis and KEGG analysis. The mode and site of action of circRNAs were predicted and analyzed by bioinformatics methods to construct a potential circRNA-miRNA-mRNA interaction regulatory network in response to tomato growth and development.【Result】A total of 532 circRNAs were obtained by high-throughput sequencing, 83% of which were exon types. The distribution of circRNA in each chromosome of flowering tomato was uneven, among which chromosome 1 produced the most circRNAs and chromosome 5 produced the least. circRNAs differentially expressed in flower, leaf and root tissues of flowering tomato showed that 79 circRNAs were differentially expressed in flower and leaf, 133 circRNAs were differentially expressed in flower and root, and 132 circRNAs were differentially expressed in leaf and root tissues. Among them, 14 circRNAs were differentially expressed in flower, leaf and root tissues. The cyclization ability of 8 circRNAs randomly selected from 14 differentially expressed circRNAs was tested, and the results showed that all 8 circRNAs had cyclization ability. GO analysis and KEGG analysis showed that circRNAs in flowering tomato were mainly related to the binding of nucleic acids, proteins and other small molecules, as well as the synthesis and metabolism of various biological macromolecules. Finally, the tomato circRNA-miRNA-mRNA interaction regulatory network composed of 14 circRNAs, 10 miRNAs and 136 mRNAs was constructed.【Conclusion】A total of 342 tissue-specific circRNAs were identified, among which 14 were significantly expressed, and 8 were successfully identified. A circRNA-miRNA-mRNA interaction regulatory network was constructed for tomato at flowering stage. This study laid a foundation for the subsequent research of circRNA in flowering period.

Key words: circRNA, tomato, flowering stages, miRNA, ceRNA

殷子贺, 杨成成, 赵钰慧, 赵丽, 吕秀荣, 杨振超, 武永军. 开花期番茄circRNA的测序分析及功能分析[J]. 中国农业科学, 2023, 56(21): 4288-4303.

YIN ZiHe, YANG ChengCheng, ZHAO YuHui, ZHAO Li, LÜ XiuRong, YANG ZhenChao, WU YongJun. Sequencing and Functional Analysis of Tomato circRNA During Flowering Stage[J]. Scientia Agricultura Sinica, 2023, 56(21): 4288-4303.

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circRNA ID		正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
Circ_97/NC_015441.2：3422764-3423369	S97	TGTTGACATTGAGAGTGTGGGGTGT	TTGAAAAGATGAAAGGGCTTTTGCT
Circ_97/NC_015441.2：3422764-3423369	F97	TCAAAATCATCACCTCCAGAAACCAG	TATCCAGCATCAGCACCGCCACTAC
Circ_473/NC_015444.2: 64279949-64300043	S473	AAAGGGAGAAGAGCAGGAGGCAGGT	CTTATGTTTCCGATGATTGAGTGTA
Circ_473/NC_015444.2: 64279949-64300043	F473	ATCATCATCTCTTCATCCTCAAACA	TGGCGGTCGTGCTCCTATT
Circ_136/NC_015444.2: 211739-220422	S136	ATTATCCATTACAGTAACACCCACC	TTAAATTAAACACATTGAAAGCCTC
Circ_136/NC_015444.2: 211739-220422	F136	GCCCATCATTGGAATAGCAAAAGAGC	CAGCTAGGAGCAGGAGGGAAGAAGC
Circ_240/NC_015441.2: 2372079-2376972	S240	CATGCTGTACGTGCTGCTGTTATAT	TCCTCTTCATCGTGTTTTGCTTTTG
Circ_240/NC_015441.2: 2372079-2376972	F240	TCACAAACCTACACCAACTCTAAACA	GGACTTCCGTACTGAAAGAAATGAG
Circ_19/NC_015447.2: 1328362-1329697	S19	TAAGAAGAAGAGGACTCACCAAAGG	AAGATCAGCATCATCCACAACCCGA
Circ_19/NC_015447.2: 1328362-1329697	F19	TGATGAGTTGATCGGTGTTCTG	TGTGCTGATCTGAAGAGGTCTCC
Circ_105/NC_015439.2: 4011485-40114693	S105	ACTTGATTCCTACTTCCTTTTTCGT	TAGTTTTAGTTGATCGTTTGCCTTG
Circ_105/NC_015439.2: 4011485-40114693	F105	CTGAAAGATTAGTTGGATGGAGGAGG	GTTGCCCTCTGTTCCGAAAATGACTC
Circ_77/NC_015438.2: 89916310-89921994	S77	CTTTACTTTCCATTTCATTTGTTGT	ATCTTTTTCTCTCCCATTTTCTTTC
Circ_77/NC_015438.2: 89916310-89921994	F77	ATGTAGTCCTGGATTGAAGGAGAC	GAACTTGTGACGATATGCCTCTC
Circ_228/NC_015449.2: 64101116-64102065	S228	ATCAAGGAGGACCAATGCTTCAGTA	CGGAGAATGTGTATGCGATTTATGT
Circ_228/NC_015449.2: 64101116-64102065	F228	TATCTCTTGTTTGTTCAACCCCACTG	ATGGAACAGATTGGTGAAAATGCCCT

