摘要
水稻是世界上最重要的粮食作物之一,也是研究禾本科植物和单子叶植物研究的模式作物。随着水稻基因组测序的完成,大量水稻突变库的创建,加快了水稻理论研究的步伐,明显缩短了基因克隆的周期,使水稻基因功能的研究进入了后基因组研究的时代。近年来,许多重要农艺性状的基因被成功的克隆,如花期、育性、叶形、抗病性等。开花时间,在水稻中又称抽穗期,是营养生长向生殖生长的过渡,所以对控制水稻开花基因的克隆,将有助于理解和完善水稻控制开花的调控网络及分子基础,扩大水稻适应性栽培的范围,为育种工作的开展提供重要的遗传基础和理论指导。
本文研究的材料基于实验室创建的10万份水稻突变体库中筛选到的一个早花突变体Osef1,基于前期侧翼序列的扩增、互补实验等证明了该突变体是Tos17插入到OsEF1基因的3’UTR失活引起的;亚细胞定位在细胞核中;构建pBDGAL4cam-EF1融合载体,利用酵母双杂交实验筛选出了35个与OsEF1互作的蛋白;明确了长日照下,OsEF1起着正调控Hd1和负调控Ehd1的作用等基础上进行深入研究的。
首先,通过田间农艺性状的调查发现在自然长日照条件下,Osef1突变体开花时间明显早于野生型,时间早30d左右,同时,突变体呈现半矮化的表型。
其次,研究了OsEF1的组织表达模式。通过转pP_(OsEF1)-GUS阳性植株的GUS活性分析,发现OsEF1在根、茎、叶、幼穗、颖壳、侧根、结节、叶鞘、叶枕都有表达,石蜡包埋切片在叶片和叶鞘的各个类型的细胞中也检测到了GUS活性,表明了OsEF1是一个组成性表达基因。同时利用Quantitative Real-Time PCR对上述组织进行相对定量分析,得出了类似的结论。
第三,通过Quantitative Real-Time PCR开展OsEF1光周期节律性分析。研究发现在短日照下,OsEF1的表达贯穿整个白天和晚上,在黑夜之前有一个最低值,在光照之前出现峰值,从而可以推断OsEF1是受光周期调控的基因,mRNA的表达呈昼夜节律性表达。
第四,为了更好的研究OsEF1蛋白的靶基因,将酵母双杂交实验筛选出的8个与OsEF1互作蛋白的基因进行RNAi干涉。主要利用Gateway系统和pTCK309载体,成功的构建了RNAi载体,其中Gateway系统构建了7个载体,以pTCK309载体为骨架的2个,将上述干涉载体通过农杆菌介导法转化日本晴,其中一个基因的转化群体中有3株表现早开花性状。
最后,原核表达和纯化OsEF1蛋白及制备多克隆抗体。将OsEF1全长cDNA融合到pET-32A载体上,经0.4mM的IPTG诱导8h,成功的表达可溶性蛋白; His标签蛋白的Western Blot检测证明了表达的总蛋白即为目的蛋白;在非变性条件下,通过His镍柱小量亲和纯化发现150mM的咪唑浓度较为适宜,大规模纯化并初定量后获得3mg蛋白,用于制备多克隆抗体,以进一步检测突变体背景下内源蛋白水平的研究。OsEF1基因的克隆及功能的深入研究,说明了水稻在光周期调控方面形成了独特和复杂的网络,与拟南芥相比,这些独特性和复杂性提供了水稻既能短日照下开花,又能适应在长日照下正常开花。
Rice (Oryza sativa L.) is one of the most important crops in the world, and also regarded as the model plant in the studies of grass family and monocotyledon. With the completion of rice genome sequencing and establishment of a large number of rice mutant libraries, the pace of theoretical studies of rice gene was accelerated greatly and the period of cloning one gene was shortened, the rice genome research went into the post-genome era. Recently, a lot of important agronomic traits were successfully cloned, such as florescence, sterile, leaf shapes and disease resistance and so on. Flowering, also known as heading time in rice, is a transition from vegetative growth to reproductive growth. Therefore, cloning of rice flowering related gene would help to understand the molecular basis of regulatory network and improve the control of flowering in rice, expand the area of rice cultivation, and provide important genetic basis and theoretical guidance for the breeding.
This article is about an early flowering mutant Osef1 which was screened from the one million mutant library of our laboratory. This work is based on the following results: According to the flanking sequence amplification result and complementation test, we proved that the Osef1 phenotype was resulted from Tos17 insertion at 3’UTR in OsEF1gene. A nuclear green fluorescence after transient expression of OsEF1 fused to GFP in onion epidermal cells revealed that OsEF1 gene was localized in the nucleus. We construct the confusion vector pBDGAL4cam-EF1, used yeast two-hybrid method to acquire thirty-five candidate proteins which interacted with OsEF1 protein. We also proved that OsEF1 positively regulated the expression of Hd1 and negatively regulated the expression of Ehd1 in the long-day condition.
Firstly, the field survey of agronomic traits found that the flowering time of Osef1 mutant is 30day earlier than the wild-type in the natural long-day conditions. Also, the mutant showed semi-dwarf phenotype.
Secondly, Expression pattern of OsEF1 gene were analyzed. Through the GUS staining in pP_(OsEF1)-GUS transgenic lines, we find OsEF1 expressed in root, stem, leaf, young spikelet, lateral root, node, leaf sheath, leaf joint. The GUS activities have been also detected in all types of leaves and leaf sheath by paraffin-embedded sections. It suggests that the OsEF1 acts as a constitutive expression gene in rice various tissue. The conclusion of Quantitative Real-Time PCR is consistence with the GUS staining result.
Thirdly, the diurnal expression patterns of OsEF1 were analyzed in wild type under short-day condition by quantitative RT-PCR. Ubq gene was selected as internal control. We found that OsEF1 was expressed throughout the light/dark cycle, with a peak at the beginning of the light. The result suggested that OsEF1 transcription was controlled by circadian clocks and exhibited a diurnal rhythm expression pattern under SD conditions.
Fourthly, in order to better research the target of OsEF1, RNAi suppression method was used to inference the expression of the eight genes which were screened by yeast two-hybrid system. Mainly use the Gateway system or pTCK309 vector to construct the RNAi vectors. Among them, they are seven vectors which are constructed by Gateway system and the other by pTCK309 vector. The construction vector was transformed to Nipponbare background by Agrobacterium-mediated method. Then there are three lines with early flowering in one of the eight transgenic groups.
Lastly, the full-length cDNA of OsEF1 was fused on PET-32A vector. This confusion vector can express soluble protein successfully in the 0.4mM IPTG under 8 hour induction. Based on the detection of His-tagged protein by Western Blot, the expressed total protein is the target protein. HIS nickel column purified this protein by a small amount under native conditions and found 150mM imidazole was more appropriate. The large-scale purified protein was used for the preparation of polyclonal antibody to further detect endogenous protein of Osef1 mutant. Detailed functional analysis and characterization of OsEF1 and compared with Arabidopsis, we find that only the unique and complex gene pathway could provide rice plants the facultative short-day nature so that they can also flower during non-inductive conditions.
引文
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