【作者】 于晶；

【导师】 郝再彬；

【作者基本信息】 东北农业大学 ， 植物学， 2009， 博士

【摘要】 小麦营养价值丰富,经济价值较高,在中国是仅次于玉米和水稻的重要粮食作物。小麦有冬小麦和春小麦两种类型。冬小麦从分蘖期到拔节期跨越整个冬季,使其种植区域受到限制,在黑龙江省等北方寒地冬季温度极低,导致冬小麦不能安全越冬,成为冬小麦种植的禁区,常年以种植春小麦为主,而春小麦分蘖能力差,成穗率低,且在生产过程中经常受到春旱、春涝、收获期高温多雨等诸多不良因素的制约,结果导致年际间产量不稳,生产成本偏高。若能种植冬小麦就能很大程度的避开春旱、春涝及收获期高温多雨对其造成的不利影响,产量比春麦可提高约30%。“东农冬麦1号”是唯一可以在黑龙江省安全越冬的冬小麦品种,田间返青率在80%以上。以往对抗寒性的研究大部分都是在室内模拟低温条件下进行品种间的抗寒性比较,与其自然生长的环境温度及光照条件(光照条件与抗寒性直接相关)存在差异,特别是不能很好实现逐级降温适应锻炼。此外,对小麦抗寒性的研究多以苗期幼嫩叶片为取材部位,而北方高寒地区小麦实现越冬的主要部位是地下器官。因此,品种室内模拟低温环境的抗寒性研究结果难以代表其在大田的实际情况,对指导作物抗寒育种及生产有一定的局限性。本研究以东农冬麦1号为试验材料(济麦22为对照,该材料来自山东),在田间自然降温条件下,观察幼苗习性及返青率;测定不同器官与抗寒相关的生理生化变化、解剖结构变化,揭示冬小麦抗寒性的生理基础和结构基础;通过不同方式不同浓度外源施用植物生长调节剂,探索小麦抗寒的激素调控机制;通过双向电泳结合质谱技术挖掘抗寒相关蛋白,揭示冬小麦抗寒的分子基础。该研究对于揭示冬小麦的抗寒机制、挖掘抗寒相关新蛋白,具有重要的理论和实践意义,为今后我国北方寒地选育抗寒小麦种质资源及冬小麦新品种的选育提供理论依据和技术支持。研究结果如下:(1)越冬麦苗的生长习性及返青率东农冬麦1号越冬期幼苗匍匐于地面生长,返青率与土壤含水量及温度直接相关,含水量在25%左右最适于该品种生长及返青,随着低温时间的延长及温度的降低,返青率略有下降,幅度在60%~100%。在一定范围内,温度与返青率间呈极显著正相关,含水量与返青率呈负相关。(2)越冬期前麦苗的生理生化变化东农冬麦1号各器官(根、分蘖节和叶片)在抗寒性上的代谢适应和物质准备表现为如下规律:相对含水量高低依次为分蘖节>根>叶片;可溶性糖含量依次为分蘖节>根>叶片;蛋白质含量依次为叶片>分蘖节>根;脯氨酸含量表现为前期为分蘖节>根>叶片,中期为根>分蘖节>叶片,后期为分蘖节>叶片>根;过氧化物歧化酶(POD)活性大小依次为分蘖节>根>叶片;超氧化物酶(SOD)活性大小表现为前期分蘖节>叶片>根,后期为叶片>分蘖节>根;相对电导率大小依次为根>分蘖节>叶片;脱落酸(ABA)含量表现为前期叶>节>根,后期节>叶>根;赤霉素(GA)含量依次为叶>根>节;生长素(IAA)含量前期为叶>节>根,后期为叶>节>根;玉米素(ZR)含量依次为节>叶>根。总体表现为分蘖节的抗寒代谢适应和物质准备能力最强,是小麦安全越冬的重要器官,对其第二年的返青具有直接影响。东农冬麦1号分蘖节中的SOD活性、可溶性蛋白、可溶性糖、脯氨酸、ABA、ZR及IAA各指标之间呈现出不同程度的相关,表明各生理指标是协同作用,共同影响小麦的抗寒性,其中可溶性糖含量与其它6项指标之间均达到了显著相关,说明可溶性糖的含量对东农冬麦1号抗寒性的影响非常重要。(3)植物生长调节剂对麦苗抗寒性的调节外源ABA对东农冬麦1号的抗冷性有很大影响,低浓度的ABA可以提高低温下麦苗的生长,而高浓度则抑制生长,ABA提高抗冷性的最适浓度为10-7M;苗期低温下,ABA可以提高叶绿素含量、可溶性糖、可溶性蛋白含量,提高SOD活性、降低膜透性。根浸ABA对提高幼苗抗冷效果要强于叶喷ABA。大田分蘖前期根际浇灌不同浓度(10-7、10-6、10-5、10-4 M)ABA、GA、6-BA可以影响东农冬麦1号的返青率,其中ABA及6-BA均提高返青率,10-5 M返青率增加效果最明显,GA降低返青率,10-4 M时返青率降为0。(4)抗寒相关蛋白在pH 4~7范围内,东农冬麦1号分蘖节蛋白质双向电泳表达图谱中发现56个蛋白质点在-30℃低温的表达量与5℃低温有明显差异(±2倍以上),通过质谱分析及数据库检索均得到了完整的肽指纹图谱。在这些蛋白中逆境蛋白占14.3%(包括抗病蛋白、类抗病蛋白、热激蛋白、热激蛋白70前体、热激蛋白70、热激蛋白90、冷诱导蛋白、干旱敏感蛋白、过氧化物酶及过氧化物酶前体等,在低温后表达量上调)、代谢相关蛋白占23.2%(包括6-磷酸葡萄糖脱氢酶、3-磷酸甘油醛脱氢酶、苹果酸脱氢酶、腺苷酸α水解酶、磷酸烯醇丙酮酸羧化酶F0-F1 ATP羧化酶α亚基、脂氧化酶、四氢叶酸还原酶、淀粉合酶、核糖核酸酶等在低温胁迫条件下它们的表达量上调或下调)、信号分子蛋白占17.9%(包括酪氨酸激酶、丝裂原活化蛋白激酶、丝苏氨酸蛋白激酶、类CBF蛋白、钙调蛋白、类钙结合蛋白、转录因子等)、未知功能蛋白占17.9%,其他类蛋白占26.8%。这些蛋白的高量或低量表达可能对东农冬麦1号抗寒具有重要作用。对蛋白液进行冰棱晶体结构观察表明,随着温度的降低,冰棱晶体结构发生明显的变化,说明已经有抗冻蛋白在分蘖节中产生。(5)冬小麦抗寒的组织细胞解剖结构东农冬麦1号各器官(根、叶、分蘖节)在-15℃左右低温下结构仍很完整。叶片细胞间隙大,根系射线细胞小,分蘖节内部交织着大量纵横交错的疏导组织。低温后叶绿体依然呈椭圆形,类囊体基粒片层和基质片层跺叠整齐,沿叶绿体的长轴平行排列,均匀分布于叶绿体中。线粒体略微肿胀,但没有嵴消失现象。这些结构特点进一步证实了东农冬麦1号的强抗寒性。更多还原

【Abstract】 Wheat is the most important food crops in China only to rice and corn and it has rich nutritional value and the high economic value. There are two types of wheat: winter wheat and spring wheat. Throughout the whole winter, the growing period of winter wheat is from its tillering stage to jointing stage. Because that winter wheat planting is restricted in the cold area of northern China, such as Heilongjiang province, the temperature in winter is very low. These areas become restricted zones of winter wheat planting, but have to plant spring wheat. Moreover, spring wheat also has its deficiencies, for example, poor tillering, low spike rate, and restricted by bad environmental factors spring drought, spring floods, high temperature and wet in harvest time, which results in unstable annual yield and high cost. If winter wheat is planted in those cold areas, bad environmental factors could be avoided of spring wheat planting and the output could be increased in 30%.