水稻体细胞根芽再生的蛋白质组学分析及相关基因功能验证

【作者】 张晓波；

【导师】 凃巨民；

【作者基本信息】 浙江大学 ， 作物学， 2010， 博士

【摘要】 植物根、芽的器官建成和生长发育的分子机理是发育生物学的一个重要研究领域。本研究通过水稻器官专化诱导,蛋白质差异显示和质谱分析,鉴定出与水稻根、芽发育密切相关的基因,并籍助cDNA克隆、过表达分析、RNAi抑制和亚细胞定位等步骤来进一步弄清候选基因的具体生物学功能及其对水稻根芽发育的调控作用。主要研究结果如下：1.根芽离体再生体系的建立以优良籼稻恢复系明恢63的成熟种子为愈伤诱导材料,获得胚性愈伤。通过不同的激素配比及其它成份的调节,配制出芽诱导培养基、根诱导培养基及植株诱导培养基,将胚性愈伤组织分别接种于这些培养基上进行诱导培养。结果显示,在芽诱导培养基上诱导出来的只有芽而没有根,其诱导频率按接种的愈伤总数为基数计算高达95.3%；在根诱导培养基上诱导的则只有根而没有芽,其诱导频率达71.3%；在植株诱导培养基上诱导出来的既有芽又有根,其诱导频率达91.3%。这些结果表明,适用于大规模蛋白质取样和双向电泳差异显示分析的体细胞根芽器官定向诱导系统构建成功。2.体细胞根芽切片观察细胞和组织学切片观察体细胞根芽的诱导形成历程发现,体细胞根芽分化是自愈伤表面的几层细胞开始；至接种后的第四天,根芽诱导培养基上的愈伤表层细胞的分化差异已清晰可见,分化也随之向内层细胞发展；至接种后的第八天,由表面几层细胞分化而来的根尖生长点已清晰可见,由里面几层细胞分化而来的芽尖生长点开始形成,同时,他们都伴随有输导组织的发育；至接种后的第12天,不同器官的分化进一步进展,至接种后的第16天,根芽器官的定向分化已经完成,但在植株诱导培养基上的分化则仍在进行,与前两者相比滞后约3-5天。3.体细胞根芽的蛋白质组学分析对处在芽诱导培养基和根诱导培养基上的不同分化时期的细胞组织,自第0天开始,每4天取一次样抽提可溶性蛋白,并用蛋白双向电泳技术进行蛋白差异表达分析。通过各分化时期不同再生样品蛋白间电泳图谱的PDQuest软件比对分析,共鉴定出102个差异蛋白点,后经蛋白质质谱分析确定出其中78个蛋白点共计83个蛋白的氨基酸序列,其中,有5个蛋白点包含2种蛋白。对这83个蛋白按其功能进行分类,一共可分为10类,前四类分别为功能未知(36%),蛋白代谢相关(15%),防御相关(14%),代谢相关(12%),占测序蛋白总数的77%。4.根芽发育相关基因的克隆及功能分析本研究克隆了对应于蛋白点5108(序号由软件PDQuest自动生成)的基因,并对其具体的生物学功能进行了初步分析和验证。结果证实蛋白点5108是一类含有一个胱氨酸残基的过氧还蛋白(1-Cys Proxiredoxin),对水稻中脉的发育起调控作用。RNAi抑制分析表明,一旦该基因的表达受到抑制,水稻叶片的中脉发育异常,中脉内的2-4个大气腔消失,因而,不能对叶片起刚性支撑作用,叶片从叶枕到叶基部处扭曲下垂,以至叶片的背面朝向阳光。从外表上看,原本在叶片背面突出的中脉消失,大小变成与侧脉一致。4. cDNA克隆过程中人工嵌合序列产生的原因分析和消除程序的提出在对具有高度同源拷贝的水稻33kDa分泌蛋白基因家族成员进行cDNA克隆时,我们发现在所获得的克隆产物中,有20%左右为基因组中不存在的嵌合序列,并发现序列的嵌合是从一个成员的5’序列转换到另一个成员的3’序列,亦或相反。随即,我们对这种人为嵌合序列产生的原因进行了系统分析。结果显示,在本实验条件下,以混合的RNA或DNA为模板的PCR扩增是这些嵌合序列的一个主要来源,其产生的机理与PCR引物的非完全延伸有关,而这种引物的非完全延伸又主要与PCR反应体系中甘油添加剂的加入与否和聚合酶种类的选用有关。另外,我们的结果还显示,在随后的cDNA克隆过程中,微生物的多种缺刻依赖型DNA修复系统对异源cDNA双链分子的修复也是上述这种人工嵌合序列的一个来源,但不是主要的来源。基于上述这些结果,我们提出了一套改进程序,用于避免RT-PCR扩增和cDNA克隆时人工嵌合序列的发生。更多还原

