怀玉山三叶青2个栽培种块根的转录组分析

洪森荣; 黄丹丹; 黄诗慧; 黄夏梦; 蒋椰; 李万平; 蔡红; 陈荣华

doi:10.19303/j.issn.1008-0384.2021.01.004

怀玉山三叶青2个栽培种块根的转录组分析

doi: 10.19303/j.issn.1008-0384.2021.01.004

洪森荣^{1, 2, 3, 4,},
黄丹丹¹,
黄诗慧¹,
黄夏梦¹,
蒋椰¹,
李万平¹,
蔡红⁵,
陈荣华⁵

1.
上饶师范学院生命科学学院，江西　上饶　334001
2.
上饶市药食同源植物资源保护与利用重点实验室，江西　上饶　334001
3.
上饶市三叶青保育与利用技术创新中心，江西　上饶　334001
4.
上饶农业技术创新研究院，江西　上饶　334001
5.
上饶市红日农业开发有限公司，江西　上饶　334700

基金项目: 国家自然科学基金项目（31960079）；江西省科技厅重点研发计划一般项目（20192BBGL70050、20202BBG73010）；江西省教育厅科学技术研究项目（GJJ201704）；上饶市科技局重点研发计划一般项目（2020C002）；上饶市科技局平台载体建设项目（2020J001）；上饶市科技局平台载体建设项目（2019I017）

详细信息

作者简介:
洪森荣（1974−），男，教授，研究方向：植物生物技术（E-mail：864035356@qq.com）

中图分类号: S 646
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出版历程
- 收稿日期: 2020-09-30
- 修回日期: 2020-11-07
- 网络出版日期: 2020-11-24
- 刊出日期: 2021-01-31

Transcriptome Analysis on Tubers of Two Huaiyushan Cultivated Varieties of Tetrastigma hemsleyanum

1.
College of Life Sciences, Shangrao Normal University, Shangrao, Jiangxi 334001, China
2.
Shangrao Key Laboratory of protection and utilization of medicinal and edible homologous plant resources, Shangrao, Jiangxi　334001, China
3.
Shangrao Tetrastigma hemsleyanum Diels et Gilg conservation and utilization technology innovation center, Shangrao, Jiangxi　334001, China
4.
Shangrao Agricultural Technology Innovation Research Institute, Shangrao, Jiangxi　334001, China
5.
Shangrao red sun Agricultural Development Co., Ltd., Shangrao, Jiangxi　334700, China

