【作者】 王云生；

【导师】 杨剑波；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 苯达松为苯并噻二唑类除草剂,可抑制杂草的光合作用,用于农田除草。野生型水稻（Oryza sativa L.）品种具有苯达松的抗性,可将苯达松快速代谢为羟基化的失活产物。水稻品种（‘N8’和‘W6154s’）经辐射诱变获得了对苯达松极其敏感的突变体,研究表明其敏感致死表型由单个bsl基因控制。bsl基因作为一个化学致死标记,在水稻杂交制种中有广阔的应用前景,主要体现在以下两方面:一是将bsl基因转育到三系恢复系,可实现杂交水稻混播制种;二是将bsl基因转育到不育系,可确保水稻杂种纯度。为了验证bsl基因的功能,为该基因在农业生产上应用提供理论依据,本文开展了水稻苯达松抗性差异的生理基础和水稻微粒体蛋白质组学等方面的研究。开展了施药对恢复系花粉活力的影响、苯达松致死剂量与环境因子的关系以及苯达松农田残留等方面的研究。并利用研究成果进一步完善杂交水稻混播制种技术,加速其应用的步伐。首先,我们以野生型水稻品种‘农林8号’（‘N8’）和‘W6154s’及相应的bsl突变体（‘农林8号m’（‘N8m’）和‘8077s’）为材料,分析了苯达松处理前后叶绿素（Chl）含量、丙二醛（MDA）含量、净光合速率（A_n）、叶绿素荧光参数及体内苯达松含量等生理生化指标的变化及水稻苯达松抗性差异的生理机制。结果表明:两个bsl水稻品种在苯达松处理后由于光合能力的丧失,进而引起氧自由基伤害的积累,最终导致Chl降解、质膜氧化加剧,植株死亡;Chl荧光参数的分析表明苯达松处理引起还原性Q_A组分积累,导致电子传递受阻是光合能力丧失的根源。在苯达松处理初期,野生型水稻品种及bsl突变体同样表现为光合能力丧失。但处理后2 h,野生型材料光合能力开始不断恢复,而bsl突变体无法恢复。体内苯达松含量分析表明,较强的苯达松代谢能力是抗性材料免受苯达松伤害的主要原因。为了寻找苯达松解毒相关蛋白,我们以‘N8’和‘N8m’为材料,通过优化条件,建立了适合于水稻微粒体蛋白的双向电泳技术。并通过双向电泳图谱的比对,共找到15个差异蛋白点,分为供试材料间差异、苯达松处理前后差异和‘N8’在苯达松处理后特异表达等3种类型。结合苯达松抗性比较及苯达松残留分析,推测材料间差异是因γ射线辐照引起基因结构变异,从而影响了基因的表达;苯达松处理前后差异与氧自由基清除酶系或系统获得抗性相关酶系有关;‘N8’在苯达松处理后特异表达与苯达松代谢有关。这为进一步开展bsl基因蛋白质组学研究奠定了基础。另外,我们通过对液体培养基中蔗糖、硼酸及钙离子浓度的优化选择,建立了适合水稻花粉离体萌发的液体培养基配方。并利用花粉萌发和碘.碘化钾（I₂-KI）染色测定法,研究了苯达松对含敏感致死基因的恢复系‘Mc526’的花粉生活力的影响。结果显示,在苯达松处理前期（施药后1～4天）,Mc526花粉活力与对照接近,处理后期（施药后5～8天）略低于对照,但差异不显著。这为苯达松敏感致死基因应用于杂交水稻混播制种的可行性提供了生理依据。再者,我们以‘混制1号’恢复系‘Mc526’为材料,通过相关生理生化指标测定,比较了在不同光照、温度条件下苯达松伤害的差异,旨在为实际制种过程中合理调整施药浓度提供理论依据。温度对苯达松药效影响的研究表明:在21℃～36℃范围内,温度与苯达松药效呈显著正相关,即温度每增加1℃,苯达松致死浓度约下降74.09mg·L^-1,药效约提高0.7%。生理生化研究表明,体内水分平衡失调引起水分胁迫和代谢旺盛引发氧自由基积累是高温下苯达松药效提高的主要原因。田间试验验证了温度对苯达松药效影响的室内研究结果。光强对苯达松药效影响的研究表明:强光(100μmol·m^-2·s^-1)和弱光(25μmol·m^-2·s^-1)条件下,苯达松药效较中光(50μmol·m^-2·s^-1)均有显著提高。相关生理生化研究证实,强光下氧自由基胁迫增加是苯达松药效加强的主要原因。最后,我们建立了苯达松、6-OH-苯达松和8-OH-苯达松高效液相色谱同柱分析方法。同时利用该方法,对杂交水稻‘混制1号’制种田苯达松最终残留和消解动态进行研究,对杂交水稻混播制种技术应用引起环境威胁的可能性进行科学评估。结果表明:不育系稻谷和稻秆、制种田土样中苯达松残留量均低于最低检测浓度,而恢复系稻谷和稻秆中苯达松残留量较大。土壤中苯达松消解动态分析表明,苯达松在土壤中的消解速度较快。更多还原

【Abstract】 Bentazon is used as a benzothiadiazole herbicide with high selectivity by an inhibition of photophythesis for killing sensitive plant.In general,common rice varieties（wild types） are all tolerant to bentazon,which can metabolize bentazon into 6-OH or 8-OH non-toxic molecules.Bentazon sensitive lethal（bsl） mutants were obtained byγ-ray radiation from wild rice varieties（’N8’ and ’W6154s’）.Previous genetic studies have confirmed that the bsl phenotype of is controlled by a single recessive gene.The bsl gene,as selective chemical marker,has a critical usage in seed production of hybrid rice（Oryza sativa.L）.In hybrid breeding system,tagging the bsl gene to the male sterile line can ensure the hybrid seeds free of false hybrids（self-pollination of sterile lines） by spraying bentazon at the seedling stage.On the other hand,if the bsl gene is transferred into a restorer line,the restorer line is easily killed by spraying bentazon immediately after pollination,while the sterile line maintains normal growth and development.In order to clarify the function of bsl gene,and provide sufficient evidance for its application in rice breeding,we researched the physiological basis on the difference of bentazon tolerance and rice microsomal proteomics in wild type rice and their bsl mutants.In addition,the bentazon influence on the pollen viability