【作者】 周芳；

【导师】 彭明；

【作者基本信息】 海南大学 ， 植物分子遗传学， 2013， 博士

【摘要】 木薯(Manihot esculenta Crantz),大戟科木薯属,是典型的热带、亚热带地区经济作物,是人类和动物的重要食物来源之一,也是工业上生产淀粉和燃料乙醇的重要原材料。在我国,木薯的主要种植区分布在热带北缘及亚热带地区,干旱和低温成为影响木薯生长发育和地理分布的主要环境胁迫因子,因此研究木薯抗旱耐寒机制具有重要的生物学及经济意义。植物的抗旱、耐寒能力不是因为单个基因或少数基因的作用,而是由复杂的基因网络协调作用的结果。本研究在转录组水平上,通过高通量数字基因表达谱测序技术分别获得木薯SC124在干旱和低温胁迫下的转录本数据,并通过系统生物学方法、利用生理生化及分子生物学分析手段,深入地探究了木薯抗旱耐寒相关基因复杂的协调作用机制。本文的主要研究结果如下：(1)分别对木薯SC124驯化后旱害处理样品及对照和驯化后寒害处理样品及对照进行数字基因表达谱测序,所得每个样品文库共产生约3.7×106个Tag数,其中丰度大于100的Tag分别约占Tag总数的1.25%和2%,丰度51-100的Tag约占Tag总数的1.5%和2%,丰度21-50的Tag约占Tag总数的3%和3.5%,丰度11-20的Tag约占Tag总数的3.25%和4%,丰度6-10的Tag约占Tag总数的5.25%和6%,丰度2-5的Tag约占Tag总数的24.25%和24.25%,拷贝数小于2的Tag约占Tag总数的60.5%和54.2%。(2)本研究最终得到木薯SC124成熟功能叶在驯化后旱害第4天、第6天和第10天共2235个差异表达基因,其中上调表达的差异基因占差异表达基因总数的59.8%,在第4、第6和第10天特异上调表达的基因分别为518、221和322个,下调表达的差异基因占差异表达基因总数的40.2%,在第4、第6和第10天特异下调表达的基因分别为262、113和183个；得到木薯SC124心叶在驯化后寒害第6小时、第24小时和第48小时共3266个差异表达基因,其中上调表达的差异基因占差异表达基因总数的54.5%,在第6、第24和第48小时特异上调表达的基因分别为521、128和473个,下调表达的差异基因占差异表达基因总数的45.5%,在第6、第24和第48小时特异下调表达的基因分别为305、88和382个。(3)差异表达基因GO功能分析得到,"response to stimulus"、"metabolic process"、"transcription regulator"、"antioxidant"、和"organelle"等与干旱、低温胁迫紧密相关的生物学途径、分子功能、细胞组成等都有明显的响应变化。其中在于旱胁迫下具有"cell killing"、"metallochaperone"、"protein tag"功能的差异基因完全上调表达；在低温胁迫下具有"virion"功能的差异基因完全上调表达,与干旱胁迫后差异基因的表达结果有很大差异的是低温胁迫下具"cell killing"、"protein tag"功能的差异基因完全下调表达。(4)差异表达基因代谢路径分析得到,干旱、低温胁迫显著影响的代谢通路包括光合作用、呼吸作用、二级代谢、三羧酸循环、细胞壁代谢、氨基酸代谢、脂类代谢和淀粉代谢等；此外旱、寒胁迫还显著影响植物的调控通路如植物激素代谢、氧化还原相关代谢、转录因子、蛋白代谢、钙调节及激酶代谢等。(5)植物抗旱耐寒过程中的各种重要生理生化活动都与气孔的运动密切相关。本研究通过扫描电镜分别观察了非驯化和驯化后干旱胁迫下抗旱品种SC124与不抗旱品种C4成熟功能叶下表面以及非驯化和驯化后低温胁迫下耐寒品种SC124与不耐寒品种KU50心叶下表面气孔的开闭状态。结果表明,旱、寒胁迫显著影响了木薯的气孔运动；旱、寒胁迫时抗旱耐寒品种SC124迅速关闭气孔,有效地减少了水分的损失,待植株逐渐适应外界胁迫环境时,气孔又逐渐打开,从而保证了植株正常基础代谢活动的进行；并且驯化后的抗性植株当再次受到逆境胁迫时能更加快速地响应且能更快地适应外界环境。(6)基于对旱、寒胁迫下调控通路中激素代谢的分析以及植物激素对气孔运动的重要调节作用,本研究利用酶联免疫吸附法测定了非驯化和驯化后干旱胁迫下抗旱品种SC124与不抗旱品种C4成熟功能叶以及非驯化和驯化后低温胁迫下耐寒品种SC124与不耐寒品种KU50心叶内四种常见激素ABA、IAA、GA和ZR的含量变化。结果表明,旱、寒害胁迫下抗性品种SC124叶内的激素总体趋势为ABA含量上升,而IAA、GA和ZR含量下降；经旱、寒驯化后的SC124植株抗性增强,激素调节幅度普遍提高。(7)渗透调节是植物抗旱、耐寒的重要手段之一,细胞内可溶性糖(主要包括葡萄糖、果糖和蔗糖)含量的变化是反应植株抗旱、耐寒性的有效指标之一。本研究通过高效液相色谱法测定了非驯化和驯化后干旱胁迫下抗旱品种SC124与不抗旱品种C4成熟功能叶以及非驯化和驯化后低温胁迫下耐寒品种SC124与不耐寒品种KU50心叶内葡萄糖、果糖和蔗糖的含量变化。结果表明,干旱胁迫可提高抗旱品种SC124成熟功能叶内葡萄糖、果糖及早期蔗糖含量,且前期干旱驯化可增加可溶性糖的积累,不抗旱品种C4中可溶性糖积累不明显；低温胁迫也能有效提高木薯心叶内葡萄糖、果糖、蔗糖含量,且前期低温驯化增加了可溶性糖的积累,提高了植株的耐寒性。(8)基于对旱、寒胁迫下木薯重要代谢通路和调控通路的分析,本研究利用实时荧光定量PCR方法,分别鉴定了多个与木薯细胞壁代谢、蛋白代谢、二级代谢、胁迫响应及转录调节相关的基因表达模式,从而进一步解释了木薯抗旱、耐寒的分子机理。(9)分析驯化后干旱和驯化后低温胁迫下差异表达基因的启动子区上游2000bp序列,寻找差异表达基因共同的顺式作用元件,最终得到与木薯抗旱、耐寒相关的反式作用因子,如ACIPVPAL2、EMBP1TAEM、SGBFGMGMAUX28、MYB1AT、 AGCBOXNPGLB、CMSRE1IBSPOA、RBENTGA3等。其中反式作用因子如ACIIPVPAL2、AGMOTIFNTMYB2、MYCATERD1等与旱、寒胁迫响应均相关。由此可以看出,当受到干旱、低温胁迫时,植物体内不同反式作用因子可通过与胁迫诱导启动子间的相互作用,使不同胁迫诱导的抗逆基因呈网络状交互作用。更多还原

【Abstract】 Cassava (ManihotesculentaCrantz), a typical cash crop in the tropics and subtropics, is not only one of the most important sources of energy for humans and animals, but also an important raw material for industrial production of starch and fuel ethanol. The major growing areas of cassava are located in the subtropical regions and northern margin of tropics in China, drought and low-temperature become the major abiotic stress to its growth and development as well as geographic distribution, so the research on drought and cold tolerance of cassava has an important biological and economic significance. The ability of plant drought and cold resistance is not because the role of a single gene or a few ones, but by the role of complex gene network. In this study, a digital gene expression profiling of cassava SC124subjected to drought and cold stress was conducted using high-throughput sequencing technology on the level of transcriptome. At the same time, the