基于转录组分析垂丝海棠响应盐碱胁迫的分子机制

Transcriptome Analysis of the Molecular Mechanism of Saline-alkali Stress Response in Malus halliana Leaves

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;NaCl+NaHCO;)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16246个DEG,其中上调7268个,下调8978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。

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.