【作者】 邓敏娟；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2009， 博士

【摘要】 逆境胁迫是影响植物生长发育的主要因素,严重影响着农作物的产量和品质。利用基因工程手段提高农作物的抗逆性已成为作物遗传改良的重要内容之一。本研究以水稻为材料,初步分析基于细胞程序性死亡（Programmed Cell Death,PCD）和根边缘细胞（RootBorder Cell,RBC）发育的抗逆机制。1.Bcl-2基因在水稻抗H₂O₂胁迫中的作用及其分子机制植物细胞程序性死亡是指在细胞生长发育或对外界刺激的反应过程中受自身基因编码、主动、有序的细胞死亡过程。研究显示,在植物中异源表达动物的抗凋亡基因能抑制PCD,从而提高植物对生物和非生物胁迫的抗性。但相关的分子调控机制还知之甚少。本研究通过农杆菌介导的转基因方法,将人类的抗凋亡基因Bcl-2导入水稻中花11（Oryza sativa L.subsp.Japonica）,获得5个水稻35S::Bcl-2纯合转基因株系,Northern blot检测表明各株系中Bcl-2均有不同程度的表达。在H₂O₂胁迫下,转基因水稻的种子萌发率（Seeds germination）、叶片叶绿素保有率（Chlorophyll retention）、根伸长量（Rootelongation）和根尖活性（Root tip viability）比野生型显著提高,表明抗凋亡基因Bcl-2的异源表达能提高水稻抗氧化胁迫的能力。DNA laddering和TUNEL的检测结果显示,在20 mM H₂O₂胁迫下,野生型水稻出现明显的细胞程序性死亡,具有明显的DNA laddering和TUNEL荧光信号,相对于野生型,转Bcl-2基因水稻中的DNA laddering和TUNEL信号明显减弱。表明Bcl-2的异源表达能抑制H₂O₂诱导的PCD发生,从而提高水稻的抗氧化胁迫能力。为了进一步探索其分子机理是否与液泡途径有关,首先通过序列比对和功能结构域分析,预测了水稻中存在4个液泡加工酶（Vacuolar Processing Enzyme,VPE）同源基因,分别为Os01g37910（OsVPE-1）、Os02g43010（OsVPE-2）、Os04g45470（OsVPE-3）、Os05g51570（OsVPE-4）;7个metacaspases同源基因,分别为Os01g58580（OsMC-1）、Os03g27170（OsMC-2）、Os03g27190（OsMC-3）、Os03g27210（OsMC-4）、Os05g41660（OsMC-5）、Os05g41670（OsMC-6）、Os11g04010（OsMC-7）,它们的酶促反应底物结合位点保守残基与动物Caspase在结构上具有高度的同源性。然后用20 mM H₂O₂处理野生型水稻中花11,通过半定量RT-PCR检测不同处理时间后OsVPEs和OsMCs的表达量,观察到OsVPE-1的表达量在处理6、8、12和24 h时均显著上调,OsVPE-2的表达在处理2 h后被显著上调,并持续至12 h,到24 h后,其表达量又恢复到未处理对照组水平。而其他的OsVPEs和OsMCs的表达量没有显著变化。认为H₂O₂诱导的水稻PCD可能是通过液泡途径（主要是OsVPE-1和OsVPE-2）执行的。进一步比较实验表明,在20 mM H₂O₂胁迫处理条件下,35S::Bcl-2转基因水稻幼苗中的OsVPE-1和OsVPE-2表达量无显著变化,而野生型中的OsVPE-1和OsVPE-2表达被明显上调。推测Bcl-2的作用可能是通过调节VPE的表达来抑制H₂O₂胁迫诱导的PCD产生,从而提高水稻对氧化胁迫的耐受性。2.水稻根边缘细胞的发育调控植物根边缘细胞是由根冠细胞发育而来,具有活性的一群细胞,其发育受遗传调控,且在多种逆境中发挥生物学功能。对其发育调控和生物学功能的研究已倍受关注。但是对水稻根边缘细胞的数目、活性、发生规律以及根边缘细胞发育相关基因的研究尚少。本研究观察水稻根边缘细胞的发生,统计细胞数目和检测细胞活性,结果显示,在水稻根形成的同时就已有边缘细胞产生,每个水稻根尖的边缘细胞数目达到1500个左右时,边缘细胞停止产生,边缘细胞产生时其活性达到95%,在离体条件下培养能保持48 h。当移去边缘细胞后,在36 h内水稻根尖又能产生一套完整的1500个左右的边缘细胞。用不同浓度的BR（油菜素内酯）、GA₃（赤霉素）、IAA（吲哚乙酸）和KT（激动素）处理水稻种子,发现这些植物激素在某一特定浓度时能促使水稻根尖产生更多边缘细胞,最高可达到2100～2500个细胞/根,是正常水平的140%～167%。表明边缘细胞的产生和发育可能受到多个激素协同调控,和根冠的细胞分裂能力密切相关。同时,检测了水稻发芽过程不同时间的根尖果胶甲基酯酶（Pectin Methylesterase,PME）活性,结果表明,PME活性随根的伸长而增强。在根长5 mm时,活性达到最高值。随后,PME活性逐渐下降,最后维持在一个较低活性水平。但当水稻根尖的整套边缘细胞移去后,根冠PME活性又迅速上升,到12 h时达到最高值,之后下降,表明水稻根边缘细胞的发生和根尖果胶甲基酯酶的活性密切相关。因此,本研究克隆了水稻果胶甲基酯酶基因OsPME-1（Os04g0458900）,序列分析表明,该基因编码568个氨基酸,含有两个保守的功能结构域PMEI domain和PME domain。并且,通过半定量RT-PCR分析,发现OsPME-1在边缘细胞产生和发育过程中的表达变化与PME活性的变化一致,说明克隆的水稻OsPME-1和边缘细胞的产生和发育可能存在密切的联系。为了进一步研究OsPME-1基因在根边缘细胞产生和发育过程中的作用,构建了4个含有OsPME-1不同结构域的植物表达载体pCAMBIA13011-OsPME-1（含OsPME-1全序列）,pCAMBIA13011-OsPME-2（含OsPME-1反义序列）,pCAMBIA13011-OsPME-3（含PMEIdomain）和pCAMBIA13011-OsPME-4（含PME domain）。并通过农杆菌介导法转入日本晴水稻,经潮霉素筛选和RT-PCR鉴定分别得到转基因株系,已获得T2代种子,为后续研究OsPME-1基因的功能以及边缘细胞在水稻抗逆中的作用提供了转基因材料。更多还原

【Abstract】 Environmental stresses are the main factors limiting plant growth and development, which seriously constraint the yields and qualities of crops. It has been an important strategy to engineer agricultural crops to improve stress resistance. In this study, the molecular mechanism of resistance to oxidative stress in rice was investigated by focusing on programmed cell death （PCD） and root border cell （ RBC ） development.1. Functional analysis of Bcl-2 in transgenic rice in response to H₂O₂-induced PCDProgrammed cell death （PCD） is a genetically controlled cell death processes during plant development or in response to environmental stresses. Previous studies have revealed that ectopic expressions of some animal anti-apoptosis genes can inhibit PCD and improve resistance to biotic and abiotic stresses in plant. However, very little is known about the underlying molecular mechanisms.Here, we transferred the human anti-apoptosis gene Bcl-2 