【作者】 董红霞；

【导师】 曾汉来；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 叶色变异是高等植物中突变频率较高且易于辨别的突变性状,在理论研究和实际应用方面都具有重要的意义。其中的一些叶色变异型以其表型明显可分辨的特点,可作为一种形态标记性状在植物遗传、育种研究中得以有效利用,在功能基因组学研究方面,它们已成为深入开展植物色素合成、叶绿体发育等相关基因功能研究的理想材料。本研究组于1996年从水稻花药培养的白化苗中获得能够自然转绿的植株,经多代选择培育成性状稳定的可自然转绿型白化苗突变体,将之与光温敏不育系培矮64S杂交,从后代中获得具有白苗复绿性状的光温敏不育株系白02S。本研究以白02S和培矮64S为材料,分别从叶色表型特征、叶绿体超微结构、蛋白质和mRNA表达水平等方面对该性状进行较为系统的研究,以期部分阐明其白化复绿机理。主要研究结果如下：1.白02S和培矮64S的形态学和超微结构观察表明,白02S苗期前三叶均为白色,第四叶及之后长出的叶片均为正常绿色,第四叶长出后,健在的白化叶有部分转绿,其白化表型明显可辨期为30天以上。培矮64S作为白02S的对照,在整个苗期都显示正常的绿色。利用透射电镜对白02S转绿前后两个时期的叶绿体超微结构进行观察,白02S的白化叶叶绿体表现发育迟缓,没有观察到完整的叶绿体双层膜及类囊体片层,四叶期复绿后,出现了完整的叶绿体结构,叶绿体数量较多,具有清晰的叶绿体双层膜结构,以及内部相互连接的基粒类囊体,与对照培矮64S无差异。结果表明白化性状产生的原因是叶绿体发育和叶绿素合成相关的基因发生改变,导致初期类囊体发育受阻,而不是其它色素改变所致。2.提取白02S和培矮64S的二、三叶期的叶片蛋白,经2D-PAGE分离后,构建了白02S突变体转绿前后的差异蛋白质的表达谱；通过比较蛋白质表达谱之间的差异蛋白,再经MALDI-TOF/MS分析和数据库检索鉴定发现白02S突变体的白化期有62个差异蛋白质点,其中44个上调蛋白,18个下调蛋白；对差异蛋白进行了功能分类和生物信息学分析发现,除了部分未知蛋白质,大部分已知蛋白参与了信号转导、植物的光反应、渗透胁迫调节、糖酵解--三羧酸循环--e-传递系统(EMP-TCA-ETS)代谢途径、光合作用、光呼吸和光合磷酸化等的生理过程。这表明白化过程是一个多种信号和代谢途径的调节的结果。3.以白02S突变体转绿前后的两个时期为实验材料,通过比较分析转绿前后的基因表达图谱的变化,得到差异表达2倍以上的探针组5167个,形成了白02S突变体转绿前后的差异基因表达谱,这为研究叶色突变的发生机制奠定了基础；对差异基因进行功能鉴定和分类分析后显示差异基因涉及到多种代谢途径以及信号转导,与对白02S蛋白质组学研究的结果相一致。4.将基因芯片表达谱数据和蛋白质质谱数据进行综合分析,结果显示：在白02S突变体白化期的18个下调蛋白中与芯片数据没有overlap基因,而上调的44个蛋白中与芯片数据结合分析找到5个overlap基因,它们分别是2个未知蛋白,一个叶绿体核糖核蛋白CP29A、一个蛋白酶体a亚基和烯醇酶；在这五个蛋白点中,四个蛋白质的表达与对应的mRNA表达趋势一致,均呈现上调趋势,只有No.37蛋白质表达和对应的mRNA表达呈相反的趋势。5.应用实时定量PCR技术对白02突变体的转绿前后两个时期的RNA表达水平进行了比较研究,旨在对差异蛋白质表达谱、差异基因表达谱以及蛋白质表达谱和基因芯片表达谱综合分析结果的验证。蛋白质表达与实时PCR验证结果的一致性、基因芯片分析结果与qRealtime-PCR分析结果一致性均在80%以上。本研究为研究水稻和其它植物的叶绿素缺失突变体生理和分子机制提供了基本数据和深入研究的线索。更多还原

【Abstract】 Leaf color mutant in the higher plant attracts much attention because it results to easily visible trait and has higher mutation frequency. These play important roles in both theoretical research and practical uses. The leaf color mutant phenotype which is clearly distinguished by its characteristics, can serve as a phenotypic marker in plant genetics and breeding studies. It can also be used as an ideal research material on chloroplast development and chlorophyll biosynthesize related gene in functional genomics.In this paper, the morphological characters, chloroplast ultrastructure, proteomics and microarray of W02S and Peiai 64S were studied. W02S is a new male sterile line with the albino green-revertible trait developed from a cross between WS and PTGMS Peiai 64S. WS was developed through anther culture by our research team in 1996 and there after showed stable expression in several generations.The results for the morphologic investigation showed that, up to three-leaf stage, the seedlings of W02S appeared apparently albino but turned into green after the 4th leaves were fully expanded. Peiai64S which was used as a control showed stable chlorophyll content and green color throughout the whole seedling stages. TEM analysis was performed for the leaves collected from the pre-and post green-revertible periods of W02S. During pre green-revertible period, W02S mutant compared to Pei’ai 64S had clear developmental defects such as lack of complete double-layer membrane and the chloroplast