南方型紫花苜蓿耐盐突变体盐胁迫响应差异基因鉴定与分析

发布时间：2019-02-20 09:16

【摘要】：我国海域辽阔,滨海盐碱地只有小部分被利用耕种,大部分还为荒地和海涂地,海岸带拥有大量的盐碱土待开发利用,是重要的后备土地资源。滨海盐碱地改良和生态绿化是我国沿海城市化进程中一项异常艰巨的重要难题。根据近年来国内外实践经验,筛选耐盐植物是进行滨海盐碱地生物改良和生态绿化最具光明前景的经济有效的方法。紫花苜蓿(Medicagosativa L.)是一种具有较强耐盐性的豆科植物,栽培历史悠久,在全球广泛种植,具有一定的观赏价值,是进行盐碱地改良和园林绿化的理想植物。本研究以高秋休眠级南方型紫花苜蓿‘Millennium’的耐盐突变体为材料,进行转录组测序分析,筛选盐胁迫响应差异基因,并对其进行GO功能显著性分析、KEGG Pathway显著性富集分析和转录因子分子,已期探究耐盐相关基因参与的主要生物学功能和代谢途径,为进一步研究紫花苜蓿耐盐分子机制,培育耐盐新品种奠定基础。本研究结果如下:1、通过对南方型紫花苜蓿耐盐突变体幼苗进行250mmol/LNaCl处理0、24、48、72和96h时生理指标的测定,我们发现在盐胁迫72h内,叶片相对含水量持续降低,脯氨酸含量、相对电导率、MDA含量和SOD活性持续升高;在盐胁迫大于72h后,叶绿素含量和SOD活性明显下降,脯氨酸含量、相对电导率和MDA含量升高幅度增大。表明250mmol/LNa Cl处理72h是紫花苜蓿耐盐突变体幼苗盐胁迫响应的最佳时间,为接下来耐盐分子机制研究提供了依据。2、利用RNA-Seq技术,经过250mmol/LNa Cl胁迫72 h,南方型紫花苜蓿耐盐突变体幼苗叶片样品中共检测到30 900个基因表达量发生改变,其中7 694个基因被定义为差异基因(4 187个基因上调表达,3 507个基因下调表达),包括隶属于50个转录家族的422个转录因子(268个转录因子上调表达,154个转录因子下调表达)。3、实时荧光定量PCR验证表明,随机选择的8个差异表达基因在盐胁迫前后的表达特点与表达谱测序结果一致,表明了转录组测序数据的可靠性。4、GO功能显著性分析显示,差异表达基因的生物学功能主要表现在催化活性、结合、细胞组分、细胞、代谢过程和细胞过程。KEGG Pathway显著性富集分析显示,差异表达基因广泛涉及代谢途径和次生代谢产物生物合成。候选出Ms GST、Ms L-APX、Ms GPX、Ms RLK、Ms SIK、Ms Sn RK、Ms CMLs、Ms CMO、Ms MIP、Ms P5CS1、Ms P5CS3、Ms ABCB、Ms WBC、Ms LEA3和Ms PP2C等重要耐盐相关基因。转录因子分析发现,应答基因数量最多的是MYB基因家族,其次是WRKY、NAC、b HLH、AP2-EREBP和GRAS等基因家族。候选出Ms Alfin-like、Ms ERF110、Ms ANT、Msb HLH36、Ms NAI1、Msb ZIP、Ms C3H、Ms GRAS、Ms NAD、Ms MYB、Ms MYB85、Ms NAC、Ms RWP-RK、Ms Trihelix和Ms WRKY等耐盐相关转录因子。
[Abstract]:China has a vast sea area, and only a small part of the coastal saline land is used for cultivation, most of which are also wasteland and coastal land. The coastal zone has a large number of saline-alkali soil to be developed and used, which is an important reserve land resource. Coastal saline-alkali land improvement and ecological greening is an extremely difficult problem in the process of coastal urbanization in China. According to the practical experience at home and abroad in recent years, screening salt-tolerant plants is the most promising economic and effective method for biological improvement and ecological greening of coastal saline-alkali land. Alfalfa (Medicagosativa L.) It is a kind of leguminous plants with strong salt tolerance. It has a long cultivation history and is widely planted in the world. It has some ornamental value. It is an ideal plant for saline-alkali land improvement and garden greening. In this study, the salt-tolerant mutants of southern alfalfa 'Millennium' with high autumn dormancy level were used as materials. The transcriptional sequence analysis was carried out to screen the different genes of salt stress response, and the significance of GO function was analyzed. Significant enrichment analysis of KEGG Pathway and transcription factor molecules have been carried out to explore the main biological functions and metabolic pathways of salt-tolerance related genes in order to lay a foundation for further study on the molecular mechanism of salt tolerance in alfalfa and the cultivation of new salt-tolerant varieties. The results were as follows: 1. The relative water content of leaves and proline content in leaves of southern type alfalfa seedlings were continuously decreased within 72 h and 96 h after 250mmol/LNaCl treatment. The relative conductivity, MDA content and SOD activity continued to increase. When salt stress was more than 72 hours, chlorophyll content and SOD activity decreased obviously, proline content, relative conductivity and MDA content increased. The results showed that 250mmol/LNa Cl treatment for 72 h was the best time for salt stress response of alfalfa salt tolerant mutants, which provided a basis for the study of molecular mechanism of salt tolerance in alfalfa seedlings. 2. Using RNA-Seq technique, 72 h of 250mmol/LNa Cl stress was used. A total of 30 900 genes were detected in the leaves of southern alfalfa salt tolerant mutants, of which 7 694 genes were defined as differential genes (4 187 genes up-regulated and 3 507 genes down-regulated). Including 422 transcription factors belonging to 50 transcription families (268 transcription factors up-regulated and 154 transcription factors down-regulated). The expression characteristics of the eight differentially expressed genes selected at random before and after salt stress were consistent with the sequencing results of the expression profiles, which indicated the reliability of the transcriptional sequence data. The biological functions of differentially expressed genes were mainly manifested in catalytic activity, binding, cell composition, cell, metabolic process and. KEGG Pathway enrichment in cellular processes. Differentially expressed genes are widely involved in metabolic pathways and biosynthesis of secondary metabolites. Important salt-tolerant genes, such as Ms GST,Ms L-APXN, GPX,Ms RLK,Ms SIK,Ms Sn RK,Ms CMLs,Ms CMO,Ms MIP,Ms P5CS1, Ms P5CS3, ABCB,Ms WBC,Ms LEA3 and Ms PP2C, were selected as candidates. Transcription factor analysis showed that the largest number of responsive genes was MYB gene family, followed by WRKY,NAC,b HLH,AP2-EREBP and GRAS gene families. Candidate transcription factors such as Ms Alfin-like,Ms ERF110,Ms ANT,Msb HLH36,Ms NAI1,Msb ZIP,Ms C _ 3H _ 2 Ms GRAS,Ms NAD,Ms MYB,Ms MYB85,Ms NAC,Ms RWP-RK,Ms Trihelix and Ms WRKY were selected.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S541.9

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