拟南芥干旱和过氧化氢突变体远红外成像技术的筛选及其初步定性
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摘要
随着人类的经济发展和人口膨胀，水资源短缺现象日趋严重，这直接导致了干旱地区的扩大与干旱化程度的加重，在许多地区已成为限制农业发展的瓶颈。开展作物抗旱机制的研究，提高作物抗旱性是解决水资源短缺、实现农业节水、保障农业可持续发展的重要选择。通过对作物抗旱分子机制的阐明可进一步揭示植物适应环境的生命本质，是最终实现人为操纵植物生命活动的基础，因此也是生命科学领域中研究的重大的科学问题。
在生长发育过程中植物往往会遭遇到各种各样的胁迫。环境胁迫会导致活性氧(ROS)的产生，主要包括H_2O_2、O_2~(·-)、·OH、~1O_2等，研究表明过氧化氢(H_2O_2)可能作为第二信使参与植物细胞的许多生理代谢过程，因而有关H_2O_2信号转H导途径的研究就愈显重要。到目前为止，对有关植物细胞中H_2O_2信号转导的细节及其与胞内其它信号分子的时空关系、作物抗旱的分子生物学和遗传学知识及其所涉及到的分子机制，特别是对作物抗旱主效基因的的表达调控及上游的信号传递知之甚少。而突变体作为剖析复杂生物学过程强有力的工具，已被广泛用于植物发育、代谢途径及细胞信号转导的研究。
远红外成像技术是利用物体自身各部分对红外热辐射的差异把红外辐射图像转换为可视图像的技术。随着红外探测器在技术上的不断突破，应用的领域也不断拓宽，如在生命科学研究中的应用就是其中的典型范例。
本实验首先是对利用远红外成像技术筛选拟南芥突变体方法的可行性进行多方面的试验，优化筛选条件、确定筛选所需幼苗的生长时间、种植密度，界定用于筛选所需的胁迫(过氧化氢和干旱)选择压力。然后用0.4％的乙酰甲基磺酸(EMS)诱变拟南芥(Arabidopsis thaliana)野生型。以8-10天的干旱和5×10~(-3)mol／LH_2O_2进行胁迫处理，用远红外成像技术检测突变体和野生型叶面温度的微小差异，对M_2代幼苗进行了大规模的筛选。当植物叶片温度与野生型即正常植株的叶片温度有明显差异，大约温差可达到0.6-1.2℃，就被确定为对过氧化氢和干旱敏感和不敏感拟
_..一.…__.__._二乡电爷贡早介呼些型丝鱼些竺困些述塑鲤竺塑竺型
南芥侯选突变体。我们对这些拟南芥侯选突变体进行进一步的鉴定和遗传学分析，
发现ro口口、doil、doi口Jlll3I等潜在突变株对ABA、过氧化氢及早胁迫有明显表型，
同时对潜在突变体的生长发育进行了详细的观察，发现多数潜在突变株与同条件下
野生型比出现了许多明显的形态改变，如:莲座基叶增多、分层、肥大，花期提前
或延迟，主茎生轮座，株型矮化，产籽量少，不育，败育等，这些生理和形态上的
差异很可能反映了它们内部某些基因的表达受到了影响、代谢调控发生了紊乱，具
体和详细的作用机制还需要进一步的研究。
With the development of economy and increase of the population, the shortage of water is becoming serious in the worldwide . These directly result in enlarging of arid regions, which limit the agriculture production. Studying the mechanism of drought-resistant and enchancing capability of drought-resistant of crops is an important measure to solve the shortage of water, realize agriculture of saving water, guarantee the sustaining development of agriculture.
Plants often encount many kinds of environmental stresses, The reactive oxygen species (ROS), including H2O2 primarily, O2- OH, 1O2 ,could be induced in plant cells in responsive to the stresses. Many phyisilogical and cell biological studies have indicates that hydrogen peroxide as second messager involved in ABA signaling in guard cells. Therefore, research on H2O2 signaling will be more important.
So far, little is known the details of H2O2 signal transduction and its complicated relation with other signal molecular, knowledge about molecular mechanism of drought-resistant and its genetics, especially to expressions of main gene of drought-resistant and controls of upstream signal transduction. Mutant, a powerful tool of analyzing genetic process , has already been extensively used for research in plant growth, metabolism and signal transduction
Infrared imaging techniques utilize every part of difference of infrared radiation of objects to obtain the details of the thermal images. With the breakthroughs of infrared thermography, upgrade of infrared imaging aparatus will be quicker; scope of application more wider continuously; precision and accuracy of measurement more accurate. Particularly application in the life science also continuous changes and improves.
Firstly we maked sure method of isolating Arabidopsisis mutant by thermal imagings in many ways on trial, optimize conditions for isolation, and confirm the good
period of seedlings and fine density for isolation, select correct isolating pressure(drought and H2O2) that is fit for isolate mutant.Secondly we obtain an Ml population derived from 0.4%ethyl methanesulfonate (EMS)-mutagenized wild type Arabidopsis thaliana. With the tiny difference of temperature leaf between mutant and wild type for index sign,We isolate Ml seedlings by 8-10 days drought-stress and 5X 10-3mol/L H2O2-stress treatment by infrared thermography in a large scale.A great deal of roi ros doi and dos Arabidopsis mutant were isolated (the difference of leaf temperature between candidate mutant and wild type is very obvious,about 0.6-1.210). The future characterization and genetic analysis for candidate mutant were carried out and find that some candidate mutant (such as roi30 doil-1 doi0311131)have good phenotype by drought H2O2 ABA -stressed treatment.At the same time we also observe the development of candidate mutant at different growth stages carefully. Many modal difference between mutant an
d wild type at the same period were found, such as more rosette layering fatty and big in leaves, advancment or delay for the flower period, rosettes living in the main stem, shorten in figure, the amount of seed little,sterilization etc. These physiological and modal changes may reflect with maladjustment in expressions of some gene and confusion on their inner control,. We will futher study concrete and detailed function mechanism.
