基于转录组测序筛选与柚裂果相关的基因

doi:10.3864/j.issn.0578-1752.2023.20.013

摘要/Abstract

摘要：

【目的】柑橘类果实生长季节内出现的裂果现象是一类生理失调病害，然而目前尚未完全揭示柑橘类果实裂果的分子机理。本研究通过对文旦柚抗裂和易裂品种果皮转录组的比较分析，筛选果实抗裂相关基因。【方法】以抗裂品种（‘度新1号文旦柚’）的正常果（此品种无裂果），以及易裂品种（‘度尾文旦柚’）的正常果和裂果为材料，果实均取自两个时期（时期A：2021年8月3日;时期B：2021年8月20日，裂果敏感期）。取果实果顶部位的果皮（以果顶为中心，30 mm半径范围内的果皮）用于转录组测序。【结果】将易裂品种裂果果皮与两品种正常果果皮转录组进行比较分析，在时期A共筛选到1 660个差异基因，易裂品种裂果果皮与两品种正常果果皮间相同的差异基因104个;在时期B共筛选到1 972个差异基因，易裂品种裂果果皮与两品种正常果果皮间相同的差异基因82个。对易裂品种裂果果皮与两品种正常果果皮间所有差异基因的GO富集分析结果显示：在生物学过程分类中，两个时期均富集到差异基因的主要亚类包括代谢过程、细胞过程、单生物过程、生物调控、刺激响应和信号。对易裂品种裂果果皮与两品种正常果果皮间所有差异基因的代谢通路分析结果显示：两个时期均富集到差异基因的主要代谢途径包括碳代谢、植物MAPK信号途径、植物激素信号转导和内质网蛋白过程，这些代谢途径富集的差异基因也最多。发现了一些与果实抗裂能力相关的重要基因：两品种正常果果皮扩张蛋白-A1基因表达量均显著高于易裂品种裂果果皮，两个时期结果一致;在时期A，两品种正常果果皮类钙调磷酸酶蛋白B亚基基因表达量显著高于易裂品种裂果果皮，但在时期B无显著差异;易裂品种裂果果皮热激转录因子、丝氨酸/苏氨酸蛋白激酶、生长素响应蛋白和脱水响应元件结合蛋白基因表达量显著高于两品种正常果果皮，两个时期结果一致。【结论】与果皮弹性、水分运动、高温和水分亏缺逆境响应相关的基因是调控文旦柚果实抗裂能力的关键基因。

关键词: 柚（Citrus grandis L. Osbeck）, 裂果, 果皮, 转录组, 抗裂基因

Abstract:

【Objective】Fruit cracking is a universal physiological disorder that occurs during growth in citrus fruits. However, the molecular mechanisms that regulate cracking in citrus fruits remain unclear. The aim of this study was to screen genes that were related to resistance to fruit cracking. 【Method】 Normal fruits from a pomelo (Citrus grandis (L). Osbeck) cultivar (Duxin 1) resistant to cracking, as well as normal and cracked fruits from Duwei, a cultivar sensitive to cracking, were collected on August 3, 2021 and August 20, 2021, respectively. The pericarp surrounding blossom ends of the fruits (the blossom end was considered the center, approximate 30 millimeters radius) were sampled for RNA-seq. 【Result】 The differentially expressed genes (DEGs) in each stage were screened based on the comparisons of a transcriptome between cracked fruits from the cracking-sensitive cultivar and normal fruits from both cultivars. In the stage A, 1 660 DEGs were obtained, and 104 DEGs were common between the comparison. A total of 1 972 DEGs were screened in stage B, and 82 were common in the comparison. All the DEGs screened at both stages were used for a Gene Ontology enrichment analysis. In the classification of biological process, the major common sub-classifications, including ‘metabolic process’, ‘cellular process’, ‘single-organism process’, ‘biological regulation’, ‘response to stimulus’, and ‘signaling’ were identified in both stages. All the screened DEGs were also analyzed using Kyoto Encyclopedia of Genes and Genomes enrichment. Many genes were enriched in several metabolic pathways, including ‘carbon metabolism’, ‘MAPK signaling pathway-plant’, ‘plant hormone signal transduction’ and ‘protein processing in endoplasmic reticulum’. In addition, these pathways were identified in both stages. Several genes related to resistance to fruit cracking were identified in this study. The levels of transcription of Expansin-A1 were significantly higher in the pericarp of normal fruits from the two cultivars than that in the pericarp of cracked fruits from the sensitive cultivar. Calcineurin B-like protein gene was highly expressed in the pericarp of normal fruits from both cultivars when compared with the pericarp of cracked fruits from the sensitive cultivar. However, this difference disappeared at the stage B. The genes for heat stress transcription factor, serine/threonine-protein kinase, auxin-responsive protein, and dehydration-responsive element-binding protein were upregulated in the pericarp of cracked fruits from the sensitive cultivar compared with the pericarp of normal fruits from the two cultivars in both stages. 【Conclusion】These findings suggested that the genes related to strength of pericarp, water movement, and responsing to high temperature and water deficiency stresses were critical to regulating resistance to fruit cracking.