摘要

摘要: 目的筛选怀玉山三叶青2个栽培种：怀玉1号（HY1组）和怀玉2号（HY2组）与黄酮类化合物合成相关差异表达基因。方法以怀玉山三叶青怀玉1号（HY1组）和怀玉2号（HY2组）的块根为试验材料进行转录组分析。结果 HY1组和HY2组的Clean reads分别为42311662和41411202。2组样品Q30碱基百分比均不小于95.75%。HY1组和HY2组的转录因子家族多为MYB-superfamily、bHLH、AP2/ERF、NAC、C2C2、WRKY等。HY1组和HY2组表达量FPKM的对数值在0-2。HY1组和HY2组表达量密度在0-0.7。HY1组和HY2组表达的共有基因数为22367，HY1组单独表达的基因数为18196，HY2组单独表达的基因数为8137。HY1组和HY2组表达量的相关系数为0.913，样本间相关性好。HY1组和HY2组共产生差异表达基因12199 个。与HY1组比较，HY2组上调基因数为3551，下调基因数为8648。GO富集分析显示，差异基因主要注释到光合作用光系统I光捕获、光合作用捕获、叶绿素代谢过程、蛋白质发色团连锁、前体代谢产物和能量的产生、叶绿素生物合成过程、氧化应激反应、α-氨基酸代谢过程、光合作用、质体小叶、光系统I、光系统II、质体类核仁、光系统、叶绿素结合、单加氧酶活性、铁离子结合、血红素结合、裂解酶活性功能。KEGG富集分析显示，差异基因主要注释到光合作用-天线蛋白、核糖体、乙醛酸和二元酸代谢、苯丙酸生物合成、二苯乙烯类、二芳基庚烷类和姜辣素的生物合成、类黄酮生物合成、光合作用、光合生物的固碳作用、甘氨酸、丝氨酸和苏氨酸代谢、植物激素信号转导、谷胱甘肽代谢、丙酮酸代谢、苯丙氨酸代谢、植物昼夜节律、黄酮和黄酮醇的生物合成、半胱氨酸与蛋氨酸代谢、氰胺酸代谢、类胡萝卜素生物合成、α-亚麻酸代谢、卟啉与叶绿素代谢等代谢途径。结论与黄酮类化合物相关差异表达基因如芪合酶（stilbene synthase）、无色花色素双加氧酶（leucoanthocyanidin dioxygenase）、查尔酮异构酶蛋白（CHI protein）、查尔酮合酶2（chalcone synthase 2）、黄烷酮3-羟化酶（flavanone 3-hydroxylase）、无色花色素还原酶（1leucoanthocyanidin reductase 1）、类黄酮3'-羟化酶（flavonoid 3'-hydroxylase）基因在怀玉2号（HY2组）块根中上调，而查尔酮合酶（chalcone synthase）、黄酮醇合酶（flavonol synthase）、类黄酮3'，5'-甲基转移酶（flavonoid 3', 5'-methyltransferase）基因在怀玉2号（HY2组）块根中下调，导致怀玉山三叶青怀玉1号（HY1组）和怀玉2号（HY2组）块根总黄酮含量的差异。
- 怀玉山三叶青 /
- 栽培种 /
- 块根 /
- 转录组分析
Abstract: Objective Transcriptomes of differentially expressed genes related to flavonoids synthesis in tubers of two Huaiyushan cultivated varieties of Tetrastigma hemsleyanum Diels et Gilg were compared. Method Tubers from Huaiyu 1 (HY1) and Huaiyu 2 (HY2) were used for the transcriptome analysis. Result HY1 and HY2 had clean reads of 42 311 662 and 41 411 202, respectively, and no less than 95.75% of Q30 base. Their transcription factors basically belonged to the MYB-superfamily, bHLH, AP2/ERF, NAC, C2C2, WRKY, etc. The paired values of FPKM in HY1 and HY2 were between 0 and 2; the expression densities, between 0 and 0.7; the number of commonly expressed genes, 22 367; and, the number of uniquely expressed genes in HY1, 18 196, while 8 137 in HY2. The correlation between the expressions of the two had a coefficient of 0.913, and that between the samples was high. There were 12 199 differentially expressed genes between the two, with 3 551 upregulated and 8 648 downregulated in HY2 as compared to HY1. The GO enrichment analysis showed that the differential genes were mainly annotated into photosynthesis, light harvesting in photosystem I, photosynthesis, light harvesting, chlorophyll metabolic process, protein-chromophore linkage, generation of precursor metabolites and energy, chlorophyll biosynthetic process, response to oxidative stress, alpha-amino acid metabolic process, photosynthesis, plastoglobule, photosystem I, photosystem II, plastid nucleoid, photosystem, chlorophyll binding, monooxygenase activity, iron ion binding, heme binding, lyase activity, etc. Whereas, the KEGG enrichment analysis indicated the differential genes to be mainly annotated into photosynthesis-antenna proteins, ribosome, glyoxylate and dicarboxylate metabolism, phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, flavonoid biosynthesis, photosynthesis, carbon fixation in photosynthetic organisms, glycine, serine and threonine metabolism, plant hormone signal transduction, glutathione metabolism, pyruvate metabolism, phenylalanine metabolism, circadian rhythm-plant, flavone and flavonol biosynthesis, cysteine and methionine metabolism, cyanoamino acid metabolism, carotenoid biosynthesis, alpha-linolenic acid metabolism, porphyrin and chlorophyll metabolism, and other metabolic pathways. Conclusion The differentially expressed genes related to flavonoids synthesis, such as stilbene synthase, leucoanthocyanidin dioxygenase, CHI protein, chalcone synthase 2, flavanone 3-hydroxylase and lleucoanthocyanidin reductase 1 and flavonoid 3’- hydroxylase gene were upregulated in HY2, while chalcone synthase, flavonol synthase and flavonoid 3’, 5’-methyltransferase downregulated. The variations apparently resulted in the differences shown on the total flavonoid content between the HY1 and HY2 tubers.
- Tetrastigma hemsleyanum Diels et Gilg /
- Huaiyushan cultivated varieties /
- tuber /
- transcriptome analysis

