大白菜花茎蜡粉近等基因系转录组分析

doi:10.16420/j.issn.0513-353x.2020-1058

摘要/Abstract

摘要：

为研究大白菜花茎蜡粉分子调控机制,针对大白菜一对花茎蜡粉差异的近等基因系进行了转录组比较分析。在有蜡粉材料RHL065_1和无蜡粉材料RHL065_2之间总共鉴定到7 237个基因存在显著差异表达,主要富集在碳水化合物生物过程、原生质膜的组成成分和四吡咯结合分子功能。结合基因功能注释,筛选到17个蜡粉相关基因,包括蜡粉合成基因MAH1、CYTB5-B（Bra022898,Bra021809）、KCS6/CER6/CUT1、KCS9、FAR3/CER4、KCS1、CER2、KCD/PAS2、CYTB5-C（Bra039268,Bra004518）、CER2-LIKE,蜡粉转运基因LTPG1（Bra010912,Bra030067）和转录因子基因WIN1/SHINE1、MYB30、DEWAX在两自交系间存在显著差异表达,其可能参与了大白菜花茎表皮蜡粉形成。利用qRT-PCR验证了转录组分析的可靠性以及差异表达基因在不同组织的表达模式。研究结果有助于进一步克隆大白菜蜡粉调控基因和开发相关功能分子标记。

关键词: 大白菜, 蜡粉, 转录组, 候选基因

Abstract:

In order to study the molecular mechanism of floral axis waxiness in Chinese cabbage,a comparative transcriptome analysis was carried out on a pair of near isogenic lines with difference in floral axis waxiness. In total,7 237 differentially expressed genes（DEGs）were identified between waxy material （RHL065_1）and non-waxy material（RHL065_2）,mainly enriched in carbohydrate biological processes,cellular components of plasma membrane and tetrapyrrole binding molecular functions. Combined with gene function annotation,17 DEGs,involved in wax synthesis MAH1,CYTB5-B （Bra022898,Bra021809）,KCS6/CER6/CUT1,KCS9,FAR3/CER4,KCS1,CER2,KCD/PAS2,CYTB5-C（Bra039268,Bra004518）,CER2-LIKE,wax transport LTPG1（Bra010912,Bra030067）and transcription factors WIN1/SHINE1,MYB30,DEWAX were proposed to contribute to surface waxiness formation in Chinese cabbage floral axis. A further qRT-PCR analysis was used to verify the transcriptome analysis and survey the expression pattern of DEGs. These results will be helpful for further gene cloning,developing functional molecular markers and understanding the genetic architecture for waxiness.

Key words: Chinese cabbage, waxy, transcriptome, candidate gene

中图分类号:

S634.1

王荣花, 王树彬, 刘栓桃, 李巧云, 张志刚, 王立华, 赵智中. 大白菜花茎蜡粉近等基因系转录组分析[J]. 园艺学报, 2022, 49(1): 62-72.

WANG Ronghua, WANG Shubin, LIU Shuantao, LI Qiaoyun, ZHANG Zhigang, WANG Lihua, ZHAO Zhizhong. Transcriptome Analysis of Waxy Near-isogenic Lines in Chinese Cabbage Floral Axis[J]. Acta Horticulturae Sinica, 2022, 49(1): 62-72.

