基于转录组测序的香水月季花香代谢基因研究

doi:10.16420/j.issn.0513-353x.2020-0944

摘要/Abstract

摘要：

通过转录组测序技术,探究大花香水月季及单瓣香水月季两个变种的花香代谢通路及花香差异形成的相关基因。在两个材料花蕾期、初开期及盛开期对比组中分别检测出8 339、8 849、7 572个差异表达基因,其中3个阶段共有的差异基因3 762个。GO和KEGG聚类分析显示,差异基因主要参与代谢过程、细胞过程、单一有机体过程、催化活性等生物学过程;主要富集于代谢途径、次生代谢物的生物合成、碳代谢等途径。在3组共有差异基因中共发现富集于6条重要花香物质合成通路（萜类骨架生物合成、苯丙素生物合成、脂肪酸生物合成、单萜类生物合成、双萜类生物合成及倍半萜和三萜类生物合成）中的差异基因27个。认为这27个基因对两个变种花香释放过程中的差异形成有较大影响。分析特征花香物质合成酶基因Rc_0128091及Rc_0119291在转录组数据中的表达,发现其表达量与相应的花香物质3,5-二甲氧基苯及1,3,5-三甲氧基苯释放量趋势基本一致,推测对特征花香物质的合成有决定性作用。随机选取10个差异表达基因进行qRT-PCR和RNA-seq的相关性验证,结果显示二者相关性较高,认为RNA-seq测序结果具有较高的准确性。

关键词: 月季, 花香, 转录组分析, 差异表达基因, 代谢通路

Abstract:

By using the technique of transcriptome sequencing,floral fragrance metabolic pathways and genes related to the unique floral fragrance of Rosa odorata var. gigantea and R. odorata var. odorata were investigated. The results showed that 8 339,8 849,and 7 572 differentially expressed genes were detected in these two plant species at the flower budding stage,early flowering stage,and blooming stage, including 3 762 differentially expressed genes that were detected in all three stages. Cluster analysis of GO and KEGG showed that the differentially expressed genes were mainly involved in the terms of metabolic process,cell process,single organism process,catalytic activity,and other biological processes. They were also largely enriched in the metabolic pathway,secondary metabolites biosynthesis,carbon metabolism,and other pathways. A total of 27 differentially expressed genes were predicted to be associated with six important floral fragrance biosynthesis pathways（terpenoid backbone biosynthesis,phenylpropanoid biosynthesis,fatty acid biosynthesis,monoterpenoid biosynthesis,diterpenoid biosynthesis,sesquiterpene,and triterpene biosynthesis）. The results showed that these 27 genes had a significant effect on the difference of floral fragrance emission between the two species,and the difference was consistent in the three stages. Furthermore,the expression of Rc_0128091 and Rc_0119291,which are the characteristic floral substance synthetase genes,were investigated. It was found that the expression of Rc_0128091 and Rc_0119291 in the transcriptional data was consistent with the emission of 3,5-dimethoxybenzene and 1,3,5-trimethoxybenzene,suggesting that they are decisive for the synthesis of the characteristic aroma compounds. By randomly selecting 10 differentially expressed genes,the correlation between qRT-PCR and RNA-seq results was verified. The highly correlated results indicate that the RNA-seq results are of high accuracy.

Key words: Rosa odorata, floral, RNA-Seq, differentially expression genes, metabolic pathway

中图分类号:

S685.12

庄玥莹, 周利君, 程璧瑄, 于超, 罗乐, 潘会堂, 张启翔. 基于转录组测序的香水月季花香代谢基因研究[J]. 园艺学报, 2021, 48(11): 2262-2274.

ZHUANG Yueying, ZHOU Lijun, CHENG Bixuan, YU Chao, LUO Le, PAN Huitang, ZHANG Qixiang. Study on the Fragrance Metabolic Genes of Rosa odorata Based on Transcriptome Sequencing[J]. Acta Horticulturae Sinica, 2021, 48(11): 2262-2274.