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图 1 HY1组和HY2组转录因子家族统计

Figure 1. Statistics on transcription factor families of HY1 and HY2

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图 2 HY1组和HY2组表达量分布盒形图（A）和密度图（B）

Figure 2. Diagrams of box (A) and density (B) on quantitative distribution of gene expressions in HY1 and HY2

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图 3 HY1组和HY2组样本间Venn图

Figure 3. Venn diagram on samples of HY1 and HY2

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图 4 HY1组和HY2组表达量样本间相关性热图

Figure 4. Thermogram of correlation on expressions between HY1 and HY2

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图 5 HY1组和HY2组表达量差异统计柱状图（A）以及表达量差异火山图（B）和表达量差异散点图（C）

Figure 5. Statistic histogram on expression difference (A), volcano map (B), and scatter map (C) of HY1 and HY2

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图 6 HY1组和HY2组差异表达基因GO富集分析

Figure 6. Go enrichment analysis on differentially expressed genes in HY1 and HY2

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图 7 HY1组和HY2组差异表达基因KEGG富集分析柱形图（A）和气泡图（B）

Figure 7. KEGG enrichment column chart (A) and bubble chart (B) on differentially expressed genes in HY1 and HY2

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表 1 HY1组和HY2组前15个表达量差异基因（HY1 vs HY2）

Table 1. Top 15 genes in HY1 and HY2 with greatest differences on gene expression (HY1 vs. HY2)

序列编号 Seq_id	长度 Length	P值 P value	校正 P值 P adjust	显著性 Significant	调节 Regulate	基因名称 Gene name
TRINITY_DN2864_c0_g1	4161	6.01E-34	3.05E-29	是 yes	下调 down	未注释 No
TRINITY_DN10874_c0_g1	1930	3.00E-27	7.61E-23	是 yes	上调 up	类核糖体失活蛋白 SNAIfribosome-inactivating protein SNAIf-like
TRINITY_DN4409_c0_g1	721	2.32E-26	2.94E-22	是 yes	下调 down	LHCII类1型叶绿素 a-b结合蛋白 chlorophyll a-b binding protein of LHCII type 1-like
TRINITY_DN1538_c0_g1	562	3.20E-26	3.25E-22	是 yes	上调 up	未注释 No
TRINITY_DN6900_c0_g1	1888	2.26E-26	3.82E-22	是 yes	下调 down	叶绿体核酮糖二磷酸羧化酶/加氧酶激活酶 ARibulose bisphosphate carboxylase/oxygenase activase A, chloroplastic
TRINITY_DN2027_c0_g1	1216	1.68E-25	1.42E-21	是 yes	下调 down	叶绿体产氧增强蛋白1 oxygen-evolving enhancer protein 1, chloroplastic
TRINITY_DN151_c0_g1	1068	3.65E-25	2.64E-21	是 yes	下调 down	核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基 ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit
TRINITY_DN119_c0_g2	1489	8.14E-25	5.16E-21	是 yes	下调 down	叶绿体甘油醛-3-磷酸脱氢酶 Aglyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
TRINITY_DN365_c0_g1	2576	1.08E-24	6.11E-21	是 yes	下调 down	叶绿体转酮酶 transketolase, chloroplastic
TRINITY_DN178_c0_g1	896	3.20E-24	1.48E-20	是 yes	下调 down	部分预测蛋白 predicted protein, partial
TRINITY_DN640_c0_g1	1411	3.11E-24	1.58E-20	是 yes	下调 down	叶绿体硫胺素噻唑合酶2 thiamine thiazole synthase 2, chloroplastic
TRINITY_DN5576_c0_g1	7575	3.75E-24	1.58E-20	是 yes	下调 down	转座子 Tf-2多蛋白 Transposon Tf2-2 polyprotein [Vitis vinifera]
TRINITY_DN757_c0_g1	1330	4.58E-24	1.79E-20	是 yes	下调 down	假定碳酸酐酶 putative carbonic anhydrase
TRINITY_DN75_c0_g2	1595	8.89E-24	3.22E-20	是 yes	下调 down	果糖-1,6-二磷酸醛缩酶8 fructose-1,6-bisphosphate aldolase 8
TRINITY_DN922_c0_g1	1199	9.98E-24	3.38E-20	是 yes	下调 down	LHCIIⅢ型叶绿素 a-b结合蛋白 chlorophyll a-b binding protein of LHCII type III, chloroplastic

