【作者】 于澄宇；

【导师】 何蓓如；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2009， 博士

【摘要】 用化学杂交剂(CHA)诱导植物雄性不育是杂种优势利用的重要方式之一,还可用于辅助育种等多种用途,但关键限制因素是新型高效CHA及其作用机理。本文以油菜为主要研究对象,验证乙酰乳酸合成酶(ALS,EC4.1.3.18)抑制型除草剂能筛选新型化学杂交剂的推断,并从生理机制上探讨化学杂交剂的作用特征及毒性机理,为开发新型CHA提供理论基础。得到的主要结果如下:1.CHA的筛选及效果评价1.1对于敏感性较高的油菜,本文所用26种除草剂中,22种可不同程度地诱导油菜雄性不育。从中筛选出2种效果最好的油菜化学杂交剂苯磺隆和酰嘧磺隆(发明专利ZL200610042886.1)。以0.2 mg/L-0.4 mg/L剂量处理甘蓝型油菜,产生95%-100%雄性不育率,两次喷药实现整个花期不育,药害程度较轻。苯磺隆或酰嘧磺隆处理能使3个环境敏感不育系达到完全不育,无明显药害。网室模拟化学杀雄制种试验所得杂交种的杂交率为98.2%。用CHA处理细胞质雄性不育系后制种种子纯度达到99.9%,表明CHA在化学杀雄制种和辅助三系制种方面具有很大应用潜力。苯磺隆也可以诱导芸薹属以及其他十字花科植物雄性不育,杀雄率能达到85%-100%。1.2对于单子叶植物小麦,本文所用多数麦田除草剂不能诱导雄性不育,只有咪草烟(专利申请号:200810231884.6)和草甘膦诱导雄性不育率均超过95%,但是草甘膦副作用较大。咪草烟对18个参试品种(系)的诱导雄性不育率都大于95%,剂量与品种之间有一定互作效应。咪草烟也能诱导禾本科的大麦、玉米达到100%不育。以上3种CHA的发现说明从磺酰脲类、咪唑啉酮类除草剂中筛选超高效CHA是可行的。2.化学杂交剂的靶标研究2.1酰嘧磺隆剂量与油菜下胚轴生长、ALS活性呈现显著负相关。在酰嘧磺隆处理油菜之后,ALS酶离体活性首先有小幅度升高,然后开始下降。表明ALS基因表达水平应激性提高,但随后蛋白合成整体水平下降导致ALS酶蛋白减少。不敏感品种82089酶活性稍高于敏感品种Qinyou3,且酶活力恢复快于Qinyou3。花蕾和雄蕊的ALS活力高于成熟叶片,但受酰嘧磺隆处理后下降幅度更大,这可能是CHA具有部位选择性的原因。采用不同ALS抑制剂处理油菜、小麦,杀雄活性高的除草剂对ALS活性抑制持续性大于杀雄效果差的除草剂,而高剂量的抑制效应又大于低剂量。表明这些CHA的作用靶标就是ALS。由于ALS是植物三种支链氨基酸合成途径的关键酶,通过ALS抑制剂中筛选、合成新型超高效CHA是可行的。2.2根据油菜乙酰乳酸合成酶基因设计16条引物,对16个品种的DNA模板进行扩增,发现ALS基因有多态性,但聚类结果不能清楚反映品种之间的遗传距离,也没有找到决定酰嘧磺隆敏感性的ALS基因位点。2.3酰嘧磺隆、酰嘧磺隆+支链氨基酸、环丙二羧酸(酮醇酸还原异构酶的特异抑制剂)三种处理都使得油菜花蕾游离氨基酸总量比对照提高1倍以上。说明CHA处理导致生长抑制,蛋白质的转换引起游离氨基酸总量变大,氨基酸比例失衡。对经过CHA处理的油菜植株,从叶面、花蕾等不同部位持续补给支链氨基酸,不能使育性恢复。3.酰嘧磺隆诱导油菜雄性不育的细胞学特征3.1用整体透明技术观察,酰嘧磺隆处理油菜具有接近正常的胚珠数量和发达的乳突结构,败育花药内小孢子数目减少,而且大都破裂变形。3.2诱导不育油菜的显微结构特征为:败育花药在小孢子发育早期不同阶段都有异常;花粉母细胞变形,细胞质收缩,出现空腔;四分体残缺不全;单核期小孢子畸形,细胞内容物基本消失。也有绒毡层异常膨大、绒毡层液泡化、提前解体或异常增生等现象。3.3超微结构显示:诱导不育油菜的部分花粉母细胞核膜破裂,核仁散碎,细胞质分解,体积收缩。部分花粉母细胞可以通过减数分裂,但产生的小孢子内有明显的线粒体、质体降解、吞噬等现象。绒毡层结构混乱,内容物残缺不全,没有绒毡层小体,造油体中颗粒呈破裂或者空瘪状。而且叶片、花药表皮、内壁细胞的叶绿体和质体空瘪,基粒片层破坏。4酰嘧磺隆诱导雄性不育的生理生化特征4.1酰嘧磺隆对油菜保护酶POD、CAT活性、以及体内毒素过氧化氢、丙二醛含量产生影响,敏感品种秦油3号和非敏感品种82089的反应不同。除草剂解毒相关的谷胱甘肽转移酶在酰嘧磺隆处理之后活性下降,然后有所回升并超过初始水平。非敏感品种82089不论叶片还是花蕾,活性降低水平低于敏感性品种Qinyou3,后来提高幅度又大于Qinyou3。这些区别有可能是形成品种间敏感度差异的原因之一。4.2酰嘧磺隆处理花蕾的丙酮酸、可溶性糖、叶绿素含量、类胡萝卜素含量、可溶性蛋白、DNA含量下降,花药DNA呈现弥散状凝胶电泳图谱。处理过的油菜光合速率有下降趋势,但是非敏感品种82089和D89比敏感品种Qinyou3和656下降缓慢,基本保持在原水平,表明光合作用下降也是雄性不育发生的特征之一。4.3在酰嘧磺隆处理油菜后喷施抗坏血酸、亚硫酸氢钠、硫代硫酸钠、过氧化氢、水杨酸、硝酸银、尿囊素、赤霉素等,均没有改变育性。补加乙烯利有助于缩短花期。利用酰嘧磺隆+PV+CMCNa+助剂配制的缓释剂处理油菜,提高药物使用剂量,延长了杀雄效果。综合以上结果可以得出本文化学杂交剂的基本作用模式为:酰嘧磺隆、苯磺隆、咪草烟等化学杂交剂的作用靶标是乙酰乳酸合成酶,毒性作用体现在多个层面,包括破坏氨基酸平衡、抑制细胞分裂,干扰DNA和蛋白质代谢、破坏叶绿体和质体结构、抑制光合作用、产生自由基等,最终对小孢子发育过程产生抑制作用。更多还原

【Abstract】 Although male sterility induced by chemical hybridizing agents provides a practicable approach for hybrid production, there are few good chemical hybridizing agents（CHAs）available for commercial utility. Attempt was made in the paper to select and evaluate chemical hybridizing agents from sulfonylurea and similar herbicides, which can inhibit acetolactate synthase （ALS）, the key enzyme catalyzes the common reaction to synthesize leucine, valine, and isoleucine in plants. The characterization and mechanism of action of novel and effective CHAs founded in the paper were studied. The main results obtained are as following:1. Selection and evaluation of CHAs1.1 Among twenty-eight chemicals tested, twenty-two herbicides elicited male sterility in Brassica napus. The best CHAs for Brassica napus were Tribenuron-methyl and Amidosulfuron （China