基于转录组分析垂丝海棠响应盐碱胁迫的分子机制

doi:10.16420/j.issn.0513-353x.2021-0077

摘要/Abstract

摘要：

以干旱盐碱生境苹果属垂丝海棠（Malus halliana Koehen）为试验材料,通过RNA-Seq测序筛选响应盐碱复合胁迫的差异表达基因,为苹果砧木耐盐碱性机制提供理论依据。以正常供水（Hoagland营养液）为对照,进行混合盐碱胁迫（Hoagland营养液 + 100 mmol · L^-1 NaCl + NaHCO₃）处理,RNA-Seq测序筛选差异表达基因（differentially expression gene,DEG）,并对 DEG进行GO（gene ontology）和 KEGG （Kyoto Encyclopedia of Genes and Genomes）功能富集分析。应用实时荧光定量qRT-PCR对主要通路的DEG表达模式进行验证。共鉴定出16 246个DEG,其中上调7 268个,下调8 978个。GO分析表明,在遭受盐碱胁迫时生物过程、细胞组分和分子功能3个方面均存在较大差异。KEGG 聚类分析显示,参与信号传导、碳代谢、氨基酸合成及次生代谢等相关的DEG在响应胁迫中具有关键作用,且主要集中在钙信号通路、植物激素信号传导、苯丙氨酸代谢、类胡萝卜素的合成等过程。对上述主要通路的20个DEG 表达模式的qRT-PCR检测结果与RNA-seq 结果类似。其中天门冬氨酸氨基转移酶（aspartate aminotransferase,GOT1）、抗坏血酸还原酶（NADH）、查尔酮异构酶（chalcone isomerase,CHI）、β-胡萝卜素羟化酶（CrtZ）基因在盐碱胁迫过程中表达量显著升高。垂丝海棠叶片对盐碱胁迫的耐受能力主要与基因对胁迫的响应程度相关,调控基因表达涉及钙信号通路、植物激素信号传导、氨基酸生物合成、类胡萝卜素生物合成等次生代谢物途径。

关键词: 垂丝海棠, 盐碱胁迫, 转录组学分析, 差异基因表达

Abstract:

Malus halliana is a highly saline-alkali-resistant apple rootstock in north-western China. The genes and main pathways which respond to saline-alkali stress were identified by RNA-Seq. This study revealed the mechanism of the response to salt damage and provided theoretical basis for the salt tolerance mechanism of apple rootstocks. Normal water（Hoagland nutrient solution）（Control）supply and mixed saline-alkali stress（Hoagland nutrient solution + 100 mmol · L^-1 NaCl + NaHCO₃）（Treatment）were set up to screen the saline-alkali resistance of each treatment in the growth period. These two samples before and after saline-alkali treatment were analyzed by RNA-seq. The function and pathway enrichment of differentially expressed genes（DEG）were also performed by GO and KEGG analysis. Twenty DEG were randomly selected for further qRT-PCR analysis to verify the RNA-seq data. A total of 16 246 DEGs were determined,7 268 genes were up-regulated and 8 978 genes down-regulated under saline-alkali stress. GO analysis fund that the DEGs were considerably differenct in the biological processes,cell components and molecular functions. KEGG analysis indicated that most enriched saline-alkali-responsive genes were mainly involved in signal transduction,carbon metabolism,biosynthesis of amino acids,and other secondary metabolites. DEG mainly focused on calcium signaling pathway,plant hormone signal transduction,phenylalanine metabolism,and carotenoid biosynthesis. qRT-PCR detection results of 20 DEG expression patterns were similar to those of RNA-seq. Among them,aspartate aminotransferase（GOT1）,monodehydroascorbate reductase（NADH）,chalcone isomerase（CHI,E5.5.1.6）,β-carotene 3-hydroxylase（CrtZ）genes were highly expressed during saline-alkali stress. The results indicated that they might play a role in the leaf response to saline-alkali stress. M. halliana is mainly regulated by calcium signal pathway,plant hormone signal transduction,biosynthesis of amino acids, carotenoid biosynthesis and other secondary metabolites to respond to saline-alkali stress.

Key words: Malus halliana, saline-alkali stress, transcriptomic profiles analysis, differential gene expression

中图分类号:

S661.4

张瑞, 张夏燚, 赵婷, 王双成, 张仲兴, 刘博, 张德, 王延秀. 基于转录组分析垂丝海棠响应盐碱胁迫的分子机制[J]. 园艺学报, 2022, 49(2): 237-251.