图/表 7

参考文献 33

[1]	Aharoni A, Dixit S, Jetter R, Thoenes E, Pereira A A. 2004. The SHINE clade of AP2 domain transcription factors activates wax biosynthesis,alters cuticle properties,and confers drought tolerance when overexpressed in Arabidopsis. The Plant Cell, 16:2463-2480. doi: 10.1105/tpc.104.022897 URL
[2]	Barthlott W, Neinhuis C, Cutler D, Ditsch F, Meusel I, Theisen I, Wilhelmi H. 1998. Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society, 126 (3):237-260. doi: 10.1111/boj.1998.126.issue-3 URL
[3]	Bernard A, Domergue F, Pascal S, Jetter R, Renne C, Faure J D, Haslam R P, Napier J A, Lessire, Joubes J. 2012. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. The Plant Cell, 24:3106-3118. doi: 10.1105/tpc.112.099796 pmid: 22773744
[4]	Bourdenx B, Bernard A, Domergue F, Pascal S, Leger A. 2011. Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiolgy, 156:29-45.
[5]	Busta L, Hegebarth D, Kroc E, Jetter R. 2017. Changes in cuticular wax coverage and composition on developing Arabidopsis leaves are influenced by wax biosynthesis gene expression levels and trichome density. Planta, 245:297-311. doi: 10.1007/s00425-016-2603-6 URL
[6]	Debono A, Yeats T H, Rose J K, Bird D, Jetter R, Kunst L, Samuels L. 2009. Arabidopsis LTPG is a glycosylphosphatidylinositol-anchored lipid transfer protein required for export of lipids to the plant surface. Plant Cell, 21 (4):1230-1238. doi: 10.1105/tpc.108.064451 pmid: 19366900
[7]	Go Y S, Kim H, Kim H J, Suh M C. 2014. Arabidopsis cuticular wax biosynthesis is negatively regulated by the DEWAX gene encoding an AP2/ERF-type transcription factor. The Plant Cell, 26 (4):1666-1680. doi: 10.1105/tpc.114.123307 URL
[8]	Kannangara R, Branigan C, Liu Y, Penfield T, Rao V, Mouille G, Hofte H, Pauly M, Riechmann J L, Broun P. 2007. The transcription factor WIN1/SHN1 regulates cutin biosynthesis in Arabidopsis thaliana. The Plant Cell, 19 (4):1278-1294. doi: 10.1105/tpc.106.047076 URL
[9]	Kim H. 2012. Characterization of glycosylphosphatidylinositol-anchored lipid transfer protein 2(LTPG2) and overlapping function between LTPG/LTPG1 and LTPG 2 in cuticular wax export or accumulation in Arabidopsis thaliana. Plant and Cell Physiology, 53:1391-1403. doi: 10.1093/pcp/pcs083 URL
[10]	Kim H, Choi D, Suh M C. 2017. Cuticle ultrastructure,cuticular lipid composition,and gene expression in hypoxia-stressed Arabidopsis stems and leaves. Plant Cell Reports, 36 (6):815-827. doi: 10.1007/s00299-017-2112-5 URL
[11]	Kim J, Jung J H, Lee S B, Go Y S, Kim H J, Cahoon R, Markham J E, Cahoon E B, Suh M C. 2013. Arabidopsis 3-ketoacyl-coenzyme A synthase 9 is involved in the synthesis of tetracosanoic acids as precursors of cuticular waxes,suberins,sphingolipids,and phospholipids. Plant Physiology, 162:567-580. doi: 10.1104/pp.112.210450 URL
[12]	Lam P, Zhao L, Eveleigh N, Yu X, Chen L. 2015. The exosome and trans-acting small interfering RNAs regulate cuticular wax biosynthesis during Arabidopsis inflorescence stem development. Plant Physiolgy, 167:323-336.
[13]	Lee S, Go Y, Bae H, Park J, Cho S, Cho H, Lee D, Park O, Hwang I, Suh M. 2009. Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition,increased plastoglobules,and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Physiology, 150:42-54. doi: 10.1104/pp.109.137745 URL
[14]	Lee S B, Kim H U, Suh M C. 2016. MYB94 and MYB96 additively activate cuticular wax biosynthesis in Arabidopsis. Plant and Cell Physiology, 57:2300-2311. doi: 10.1093/pcp/pcw147 URL
[15]	Lee S B, Suh M C. 2015. Advances in the understanding of cuticular waxes in Arabidopsis thaliana and crop species. Plant Cell Reports, 34:557-572. doi: 10.1007/s00299-015-1772-2 URL
[16]	Liu S, Wang R, Zhang Z, Li Q, Wang L, Wang Y, Zhao Z. 2019. High-resolution mapping of quantitative trait loci controlling main floral stalk length in Chinese cabbage(Brassica rapa L. ssp. pekinensis). BMC Genomics, 20:437. doi: 10.1186/s12864-019-5810-2 URL
[17]	Liu Z, Fang Z, Zhuang M, Zhang Y, Lv H, Liu Y, Li Z, Sun P, Tang J, Liu D, Zhang Z, Yang L. 2017. Fine-mapping and analysis of Cgl1,a gene conferring glossy trait in cabbage(Brassica oleracea L. var. capitata). Frontiers in Plant Science, 8 (14024):239.
[18]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25:402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	McFarlane H E, Shin J J H, Bird D A, Samuels A L. 2010. Arabidopsis ABCG transporters,which are required for export of diverse cuticular lipids,dimerize in different combinations. The Plant Cell, 22:3066-3075. doi: 10.1105/tpc.110.077974 pmid: 20870961
[20]	McFarlane H E, Watanabe Y, Yang W, Huang Y, Ohlrogge J, Samuels A L. 2014. Golgi- and trans-Golgi network-mediated vesicle trafficking is required for wax secretion from epidermal cells. Plant Physiolgy, 164:1250-1260.
[21]	Mu Xiangli, Wang Chao, Wang Shuai. 2013. Observation of ultra microstructure of wax-less mutant epicuticular wax on Cabbage. Chinese Vegetable,(4):32. (in Chinese)
	牟香丽, 王超, 王帅. 2013. 甘蓝无蜡质突变体叶表皮蜡质超微结构观察. 中国蔬菜,(4):32.
[22]	Ni Z, Xin M, Hu Z, Yao Y, Wang T, Liu X, Xing J, Peng H, Zhang Y, Zhou D. 2018. GCN5 contributes to stem cuticular wax biosynthesis by histone acetylation of CER3 in Arabidopsis. Journal of Experimental Botany, 69 (12):2911-2922. doi: 10.1093/jxb/ery077 URL
[23]	Oshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M. 2013. MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. The Plant Cell, 25:1609-1624. doi: 10.1105/tpc.113.110783 URL
[24]	Park C S, Go Y S, Suh M C. 2016. Cuticular wax biosynthesis is positively regulated by WRINKLED4,an AP2/ERF-type transcription factor,in Arabidopsis stems. The Plant Journal, 88:257-270. doi: 10.1111/tpj.2016.88.issue-2 URL
[25]	Pu Y, Gao J, Guo Y, Liu T, Zhu L, Xu P, Yi B, Wen J, Tu J, Ma C, Fu T, Zou J, Shen J. 2013. A novel dominant glossy mutation causes suppression of wax biosynthesis pathway and deficiency of cuticular wax in Brassica napus. BMC Plant Biology, 13:215. doi: 10.1186/1471-2229-13-215 URL
[26]	Raffaele S, Vailleau F, Léger A, Joubes J, Miersch O, Huard C, Blee E, Mongrand S, Domergue F, Roby D. 2008. A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis. The Plant Cell, 20:752-767. doi: 10.1105/tpc.107.054858 URL
[27]	Rowland O, Domergue F. 2012. Plant fatty acyl reductases:enzymes generating fatty alcohols for protective layers with potential for industrial applications. Plant Science, 193-194:28-38. doi: S0168-9452(12)00095-7 pmid: 22794916
[28]	Shao Meini, Hao Xin, Cui Na, Qu Bo, Guan Ping, Jia Weikang, Xu Yufeng. 2020. Effects of epicuticular waxes on the physiological characteristics of blue-leaf Hosta. Acta Horticulturae Sinica, 47 (7):1401-1411. (in Chinese)
	邵美妮, 郝鑫, 崔娜, 曲波, 关萍, 贾伟康, 许玉凤. 2020. 蓝叶类型玉簪叶片表皮蜡质对光合生理的影响. 园艺学报, 47 (7):1401-1411.
[29]	Todd J, Post-Beittenmiller D, Jaworski J G. 1999. KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. The Plant Journal, 17:119-130. doi: 10.1046/j.1365-313X.1999.00352.x URL
[30]	Xue Y, Xiao S, Kim J, Lung S, Chen L, Tanner J A, Suh M C, Chye M L. 2014. Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation. Journal of Experimental Botany, 65:5473-5483. doi: 10.1093/jxb/eru304 URL
[31]	Yeats T H, Rose J K. 2008. The biochemistry and biology of extracellular plant lipid-transfer proteins(LTPs). Protein Science, 17 (2):191-198. doi: 10.1110/ps.073300108 URL
[32]	Zhang X, Liu Z, Wang P, Wang Q, Yang S, Feng H. 2013. Fine mapping of BrWax1,a gene controlling cuticular wax biosynthesis in Chinese cabbage(Brassica rapa L. ssp. pekinensis). Molecular Breeding, 32 (4):867-874. doi: 10.1007/s11032-013-9914-0 URL
[33]	Zhao L, Kunst L. 2016. SUPERKILLER complex components are required for the RNA exosome-mediated control of cuticular wax biosynthesis in Arabidopsis inflorescence stems. Plant Physiology, 171:960-973.