thylakoid lamellae. However, during the greening period, complete chloroplast structure, double membrane structure, and internal interconnected grana thylakoids observed in the W02S were the same as that of the control Pei’ai 64S. These results indicated that the cause of albino trait was the changes of the genes related to chloroplast development and chlorophyll biosynthesis that led to the initial thylakoid retardation, rather than changes in other pigments.The protein for analysis of differential protein expression profile between W02S and Peiai64S was extracted at seedling stage. In total 62 proteins were analyzed and identified in the W02SW through MALDI-TOF MS and database searching. Among them 44 were up-regulated proteins and 18 down-regulated proteins. The identified proteins were sorted according to their function. Most of them were involved in signal transduction, plant light responses, osmotic stress regulation, EMP-TCA-ETS metabolic pathways, photosynthesis, photorespiration photophosphorylation etc. This indicated that the albino green-revertible process was regulated by multi-signals and metabolic pathways.To study the differentially expressed genes between the pre-and post green-revertible period of W02S, we examined the mRNA expression changes between the two stages, W02Sw and W02Sg. A total of 5167 rice genes were found to be differentially expressed at their mRNA levels between the two stages. Differential genes expression profile was simultaneously constructed though comparative analysis of gene expression profiles. According to effective classification of the Gene Ontology, these genes could be classified into 17 categories, suggesting that the etiolating process is a regulatory consequence of multiple signal and metabolism pathways.To compare the correlations between the changes in corresponding mRNA and protein expressions, we comprehensively analyzed proteins and mRNA that showed significant differential expressions between W02Sw and W02Sg. We found 5 overlap genes that encode two unknown proteins, one 29 kDa ribonucleoprotein A(cp29A), proteasome alpha subunit and enolase. The expression of four proteins and their corresponding mRNAs showed an upward trend. The only protein and its corresponding mRNA that showed the opposite expression trend was No.37.To obtain complete expression data for the 62 proteins, the mRNA expressions of the 57 proteins, which were not detectable by microarray, were further analyzed by real-time PCR. The results from proteomic analysis, microarray assay and real-time PCR demonstrated a good correlation of the protein differences between W02Sw and W02Sg which in turn corresponded to mRNA differences. Overall, of the 62 proteins, less 20% with upward/downward trends had an opposite mRNA trend; these discrepancies might be due to the modifications of the post-transcription and post-translation processes. The reliability of the experimental results is very high.These new findings provide the basic data that may lead towards the understanding of mechanisms of chlorophyll-deficient phenotype in rice and other plants.更多还原