“Dongnongdongmai 1”is the only cultivar of winter wheat which could pass the winter securely in Heilongjiang province, and its greening rate is more than 85%.The previous researches on cold tolerance are mainly about comparing cold tolerance among species at the simulative low temperature indoor conditions, which is different from temperature and light (which related to the cold tolerance directly) of the natural growth conditions, especially can not realize the cooling exercise at the continually low temperature. In addition, the studies of cold tolerance of wheat seedling may use the young leaves as the materials, while in the northern cold area, underground organs are main part of winter wheat to pass the whole winter. Therefore, the studies simulated cold resistance under low-temperature indoor is difficult to represent their real findings at the actual situation and has limitations in guiding cold crop breeding and production.In this study, cold resistant variety Dongnongdongmai 1 (Jimai22, from Shan-dong province, as control) was used as experimental material. The seedling habit, greening rate, physiological and biochemical characteristics, and anatomical structure were investigated to reveal the physiological and structural mechanisms for cold resistance of winter wheat. In addition, the regulatory mechanism of hormone was also explored by using exogenous hormone at different ways and different concentrations. Moreover, cold proteins were obtained through the two-dimensional gel electrophoresis and mass spectrometry to reveal the molecular mechanism of cold resistance of winter wheat. This research provided the theoretical bases for revealing the mechanism of cold tolerance and finding the cold-related proteins of winter wheat, and had important theoretical and practical significance in selecting cold-resistant winter wheat germplasm resources and breeding new varieties in northern cold area of China.The main experimental results were as follows. (1)Seedling habits and greening rateThe seedlings of Donongdongmai 1 grew prostratly in the field overwintering , and theirs greening rate had significant correlation with soil water content and temperature. The soil water content of 25% was suitable environment for growing and greening of Donongdongmai 1. Under low temperature and long time of duration of low temperature, the greening rate decreased about 60%~100%. In certain extent, greening rate had a significantly positive correlation with temperature, and negative correlation with water content.(2)The characteristics of physiological and biochemical before overwinteringThe organs (root, tillering node and leaf) of Dongnongdongmai1 showed the following laws in metabolic adaptation and material preparations for resisting cold: Relative water content, tillering node> root> leaf; soluble sugar content , tillering node> root> leaf; protein content, leaf> tillering node > root; proline content, tillering node > root> leaf in early stage , root> tillering node > leaf in middle stage, and tillering node > leaf> root in late stage; POD activity, tillering node > root> leaf; SOD activity, tillering node > leaves> roots in early stage, leaf > tillering node > root in late stage; relative conductivity, root> tillering node > leaf; ABA content, leaf> tillering node > root in early stage, tillering node > leaf> root in late stage; GA content, leaf> root> tillering node; IAA content, leaf> tillering node > root in early stage, leaf > tillering node > root in late stage; ZR content, tillering node > leaf> root. The general results showed that tillering node was the most important organ of winter wheat for safely overwintering because of high cold resistant metabolic adaptation and strong ability for material preparations. And it directly affected greening rate of winter wheat in next year.The SOD activity, soluble sugar, soluble protein, proline, ABA, ZR and IAA content in the tillering node of Dongnongdongmai 1 showed the correlation at different levels, indicating that these physiological indices had synergistic effect to cold resistance of wheat. The soluble sugar had significant correlation with other six indices and it was the important index to show cold resistance of winter wheat dongnongdongmai 1.