invention patent ZL200610042886.1）. Double foliar application of them at the concentration of 0.2-0.4 mg/L and at 12days interval resulted in nearly 100% male sterility. They could also make three environment-sensitive male sterile lines Zhong 9A （Shaan 2A CMS）, Y88A （Polima CMS）, and Hy50S （recessive genic male sterility） to be completely sterile. The hybridity of seeds from two mimic hybrid production trails via CHAs and CHA-aided CMS were 98.4% and 99.9% respectively. Tribenuron-methyl and Amidosulfuron could also be applied on other Brassica and cruciferous species.1.2 Most of herbicides used in this experiment had not or very weak gametocidal effect on wheat （Triticum aestivum）. Only Imazethapyr and Glyphosate could induce high male sterility but phytotocxicity of the later was very serious. Imazethapyr resulted in over 95% male sterility in 18 different cultivars at 7g/ha and 10 g/ha rate. The effect of CHA×cultivar interaction was found in some cultivars. Application of Imazethapyr also resulted in 100% male sterility in Barley （Hordeum vulgare） and Zea mays. Glyphosate had good gametocidal effect on cotton （Gossypium hirsutum）and pepper （Capsicum annuum）.2. Studies of the target of CHAs2.1 The dose of Amidosulfuron was negatively correlated with hypocotyls growth and ALS activity of B.napus. After Amidosulfuron treatment, the in vitro activity of ALS was raised little and then went down, this suggested the expression of ALS gene was promoted firstly by feedback regulation but then the protein synthesis was inhibited. The in vivo activity of ALS of non-sensitive cultivar 82089 was somewhat higher and recovered fast than sensitive cultivar Qinyou3. The in vivo activity of ALS of flora buds was higher than mature leaf but decrease more quickly. This was the first evidence to interpreter the selection of CHAs among different cultivars and different tissues. Brassica napus and Triticum aestivum were treated with different ALS-inhibitors. The one with good gametocidal efficiency had stronger inhibiting ability than those with lower efficiency. These indicated that ALS was the target of these novel CHAs.2.3 ALS genes fragments was amplified against 16 different cultivars with 16 primers designed based on ALS genes of B.napus. Some polymorphic bands were found but the cluster results based them could not reflex the relationships among these cultivars. The sites in ALS genes determining the sensitivity to amidosulfuron was not found.2.4 The contents of total and individual free amino acid in the buds of B.napus from three treatments, i.e. amidosulfuron, amidosulfuron+ branched-chain amino acids, and 1,1-cyclopropanedicarboxylate（specific inhibitors of ketol-acid reductoisomerase）, increased by one fold. This was the results of the turn over of protein. Branched-chain amino acids were supplemented to the treated B.napus from leaf and bud, but this treatment could not restore the fertility.3. Cytological characters of male sterility in Brassica napus induced by Amidosulfuron3.1 The number and structure of ovum and the papilla cells in the stigma of Amidosulfuron treatment were similar to the contrast. However, the number of pollen grains in the CHA-treatment decreased and most pollen grains