在生长发育过程中植物往往会遭遇到各种各样的胁迫。环境胁迫会导致活性氧(ROS)的产生，主要包括H_2O_2、O_2~(·-)、·OH、~1O_2等，研究表明过氧化氢(H_2O_2)可能作为第二信使参与植物细胞的许多生理代谢过程，因而有关H_2O_2信号转H导途径的研究就愈显重要。到目前为止，对有关植物细胞中H_2O_2信号转导的细节及其与胞内其它信号分子的时空关系、作物抗旱的分子生物学和遗传学知识及其所涉及到的分子机制，特别是对作物抗旱主效基因的的表达调控及上游的信号传递知之甚少。而突变体作为剖析复杂生物学过程强有力的工具，已被广泛用于植物发育、代谢途径及细胞信号转导的研究。
远红外成像技术是利用物体自身各部分对红外热辐射的差异把红外辐射图像转换为可视图像的技术。随着红外探测器在技术上的不断突破，应用的领域也不断拓宽，如在生命科学研究中的应用就是其中的典型范例。
本实验首先是对利用远红外成像技术筛选拟南芥突变体方法的可行性进行多方面的试验，优化筛选条件、确定筛选所需幼苗的生长时间、种植密度，界定用于筛选所需的胁迫(过氧化氢和干旱)选择压力。然后用0.4％的乙酰甲基磺酸(EMS)诱变拟南芥(Arabidopsis thaliana)野生型。以8-10天的干旱和5×10~(-3)mol／LH_2O_2进行胁迫处理，用远红外成像技术检测突变体和野生型叶面温度的微小差异，对M_2代幼苗进行了大规模的筛选。当植物叶片温度与野生型即正常植株的叶片温度有明显差异，大约温差可达到0.6-1.2℃，就被确定为对过氧化氢和干旱敏感和不敏感拟
_..一.…__.__._二乡电爷贡早介呼些型丝鱼些竺困些述塑鲤竺塑竺型
南芥侯选突变体。我们对这些拟南芥侯选突变体进行进一步的鉴定和遗传学分析，
发现ro口口、doil、doi口Jlll3I等潜在突变株对ABA、过氧化氢及早胁迫有明显表型，
同时对潜在突变体的生长发育进行了详细的观察，发现多数潜在突变株与同条件下
野生型比出现了许多明显的形态改变，如:莲座基叶增多、分层、肥大，花期提前
或延迟，主茎生轮座，株型矮化，产籽量少，不育，败育等，这些生理和形态上的
差异很可能反映了它们内部某些基因的表达受到了影响、代谢调控发生了紊乱，具
体和详细的作用机制还需要进一步的研究。
With the development of economy and increase of the population, the shortage of water is becoming serious in the worldwide . These directly result in enlarging of arid regions, which limit the agriculture production. Studying the mechanism of drought-resistant and enchancing capability of drought-resistant of crops is an important measure to solve the shortage of water, realize agriculture of saving water, guarantee the sustaining development of agriculture.
Plants often encount many kinds of environmental stresses, The reactive oxygen species (ROS), including H2O2 primarily, O2- OH, 1O2 ,could be induced in plant cells in responsive to the stresses. Many phyisilogical and cell biological studies have indicates that hydrogen peroxide as second messager involved in ABA signaling in guard cells. Therefore, research on H2O2 signaling will be more important.
So far, little is known the details of H2O2 signal transduction and its complicated relation with other signal molecular, knowledge about molecular mechanism of drought-resistant and its genetics, especially to expressions of main gene of drought-resistant and controls of upstream signal transduction. Mutant, a powerful tool of analyzing genetic process , has already been extensively used for research in plant growth, metabolism and signal transduction
Infrared imaging techniques utilize every part of difference of infrared radiation of objects to obtain the details of the thermal images. With the breakthroughs of infrared thermography, upgrade of infrared imaging aparatus will be quicker; scope of application more wider continuously; precision and accuracy of measurement more accurate. Particularly application in the life science also continuous changes and improves.
Firstly we maked sure method of isolating Arabidopsisis mutant by thermal imagings in many ways on trial, optimize conditions for isolation, and confirm the good
period of seedlings and fine density for isolation, select correct isolating pressure(drought and H2O2) that is fit for isolate mutant.Secondly we obtain an Ml population derived from 0.4%ethyl methanesulfonate (EMS)-mutagenized wild type Arabidopsis thaliana. With the tiny difference of temperature leaf between mutant and wild type for index sign,We isolate Ml seedlings by 8-10 days drought-stress and 5X 10-3mol/L H2O2-stress treatment by infrared thermography in a large scale.A great deal of roi ros doi and dos Arabidopsis mutant were isolated (the difference of leaf temperature between candidate mutant and wild type is very obvious,about 0.6-1.210). The future characterization and genetic analysis for candidate mutant were carried out and find that some candidate mutant (such as roi30 doil-1 doi0311131)have good phenotype by drought H2O2 ABA -stressed treatment.At the same time we also observe the development of candidate mutant at different growth stages carefully. Many modal difference between mutant an
d wild type at the same period were found, such as more rosette layering fatty and big in leaves, advancment or delay for the flower period, rosettes living in the main stem, shorten in figure, the amount of seed little,sterilization etc. These physiological and modal changes may reflect with maladjustment in expressions of some gene and confusion on their inner control,. We will futher study concrete and detailed function mechanism.
引文
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