基因	基因号	正向引物（5′-3′）	反向引物（5′-3′）
Gene	ID	Forward primer	Reverse primer
CER2*	Bra013809	TGAATATAGTGTGAACGCATTAGC	GCCCTAGTGATAACCCACCA
MAH1	Bra027904	AGGTTCATGAGGCCAACGAT	CGGAAATTTTGAGCTGAGCAT
KCS9	Bra001974	TAAGAAGGCGATGCTGATCC	ACGCCTTCTCATCAGCTCCT
KCS1	Bra033283	CTCTATCGGCGATGATCGTG	GTTGGCGAGTTCGATTGAGA
CER2	Bra013809	GCTCGACCTCCAGTGTTATGA	ACTGTCGTTGCAGCGAATGT
CYTB5-B	Bra021809	GCACAATCACGCTCATGACTG	TCATCCGTTGCATCCTTACC
CYTB5-C	Bra039268	GCGGTGACAATGTTCTCCTC	TGGAATATACTTCGCCGTCAC
MYB30	Bra033067	TGCCGTAGCACCGATCAAT	TCGCTATGTTCTCGGTGTTTG
LTPG1	Bra030067	AACGCTAGCATCTCCAACTGTC	TTGGAGTTGCCGGAGACTTC
G6PD		GGGTATGCCAGGACTAAGCTC	GAATCATAAGGGCCACTCACAT