had abnormal shape observed via a whole flower clearing technique.3.2 Light microscopy studies revealed that pollen abortion in CHA treated plants was associated with abnormal behavior of tapetum, endothecium and microspore. CHA treatment made the cytoplasm of pollen mother cell shrunk. The tetrads were malformed and thus most microspores derived from them were destructured. Some tapetal cells elongated radially, however, sometimes they failed to degenerate.3.3 Ultrastructural studies revealed the number of vacuole and organelle in the tapetal cells in Amidosulfuron-treated rapeseed decreased. The pollen mother cell are destroyed with the karyotheca and nucleolus damaged, and the cytoplasm degenerated. It is observed that the presence of plastid and mitochondria degenerated and phagocytosis in the microspore and tapetal cell in the anthers of amidosulfuron treated plants. The tapetal cells were damaged with tapetosomes absent and elaioplasts malformed. One significant feature was the plastids and chloroplasts in leaf, epidermis and endothecium were destroyed.4 Physiological and biochemical characters of male sterility induced by amidosulfuron4.1 Application of amidosulfuron influenced the activity of peroxidase and catalase, and the content of endotoxin hydrogen peroxide and malondialdehyde. The trend of these influences was different between amidosulfuron-sensitive cultivar Qinyou3 and non-sensitive cultivar 82089. The activity of glutathione S-transferase, which is an important detoxifying enzyme, decreased abruptly but then rose to surpass the normal level. Its activity in cultivar 82089 was higher than in Qinyou3. These differences in defense systems might be the reason for manipulating of amidosulfuron-sensitivity.4.2 Application of amidosulfuron resulted in decrease of content of pyruvate acid, soluble sugar, chlorophyll carotene soluble protein and DNA. The genomic DNA from aborted anther showed smear band by agarose gel electrophoresis. The decrease of photosynthesis of rapeseed was induced, but cultivar 82089 can maintain higher photosynthesis than sensitive cultivar qingyou3 and 656. The decrease of photosynthesis was also an important reason for male sterility.4.3 Supplementary of ascorbic acid, NaHSO₃, Na₂S₂O₃, hydrogen peroxide, salicylic acid、AgNO₃, Allantoin, and GA3, could not aggravate or relieve the effect of amidosulfuron. However, addition of ethephon shorten the flowering period, this was useful to keep complete male sterility during the whole flowering period. A slow-release formulation （Amidosulfuron+PV+CMCNa+adjuvants） could enhance the endurance to doubled dose and prolonged the gametocidal effect of Amidosulfuron on B.napus.In conclusion, the primary model of CHAs used in this paper could be established. CHAs including Amidosulfuron, Tribenuron-methyl, and Imazethapyr, inhibited the activity of their target—acetolactate synthase. The toxicity embodied from various aspects: imbalance of amino acid, abnormal cell split, disturbance of DNA and protein biosynthesis, destruction of chloroplasts and plastid, restriction of the photosynthesis, production of free radical etc. The development of microspore was suppressed because it was most sensitive to these changes.更多还原