ZHANG Rui, ZHANG Xiayi, ZHAO Ting, WANG Shuangcheng, ZHANG Zhongxing, LIU Bo, ZHANG De, WANG Yanxiu. Transcriptome Analysis of the Molecular Mechanism of Saline-alkali Stress Response in Malus halliana Leaves[J]. Acta Horticulturae Sinica, 2022, 49(2): 237-251.

图/表 9

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基因 Gene	名称 Description	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
ANT	溶质载体家族25 Solute carrier family 25	CCAACTTCCACCGTGATCTGATG	TCCTTCCACCGCCGAGAATG
ATP2A	钙转运体 Calcium transporter	GGTACAGTAGTCGTGGCTGGTAG	TTCAATGGCGTCACTTCATCTTCC
PLCD	磷脂酰肌醇磷脂酶C Phosphatidylinositol phospholipase C,delta	GGTGTTCGCCAAGTACGCTCAG	GGGTCGTGAACTGGAGGAGGATAG
SPHK	鞘氨醇激酶Sphingosine kinase	CTCATAAGCGGCGTCGTCACAC	CGAACCCCAGGACCTCCTTCTC
VDAC1	赖性阴离子通道蛋白1 Voltage-dependent anion channel protein 1	TGCCGAACTGACCCACAGATTTTC	GGTCTCCATTCACGCTGCCATAG
ABF	ABA响应元件结合因子 ABA responsive element binding factor	TGACAATGTTGAGTTCCGTGAGC	TCGACGACTGCCTCGCTAAC
PR1	病程相关蛋白1 Pathogenesis-related protein 1	ATGGGGTTGTGCAATATTTC	CTAGTAAGGCTTCTCCCCAAC
PYL	脱落酸受体PYR/PYL家族 Abscisic acid receptor PYR/PYL family	ATGTCTTCACCAATCCAGTTTC	AGTACTCCCAAAGAACCACAAA
COI-1	冠菌素不敏感蛋白1 Coronatine-insensitive protein 1	CGAGGGCGGCGATGTTCAATC	ACTTCAAGCGGTGGAACGAGTTG
GOT1	细胞质天冬氨酸转氨酶 Aspartate aminotransferase,cytoplasmic	ACAACGCCTCCAAGCTCCTC	TCTGAGTTCGCCGAGTCCTTC
CHI	查耳酮异构酶Chalcone isomerase	ATGGCTGACGGTGACCAAC	TTATCTAGTGATATGAGCAATAAA
ANS	花青素合成酶Anthocyanidin synthase	ATGGTGAGCTCTGATTCAG	TCACTTGGGGAGCAAAGC
ANR	花青素还原酶Anthocyanidin reductase	ATGGCCACCCAACAACCC	CTAGTTCTGCAGCAGCCC
NADH	单脱水抗坏血酸还原酶 Monodehydroascorbate reductase（NADH）	ATGGGAAGGGCATTTGTG	TCACCACCTCCGGCGCTT
CrtY	番茄红素Lycopene beta-cyclase	ATGGATACATTGCTTAAAACGC	GATTAGAGATAGGACATAGTTCAAC
CrtZ	β-胡萝卜素β-carotene 3-hydroxylase	GCAAGGTGGGCTCATAGAACTCTG	AAGAAGGGCAATAGCAGGAACAGC
HPD	羟苯丙酮酸加双氧酶 Hydroxyphenylpyruvate dioxygenase	TGTACCACAGAGACCTGAAGCC	CAGCAGCAGAGCCATCATAACC
pckA	膜伯胺氧化酶 Primary-amine oxidase	CGACGCCTTCCTGTTGCTTCC	CGGTGACGCCAGTCCAATCTTG
VPS35	液泡蛋白分选相关蛋白35 Vacuolar protein sorting-associated protein 35	TGAAGATTGGCTGCACTTGACTGG	CCGACAACTGCCCATTGAGGAAG
VTA1	液泡蛋白分选相关蛋白 Vacuolar protein sorting-associated protein	GGGCTCTGATCCTTCCTACTCTCC	AGTCCCATTTCTGTTGCTCGTCTG