of sensitive restorer line,the relation between bentazon lethal dosage and environmental factor,and the bentazon residues in mixed-planting seed production were studied to further perfect the technology of hybrid rice mixed-planting seed production and accelerate its application.Firstly,this study was to elucidate the physiological mechanism of bentazon tolerance in rice.Tolerant rice varieties（W6154S and Norin 8） as well as their corresponding mutants,sensitive to bentazon,were employed in this study.Plant net photosynthetic rate （P_n）,chlorophyll fluorescence,and the level of superoxide radical（O₂^·） as well as the contents of chlorophyll（Chl） and malondialdehyde（MDA）,were analyzed for both tolerant and sensitive rice plants treated with bentazon.After treatment,the two sensitive mutants showed a significant reduction in P_n at 0.5 h.A continuous decrease of Chl contents was found over the first 3 d whereas a significant increase of MDA contents was noticed on the 3rd day and thereafter.Analysis of chlorophyll fluorescence revealed a bentazon-induced increase in the proportion of the reduced state of Q_A.In the early stage after bentazon treatment,wild types and their mutants showed no significant difference in the alteration of P_n and chlorophyll fluorescence.While these two parameters then increased progressively in both wild types and remained low in the mutants.A significant generation of O₂^·was found over the 5 d period in the mutants.Both wild types and mutants contained the same level of bentazon after 2 h of treatment.Bentazon content dropped to barely detectable amount in the wild type varieties at 1 d.However,the mutants retained a substantial amount of the herbicide after 5 d.It is proposed that the herbicide might inhibit rice photophythesis and accumulation of oxidative stress with the treatment of bentazon in both lethal mutants.The damaging effect on PSⅡsystem can be significantly alleviated in the wild type varieties due to a higher rate of catabolism of the herbicide.Secondly,we established a modified two-dimensional gel electrophoresis（2-DE） protocol suitable for rice microsomal proteins by using differential centrifugation in sample preparation,optimizing length and pH range of immobilized pH gradient（IPG） strips,and modifying the electrophoresis program.In order to search bentazon tolerance-related proteins,we used the 2-DE technique to detect the change of rice microsomal proteins expression induced by bentazon in N8 as well as its bentazon sensitive lethal mutant N8m. Results showed that 15 differentially expressed protein spots were found after bentazon treatment between or within the two lines.The proteins in these spots were classified into three types.TypeⅠproteins showed differential expression between the two lines while typeⅡproteins differed between the treated and untreated samples,and typeⅢproteins were only induced in N8 after the treatment.TypeⅠproteins arise from rice DNA damage byγ-ray radiation,based on the fact that N8m is a new strain from N8 mutated byγ-ray. Our previous study indicated that N8 and N8m showed no significant difference in the alteration of photosynthesis in the early stage after bentazon treatment,which induced accumulation of oxidative stress.It