【作者】 王俊生；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 博士

【摘要】 化学杂交剂诱导的小麦生理型雄性不育及其杂种利用体系是目前小麦杂种优势利用最成功的典范之一。采用化杀途径利用小麦杂种优势具有诸多优点,尤其可以把育种起点建立在小麦常规育种的最新成果之上。SQ-1是一种新型小麦杀雄剂,具有杀雄彻底、喷药窗口宽、对农艺性状无明显副效应等优点,是目前国内外最优良小麦化杀剂之一。然而,其诱导小麦雄性不育的机理尚不清楚,尤其在基因表达水平和蛋白质表达水平上是否存在差异,差异如何均需深入探索。因此,本文在研究小麦不同组织部位对化杀剂SQ-1吸收转运效果基础上,观察了败育花粉粒细胞形态变化,采用动态取样测定了花药发育过程中的活性氧和抗氧化酶变化;其次利用cDNA-AFLP技术分析了败育关键时期的特异基因的表达差异,筛选出一些可能的不育相关基因,并对其进行电子延伸,设计PCR引物克隆其cDNA序列;最后利用RT-PCR技术验证和分析了育性相关基因的表达模式,探讨了育性相关基因的表达与雄性不育的关系,获得的主要结果如下:1在小麦发育到Feeke’s8.5～Feeke’s9.0时期,对旗叶,倒二叶和倒三叶进行单叶片或多叶片涂抹一定面积(剂量)的化学杂交剂,均能引起主茎穗高度雄性不育和分蘖穗部分雄性不育,其中发育完全的旗叶吸收运输能力最强,倒三叶最弱,同一叶片正面比背面吸收转运效果好;用化学杂交剂直接涂抹单核后期的小穗,也能诱导较高程度的雄性不育,但涂抹二核期的穗子仅诱导一定程度的不育率,并且发现化学杂交剂可横向影响对侧小穗花粉粒的育性,而不能从穗顶部运输到基部小穗影响其花粉粒育性。对主茎叶片涂抹足量化杀剂不但可以诱导主茎穗高度雄性不育,而且可以诱导分蘖穗产生较高程度的雄性不育;常规喷施条件下,花粉粒败育的主要时期为单核期到二核初期,以单核后期败育花粉粒比例最高,败育花粉粒呈畸形,不积累或积累极少量淀粉粒。2活性氧代谢研究表明,在幼穗期,o ?2.生成速率、H2O2和MDA含量、超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性均高于相应对照,而过氧化物酶(POD)和过氧化氢酶(CAT)活性则低于或显著低于对照;在单核早期到二核初期,o ?2.生成速率、H2O2和MDA含量极显著高于对照,而SOD、POD、CAT和APX酶活性却极显著低于对照;在败育后的花药中,o ?2.生成速率和H2O2含量与对照之间差异幅度缩小,但MDA含量依然加大,同期的几种抗氧化酶活性依然极显著低于对照。相同剂量喷施处理下,不同品种花药内活性氧和抗氧化酶变化存在差异。杀雄剂SQ-1能诱导小麦花药中o ?2.和H2O2大量积累以及SOD、POD、CAT和APX活性的极显著降低,引起花粉关键败育期花药活性氧代谢严重失衡和严重膜脂过氧化,是导致大量花粉细胞败育的主要生理原因。3对西农1376生理型不育和可育花药关键败育期基因表达分析,共获得了144个非冗余的差异基因,这些基因编码的蛋白主要涉及氧化胁迫或自身防御反应(5.6%),信号转导和转录调节(15.3%),核酸代谢(2.8%),细胞内物质运输(5.6%),能量代谢(12.5%),蛋白质代谢(13.2%),细胞组成和发育(2.8%),27.1%的差异基因与已知基因或蛋白具有较弱的同源性;11.1%的差异基因没有同源基因或蛋白,仅存在同源EST序列,最后有6个TDFs没有任何同源序列,可能与目前数据库数据不足有关,或者该基因是尚未报道新的基因。化学杂交剂SQ-1诱导小麦雄性不育败育过程中,涉及了多个生理生化代谢途径,其中转录表达调节、蛋白质代谢和能量代谢途径中差异表达基因最多。不同转录因子调节的功能蛋白、参与的生化途径和不同途径中基因或蛋白之间的关系有待深入研究。4对小麦Urm1,Ubiquitin-S27a,U-box域蛋白,丙酮酸脱氢酶E1 alpha亚基,Arf GTPase激活蛋白家族蛋白,细胞色素P450家族蛋白,顺乌头酸脱氢酶进行了电子克隆,获得了这些基因的cDNA序列,并对其序列特性进行了分析。利用RT-PCR技术成功克隆了小麦Urm1和Ubiquitin-S27a的cDNA基因,验证了电子克隆的正确性。5对抗氧化胁迫和防御相关的细胞质型的APX和嘧啶核酸二硫化物氧化还原酶基因、与泛素/26S蛋白酶体降解途径有关的Urm1、Ubiquitin- S27a、F-box域蛋白以及与能量代谢相关的GAPDH、顺乌头酸酶、NFU域蛋白4共9个基因进行了半定量表达分析,结果表明,花药败育过程中,嘧啶核酸二硫化物氧化还原酶以及抗坏血酸过氧化物酶基因下调表达,导致细胞内处于较高的氧化态,细胞内发生严重膜脂过氧化作用。泛素蛋白酶体途径相关的Urm1、Ubiquitin-S27a、F-box域蛋白(与水稻UFO基因高度同源)在两个品种不育花药中不同时期均上调表达,且两个F-box域蛋白均表现出瞬时表达特点;TDF362代表的F-box域蛋白在不育花药单核后期和二核初期表达受到抑制,推测该F-box蛋白的靶标蛋白是正常花药发育的负调控因子。对F-box域蛋白调控的靶标蛋白有待深入研究。以上分析认为,控制正常花粉发育过程中的某些或某类关键蛋白的降解是由泛素蛋白酶体途径精确控制的,化学杂交剂SQ-1通过促进或抑制这些功能蛋白的调节蛋白基因表达来影响花药发育的正常行为。6 GAPDH和提供铁硫族蛋白分子骨架的NFU域蛋白4,与同期对照相比,在不育花药单核期到三核期均下调表达,其中在大量花粉粒败育的单核期,GAPDH下调表达尤为显著,使糖酵解过程受到抑制,降低了能量的供应;另外,顺乌头酸酶作为三羧酸循环的关键酶之一,也表现为下调表达,从而使三羧酸循环受阻或能量代谢途径改变,导致能量供应不足引起雄性不育。更多还原

【Abstract】 Physiological male sterility (PMS) induced by chemical hybrid agents (CHA) and its utilization are more successful system in wheat heterosis today. Utilization of CHA hybrids has many merits, the latest released varieties can be directly used for producing hybrid specially; SQ-1 is a new type of wheat CHAs, also the best wheat CHAs at home and abroad. However, understanding of its sterile mechanism is still poor, specially on the levels of gene and protein expression. In the paper, the sterile effects and absorbency of different leaves and spikes to CHA SQ-1 were studied, the cytological observation of pollen and ROS (reactive oxygen species) metabolism of anthers at different developmental stages were performed, then the differential gene expression was also compared using cDNA-AFLP technique, the genes related to male sterility induced by SQ-1 were identified and cloned, the relationship between genes expression and sterility was studied, the main results were as follows:1 