图/表 12

参考文献 28

[1]	Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp:an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 34 (17):i884-i890.
[2]	Davis A P, Sherlock G, Botstein D, Eppig J T, Matese J C, Harris M A, Kasarskis A, Blake J A, Dolinski K, Dwight S S, Issel-Tarver L, Ringwald M, Ball C A, Richardson J E, Rubin G M, Cherry J M, Ashburner M, Lewis S, Butler H, Hill D P. 2000. Gene Ontology:tool for the unification of biology. Nature Genetics, 25 (1):25-29. pmid: 10802651
[3]	Dexter R, Qualley A, Kish C M, Ma C J, Koeduka T, Nagegowda D A, Dudareva N, Pichersky E, Clark D. 2007. Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol. Plant Journal, 49 (2):265-275. doi: 10.1111/j.1365-313X.2006.02954.x URL
[4]	Fan Li. 2013. Bioinformatic and functional analysis of mulberry genes involved in lignin biosynthesis[Ph. D. Dissertation]. Chongqing:Southwest University. (in Chinese)
	范丽. 2013. 桑树木质素合成基因的生物信息和功能分析[博士论文]. 重庆: 西南大学.
[5]	Guo Guang-yan, Bo Feng, Liu Wei, Mi Cai-li. 2015. Advances in research of the regulation of transcription factors of lignin biosynthesis. Scientia Agricultura Sinica, 48 (7):1277-1287. (in Chinese)
	郭光艳, 柏峰, 刘伟, 秘彩莉. 2015. 转录因子对木质素生物合成调控的研究进展. 中国农业科学, 48 (7):1277-1287.
[6]	Kanehisa M. 2000. KEGG:Kyoto encyclopedia of genes and genomes. Nucleic Acids Research, 28 (1):27-30. pmid: 10592173
[7]	Kim D, Langmead B, Salzberg S L. 2015. HISAT:a fast spliced aligner with low memory requirements. Nature Methods, 12 (4):357-360. doi: 10.1038/NMETH.3317
[8]	Langmead B, Salzberg S L. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9 (4):357-359. doi: 10.1038/nmeth.1923 pmid: 22388286
[9]	Lavid N, Wang J, Shalit M, Guterman I, Bar E, Beuerle T, Menda N, Shafir S, Zamir D, Adam Z, Vainstein A, Weiss D, Pichersky E, Lewinsohn E. 2002. O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiology (Bethesda), 129 (4):1899-1907. doi: 10.1104/pp.005330 URL
[10]	Li Jin-hua, Yan Hui-jun, Yang Jin-hong, Jian Hong-ying, Zhang Hao, Chen Min, Tang Kai-xue. 2018. Analysis of volatile components from Rosa odorata complex by SPME-GC/MS. Southwest China Journal of Agricultural Sciences, 31 (3):587-591. (in Chinese)
	李晋华, 晏慧君, 杨锦红, 蹇洪英, 张颢, 陈敏, 唐开学. 2018. 香水月季复合群(Rosa odorata Complex)花香成分分析. 西南农业学报, 31 (3):587-591.
[11]	Liu Wei. 2019. Functional research of CmCADs in lignin biosynthesis in oriental melon(Cucumis melo L.)under abiotic stresses[Ph. D. Dissertation]. Shenyang:Shenyang Agricultural University. (in Chinese)
	刘威. 2019. 薄皮甜瓜肉桂醇脱氢酶(CAD)在非生物胁迫下参与木质素合成的功能探究[博士论文]. 沈阳: 沈阳农业大学.
[12]	Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15 (12):550. doi: 10.1186/s13059-014-0550-8 URL
[13]	Ma Zhuan-zhuan, Pang Xiao-qing, Chen Rong, Pei Xiao-lin, Wang Qiu-yan, Xie Tian, Yin Xiao-pu. 2015. Research progress of key enzymes in biosynthesis pathway of terpenes. Journal of Hangzhou Normal University(Natural Science Edition), 14 (6):608-615. (in Chinese)
	马转转, 庞潇卿, 谌容, 裴晓林, 王秋岩, 谢恬, 殷晓浦. 2015. 萜类化合物生物合成途径中关键酶的研究进展. 杭州师范大学学报(自然科学版), 14 (6):608-615.
[14]	Min Dan-dan, Tang Mei-qiong, Li Gang, Qu Xiao-sheng, Mou Jian-hua. 2015. Cloning and expression analysis of geranylgeranyl diphosphate synthase gene of Panax notoginseng. Chinese Journal of Traditional Chinese Medicine, 40 (11):2090-2095. (in Chinese)
	闵丹丹, 唐美琼, 李刚, 屈啸声, 缪剑华. 2015. 三七香叶基香叶基焦磷酸合酶基因的克隆及表达分析. 中国中药杂志, 40 (11):2090-2095.
[15]	Mortazavi A, Williams B A, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods, 5 (7):621-628. doi: 10.1038/nmeth.1226 pmid: 18516045
[16]	Pertea M, Pertea G M, Antonescu C M, Chang T, Mendell J T, Salzberg S L. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33 (3):290-295. doi: 10.1038/nbt.3122 URL
