短期盐胁迫下中国石竹幼苗响应的转录组测序、组装和分析

发布时间：2018-04-10 23:00

本文选题：中国石竹 + 盐胁迫　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：盐害是植物生长面临的重要逆境危害之一,研究耐盐植物的耐盐机制可为植物改良和盐地种植技术提供理论依据。本试验以耐盐性观赏植物中国石竹的幼苗为材料,通过在100mMNaCl胁迫的Oh、1h、3h、6h及去除盐胁迫的1h、3h分别收集幼苗叶片和根,提取总RNA,然后进行转录组测序、组装、分析,从基因表达水平深入了解中国石竹的耐盐机理,获得耐盐基因,进而达到在石竹属植物中进行抗盐新品种培育的目的。取得的研究成果有:1.测序、组装后获得 62,371 unigenes,其中 31863 unigenes(51.08%)成功注释。其中22662个unigenes可富集到46个GO terms,1 11810个unigenes可分配到KOG的26个groups上,经KEGG注释,10265个unigenes共预测了 272个途径;2.盐胁迫下叶中共获得9139个盐诱导基因,根中共获得19280个盐诱导基因;在盐胁迫的1h、3h和6h,根中的DEGs分别是叶中的19.90、1.25和2.6倍;分别对叶和根中的盐诱导基因进行GO富集分析,发现盐胁迫影响根部的纤维素代谢,叶中比根部延后,主要影响到转录、翻译过程;KEGG分析表明,盐胁迫下根部通过应激反应,产生信号物质,影响糖代谢、苯丙素代谢、脂肪代谢,叶中比根中延后,在盐胁迫6h开始产生信号物质。3.根据GO富集中的BP途径,筛选出与氧化还原过程或细胞氧化还原平衡有关的盐诱导基因,经RT-PCR验证,发现在短期盐胁迫下叶中的DchAAO1、DchPOD1基因和根中的DchAAO2、DchPOD2基因表达量下调,叶中的DchTrx基因和根中的DchGrx1、DchGrx2、DchTrx基因表达量上调,去除盐胁迫后则相反,所有的被验证基因的表达模式与RNA-Seq的数据吻合,表明试验结果可靠。4.延长盐胁迫时间后,对筛选出的差异基因进行RT-PCR验证,发现中国石竹叶片中基因DchAAO1、DchPOD1、DchTrx的表达量从9h到12h下降,12h后下降平缓,且在盐胁迫同一时间点下均低于对照;根中基因DchPOD2的表达量随胁迫时间的延长变化不大,在胁迫12、24h后其表达量低于对照,基因DchAAO2的表达量从9h到12h上升,12h后下降,盐胁迫12、24h后基因表达量高于对照,基因DchGrx1、DchGrx2的表达量从9h到12h上调且胁迫下的高于对照,12h后下调且胁迫下的低于对照,基因DchTrx的表达量呈先升高后降低趋势,在盐胁迫下其基因表达量均低于对照。
[Abstract]:Salt damage is one of the important stresses on plant growth. The study of salt tolerance mechanism of salt-tolerant plants can provide theoretical basis for plant improvement and salt planting techniques.In this experiment, the seedlings of Chinese carnation, a salt-tolerant ornamental plant, were collected from the leaves and roots of the plants under 100mMNaCl stress for 3 h and 1 h for 3 h, respectively, and the total RNAs were extracted, then sequenced, assembled and analyzed.The salt-tolerant genes were obtained from the level of gene expression in Chinese carnation, and the purpose of breeding new salt-tolerant varieties in Carnation was achieved.The results of the research are as follows: 1: 1.Sequencing, assembled 62371 unigenes, of which 31863 unigenes 51.08) successfully annotated.Among them, 22662 unigenes can be enriched to 46 go termsl 11810 unigenes can be distributed to 26 groups of KOG. According to KEGG annotation, a total of 272 pathways are predicted by 10265 unigenes.A total of 9139 salt-inducible genes were obtained in leaves and 19280 in roots under salt stress, and the DEGs in roots was 19.90,1.25 and 2.6-fold higher than that in leaves at 1h and 6h, respectively. Go enrichment analysis was carried out in leaves and roots, respectively.It was found that salt stress affected cellulose metabolism in roots, and that in leaves was later than that in roots, which mainly affected transcription. KEGG analysis showed that under salt stress, the roots produced signal substances, sugar metabolism and phenylpropanin metabolism through stress response.Fat metabolism, delayed in leaves than in roots, began to produce signal substance. 3 at 6 h of salt stress.According to BP pathway in go enrichment, salt-induced genes related to redox process or redox balance of cells were screened. After RT-PCR verification, it was found that DchAAO1DchPOD1 gene in leaves and DchAAO2mDchPOD2 gene in roots were down-regulated under short-term salt stress.The expression of DchTrx gene in leaves and DchGrx1 + DchGrx2DchTrx gene in root was up-regulated, but the expression pattern of all the verified genes was consistent with that of RNA-Seq after salt stress was removed, indicating that the results of the experiment were reliable. 4.After prolonging the time of salt stress, the differentially screened genes were verified by RT-PCR. It was found that the expression of DchAAO1, DchPOD1, DchTrx in Chinese carnation leaves decreased slowly from 9h to 12h, and the expression of DchPOD1DchTrx was lower than that of control at the same time point of salt stress.The expression of gene DchPOD2 in roots did not change with the prolongation of stress time. After 1224 hours of stress, the expression of gene DchPOD2 was lower than that of control. The expression of gene DchAAO2 increased from 9 h to 12 h and decreased after 12 h of salt stress, and the amount of gene expression was higher after 1224 h of salt stress than that of control.The expression of DchGrx1 and DchGrx2 was up-regulated from 9 h to 12 h, and decreased after 12 h of stress, and the expression of DchTrx increased first and then decreased. The expression of DchGrx2 was lower than that of control under salt stress.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S681.5

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