At Feeke’s 8.5～9.0 stage, daubing sufficient CHA SQ-1 on flag leaf, penultimate leaf, the third reciprocal leaf separately and two or three leaves simultaneous (application concentration: 1.5kg/300kg water ) can cause complete male sterility of wheat spikes; for the ability of absorbency and transfer, flag leaf is the best, the third reciprocal leaf is bad, the adaxial surface is better than that of abaxial surface of same leaf; daubing directly the spikelet with 5.01ug or 8.3ug SQ-1 at late mononuclear pollen stage will cause higher male sterility of daubed spikelet, but at late binuclear pollen stage only caused partial male sterility of daubed spikelet. Further, SQ-1 might be transported at regular direction in wheat spikes from male sterility of treatments D7～D10 and CK3. Daubing sufficient SQ-1 on the caulis leaf caused complete male sterility of caulis spike and partial male sterility of tillering spikes. The pollens abortion can be happen at any developmental stages from mononuclear to trinucleated cell, but the majority of pollens aborted stayed at late mononuclear to initial stage of binuclear cell, and the ration of aborted pollen at late mononuclear pollens was the most. Aborted pollen showed abnormal shape, without starch grains. Abortion-occurring stage was associated with spraying or daubing dose and developmental stage of pollen.2 The study on ROS metabolism of PMS showed:①During the young spike stage, o ?2. production rate and contents of H2O2 and malondialdehyde (MDA) were higher than those of corresponding controls, the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were also higher than those of corresponding controls, but the activities of peroxidase (POD)and catalase (CAT) were lower or significantly lower than those of their corresponding controls, so the susceptibility of different antioxidase to CHA stress before pollen abortion was different②From early mononuclear pollen stage to initial stage of binuclear pollen, on the one hand, o ?2. production rate and contents of H2O2 and MDA were significantly higher than those of their corresponding controls, but then the activities of SOD, POD, CAT and APX (except for early mononuclear pollen) were significantly lower than those of the corresponding controls, hence the metabolism balance of ROS in treated anthers was seriously destroyed, violent membrane lipid peroxidation was caused, subsequently the microspore developing was inhibited. At trinucleated pollen stage, the gaps of the o ?2. production rate and content of H2O2 were reduced, but that of MDA was still increased, at the same time the activities of four antioxidases between treatments and controls remained significant different. Therefore, ROS of aborted anthers still caused damage to themselves. During the key abortion stage, the increment of ROS in 1376-CHA anther were higher, but the decrement of activities of antioxidase was lower than those of 2611-CHA, which may be the reasons why the male sterility rate of xinong 1376-CHA was higher than that of xinong 2611-CHA. Excessive accumulation of o ?2.,H2O2 and MDA and significant reduction of SOD, POD and CAT antioxidase activities happened ahead of pollen abortion and went on during pollen abortion stage in the anthers of wheat PMS, the imbalance of ROS metabolism was the main physiological reason of pollen abortion in PMS.3 cDNA-AFLP analysis was conducted in PMS and normal anthers at key abortion stage. Total 144 