[17]	Raymond O, Gouzy J, Just J, Badouin H, Verdenaud M, Lemainque A, Vergne P, Moja S, Choisne N, Pont C, Carrere S, Caissard J, Couloux A, Cottret L, Aury J, Szecsi J, Latrasse D, Madoui M, Francois L, Fu X, Yang S, Dubois A, Piola F, Larrieu A, Perez M, Labadie K, Perrier L, Govetto B, Labrousse Y, Villand P, Bardoux C, Boltz V, Lopez-Roques C, Heitzler P, Vernoux T, Vandenbussche M, Quesneville H, Boualem A, Bendahmane A, Liu C, Le Bris M, Salse J, Baudino S, Benhamed M, Wincker P, Bendahmane M. 2018. The Rosa genome provides new insights into the domestication of modern roses. Nature Genetics, 50 (6):772. doi: 10.1038/s41588-018-0110-3 URL
[18]	Scalliet G, Journot N, Jullien F, Baudino S, Magnard J L, Channeliere S, Vergne P, Dumas C, Bendahmane M, Cock J M, Hugueney P. 2002. Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases. FEBS Letters, 523 (1-3):113-118. pmid: 12123815
[19]	Tang Yun. 2019. Molecular identification of the biosynthesis pathway of sesquiterpenes and functional analysis of PatFPPS[Ph. D. Disseration]. Guangzhou:Guangzhou University of Chinese Medica. (in Chinese)
	唐云. 2019. 广藿香倍半萜合成通路关键酶基因鉴定和PatFPPS的功能分析[博士论文]. 广州: 广州中医药大学.
[20]	Wang Hua-mei, Yu Yan-chong, Fu Chun-xiang, Zhou Gong-ke, Gao Huan-huan. 2014. Research progress of caffeoyl-CoA 3-O-methyltransferase key enzyme in Lignin synthesis. Genomics and Applied Biology, 33 (2):458-466. (in Chinese)
	王华美, 于延冲, 付春祥, 周功克, 高欢欢. 2014. 木质素合成关键酶咖啡酰辅酶A氧甲基转移酶的研究进展. 基因组学与应用生物学, 33 (2):458-466.
[21]	Wang Jia. 2016. Construction of over-expression vector of RrDXR and RrAAT and genetic transformaton study in Rosa rugosa[Ph. D. Dissertation]. Yangzhou:Yangzhou University. (in Chinese)
	王佳. 2016. 玫瑰花香相关基因RrDXR和RrAAT超表达载体构建及遗传转化研究[博士论文]. 扬州: 扬州大学.
[22]	Wang Jian-yong. 2014. The study on cloning and function of seven genes involved in the fatty acid metabolism of Camellia oleifera seeds[Ph. D. Dissertation]. Changsha: Central South University of Forestry and Technology. (in Chinese)
	王建勇. 2014. 油茶种子脂肪酸代谢过程7个关键酶基因的克隆与功能研究[博士论文]. 长沙: 中南林业科技大学.
[23]	Wu Shao-yan, Li Jin, Zhou Shun, Lu Xiao-ying. 2019. Study on synthesis and application of coumarin and its derivatives. Guangdong Chemical Industry, 46 (24):127-128. (in Chinese)
	吴绍艳, 李进, 周顺, 卢小英. 2019. 香豆素及其衍生物的合成及应用研究. 广东化工, 46 (24):127-128.
[24]	Wu S, Watanabe N, Mita S, Dohra H. 2004. The key role of phloroglucinol O-Methyltransferase in the biosynthesis of Rosa chinensis volatile 1,3,5-trimethoxybenzene1. Plant Physiology (Bethesda), 135 (1):95-102. doi: 10.1104/pp.103.037051 URL
[25]	Zhang Xu, Wang Xiao-jia, Li Si-chen, Dong Tian-tian, Wang Zhi-hui. 2019. Research progress of lignin biosynthesis and regulation during granulation of citrus. Acta Agriculturae Zhejiangensis, 31 (12):2131-2140. (in Chinese)
	张旭, 王小佳, 黎思辰, 董甜甜, 汪志辉. 2019. 柑橘果实粒化过程中木质素生物合成与调控研究进展. 浙江农业学报, 31 (12):2131-2140.
[26]	Zhou Han-chen, Lei Pan-deng, Ding Yong. 2016. Research advance on β-glucosidase of tea plant. Journal of Tea Science, 36 (2):111-118. (in Chinese)
	周汉琛, 雷攀登, 丁勇. 2016. 茶树β-葡萄糖苷酶研究进展. 茶叶科学, 36 (02):111-118.
[27]	Zhou L, Yu C, Cheng B, Han Y, Luo L, Pan H, Zhang Q. 2020a. Studies on the volatile compounds in flower extracts of Rosa odorata and R. chinensis. Industrial Crops and Products, 146:112143. doi: 10.1016/j.indcrop.2020.112143 URL
[28]	Zhou L, Yu C, Cheng B, Wan H, Luo L, Pan H, Zhang Q. 2020b. Volatile compound analysis and aroma evaluation of tea-scented roses in China. Industrial Crops and Products, 155:112735. doi: 10.1016/j.indcrop.2020.112735 URL

