【作者】 周小云；

【导师】 陈信波；

【作者基本信息】 湖南农业大学 ， 植物学， 2007， 博士

【摘要】 植物能够适应外界环境的变化并生存下来的一个重要机制是分泌蜡质到植物角质层表面,充当植物阻挡紫外辐射及病虫害侵蚀的第一道屏障,并能降低植物的非气孔性水分丧失,有研究表明,表皮蜡质含量与植物抗逆性呈正相关。而转录因子是调控植物生长发育和逆境应答过程中的重要成员,能同时调控同一性状相关多个基因的表达,操纵一个转录因子就可通过它促使多个功能基因发挥作用从而使植株性状获得综合改良,显示出操纵转录因子比操纵功能基因巨大的优越性,因而近年来对转录因子特别是逆境相关转录因子的研究成为热点。本研究对水稻叶片发育过程中表皮蜡质的积累进程进行了系统的观察,发现在水稻各部位暴露到外界环境之前,水稻的表皮蜡质就基本完成其发育以备适应外界的环境。水稻叶片表皮蜡质的部位发育程序为由叶尖往下依次发育的顺序;叶腹面比叶背面发育早,蜡质晶体积累多;全展叶期叶片蜡质晶体积累达到高峰,与全展叶开始全面行使叶片的功能相适应。到了成熟末期,叶片表皮蜡质从叶片边沿向中心开始崩溃退化。为了研究水稻表皮蜡质与水稻抗逆性的关系,本研究总结了一套简单的水稻蜡质提取方法并对不同品种水稻在正常生长条件的表皮蜡质积累情况以及逆境条件下表皮蜡质的变化情况进行了比较研究。发现以氯仿为溶剂,在60℃,30s的条件下高温短时间内浸提是非常有效的水稻表皮蜡质提取方法。正常生长条件下,表皮蜡质分布的密集程度为旱209>日本晴>9311,蜡质晶体的大小为9311>旱209>日本晴,而提取的蜡质含量为旱209>9311>日本晴。说明水稻叶片表皮蜡质在叶面上分布越广泛,覆盖率越高,则水稻的保水能力越强,水稻的抗旱性也越强。适当剂量的紫外和干旱处理能提高水稻表皮蜡质的含量,表明水稻叶片表皮蜡质的产生是一个积极的、可调控的过程。通过与拟南芥蜡质代谢转录因子基因的序列同源性比较,找到两个在水稻中同源性较高的基因序列,命名为OsWTF1和OsWTF2。生物信息学分析表明,OsWTF1和OsWTF2都含有单个AP2保守结构域,归属于转录因子家族AP2/ERF超家族ERF亚家族成员;启动子部位包含大量调控植物部位特异基因表达和逆境应答基因表达的顺式作用元件,推测OsWTF1和OsWTF2与水稻的逆境应答调控相关。利用GUS融合表达研究OsWTF1和OsWTF2的部位表达,发现OsWTF1和OsWTF2在水稻的根、茎、叶、鞘、幼穗中都有表达;比较而言,OsWTF1在生殖生长期的表达强,在营养生长期表达弱,而OsWTF2在水稻营养生长期的表达强,在生殖生长期表达弱。利用RACE方法获得OsWTF1全长cDNA,从日本购得OsWTF2全长cDNA,并构建OsWTF1和OsWTF2过表达载体和OsWTF2的RNAi表达载体,分别转入水稻中获得转基因植株。发现转OsWTF1、OsWTF2的过表达植株的株型矮小,育性降低,花粉形态发生改变,叶片表皮蜡质晶体明显增厚,表皮刺毛数量减少,叶绿素浸提率降低,表明OsWTF1和OsWTF2可能参与水稻表皮蜡质的生物合成并对水稻抗旱性具有积极作用;而导致表皮蜡质表达沉默的转基因植株暂未得到,可能表皮蜡质的表达是一个多基因控制的复杂体系,对其中一个基因的沉默达不到使表皮蜡质表达缺失的效果。更多还原