non-redundant transcript-derived fragments (TDFs) were cloned and identified, the majority of these cloned genes were complete presence or absence (qualitative variants), and the others were differentially expressed (quantitative variants). The biological role of these TDFs mainly involved in defense system against oxidation stress(5.6%), signal transduction and transcriptional regulation(15.3%), nucleic acid metabolism(2.8%), energy and protein metabolism(12.5% and 13.2%), intracellular transporters (5.6%), cell wall and cytoskeleton components(2.8%) , whereas 35.2% of them either had lower similarity with known function gene (or protein) or their function were unknown.The results showed that CHA SQ-1 triggered dynamic changes at transcriptional regulation levels when uninucleate pollen developed to bi-nuclear pollen. The course of pollen abortion involved in intricate physiological and biochemical metabolism. The majority of genes were involved transcriptional regulation, energy and protein metabolism. But the relationship between transcriptional factors and other genes remained to be studied. 4 Seven interested TDFs were extended by electronic cloning, six of them were obtained cDNA sequences with entire ORF, their encoded amino acid had typical conserved domains, they were Urm1(ubiquitin related modifier 1), ubiquitin-S27a, U-box domain protein, pyruvate dehydrogenase E1 alpha subunit, Arf GTPase activating protein, Cytochrome P450 family protein and aconitase, respectively. The cDNA and amino acid sequences of these genes were compared with some homology genes. The partial cDNA sequences of Urm1 and ubiqutin-S27a were amplified and cloned.5 Two genes related to oxidation stress, four key genes related to ubiquitin-26S proteasome pathway (UPP) and three genes related to energy metabolism were selected to conduct RT-PCR analysis for revealing the relationship between them and male sterility induced by CHA SQ-1, the results indicated that cytoplasmic APX and Pyridine nucleotide-disulphide oxidoreductase were down-regulated in PMS anthers at key abortion period, and caused the imbalance of ROS production and scavenging, some key enzymes were oxidized and resulted in pollen abortion. The result was accordant with ROS determination. Urm1, ubiquitin-S27a and F-box domain protein (TDF360) showed up-regulated expression at key abortion stage, but the other F-box domain protein (TDF362) showed significantly down-regulated in PMS anther, the two F-box domain proteins were obviously expressed during instantaneous period. So the degradation of some key enzyme or protein related to anther development was accurately controlled by ubiquitin-26S proteasome pathway, CHA SQ-1 leaded to PMS in wheat through affecting expression of regulating proteins of the key enzymes.6 GAPDH, a key enzyme of glycolysis pathway, aconitase, a key enzyme of tricarboxylic acid cycle (TCA cycle), they all showed down-regulated expression in PMS anther at different developmental stage, especially at key stage of pollen abortion, so ATP production was impeded; NFU domain protein4 mainly provides molecule scaffolds of Fe-S cluster biogenesis located in mitochondria such as aconitase and succinate dehydrogenase, and involved in electron transfer, its down-regulated expression in sterile anther maybe caused male sterility by repressing the efficiency of electron transfer and affecting energy supply. Further study about the gene is underway.更多还原