基因	基因号	正向引物（5′-3′）	反向引物（5′-3′）
Gene	ID	Forward primer	Reverse primer
CER2*	Bra013809	TGAATATAGTGTGAACGCATTAGC	GCCCTAGTGATAACCCACCA
MAH1	Bra027904	AGGTTCATGAGGCCAACGAT	CGGAAATTTTGAGCTGAGCAT
KCS9	Bra001974	TAAGAAGGCGATGCTGATCC	ACGCCTTCTCATCAGCTCCT
KCS1	Bra033283	CTCTATCGGCGATGATCGTG	GTTGGCGAGTTCGATTGAGA
CER2	Bra013809	GCTCGACCTCCAGTGTTATGA	ACTGTCGTTGCAGCGAATGT
CYTB5-B	Bra021809	GCACAATCACGCTCATGACTG	TCATCCGTTGCATCCTTACC
CYTB5-C	Bra039268	GCGGTGACAATGTTCTCCTC	TGGAATATACTTCGCCGTCAC
MYB30	Bra033067	TGCCGTAGCACCGATCAAT	TCGCTATGTTCTCGGTGTTTG
LTPG1	Bra030067	AACGCTAGCATCTCCAACTGTC	TTGGAGTTGCCGGAGACTTC
G6PD		GGGTATGCCAGGACTAAGCTC	GAATCATAAGGGCCACTCACAT

样品名称	原始序列	高质量序列	高质量碱基数/Gb	错误率/%	Q20/%	Q30/%	GC含量/%
Sample name	Raw reads	Clean reads	Clean base	Error rate	Q20/%	Q30/%	GC content
RHL065_1_T1	42628776	41061052	6.16	0.02	98.53	95.36	47.34
RHL065_1_T2	59538100	56577062	8.49	0.02	98.22	94.59	47.28
RHL065_1_T3	45954482	43800974	6.57	0.02	98.36	94.96	47.24
RHL065_2_T1	61069026	57685276	8.65	0.02	98.42	95.12	47.15
RHL065_2_T2	59286084	56360648	8.45	0.02	98.26	94.74	47.11
RHL065_2_T3	61847196	58688916	8.80	0.02	98.42	95.12	46.79

样品名称	原始序列	高质量序列	高质量碱基数/Gb	错误率/%	Q20/%	Q30/%	GC含量/%
Sample name	Raw reads	Clean reads	Clean base	Error rate	Q20/%	Q30/%	GC content
RHL065_1_T1	42628776	41061052	6.16	0.02	98.53	95.36	47.34
RHL065_1_T2	59538100	56577062	8.49	0.02	98.22	94.59	47.28
RHL065_1_T3	45954482	43800974	6.57	0.02	98.36	94.96	47.24
RHL065_2_T1	61069026	57685276	8.65	0.02	98.42	95.12	47.15
RHL065_2_T2	59286084	56360648	8.45	0.02	98.26	94.74	47.11
RHL065_2_T3	61847196	58688916	8.80	0.02	98.42	95.12	46.79

通路	基因号	基因名称	差异倍数	显著性	调控模式
Pathway	Gene ID	Gene name	log₂Fold Change	P value	Regulated mode
表皮蜡粉生物合成 Cuticular wax biosynthesis	Bra027904	MAH1	3.342	1.1326E-07	上调 Up
	Bra022898	CYTB5-B	2.732	4.7382E-17	上调 Up
	Bra004034	KCS6/CER6/CUT1	-1.193	1.2047E-38	下调 Down
	Bra001974	KCS9	-1.250	7.6776E-63	下调 Down
	Bra034583	FAR3/CER4	-1.372	7.6944E-35	下调 Down
	Bra033283	KCS1	-1.399	8.2146E-66	下调 Down
	Bra013809	CER2	-1.691	7.229E-73	下调 Down
	Bra021809	CYTB5-B	-1.715	5.039E-135	下调 Down
	Bra006062	KCD/PAS2	-2.222	5.411E-240	下调 Down
	Bra039268	CYTB5-C	-2.239	4.6399E-50	下调 Down
	Bra004518	CYTB5-C	-5.226	4.6843E-12	下调 Down
	Bra014988	CER2-LIKE	-6.478	1.1694E-05	下调 Down
转录调控 Transcriptional regulation	Bra026140	WIN1/SHINE1	1.451	0.0011061	上调 Up
	Bra033067	MYB30	-1.063	0.00044649	下调 Down
	Bra029319	DEWAX	-2.482	2.287E-15	下调 Down
表皮蜡粉转运 Cuticular wax transport	Bra010912	LTPG1	-1.024	3.5614E-13	下调 Down
表皮蜡粉转运 Cuticular wax transport	Bra030067	LTPG1	2.232	9.9446E-09	上调 Up