基于转录组测序的湖北海棠抗白粉病机制分析

doi:10.16420/j.issn.0513-353x.2020-0578

摘要/Abstract

摘要：

为研究湖北海棠[Malus hupehensis(Pamp)Rehd.]响应白粉病菌胁迫过程中相关基因的表达及分子机制,取同一时期爆发白粉病的‘青砧1号’、‘青砧1号’/湖北海棠嫁接苗的接穗部分和湖北海棠等3份材料的叶片,使用BGISEQ-500测序技术对其进行转录组测序分析。结果表明:青砧1号、青砧1号/湖北海棠和湖北海棠对白粉病的抗性依次增加;GO富集显示代谢过程和催化活性相关的GO term在青砧1号、青砧1号/湖北海棠和湖北海棠中被显著富集;KEGG分析显示亚油酸代谢、植物病原体相互作用、磷酸肌醇代谢、油菜素类固醇生物合成和磷脂酰肌醇信号系统等5条代谢途径在‘青砧1号’/湖北海棠和湖北海棠中均显著富集,碳代谢途径只在抗性强的湖北海棠中显著富集。与白粉病抗性相关基因家族表达分析显示,WRKY70、WRKY33、NPR3和WRKY40等转录因子基因在青砧1号/湖北海棠和湖北海棠中较青砧1号显著上调表达。实时荧光定量PCR结果证实了转录组测序结果的准确性。

关键词: 苹果, 湖北海棠, 砧木, 砧穗互作, 白粉病, 转录组, 差异表达

Abstract:

To explore the pathways and mechanism of response to powdery mildew in Malus hupehensis(Pamp.)Rehder,we used the BGISEQ-500 sequencing technology and de novo transcriptome assembly to gain a comprehensive overview of the transcriptome of the leaves of‘Qingzhen 1’, ‘Qingzhen 1’/Malus hupehensis and M. hupehensis after the outbreak of powdery mildew in the same period. The resistance to powdery mildew of three materials increased in turn. Gene Ontology(GO)enrichment showed significant enrichment of GO term associated with metabolic process and catalytic activity in Qingzhen 1,Qingzhen 1/Malus hupehensis and M. hupehensis. KEGG analysis showed linoleic acid metabolism,plant-pathogen interaction,inositol phosphate metabolism,brassinosteroid biosynthesis and phosphatidylinositol signaling system etc. were significantly enriched in Qingzhen 1/Malus hupehensis and resistance to powdery mildew,respectively. And the carbon metabolism pathway was only significantly enriched in M. hupehensis. Expression analysis of genes related to the resistance to powdery mildew showed that WRKY70,WRKY33,NPR3and WRKY40 were significantly up-regulated in Qingzhen 1/Malus hupehensis and M. hupehensis compared to Qingzhen 1. The accuracy of sequencing were confirmed by qRT-PCR experiment.

Key words: apple, Malus hupehensis, rootstock, rootstock-scion interaction, powdery mildew, transcriptome, differentially expressed

中图分类号:

S661.4

兰黎明, 罗昌国, 王三红. 基于转录组测序的湖北海棠抗白粉病机制分析[J]. 园艺学报, 2021, 48(5): 860-872.

LAN Liming, LUO Changguo, WANG Sanhong. Analysis of Resistance Mechanism to Powdery Mildew Based on Transcriptome Sequencing in Malus hupehensis[J]. Acta Horticulturae Sinica, 2021, 48(5): 860-872.

图/表 7

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样品名称 Sample name	clean reads数量/Mb Number of clean reads	GC含量/% GC content	Q30/%	低质量含量/% Low quality content
青砧1号Qingzhen 1	46.17(96.40%)	49.40	93.31	3.09
青砧1号/湖北海棠 Qingzhen 1/Malus hupehensis	56.53(97.25%)	47.33	93.98	2.34
湖北海棠 M. hupehensis	52.78(96.64%)	48.53	93.50	2.87

样品名称 Sample name	clean reads数量/Mb Number of clean reads	GC含量/% GC content	Q30/%	低质量含量/% Low quality content
青砧1号Qingzhen 1	46.17(96.40%)	49.40	93.31	3.09
青砧1号/湖北海棠 Qingzhen 1/Malus hupehensis	56.53(97.25%)	47.33	93.98	2.34
湖北海棠 M. hupehensis	52.78(96.64%)	48.53	93.50	2.87