transcripts data was analyzed by system biology methods and the means of physiological, biochemical and molecular analysis. As a result, the complex cassava drought and cold resistance coordination mechanism was explored in depth. The main results are as follows:(1) The gene expression profiling of cassava SC124under the treatment of drought acclimation hurt and control, cold acclimation injury and control, was conducted in the study. In total, around3.7×106tags were obtained from each sample library. Among the gene expression profiling data, the copy number which is above100accounts for about1.25%and2%of the total tags, respectively; the copy number which is51to100accounts for about1.5%and2%of the total tags, respectively; the copy number which is21to50accounts for about3%and3.5%of the total tags, respectively; the copy number which is11to20accounts for about3.25%and4%of the total tags, respectively; the copy number which is6to10accounts for about5.25%and6%of the total tags, respectively; the copy number which is2to5accounts for about24.25%and24.25%of the total tags, respectively; the copy number which is below2accounts for about60.5%and54.2%of the total tags, respectively.(2) In this study,2235differentially expressed genes were identified from the gene expression profiling of SC124unfolded leaves subjected to drought acclimation hurt at the4th,6th and10th day;the up-regulated differentially expressed genes accounts for59.8%of the total differentially expressed genes, and the number of specific expressed gene is518,221,322corresponds to the4th,6th and10th day; the down-regulated differentially expressed genes accounts for40.2%of the total differentially expressed genes, and the number of specific expressed gene is262,113,183corresponds to the4th,6th and10th day.3266differentially expressed genes were identified from the gene expression profiling of SC124folded leaves subjected to cold acclimation injury at the6th,24th and48th hour; the up-regulated differentially expressed genes accounts for54.5%of the total differentially expressed genes, and the number of specific expressed gene is521,128,473corresponds to the6th,24th and48th hour; the down-regulated differentially expressed genes accounts for45.5%of the total differentially expressed genes, and the number of specific expressed gene is305,88,382corresponds to the6th,24th and48th hour.(3)GO functional analysis indicated that drought-stress and cold-stress related biological pathways, molecular function, cell composition, such as "response to stimulus","metabolic process","transcription regulator","antioxidant" and "organelle", have obvious response to the stress. The drought-stress related differentially expressed genes with the "cell killing","metallochaperone","protein tag" function are up-regulated completely."Virion" responses only in the up-regulated differentially expressed genes to chilling injury stress. The cold-stress related differentially expressed genes with the "cell killing","protein tag"function are down-regulated completely, unlike the result of drought stress.(4) Metabolic pathway analysis showed that drought stress and cold stress can significantly influence metabolic pathways including photosynthesis, respiration, secondary metabolites, citric acid cycle, cell wall metabolism, amino acid metabolism, lipid metabolism, starch metabolism and so on. In addition, drought stress and cold stress significantly affected regulatory pathways such as phytohormone metabolism, redox metabolism, transcription factors, protein metabolism, calcium regulation and kinase metabolism.