Key words: pummelo (Citrus grandis L. Osbeck), fruit cracking, pericarp, transcriptome, cracking-resistant gene

卢艳清, 林燕金, 王贤达, 卢新坤. 基于转录组测序筛选与柚裂果相关的基因[J]. 中国农业科学, 2023, 56(20): 4087-4101.

LU YanQing, LIN YanJin, WANG XianDa, LU XinKun. A Transcriptome Analysis Identifies Candidate Genes Related to Fruit Cracking in Pomelo Fruits[J]. Scientia Agricultura Sinica, 2023, 56(20): 4087-4101.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.20.013

https://www.chinaagrisci.com/CN/Y2023/V56/I20/4087

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基于转录组比较筛选到的所有差异基因富集到相同的KEGG代谢途径"

代谢途径 Pathway	代谢途径编号 Ko ID	差异基因数量 DEG number
代谢途径 Pathway	代谢途径编号 Ko ID	A1 vs A3	B1 vs B3	A2 vs A3	B2 vs B3
植物病原菌互作Plant-pathogen interaction	ko04626	34	95	48	7
植物激素信号转导Plant hormone signal transduction	ko04075	15	43	37	5
碳代谢Carbon metabolism	ko01200	14	40	8	1
植物MAPK信号途径MAPK signaling pathway - plant	ko04016	12	32	27	2
内质网蛋白加工Protein processing in endoplasmic reticulum	ko04141	12	32	10	1
糖酵解/糖异生Glycolysis / Gluconeogenesis	ko00010	7	21	6	2
磷酸戊糖途径Pentose phosphate pathway	ko00030	3	7	1	1
果糖和甘露糖代谢Fructose and mannose metabolism	ko00051	3	7	1	1
半乳糖代谢Galactose metabolism	ko00052	4	18	6	1
抗坏血酸和醛酸代谢Ascorbate and aldarate metabolism	ko00053	3	9	1	1
类固醇代谢Steroid biosynthesis	ko00100	1	6	1	2
氰基氨基酸代谢Cyanoamino acid metabolism	ko00460	1	9	1	2
谷胱甘肽代谢Glutathione metabolism	ko00480	4	11	6	1
磷酸肌醇代谢Inositol phosphate metabolism	ko00562	4	16	3	2
甘油磷脂代谢Glycerophospholipid metabolism	ko00564	2	12	5	2
亚油酸代谢Linoleic acid metabolism	ko00591	2	3	4	2
ɑ-亚油酸代谢alpha-Linolenic acid metabolism	ko00592	5	11	8	2
丙酮酸代谢Pyruvate metabolism	ko00620	5	11	3	1
光合组织碳固定Carbon fixation in photosynthetic organisms	ko00710	1	10	2	1
类倍半萜烯和三萜烯合成Sesquiterpenoid and triterpenoid biosynthesis	ko00909	2	10	4	1
苯丙烷类生物合成Phenylpropanoid biosynthesis	ko00940	17	28	6	4
类黄酮合成Flavonoid biosynthesis	ko00941	9	12	1	2
异黄酮合成Isoflavonoid biosynthesis	ko00943	2	2	1	1
芪类化合物、二芳基庚烷和姜辣素合成 Stilbenoid, diarylheptanoid and gingerol biosynthesis	ko00945	7	12	1	2
ABC转运体ABC transporters	ko02010	6	12	6	1
剪接体Spliceosome	ko03040	5	11	6	1
玉米素合成Zeatin biosynthesis	ko00908	1	9	4	1
泛素介导的蛋白质降解过程Ubiquitin mediated proteolysis	ko04120	9	12	7	1
吞噬体Phagosome	ko04145	1	5	2	1
植物昼夜节律Circadian rhythm-plant	ko04712	2	17	5	3