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表 2 HY1组和HY2组部分差异表达基因GO富集结果

Table 2. GO enrichment on certain differentially expressed genes in HY1 and HY2

富集到该 GO term的基因/转录本数目 Number of genes enriched to the GO term	GO Term对应的编号 ID corresponding to go term	GO三大分类 Three categories of GO	GO功能描述 Go function description	该 GO在目标基因集中占有的比例 Proportion of the GO in the target gene set/%	该 GO在背景基因/转录本中占有的比例 Proportion of the go in the background gene/%	矫正后的P值 P value_ corrected
46	GO:0009768	生物学过程 BP	光合作用光系统 I光捕获 photosynthesis, light harvesting in photosystem I	0.42	0.18	0.0009
57	GO:0009765	生物学过程 BP	光合作用光捕获 photosynthesis, light harvesting	0.52	0.23	0.0009
44	GO:0015994	生物学过程 BP	叶绿素代谢过程 chlorophyll metabolic process	0.40	0.21	0.0009
65	GO:0018298	生物学过程 BP	蛋白质发色团连锁 protein-chromophore linkage	0.59	0.33	0.0009
213	GO:0006091	生物学过程 BP	前体代谢产物和能量的产生 generation of precursor metabolites and energy	1.95	1.35	0.0009
31	GO:0015995	生物学过程 BP	叶绿素生物合成过程 chlorophyll biosynthetic process	0.28	0.14	0.0009
139	GO:0006979	生物学过程 BP	氧化应激反应 response to oxidative stress	1.27	0.90	0.0009
227	GO:1901605	生物学过程 BP	α-氨基酸代谢过程 alpha-amino acid metabolic process	2.08	1.57	0.0009
87	GO:0015979	生物学过程 BP	光合作用 photosynthesis	0.80	0.52	0.0009
69	GO:0010287	细胞组分 CC	质体小叶 plastoglobule	0.63	0.29	0.0009
65	GO:0009522	细胞组分 CC	光系统 I photosystem I	0.59	0.31	0.0009
68	GO:0009523	细胞组分 CC	光系统 II photosystem II	0.62	0.30	0.0009
36	GO:0042646	细胞组分 CC	质体类核仁 plastid nucleoid	0.33	0.17	0.0009
78	GO:0009521	细胞组分 CC	光系统 photosystem	0.71	0.39	0.0009
61	GO:0016168	分子功能 MF	叶绿素结合 chlorophyll binding	0.56	0.29	0.0009
188	GO:0004497	分子功能 MF	单加氧酶活性 monooxygenase activity	1.72	1.15	0.0009
208	GO:0005506	分子功能 MF	铁离子结合 iron ion binding	1.90	1.39	0.0009
228	GO:0020037	分子功能 MF	血红素结合 heme binding	2.08	1.53	0.0009
276	GO:0016829	分子功能 MF	裂解酶活性 lyase activity	2.52	1.91	0.0009

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表 3 HY1组和HY2组差异表达基因KEGG富集分析（前20）