(5) The plant important physiological and biochemical activities during drought and cold resistance process are closely related to stomatal movement. Stomatal opening and closing on the lower unfolded leaf surface of SC124(drought-resistant) and C4(not drought-resistant), also on the lower folded leaf surface of SC124(cold-resistant) and KU50(not cold-resistant), were observed by scanning electron microscopy. The results showed cassava stomatal movement was significantly affected by drought and cold stress. The drought-resistant and cold-resistant SC124closed the stomata quickly when plant felt the stress to reduce the loss of water effectively, gradually opened when the plant adapt to the environment in order that the plant can ensure the normal basal metabolic activities. The drought and cold acclimation plant can respond to stress and adapt to external environment more quickly.(6) The content of endogenous phytohormone (ABA, IAA, GA, ZR) in the unfolded leaves of SC124(drought-resistant) and C4(not drought-resistant), also in the folded leaves of SC124(cold-resistant) and KU50(not cold-resistant), both under acclimation and non-acclimation treatment, was determined by enzyme-linked immunosorbent assay (ELISA) based on the analysis of phytohormone metabolic pathway and the importance of hormone to regulate stomatal movement. The result showed the content of ABA in drought and cold resistant SC124unfolded leaves increased, while the content of IAA, GA, ZR decreased under drought and cold stress. Drought and cold acclimation enhanced SC124tolerance to stress, and increased the amplitude of accommodation.(7) Osmotic adjustment plays an important role in plant drought and cold resistance, the content of soluble sugar including glucose, fructose, sucrose and so on is an effective indicator of drought and cold resistance. The content of glucose, fructose and sucrose in the unfolded leaves of SC124(drought-resistant) and C4(not drought-resistant), also in the folded leaves of SC124(cold-resistant) and KU50(not cold-resistant), both under acclimation and non-acclimation treatment, was determined by HPLC-ELSD. The result showed that the content of glucose, fructose and sucrose (in the early stage) increased in drought and cold resistant SC124unfolded leaves under drought and cold stress, drought and cold acclimation can enhance the accumulation of glucose and fructose, the accumulation of soluble sugar in C4(not drought-resistant) was not obvious. Cold stress can also effectively improve the content of glucose, fructose and sucrose in cassava folded leaves, and cold acclimation can enhance the accumulation of soluble and cold tolerance.(8) Multiple of gene expression patterns related to cassava cell wall metabolism, protein metabolism, secondary metabolism, stress-responsive and important transcriptional factors were identified by real-time quantitative PCR based on the analysis of metabolic pathways and regulatory pathways under drought and cold stress, to further explain the molecular mechanism of cassava drought and cold resistance.(9) The2,000bp upstream sequences relative to the transcription starting site of all of the differentially expressed genes from each co-expression module under drought acclimation hurt and cold acclimation injury was analyzed to find out the common cis-acting elements. Finally, we obtained drought and cold resistance related trans-acting factors like ACIPVPAL2, EMBP1TAEM, SGBFGMGMAUX28, MYB1AT, AGCBOXNPGLB,CMSRE1IBSPOA, RBENTGA3, etc.Some transcription factors like ACIIPVPAL2. AGMOTIFNTMYB2、 MYCATERDlwere related to drought and cold resistance at the same time, which proved the cross talk between the drought-resistance and cold-resistance mechanism.更多还原