基因 Gene	名称 Description	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
ANT	溶质载体家族25 Solute carrier family 25	CCAACTTCCACCGTGATCTGATG	TCCTTCCACCGCCGAGAATG
ATP2A	钙转运体 Calcium transporter	GGTACAGTAGTCGTGGCTGGTAG	TTCAATGGCGTCACTTCATCTTCC
PLCD	磷脂酰肌醇磷脂酶C Phosphatidylinositol phospholipase C,delta	GGTGTTCGCCAAGTACGCTCAG	GGGTCGTGAACTGGAGGAGGATAG
SPHK	鞘氨醇激酶Sphingosine kinase	CTCATAAGCGGCGTCGTCACAC	CGAACCCCAGGACCTCCTTCTC
VDAC1	赖性阴离子通道蛋白1 Voltage-dependent anion channel protein 1	TGCCGAACTGACCCACAGATTTTC	GGTCTCCATTCACGCTGCCATAG
ABF	ABA响应元件结合因子 ABA responsive element binding factor	TGACAATGTTGAGTTCCGTGAGC	TCGACGACTGCCTCGCTAAC
PR1	病程相关蛋白1 Pathogenesis-related protein 1	ATGGGGTTGTGCAATATTTC	CTAGTAAGGCTTCTCCCCAAC
PYL	脱落酸受体PYR/PYL家族 Abscisic acid receptor PYR/PYL family	ATGTCTTCACCAATCCAGTTTC	AGTACTCCCAAAGAACCACAAA
COI-1	冠菌素不敏感蛋白1 Coronatine-insensitive protein 1	CGAGGGCGGCGATGTTCAATC	ACTTCAAGCGGTGGAACGAGTTG
GOT1	细胞质天冬氨酸转氨酶 Aspartate aminotransferase,cytoplasmic	ACAACGCCTCCAAGCTCCTC	TCTGAGTTCGCCGAGTCCTTC
CHI	查耳酮异构酶Chalcone isomerase	ATGGCTGACGGTGACCAAC	TTATCTAGTGATATGAGCAATAAA
ANS	花青素合成酶Anthocyanidin synthase	ATGGTGAGCTCTGATTCAG	TCACTTGGGGAGCAAAGC
ANR	花青素还原酶Anthocyanidin reductase	ATGGCCACCCAACAACCC	CTAGTTCTGCAGCAGCCC
NADH	单脱水抗坏血酸还原酶 Monodehydroascorbate reductase（NADH）	ATGGGAAGGGCATTTGTG	TCACCACCTCCGGCGCTT
CrtY	番茄红素Lycopene beta-cyclase	ATGGATACATTGCTTAAAACGC	GATTAGAGATAGGACATAGTTCAAC
CrtZ	β-胡萝卜素β-carotene 3-hydroxylase	GCAAGGTGGGCTCATAGAACTCTG	AAGAAGGGCAATAGCAGGAACAGC
HPD	羟苯丙酮酸加双氧酶 Hydroxyphenylpyruvate dioxygenase	TGTACCACAGAGACCTGAAGCC	CAGCAGCAGAGCCATCATAACC
pckA	膜伯胺氧化酶 Primary-amine oxidase	CGACGCCTTCCTGTTGCTTCC	CGGTGACGCCAGTCCAATCTTG
VPS35	液泡蛋白分选相关蛋白35 Vacuolar protein sorting-associated protein 35	TGAAGATTGGCTGCACTTGACTGG	CCGACAACTGCCCATTGAGGAAG
VTA1	液泡蛋白分选相关蛋白 Vacuolar protein sorting-associated protein	GGGCTCTGATCCTTCCTACTCTCC	AGTCCCATTTCTGTTGCTCGTCTG

样品 Sample	原始数据 Raw reads	过滤数据 Clean reads	有效碱基/% Valid bases	Q30含量/% Q30 content	GC含量/% GC content
对照 Control	49 794 146	47 025 046	90.15	93.52	47.70
胁迫处理 Stress treatment	49 258 400	47 513 110	92.50	94.63	47.90

样品 Sample	原始数据 Raw reads	过滤数据 Clean reads	有效碱基/% Valid bases	Q30含量/% Q30 content	GC含量/% GC content
对照 Control	49 794 146	47 025 046	90.15	93.52	47.70
胁迫处理 Stress treatment	49 258 400	47 513 110	92.50	94.63	47.90