基因名称 Gene name	基因ID Gene ID	Nr-注释 Nr-annotation	引物序列(5'-3') Primer sequence(5'-3')
WRKY70	XP_008388364.1^a	WRKY转录因子70[苹果] WRKY transcription factor 70[Malus × domestica]	F:AAACCACCTACATCGGCGAG
WRKY70	XP_008388364.1^a		R:ATGAGAATTGACGGTGCGGA
WRKY33	NP_001315684.1^a	WRKY转录因子26[苹果] WRKY transcription factor 26[Malus × domestica]	F:ACAACAACAGCGCCTCTACC
WRKY33	NP_001315684.1^a		R:CCCTCCCCAAAAGACAGCAAA
WRKY3	XP_008352971.1^a	WRKY转录因子3[苹果] WRKY transcription factor 3[Malus × domestica]	F:TACACAGGCAACAACAACGC
WRKY3	XP_008352971.1^a		R:TCTGATTCCTCGTGCTTGCTT
WRKY22	XP_008345081.1^a	WRKY转录因子22[苹果] WRKY transcription factor 22[Malus × domestica]	F:CTAGTAGTGCTGCCACTGCC
WRKY22	XP_008345081.1^a		R:TCCCACCCATAGTTACACCCA
WRKY2	XP_008344758.1^a	WRKY转录因子2[苹果] WRKY transcription factor 2[Malus × domestica]	F:TCTTGGTGCAACGCCTTGAT
WRKY2	XP_008344758.1^a		R:TCTCGAGCTCCCAGGAAGAA
NPR3	ANJ45443.1^a	NPR5a [苹果] NPR5a[Malus × domestica]	F:CATCTTCTCCGAAGGACGGG
NPR3	ANJ45443.1^a	NPR5a [苹果] NPR5a[Malus × domestica]	R:TCTGTTTGATAAGGAGAGAGCCA
WRKY40	NP_001315769.1^a	WRKY转录因子40[苹果] WRKY transcription factor 40[Malus × domestica]	F:TGCTCAACTCCAAGGCAATG
WRKY40	NP_001315769.1^a		R:TTCACTGTCTGCCTTTCGCT
EF1	MD09G014760^b	内参基因 Reference gene	F:TACTGGAACATCACAGGCTGAC
EF1	MD09G014760^b	内参基因 Reference gene	R:TGGACCTCTCAATCATGTTGTC

基因名称 Gene name	基因ID Gene ID	Nr-注释 Nr-annotation	引物序列(5'-3') Primer sequence(5'-3')
WRKY70	XP_008388364.1^a	WRKY转录因子70[苹果] WRKY transcription factor 70[Malus × domestica]	F:AAACCACCTACATCGGCGAG
WRKY70	XP_008388364.1^a		R:ATGAGAATTGACGGTGCGGA
WRKY33	NP_001315684.1^a	WRKY转录因子26[苹果] WRKY transcription factor 26[Malus × domestica]	F:ACAACAACAGCGCCTCTACC
WRKY33	NP_001315684.1^a		R:CCCTCCCCAAAAGACAGCAAA
WRKY3	XP_008352971.1^a	WRKY转录因子3[苹果] WRKY transcription factor 3[Malus × domestica]	F:TACACAGGCAACAACAACGC
WRKY3	XP_008352971.1^a		R:TCTGATTCCTCGTGCTTGCTT
WRKY22	XP_008345081.1^a	WRKY转录因子22[苹果] WRKY transcription factor 22[Malus × domestica]	F:CTAGTAGTGCTGCCACTGCC
WRKY22	XP_008345081.1^a		R:TCCCACCCATAGTTACACCCA
WRKY2	XP_008344758.1^a	WRKY转录因子2[苹果] WRKY transcription factor 2[Malus × domestica]	F:TCTTGGTGCAACGCCTTGAT
WRKY2	XP_008344758.1^a		R:TCTCGAGCTCCCAGGAAGAA
NPR3	ANJ45443.1^a	NPR5a [苹果] NPR5a[Malus × domestica]	F:CATCTTCTCCGAAGGACGGG
NPR3	ANJ45443.1^a	NPR5a [苹果] NPR5a[Malus × domestica]	R:TCTGTTTGATAAGGAGAGAGCCA
WRKY40	NP_001315769.1^a	WRKY转录因子40[苹果] WRKY transcription factor 40[Malus × domestica]	F:TGCTCAACTCCAAGGCAATG
WRKY40	NP_001315769.1^a		R:TTCACTGTCTGCCTTTCGCT
EF1	MD09G014760^b	内参基因 Reference gene	F:TACTGGAACATCACAGGCTGAC
EF1	MD09G014760^b	内参基因 Reference gene	R:TGGACCTCTCAATCATGTTGTC