into rice by Agrobacterium tumefaciens-mediated transformation, and obtained five lines of 35S::Bcl-2 homozygous transgenic rice. Northern blot confirmed the expression of Bcl-2 in transgenic lines. In contrast to wild type, H₂O₂-inhibition of seed germination, chlorophyll retention, root elongation and root tip viability was significantly alleviated in the transgenic rice, suggesting that over-expression of Bcl-2 could enhance the resistance to H₂O₂ toxicity.The results of DNA laddering and TUNEL test indicated that H₂O₂ induced cell death accompanied by typical hallmarks of PCD, while Bcl-2 could promote H₂O₂ tolerance in transgenic plants via inhibition of PCD.To investigate PCD associated genes in plant, we found 4 OsVPEs and 7 OsMCs in rice genome by sequence alignment and function domain analysis. The expressions of these two kinds of caspase-like genes were analyzed by semi-quantitative RT-PCR. The expression analysis showed that the expression of OsVPE-1 in root tips was significantly increased after 6, 8, 12 and 24h of H₂O₂ treatment. The level of OsVPE-2 transcript accumulated after 2h and lasted till 12h treatment, and decreased to the level of controls after 24h. The results suggested that H₂O₂-induced PCD might be regulated through vacuolar pathway. However, in Bcl-2 transgenic plants, the expression of VPE did not change after H₂O₂ treatment, implying that Bcl-2 might repress H₂O₂-induced PCD in rice by inhibiting expression of VPE.2. Development regulations of root border cells in rice.More and more evidences have revealed that root border cells, whose development is genetically regulated, are biologically viable （>90%） in the majority of higher plant species, and take multi-defense roles during root growth and development. Hence, studies on developmental regulation and biological functions of root border cells have attracted the attention of many researchers. However, the root border cells production, number and viability in rice are little known.During seed germination, the formation of the first root border cell was observed almost synchronously with root tip emergence. Our results indicated that each rice root tip owns about 1500 root border cells, and 95% of which was viable by cell counting and viability assay. Further observation showed that root tips could reproduce another set of root border cells in 36 hours after removal of existing border cells. Root tips could produce much more root border cells when stimulated by some phytohormones such as BR, GA₃, IAA and KT. It suggested that production and development of the root border cells was regulated by multiple phytohormones. The activity of PME in root caps was estimated on different developmental stages and various time points after removing root border cells in rice root. The results showed closely correlation between root border cell development and PME activity in root caps. The gene OsPME-1（Os04g0458900） was cloned from rice, which encodes 568 amino acids and contains two conserved functional domains （PMEI and PME domains）. The result of semi-quantitative analysis showed that OsPME-1 expression pattern was consistent with the production of root border cells.Four plant expression vectors （pCAMBIA13011-Ospme, pCAMBIA13011-anti-Ospme, pCAMBIA13011- Ospmei-Domain and pCAMBIA13011-Ospme-Domain） were constructed and transformed into rice by Agrobacterium tumefaciens-mediated transformation. The transgenic rice plants were verified by using hygromycin selection and RT-PCR test. Until now, the T2 generation of transgenic rice has been obtained, and detailed study of the function of OsPME-1 in transgenic plants is now in progress.更多还原