【作者】 乌凤章；

【导师】 邹学忠； 杨传平； 王柏臣；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2008， 博士

【摘要】 低温是植物最主要的非生物胁迫因子之一,影响很多重要经济植物的生长和地理分布。白桦是世界重要的经济植物,而且具有较强的抗寒性。利用蛋白质组学方法研究白桦对低温胁迫的应答机制,发掘耐寒基因和低温应答相关蛋白,对于全面揭示抗寒植物低温适应的遗传基础,揭示其抗寒机理具有重要的意义。将白桦幼苗分别置于4℃和对照(23℃)的人工气候室中,在处理当天、4d、7d、10d、14d时测定其高生长量、叶片质膜透性、叶绿素相对含量、叶绿素荧光参数PSⅡ有效光化学量子产量(Fv′/Fm′)、光化学淬灭效率(q_p)、非光化学猝灭系数(NPQ)和可溶性糖含量的变化情况。结果表明:低温胁迫导致高生长基本停止,叶绿素含量逐渐降低,14d时叶片变黄;相对电导率先升高后降低,最后恢复到对照水平;Fv′/Fm′、q_p和NPQ与对照相比均显著降低,Fv′/Fm′和NPQ则表现为先降低后有升高,最后降低的波动形式,可溶性糖含量明显提高。这些现象表明,4℃低温已造成了质膜可逆性的伤害和PSⅡ原初光能转化受到抑制,但随着胁迫时间延长,植物产生了适应性,抗寒能力得到了显著提高。用2 DE-GEL方法分析了白桦幼苗在4℃处理当天、4d和14d时的蛋白质图谱,在凝胶图上检测到37个丰度发生2倍以上变化的差异蛋白点,其中15个高丰度的差异蛋白点用ESI-MS/MS成功地得到了鉴定。鉴定的蛋白质通过Swiss Prot/rEMBL数据库检索和分析以及相关的文献报道对其功能进行了分类,大致分成4类,即参与信号转导、光合作用,碳、氮及能量代谢和应激相关蛋白。分析鉴定的蛋白质有50%以上定位于叶绿体上,表明叶绿体可能在植物应答低温胁迫机制中具有重要的作用。叶绿体RNA结合蛋白是调控叶绿体基因表达的重要因素。通过层析柱分离了白桦叶绿体蛋白中与多聚腺苷酸和ssDNA紧密结合的RNA结合蛋白。然后使用双向电泳技术分离这些蛋白,通过MALDI-TOF-MS和ESI-MS-MS分析图谱上的主要蛋白质点,成功地鉴定了13个蛋白质。鉴定的蛋白多数在Swiss-prot/TrEMBL数据库中定义了功能,其中4个已报道具有RNA结合蛋白的性质;3个具有ATP结合特性;1个具有NAD结合特性;1个具有DNA结合特性;4个蛋白没有外观的结合性质;用这种方法浓缩和鉴定了4个丰度蛋白,包括1个转录因子。此研究加深了对树木中叶绿体RNA结合蛋白功能的全面理解,也表明可以在其他植物叶绿体中开展RNA和蛋白质相互作用的研究。更多还原

【Abstract】 Low temperature stress is one of the major abiotic factors affecting plant growth and the geographical distribution of many economically important plants.birch is not only the most important plants worldwide,but also cold-resistant plant.Proteomic techniques are effective approaches that have been adopted to discover new genes related to cold resistance and cold responsive protein.Therefore,it will be very significant for to study the mechanism of birch respond to cold stress.birch seedlings were treated in the 4℃and 23℃phytotron on the same day,the fourth days,the seventh day,the tenth days,and the fourth daycrespectively.The changes of growth status,leaf plasma-membrane permeability,chlorophyll relative content,and photochemical efficiency of PSⅡin the light(Fv’/Fm’),photochemical quenching coefficient(q_p),non-photochemical quenching coefficient(NPQ) and total soluble carbohydrate Conten were measured.The results showed that the height growth was primarily suspended by low temperature stress,the Chlorophyll Content was gradually decreased and the leaves got yellow on the fourteenth day.The relative conductivity was increased at first,then it was decreased,and returned to the control level in the end.Compared with the control level,the Fv’/Fm’,q_p and NPQ were evidently decreased.The Fv’/Fm’ and NPQ were decreased at first and then increased,q_p was decreased at first and then increased,and decreased in the end..total soluble carbohydrate Conten was increased.It indicated,the low temperature of 4℃led the membrane in the reversible damage,and the conversion of primary light energy of PSⅡto be restrained. The birch have relatively high adaptability to cold stress with the prolonging of cold stress time,and increased their freezing tolerance.By using 2 DE-GEL method we analyzed their protein profiles.37 differentially expressed protein spots with higher than 2-fold spotdensity were detected in gel image.Among them,15 high abundance protein spots were successfully identified with ESI-MS/MS.According to Swiss Prot database and related references,these proteins were classified into 4 functional groups,which mainly involved in signal transduction,photosynthesis,and metabolisms of carbon,nitrogen,energy,and stress.This analysis also elucidated that 50%of proteins detected under cold stresss are located in chloroplast,suggesting that the chloroplast is likely play important role in thecold-response in plants.The RNA binding proteins in chloroplast are important factors in regulating gene expression in the organelle.We separated chloroplast proteins with affinity to polyadenylate or single stranded DNA through affinity chromatography in the birch tree.2-D Electrophoresis and MALDI-TOF-MS as well as ESI-MS/MS have been used in the present work to identify a total of 13 proteins from intact chloroplasts.Most of the identified proteins were defined as to their function inSwiss-prot/TrEMBL database.including 4 RNA-binding proteins,3 ATP-binding proteins,an NAD- and a DNA-binding protein as well as 4 proteins with no apparent binding function reported previously.With these methods four abundance proteins including a transcriptional factor were also enriched and identified.These results should advance the understanding of the function of general chloroplast RNA-binding proteins in a tree species that suggest they may have some application to the study of native RNA-protein interactions in and chloroplasts of other plants.更多还原