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基因号 Gene ID	功能注释 Functional annotation	正向和反向引物序列 Forward and reverse primer sequences (5'-3')
c22288.graph_c0	脱水响应元件结合蛋白 Dehydration-responsive element-binding protein	F: AGGTAAAGGAGGACCGGAGA
c22288.graph_c0	脱水响应元件结合蛋白 Dehydration-responsive element-binding protein	R: ATTAAGCCGTGCACAAGGAC
c39020.graph_c1	未知蛋白 Uncharacterized protein	F: CGTTTGAACCGTAGGAGCAT
c39020.graph_c1	未知蛋白 Uncharacterized protein	R: GGCATAGGGCTGTGTTTGTT
c35353.graph_c1	扩张蛋白-A10 Expansin-A10	F: TAAGGTCACCACAAGCGATG
c35353.graph_c1	扩张蛋白-A10 Expansin-A10	R: GTGGCTCTAGCGGAATTGAG
c35353.graph_c0	扩张蛋白-A1 Expansin-A1	F: AGTTTTGGCCCCAGTTTCTT
c35353.graph_c0	扩张蛋白-A1 Expansin-A1	R: GGAGGCATCAGGTTCACAAT
c19677.graph_c0	扩张蛋白-A1 Expansin-A1	F: AGGAAGGGAGGCATCAGATT
c19677.graph_c0	扩张蛋白-A1 Expansin-A1	R: AGTTCCTTGACATGGGTTGC
c17406.graph_c1	生长素响应蛋白IAA30 Auxin-responsive protein IAA30	F: AGCCAGAGCTGCAAGTTGTT
c17406.graph_c1	生长素响应蛋白IAA30 Auxin-responsive protein IAA30	R: ATCATCCCCCACACTTGGTA
c37618.graph_c0	生长素响应蛋白IAA26 Auxin-responsive protein IAA26	F: GCAGGAGCAGTCCTTTTCTG
c37618.graph_c0	生长素响应蛋白IAA26 Auxin-responsive protein IAA26	R: CTCACAGCCCTGTTGCACTA
c36022.graph_c0	丝氨酸/苏氨酸蛋白激酶 Probable serine/threonine-protein kinase	F: TCCCGAAAATCTCCATCTTG
c36022.graph_c0	丝氨酸/苏氨酸蛋白激酶 Probable serine/threonine-protein kinase	R: ATTGGAAAGGCACTGACCAC
c28883.graph_c0	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	F: TGGTCACTTTTGGGGAAGAG
c28883.graph_c0	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	R: TGTCACCATCTCCCAAATCA
c17721.graph_c0	热激响应转录因子A-4b Heat stress transcription factor A-4b	F: TGCTGTCTCATCCTCAGTGG
c17721.graph_c0	热激响应转录因子A-4b Heat stress transcription factor A-4b	R: GGGTATTGCGACCAGTGTCT
c34836.graph_c0	热激响应转录因子A-8 Heat stress transcription factor A-8	F: AAAAAGCCAGCAGCAGATGT
c34836.graph_c0	热激响应转录因子A-8 Heat stress transcription factor A-8	R: CCATGCGCCAGTTATTTTCT
GQ389668.1	肌动蛋白 Actin	F: AGAGCATCCGGTTCTTCTCA
GQ389668.1	肌动蛋白 Actin	R: TTGCACAGCAAAGAATCAGG

序列长度 Length range (bp)	转录本Transcript		Unigene
序列长度 Length range (bp)	数量 Total number	比例 Percentage (%)	数量 Total number	比例 Percentage (%)
200-300	21084	6.90	17974	33.87
300-500	17482	5.72	11546	21.75
500-1000	26848	8.78	8780	16.54
1000-2000	64480	21.09	6155	11.60
2000+	175863	57.52	8620	16.24
总数量 Total number	305757		53075
总长度 Total Length	783171572		54363867
N50长度 N50 Length	3486		2329
平均长度 Mean Length	2561		1024

比较组 Comparison	所有差异基因数量 All DEG number	上调基因 Up-regulated DEG	下调基因 Down-regulated DEG
A1 vs A3	752	441	311
A2 vs A3	908	665	243
A1 vs A2	1650	848	802
B1 vs B3	1875	1074	801
B2 vs B3	97	71	26
B1 vs B2	1073	565	508

时期 Stage	基因号 Gene ID	基因表达量 FPKM			错误发现率 FDR			功能注释 Functional annotation
时期 Stage	基因号 Gene ID	1	2	3	1 vs 3	1 vs 2	2 vs 3	功能注释 Functional annotation
A	c35353.graph_c0	723.06	1041.65	280.40	4.30E-02	-	2.87E-07	扩张蛋白-A1 Expansin-A1
B	c19677.graph_c0	1472.25	1252.20	542.02	2.37E-12	-	6.02E-10	扩张蛋白-A1 Expansin-A1

时期Stage	基因号 Gene ID	基因表达量 FPKM			错误发现率 FDR			功能注释 Functional annotation
时期Stage	基因号 Gene ID	1	2	3	1 vs 3	1 vs 2	2 vs 3	功能注释 Functional annotation
A	c17406.graph_c1	4.44	7.73	14.15	1.49E-02	-	2.45E-02	生长素响应蛋白Auxin-responsive protein IAA30
B	c37618.graph_c0	16.51	15.89	29.42	1.34E-02	-	8.54E-03	生长素响应蛋白Auxin-responsive protein IAA26