样本 ID Sample ID	总读数的数量 No. of total reads	清洁读数对比率 Percentage of mapped reads (%)	可映射到基因组上的读数率 Percentage of unique mapped reads (%)
F1	81824486	0.9999	0.9037
F2	81760358	0.9999	0.8673
F3	80496996	0.9998	0.9237
L1	80618686	0.9998	0.7741
L2	81111640	0.9998	0.7384
L3	84395574	0.9999	0.7272
R1	80571720	0.9999	0.8953
R2	80841312	0.9998	0.8781
R3	80087212	0.9999	0.7404

GO类别 GO category	GO名称 GO name	GO ID	宿主基因数量 No. of host genes
生物过程 Biological process	生物调节 Biological regulation	GO: 0065007	41
	基因表达调控 Regulation of gene expression	GO: 0010468	17
	细胞分解代谢过程 Cellular catabolic process	GO: 0044248	17
细胞组分 Cellular component	细胞质 Cytoplasm	GO: 0005737	70
	胞质部分 Cytoplasmic part	GO: 0044444	51
	质膜 Plasma membrane	GO: 0005886	21
分子功能 Molecular function	核苷酸结合 Nucleotide binding	GO: 0000166	49
	离子结合 Ion binding	GO: 0043167	45
	碳水化合物衍生物结合 Carbohydrate derivative binding	GO: 0097367	42

KEGG通路名称 Name of the KEGG pathway	KEGG编号 KEGG number	宿主基因数量 No. of host genes
丙氨酸，天冬氨酸和谷氨酸的代谢 Alanine, aspartate and glutamate metabolism	sly00250	30
精氨酸的生物合成 Arginine biosynthesis	sly00220	12
磷酸戊糖途径 Pentose phosphate pathway	sly00030	10
脂肪酸的降解 Fatty acid degradation	sly00071	6
脂肪酸的生物合成 Fatty acid biosynthesis	sly00061	5
脂肪酸代谢 Fatty acid metabolism	sly01212	3
花生四烯酸代谢 Arachidonic acid metabolism	sly00590	2

Trans ID	miR ID	靶向基因数量 No. of target genes
circ_1420/NC_015447.2: 2008132-2031198	sly-miR5303	38
circ_547/NC_015449.2: 53700671-53711562	sly-miR5303	38
circ_459/NC_015444.2: 21380917-21381396	sly-miR9476-5p	5
circ_1112/NC_015444.2: 64279962-64300043	sly-miR396b	30
circ_473/NC_015444.2: 64279949-64300043	sly-miR396b	30
circ_10/NC_015444.2: 208457-238616	sly-miR9478-3p	4
circ_1079/NC_015444.2: 230400-238546	sly-miR9478-3p	4
circ_176/NC_015444.2: 210427-211962		4
circ_437/NC_015438.2: 97435162-97451603	sly-miR156e-3p	4
circ_704/NC_015438.2: 95819397-95828905	sly-miR482d-5p	1
circ_733/NC_015438.2: 95819397-95825691	sly-miR482d-5p	1
circ_739/NC_015438.2: 90765535-90770648	sly-miR1916	6
circ_309/NC_015445.2: 2493994-2498871	sly-miR1918	3
circ_1403/NC_015439.2: 47259582-47353207	sly-miR9479-3p	1
circ_1403/NC_015439.2: 47259582-47353207	sly-miR167b-3p	2