基因ID Gene ID	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
GAPDH	ATCCATTCATCACCACCGACTACA	GCATCCTTACTTGGGGCAGAGA
Rc 0174951	TGGTTTCCCTAGGAGTCACG	GGAAGTGGTGCATAAGGGAT
Rc 0121071	AGAAAGGCCATCCATAGTGC	TTGGCCTTCTTGATTGTCTC
Rc 0059511	TGGATTTCAACCTGCTGCA	TCTCACGTCCAAGTCCTTCC
Rc 0412071	TGAAACTTGATTCAGGCAAGC	TTCTGGGGTGGAGGACCTT
Rc 0241141	ATGGCATTTAACACTGCTGGT	CGGCTCCTGCTTAAGAACCT
Rc 0083751	TCAGTGGCTATGTTGGTGGT	ACCAATCAGCCTAAAATGCC
Rc 0392511	CTCCTCCAGTCTGGCCAAAT	TGACATGTATGTTTTCCGCGT
Rc 0110711	ACTCAACATTCCTGCAACGA	AGCGGACAGGTAGTTTTTGA
Rc 0214071	CTGCTGGGGAAGAGAAGAGT	ATGGAGGACGAAACATTTGC

基因ID Gene ID	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
GAPDH	ATCCATTCATCACCACCGACTACA	GCATCCTTACTTGGGGCAGAGA
Rc 0174951	TGGTTTCCCTAGGAGTCACG	GGAAGTGGTGCATAAGGGAT
Rc 0121071	AGAAAGGCCATCCATAGTGC	TTGGCCTTCTTGATTGTCTC
Rc 0059511	TGGATTTCAACCTGCTGCA	TCTCACGTCCAAGTCCTTCC
Rc 0412071	TGAAACTTGATTCAGGCAAGC	TTCTGGGGTGGAGGACCTT
Rc 0241141	ATGGCATTTAACACTGCTGGT	CGGCTCCTGCTTAAGAACCT
Rc 0083751	TCAGTGGCTATGTTGGTGGT	ACCAATCAGCCTAAAATGCC
Rc 0392511	CTCCTCCAGTCTGGCCAAAT	TGACATGTATGTTTTCCGCGT
Rc 0110711	ACTCAACATTCCTGCAACGA	AGCGGACAGGTAGTTTTTGA
Rc 0214071	CTGCTGGGGAAGAGAAGAGT	ATGGAGGACGAAACATTTGC

样本 Sample	原始测序数据 Raw reads	过滤后测序数据 Clean reads	未比对测序数据 Unmapped reads	Q20/%	Q30/%	GC/%
T1a	62 786 040	62 674 405	60 810 922	97.83	93.80	47.62
T1b	67 274 777	67 166 924	65 797 731	98.17	94.59	47.80
T1c	61 386 237	61 283 162	60 016 880	97.85	93.82	47.71
T2a	66 064 221	65 936 595	61 819 035	97.94	94.03	47.93
T2b	65 163 741	65 017 603	61 516 931	97.48	93.01	47.94
T2c	62 902 745	62 794 761	58 409 372	98.06	94.28	48.21

样本 Sample	原始测序数据 Raw reads	过滤后测序数据 Clean reads	未比对测序数据 Unmapped reads	Q20/%	Q30/%	GC/%
T1a	62 786 040	62 674 405	60 810 922	97.83	93.80	47.62
T1b	67 274 777	67 166 924	65 797 731	98.17	94.59	47.80
T1c	61 386 237	61 283 162	60 016 880	97.85	93.82	47.71
T2a	66 064 221	65 936 595	61 819 035	97.94	94.03	47.93
T2b	65 163 741	65 017 603	61 516 931	97.48	93.01	47.94
T2c	62 902 745	62 794 761	58 409 372	98.06	94.28	48.21

样本 Sample	0 ~ 0.1（%）	0.1 ~ 1（%）	1 ~ 10（%）	10 ~ 100（%）	100 ~ 1 000（%）	> 1 000（%）
T1a	12 189（30.59）	9 640（24.21）	10 392（26.08）	6 725（16.88）	847（2.13）	48（0.12）
T1b	12 998（32.62）	9 860（24.77）	9 968（25.02）	6 144（15.42）	806（2.02）	61（0.15）
T1c	13 637（34.22）	9 464（23.75）	9 652（24.22）	6 221（15.61）	811（2.04）	61（0.15）
T2a	13 724（34.44）	8 871（22.26）	9 473（23.77）	6 755（16.95）	964（2.42）	59（0.15）
T2b	15 133（37.98）	9 288（23.31）	8 889（22.31）	5 534（13.89）	937（2.35）	65（0.16）
T2c	14 104（35.40）	9 408（23.61）	9 433（23.67）	5 816（14.60）	1 023（2.57）	62（0.16）
平均Mean	13 631（34.21）	9 422（23.65）	9 635（24.18）	6 199（15.56）	898（2.26）	59（0.15）