【Abstract】 A mechanism that plants have adopted to survive in a terrestrial, aerial environment is the production of waxes deposited into and on the cuticular layer of their outmost surface. The epicuticular wax acts as the first protective barrier against UV radiation, bacterial and fungal attacks and regulates non-stomatal water loss. Studies have showed a positive relationship between the epicuticular wax content and plant stress resistance. Stress-related transcription factors play important roles in regulating the expression of special stress-related genes that allow plants to respond optimally to their changing environment. It has been suggested that manipulating of genes encoding transcription factors might be an effective method in plant anti-stress gene engineering.In this paper, epicuticular wax deposition during rice leaf development, among different cultivars, under drought and ultraviolet-B radiation stresses was analyzed respectively. Two putative wax related rice transcription factor genes, OsWTF1 and OsWTF2 were cloned. The expressions pattern of OsWTF1 and OsWTF2 were analyzed using their promoter driven GUS transgenic lines. The functions of OsWTF1 and OsWTF2 were characterized by overexpression and RNAi transgenic plants analyses. The following results were obtained:1) Deposition and crystalloid morphology of epicuticular wax were observed by scanning electronic microscopy during the development of rice leaf. Epicuticular wax was deposited well on the leaf before it was exposed to the aerial environment. The development process of epicuticular wax is from leaf apex to leaf base, from leaf adaxial to leaf abaxial. More wax crystalloids were deposited on adaxial than on abaxial. Wax crystalloid accumulation achieved to peak when leaves were fully developed. Beginning from the late mature stage, the epicuticular wax on leaves started to collapse from leaf margin to leaf center.2) The extraction method of rice epicuticular wax was optimized. High-temperature method with solution CHCl₃ was the most effective and simplest rice wax extraction method.3) Epicuticular wax crystalloids on leaves of three different rice （Oryza sativa L.） genotypes, including two wetland varieties （Japonica cultivar Nipponbare and Indica cultivar 9311） and one upland rice variety （drought resistance variety Han 209） were observed by scanning electronic microscope. The wax contents on leaves of three rice genotypes were compared at normal growth condition and under drought or ultraviolet-B radiation stress. The results indicated that upland rice cultivar had more epicuticular wax crystalloids and higher wax contents than wetland cultivars. Wax contents and crystalloid distribution were increased in all three cultivars under ultraviolet-B radiation and drought stresses. Uniform and dense epicuticular wax distribution on rice leaves were positively related with strong resistance to drought and ultraviolet-B radiation stresse.4) By BLAST Arabidopsis transcription factor gene WIN1（SHN1）involved in wax biosynthesis to the rice genome sequences, two homologous gene sequences were found in rice and were named OsWTF1 and OsWTF2 respectively. Bioinformatics analysis showed that both OsWTF1 and OsWTF2 contained a single conserved AP2 DNA-binding domain respectively, which belonging to ERF subfamily of AP2/ERF transcriptional factor super-family. Moreover, many cis-acting elements were found in controlling the spatial and temporal expression and the stress-responsive gene expression. These results suggested that OsWTF1 and OsWTF2 might participate in regulating the stress responses.5) The expression patterns of the OsWTF1 and OsWTF2 genes were examined by their putative promoter driven GUS expression in the transgenic rice plants. GUS expression was detected in most plant organs, including epidermis, pith of the root, vascular bundle or pith of the stem, stomata apparatu, trichomes, leaf sheath and stamen. OsWTF1 expressions were stronger in nutrient organs than that in reproductive organs, but reverse to OsWTF2.6) Full length cDNA sequence of OsWTF1 was cloned by RACE technology from rice mRNA and full length of OsWTF2 were bought from rice resource centre of Japan. OsWTF1 and OsWTF2 Overexpression vectors and OsWTF2 RNAi vector were constructed and separately transformed into rice by agrobacterium-mediated method. The transformations were confirmed by PCR and RT-PCR. OsWTF1 and OsWTF2 overexpression transgenic lines showed reduced plant heights and seed setting rates,,abnormal pollen morphology, decreased trichome numbers on leaf and chlorophyll leaching rates and increased leaf epicuticular wax These phenotypes suggested that OsWTF1 and OsWTF2 were involved in the wax metabolism. However, no epicuticular wax change was detected in silenced OsWTF2 RNAi transgenic lines.更多还原