【Abstract】 Plant root and shoot development and their molecular mechanism is a hot area in development biology. The present study was attempted to identify the genes that are closely related to rice root and shoot development via in vitro individual organ specific induction, protein differential display and mass spectrum analysis. And then, to make clear the candidate genes’biological function and their effect to regulate rice root and shoot development via cDNA cloning, gene over-expression, and RNAi repression.The main results were summarized as followings:1. Shoot and root in vitro regeneration systems were successfully set upAn elite indica rice restorer line, MH63, was selected as starting material for callus induction. The medium for regeneration of individual shoot or root or whole plant were optimized by adjusting the concentration of plant hormones and other components. The calli were then inoculated onto these optimized media, respectively, for organ specific induction. The results showed that the calli induced on the shoot specific induction medium grows only shoot but no root, with the ratio up to 95.3%, while the calli induced on the root specific induction medium grows only root but no shoot, with the rate no lees than 71.0%. The higher rate up to 91.3% was also obtained for the whole plant induction. These results thus indicated that an in vitro organ specific induction system applicable for large scale protein sampling and subsequent 2-D electrophoresis has been successfully set up.2. Microscope observation of somatic root and shoot developmentThe data derived from the microscope observation indicated that somatic root and shoot differentiation is started from the several layers of cells on the callus surface. Four days after inoculation, the differentiation differences of the callus surface cells on the shoot and root specific induction media was becoming clear. At the same time, the differentiation progressed into inner cells; another four-day after that, the root meristem derived from the several layers of callus surface cells was becoming clear, while the shoot meristem derived from the several layers of callus inner cells began to develop. At the same time, the transport tissues were accompanying them to develop. Twelve days after inoculation, the differentiation of the different organs was further progressed; Up to sixteen days after inoculation, the shoot and root specific induction was finished. However, the whole plant differentiation at the moment was still continued, lagging behind about 3 to 5 days as compared with that of root and shoot.3. Proteomic analysis of somatic shoot and rootStarted from zero days, soluble proteins was extracted every four days from the differentiated calli at the different developing stages and used for protein differential express display. The numbers of 102 divergence protein spots were identified via PDQuest analysis. Among them, the sequences of 83 proteins contained in the 78 spots were determined using mass spectrum. Further analysis indicated that these proteins could be classified into 10 categories based on their function. The first four categories were function unknown (36%), protein destination/chaperor (15%), defense related (14%), and metabolism related (12%), accounting for 77% of total sequenced proteins.4. Molecular cloning of leaf midrib related gene and its functional analysisGene corresponding to protein spot 5108 with clear differences among samples and developmental stages in 2-D gel (the numbers were given by software PDQuset) were selected and cloned for further functional dissection. The results shows that the protein spot 5108 encode a 1-Cys-Proxiredoxin protein and plays an important roles in leaf midrib development. RNAi repression analysis indicates that once this gene’s expression was repressed the clear cells in the middle of leaf midrib disappear so that the midrib developed abnormally and can not act to support leaf any more. The leaf blade thus droops down. In addition, the leaf base distorts so that the leaf back side turns up.5. Analysis of causes leads to sequence chimeras during cDNA cloningWhen clone two members of a closely related rice 33-kDa secretory protein gene family, we found existence of 20% sequence chimeras in the final products. These sequence chimeras generally switched from 5’-end of one member to 3’-end of another member, or vice versa. To make clear how these chimeras were produced, we systematically analyzed their occurrence causes. The data indicate that the mix template amplification is the major source leading to formation of these sequence chimeras. Their occurrences mechanism was confirmed mostly due to incomplete DNA amplification. While this incomplete DNA amplification was largely affected by PCR buffer additive and DNA polymerase species. In addition, our data also reveal that heteroduplex repairs by nick-dependent microbial DNA repair systems in the cDNA cloning step were found to produce chimeras, although it was not the major source under the present conditions. Based on these results, several simple strategies have been suggested to reduce the formation of the chimeras in the mixed-template amplification and subsequent cDNA cloning.更多还原