论文部分内容阅读
【目的】探明云南割手密82-114受干旱胁迫的内在分子机制,挖掘与抗旱密切相关的功能基因,提高割手密抗旱基因的研究与利用。【方法】以干旱胁迫24、48和72 h的云南割手密82-114的根系进行Illumina Hi Seq TM 4000高通量转录组测序分析,将组装得到的Unigene分别与Swiss-Prot、Nr、KOG、Pfam和KEGG数据库进行比对,利用RPKM来衡量各样本间的基因表达丰度,以FDR≤0.05和|log2 fold change|≥1来评估样本间的差异表达基因,通过Gene Ontology(GO)数据库、KEGG pathway数据库对不同干旱胁迫样本差异表达基因的功能和参与的调控路径进行分析。【结果】未胁迫处理的CK与胁迫24、48和72 h后的样本转录组分别检测到134 724、130 368、133 564和131 321个表达基因,与CK相比,各胁迫样本显著上调(或下调)的差异表达基因分别有3 061(1 302)、2 304(2 841)和3 236(2 525)个;以FDR值=0为筛选条件,3个样本的极显著差异表达基因主要参与转录激活、水分运输、DNA结合、ATP结合及细胞膜、跨膜运输和防御反应等有关代谢活动。GO富集分析表明:3个胁迫处理的样本在生物学途径中富集最多的类别并不完全相同,其中,胁迫24 h与48 h的样本富集最多的类别是与DNA依赖型转录和转录调节子相关的基因,胁迫72 h样本富集最多的类别是与翻译相关的基因;而在细胞组件和分子功能中,3个胁迫样本富集最多的基因类别均是与内在的膜、核和ATP结合相关的基因。KEGG富集分析表明:胁迫24 h的样本有2 248个差异表达基因参与了107条代谢途径,胁迫48 h的样本有2 114个差异表达基因参与了130条代谢途径,胁迫72 h的样本有2 392个差异表达基因参与了144条代谢途径;经KEGG显著性富集分析,筛选到鞘糖脂生物合成途径、MAP激酶信号途径和ABC转运蛋白等与非生物胁迫或逆境相关。【结论】获得3个干旱胁迫样本与CK样本间差异表达基因的变化及其功能信息,发现参与云南割手密82-114干旱胁迫响应相关的细胞外信号调节激酶、磷脂酶D、β-氨基己糖苷酶和ATP结合盒B亚族(MDR/TAP)-1等,获得在整个干旱胁迫时期稳定上调表达基因70个,稳定下调表达基因11个,通过同源基因序列的比对分析,阐明这些基因在各自的代谢通路中被强烈诱导或抑制表达,显示与干旱胁迫存在密切关系。
【Objective】 To explore the intrinsic molecular mechanism of drought stress induced by drought in Mianyang 82-114 and to explore the functional genes closely related to drought resistance and to improve the research and utilization of drought resistance genes in Mudhoumi. 【Method】 The Illumina Hi Seq TM 4000 high-throughput transcriptome sequencing analysis was carried out at 24, 48 and 72 h drought stress in roots of Yunnan Mudami 82-114. Unigene was assembled with Swiss-Prot, Nr, KOG , Pfam and KEGG databases, RPKM was used to measure the abundance of gene expression in each sample. Differentially expressed genes were evaluated by FDR ≤0.05 and | log2 fold change | ≥1. Gene Ontology (GO) database , KEGG pathway database were used to analyze the regulatory pathway of differentially expressed genes in different drought stress samples. 【Result】 The results showed that 134 724, 130 368, 133 564 and 131 321 expression genes were detected in untreated CK and 24, 48 and 72 h after stress, respectively. Compared with CK, all stressed samples were significantly up-regulated Or down-regulated) were 3 061 (1 302), 2 304 (2 841) and 3 236 (2 525), respectively. For FDR value = 0, the most significantly differentially expressed genes in 3 samples Participate in transcriptional activation, water transport, DNA binding, ATP binding and cell membrane, transmembrane transport and defense response and other related metabolic activity. The results of GO enrichment analysis showed that the three most abundant samples were not completely the same in the biological pathway. Among them, the most enriched samples at 24h and 48h were DNA-dependent transcription and transcription Regulator-related genes, the most enriched 72-h samples were translating-related genes, whereas the most abundant genes among the three stress samples in cellular components and molecular functions were all related to intrinsic membrane, nuclear and ATP binding related genes. KEGG enrichment analysis showed that there were 2 248 differentially expressed genes involved in 107 metabolic pathways in 24 h stress samples, 2 114 differentially expressed genes involved in 130 metabolic pathways in 48 h stress samples, and 72 stress samples A total of 2 392 differentially expressed genes were involved in 144 metabolic pathways. After significant enrichment analysis by KEGG, the pathways of glycosphingolipid biosynthesis, MAP kinase and ABC transporters were screened for abiotic stress or stress. 【Conclusion】 The changes of differentially expressed genes and their functional information among three drought-stress samples and CK samples were observed. It was found that extracellular signal-regulated kinase, phospholipase D, β-amino The results showed that 70 genes were up-regulated steadily and 11 genes were down-regulated steadily during drought stress, and the homologous gene sequences were aligned to elucidate the relationship between MDR / TAP and ATP-binding cassette subunit B (MDR / TAP) These genes are strongly induced or repressed in their respective metabolic pathways and show a close relationship with drought stress.