摘要
短日照植物水稻,是世界上最重要的粮食作物之一,也是典型的单子叶模式植物,随着水稻基因组测序的完成,对于水稻基因组中功能基因的研究是当前迫在眉睫的任务。
高等植物的成花诱导过程由自身遗传因子和外界环境因素两方面决定,开花的引发是个动态复杂的空间模式,开花时间,在禾谷类作物中也叫抽穗期,水稻的开花是水稻由营养生长向生殖生长转变的标志,培育早开花或光周期不敏感栽培种是几十年来水稻育种的主要目标,通过控制开花相关基因的克隆,利用分子遗传学手段和转基因技术,可以加快育种的进程,研究水稻早熟品种内在分子机理,T-DNA标签法、Ac/Ds和Tos17逆转座子等可以很容易获得突变体,并且迅速地找到突变基因所在位点,明显缩短克隆基因的周期,进而有利用于加快育种进程。
本文依托本实验室创制的10万份水稻突变体库中已筛选到的不育及早熟性状突变体300多份为研究材料,采用PCR-Walking的策略对其进行插入位点侧翼序列的扩增,利用生物信息学分析确定候选基因的插入位点,并根据表型与候选基因的共分离分析,同时通过功能互补实验证实候选基因功能。
在长日照下Osef1突变体可以引起水稻较野生型早开花近30天,通过共分离分析及功能互补实验,结合生物信息学表明,此突变体中Tos17序列插入到基因OsEF1的3’UTR,该基因编码与小粒碗藓控制光下配子托发育所相似的蛋白,基因枪轰击洋葱表皮进行亚细胞定位,该基因的蛋白产物定位于细胞核内,通过酵母双杂交分析,OsEF1在酵母细胞中具有较强的自激活活性,同时从水稻cDNA文库中筛选到候选互作蛋白35个,利用回复杂交证明OSK4与OsEF1间的相互作用,此外,我们也证明了与OSK4高度相似的OSK3(约98%)与OsEF1也具有相互作用。
构建的融合标签基因载体在IPTG诱导下可以成功的在原核细胞中表达OsEF1和OSK3及OSK4,Western Blot进一步证实了表达的目的蛋白,为后续的功能研究提供实验支持和依据。
利用RT-PCR及Real-Time PCR进行基因的表达分析,在长日照下,水稻基因OsEF1在不同的组织表达具有明显的差异性,尤其是在开花前基因OsEF1在叶片及叶鞘中的表达明显较其它组织高,长日照下,突变体Osef1的生物钟组分基因OsLhy、OsPRR和生物钟相关基因OsGI的表达均不受影响,然而在光周期控制水稻开花中起重要作用的基因Hd1在Osef1中的表达却明显降低,同时hd1突变体中,OsEF1基因的表达几乎不受影响,这表明基因Hd1位于OsEF1的下游,同时受OsEF1的正调控,另一个光周期控制开花的独立途径基因Ehd1,在长日照下,突变体Osef1中表达明显升高,说明基因OsEF1可能具有下调Ehd1表达的作用,所有结果表明,OsEF1通过在长日照下,对光周期途径中的Hd1正调控以及对Ehd1的负调控从而对抑制水稻开花。
Rice (Oryza sativa L.), a short-day (SD) plant, is one of the most important crops, it is also an model of the monocot plant, with the complete sequences of rice available, subsequently, it is urgent to study on the functional analysis of the rice genome.
The floral induction is determined by internal genetic factor and external environment signal in higher plant, and triggering flowering is a dynamic and complicated spatial pattern.The flowering time, often termed as heading time in grains, the flowering is an symbol of the transition from vegetative phase to reproductive phase, so the major objective has been set up for decades is that several new early flowering or photoperiod-insensitive varieties of rice are cultivated.Take advantage of the clone the gene which is related to regulation of the flowering, combined with the molecular genetics and transgenic technology, accerlate the breeding process,Studying the internal molecular mechanism of early flowering species, as well as the mutants were easily obtained and located candidate genes’site where led to mutation by T-DNA tagging technology or Ac/Ds system and Tos17 retrotransposon,etc, thus it is obvious to shorten the period for cloning gene, thus it is convenientuseful to speed the breeding.
In my dissertation, based on the total 300 sterile and early flowering mutants screened by one million rice mutation library, we amplified the flanking sequences of the insertaion site through PCR-Walking strategy, and located the instertion sites of the candidate genes with bioinformatics analysis, in addition, in terms of the co-segregation analysis between phenotype and candidate genes, we further confirmed the function of candidate gene by complementation test.
In long day (LD),the rice mutant Osef1 cause the flower nearly 30 days earlier than wild type plant, according to the co-segregation analysis and complementation test combined with bioinformatics, it is indicated that this Osef1 mutagenesis resulted from Tos17 sequences insertation at 3’UTR in OsEF1, and OsEF1 encoded an protein which is similar to protein that functions gametophores development in Physcomitrella patens under light conditions, and this OsEF1 protein product is localized to the nucleus by making use of onion epidermal by particle bombardment. The OsEF1 held the strong auto-activation effect in yeast cell , we constructed the mutual reverse vector which is comprised of the candidate interation protein as bait and OsEF1 as prey co-transform the AH109, and we confirmed that there is real interaction between OsEF1 and OSK4, what is more , we found that OSK3 and OsEF1 existed interaction during the yeast system analysis.
The recombinant vector fused GST or His tag gene successfully expressed OsEF1、OSK3 and OSK4 in Transetta(DE3) induced with IPTG, Western Blot further tesfied them, which provided the experimental ground for further study.
We performed the RT-PCR and Real-Time PCR to analyse the expression of genes, in LD, expression of OsEF1 in various tissues is different, and the OsEF1 expressed in a more level in leaf blade and shleath than other tissues. The Osef1 had no effect on the expression of the OsLhy and OsPRR which are circadian clock components, whereas, Hd1, a gene had an important role in photoperiod pathway, expressed in a rather low level. Meantime, hd1 had no effect on the expression of OsEF1,thus we concluded that Hd1 is upstream of OsEF1 and controlled positively by OsEF1.Ehd1, another gene controlled flowering independently of Hd1 , Ehd1 epression increased in mutant Osef1,this indicated OsEF1 might down-regulated the Ehd1 epression. To sum up, our result demonstrated that in LD, OsEF1 inhibit flowering in rice through both Hd1 and Ehd1 flowering pathways.
引文
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