can be confirmed that typeⅡproteins are related to the reactive oxygen scavenging system or systemic acquired resistance.N8 could metabolize the absorbed bentazon thoroughly while residual bentazon was present in the treated leaves of N8m.Therefore,we predict the typeⅢproteins to be related to bentazon metabolism.Thirdly,A culture solution for rice’s pollen germination in vitro was established through Studying on the concentration of sucrose,boric acid and calcium chloride.The influence of bentazon treatment on the pollen viability of Mc526,which included bsl gene, was researched by using pollen bourgeoned and I₂-KI pigmentation’s measurement.The results shown that in early period（1～4 days） after bentazon treatment Mc526’s pollen viability was close to comparison and in later period（5～8 days） Mc526’s pollen viability was a bit lower than comparison,but the difference was not salience.The setting rate results showed that bentazon treatment had no salient influence on the setting rate of sterile line.This indicates that bentazon does not effect on bsl rice’s pollen viability immediately, which provides the physiological basis for the feasibility of applying the method of mixed-planting seeds production in hybridize rice.In addition,this study investigated the differences of bentazon injure at different light intensities and temperatures in order to provide theoretical basis for properly regulating bentazon dosage in hybridize rice seeds production.’Mc526’ was employed in this study. The research of the influence of temperature on bentazon injure indicated that there was significant positive correlation between temperature and bentazon injure degree in the range of 21℃～36℃.As for 1℃increase of temperature,the lethal concentration of bentazon declined 74.09 mg·L^-1,namely bentazon injure degree increased 0.7%.Related physiological research showed that the increases of water stress and Oxygen free radical stress were the main cause for the increase of bentazon injures in high temperature.The same results were obtained from the field experiments.The research of the influence of light intensity on bentazon injure indicated that bentazon injures in strong light(100μmol·m^-2·s^-1) or weak light(25μmol·m^-2·s^-1) were significantly increased compared with bentazon injure in medium light(50μmol·m^-2·s^-1).Related physiological research showed that the increases of Oxygen free radical stress was the main cause for the increasing bentazon injure degree in strong light.Finally,a reversed phase HPLC method was determined for analyzing bentazon and its metabolites—6-hydroxy-bentazon and 8-hydroxy-bentazon in this study.We used the HPLC method to detect the bentazon residue and degradeation behaveior in the field of hybrid rice maxed-planting seed production,in order to scientifically evaluate the possibility of environmental threat induced by the application of hybrid rice maxed-planting seed production.The results showed that the herbicide residues in the grain and plant of sterile line were lower than the minimum detectable concentrateion. However,there were more residues in the grain and plant of restorer line.The analysis of bentazon degradeation behaveior indicated that bentazon in soil could be rapidly degraded.更多还原