Table 3. KEGG enrichment on top 20 differentially expressed genes in HY1 and HY2

数量 Number	途径编号 Pathway ID	描述 Description	KEGG在目标基因集中占有的比例 Ratio in study/%	KEGG在背景中占有的比例 Ratio in pop/%	校正后 P值 P value corrected
57	map00196	光合作用-天线蛋白 Photosynthesis-antenna proteins	1.00	0.46	0.0000
306	map03010	核糖体 Ribosome	5.41	4.42	0.0002
90	map00630	乙醛酸和二元酸代谢 Glyoxylate and dicarboxylate metabolism	1.59	1.08	0.0003
100	map00940	苯丙酸生物合成 Phenylpropanoid biosynthesis	1.77	1.24	0.0003
31	map00945	二苯乙烯类、二芳基庚烷类和姜辣素的生物合成 Stilbenoid, diarylheptanoid and gingerol biosynthesis	0.55	0.29	0.0003
34	map00941	类黄酮生物合成 Flavonoid biosynthesis	0.60	0.35	0.0018
64	map00195	光合作用 Photosynthesis	1.13	0.77	0.0019
82	map00710	光合生物的固碳作用 Carbon fixation in photosynthetic organisms	1.45	1.04	0.0024
71	map00260	甘氨酸、丝氨酸和苏氨酸代谢 Glycine, serine and threonine metabolism	1.26	0.89	0.0030
132	map04075	植物激素信号转导 Plant hormone signal transduction	2.34	1.85	0.0075
77	map00480	谷胱甘肽代谢 Glutathione metabolism	1.36	1.02	0.0119
92	map00620	丙酮酸代谢 Pyruvate metabolism	1.63	1.25	0.0120
41	map00360	苯丙氨酸代谢 Phenylalanine metabolism	0.73	0.49	0.0165
50	map04712	植物昼夜节律 Circadian rhythm - plant	0.88	0.63	0.0187
6	map00944	黄酮和黄酮醇的生物合成 Flavone and flavonol biosynthesis	0.11	0.04	0.0217
89	map00270	半胱氨酸与蛋氨酸代谢 Cysteine and methionine metabolism	1.57	1.23	0.0221
42	map00460	氰胺酸代谢 Cyanoamino acid metabolism	0.74	0.52	0.0222
36	map00906	类胡萝卜素生物合成 Carotenoid biosynthesis	0.64	0.44	0.0343
44	map00592	α-亚麻酸代谢 alpha-Linolenic acid metabolism	0.78	0.56	0.0398
46	map00860	卟啉与叶绿素代谢 Porphyrin and chlorophyll metabolism	0.81	0.60	0.0445

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表 4 与次生代谢物合成相关差异基因的表达

Table 4. Expressions of differentially expressed genes related to secondary metabolites synthesis

基因ID Gene id	基因名称 Gene name	HY1	HY2	调节 Regulate
TRINITY_DN10561_c0_g1	芪合酶 stilbene synthase	4.64	23.15	上调 up
TRINITY_DN12750_c0_g1	无色花色素双加氧酶 leucoanthocyanidin dioxygenase	14.07	50.17	上调 up
TRINITY_DN1644_c0_g1	查尔酮异构酶蛋白 CHI protein	82.09	160.88	上调 up
TRINITY_DN16981_c0_g4	查尔酮合酶 chalcone synthase	7.15	0	下调 down
TRINITY_DN30140_c0_g1	黄酮醇合酶 flavonol synthase	5.44	2.92	下调 down
TRINITY_DN43_c0_g1	查尔酮合酶2chalcone synthase 2	32.52	281.84	上调 up
TRINITY_DN6146_c0_g1	黄烷酮3-羟化酶 flavanone 3-hydroxylase	54.15	64.75	上调 up
TRINITY_DN6563_c0_g1	无色花色素还原酶1 leucoanthocyanidin reductase 1	4.91	25.68	上调 up
TRINITY_DN22758_c0_g2	类黄酮3′，5′-甲基转移酶 flavonoid 3′,5′-methyltransferase	7.41	1.15	下调 down
TRINITY_DN4109_c0_g1	类黄酮3′-羟化酶 flavonoid 3′-hydroxylase	18.35	82.99	上调 up

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