(3)Regulation of plant growth regulatorABA had significant effects on cold resistance of dongnongdongmai 1,the low concentration could promote the growth of wheat under low temperature,but the high concentrations inhibit that. For increasing the wheat cold resistance,the optimal concentration of ABA was 10-7M. Under low temperature at seeding stage, the ABA could increase chlorophyll content, soluble sugar content, and soluble protein content, improve the activity of SOD and decrease membrane permeability. The root absorption of ABA was better than leaf-spraying. In addition, the next year greening rate was increased by watering rhizosphere with different concentrations (0, 10-4, 10-5, 10-6, 10-7 M) of the ABA, GA and 6-BA in the field at early tillering seedlings. The general results showed that ABA and 6-BA could increase the greening rate at 10-5 M, but GA had the contrary effect, and decrease the greening rate to 0 at 10-4 M. (4)Cold resistance ccorelated proteinThe expression level of 55 protein spots were significantly different (±more than 2 folds) after low temperature stress -30℃in protein profiles of tillering node Dongnongdongmai 1 from pH4 to pH7. The high expressed protein spots were detected by matrix-assisted laser desorption ionization time-of -flight mass spectrometry (MALDI-TOF MS), and analyzed in retrieval database. According to the peptide mass fingerprints, 14.3% were stress protein(TIR-NBS-LRR-TIR type disease resistance protein, heat shock protein 70 precursor, non-cell-autonomous heat shock cognate protein 70, chilling-inducible protein,heat shock protein, 23.5 kDa heat-shock protein, heat shock protein 90, disease resistance protein-like, resposive to dessication 22, ascorbate peroxidase and class III peroxidase 59 precursor ) ; 23.2% were metabolism correlated protein(glucose-6-phosphate dehydrogenase, subfamily of Adenine nucleotide alpha hydrolases superfamily, phosphoenolpyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase subunit B, ribulose-1,5- bisphosphate carboxylase, F0-F1 ATPase alpha subunit, lypoxygenase, alpha-glucosidase, 5,10-methylene-tetrahydrofolate reductase, starch synthase II, cytosolic malate dehydrogenase, self-incompatibility ribonuclease); 17.9% were signaling molecule(incle Protein tyrosine kinase, CBF-like protein, calmodulin,calmodulin binding protein-like, mitigen activated protein kinase, serin/threonine protein kinase, transcription factor< PWWP domain protein> -like protein); 17.9% were un-known function protein, and other protein took up 26.8%. The results indicated that these high or low expressed proteins might play an important role in dongnongdongmai1 cold resistance. Moreover, the shape of ice crystals significant changed as the temperature decreased, which indicated that antifreeze proteins were produced in tillering node.(5)Anatomic structure of tissue and cell for cold resistance of winter wheatStructure of Dongnongdongmai 1 (leaf, root and tillering node) was integrity at about -15℃. The inter-space among the leaf cells was big, and the root ray cells were small, and there are many interlace bulks arranged in a crisscross pattern in the tillering node. After low temperature, the chloroplast was ellipse, and grana lamellae and stroma lamellae of thylakoid arranged regularly and parallel-aligned among long axis of chloroplast, distributed uniformly in chloroplast. The mitochondria were a little intumesces, but the cristae were not absent. These structural features furtherly confirmed the high cold resistance of Dongnongdongmai 1.更多还原