【作者】 胡瑞波；

【导师】 林辰涛； 傅永福；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 磷脂酰乙醇胺结合蛋白基因家族(Phosphatidylethanolamine-binding Protein,PEBP)编码一类对植物开花时间起重要调控作用的蛋白,其主要成员之一FT(FLOWERING LOCUS T)蛋白或(和)mRNA是成花素的重要组分。对大豆PEBP基因家族进行研究,明确其在大豆开花调控网络中的地位和作用,阐明其基因功能,将有助于揭示大豆开花调控的分子机制。本研究中克隆到了大豆PEBP基因家族的19个基因,对其进行了生物信息学分析,用实时荧光定量PCR(QuantitativeReal-time RT-PCR)分析了其在不同组织器官和发育时期的时空表达模式,并将部分基因在拟南芥中过表达研究其功能。由于qRT-PCR结果的准确性在很大程度上取决于合适内参基因的选择,所以,为了更好地研究大豆开花基因的表达模式,本研究还评价了14个大豆候选内参基因在116个不同发育时期、组织器官、光周期和品种样品间的表达稳定性。主要研究结果如下:1.ACT11,UKN1和UKN2在不同组织器官样品中的稳定性最好,SKIP16,UKN1和MTP在不同发育时期样品中的表达最为稳定。在长日和短日光周期样品中,ACT11,TUA5和TIP41稳定性最好。TIP41,UKN1和UKN2在蓝光和红光光周期样品中的稳定性最好,不同品种间ACT11,UKN2和TUB4稳定性最好。整体而言,SKIP16,UKN1和UKN2的表达稳定性最好。2.确定了大豆不同类别样品基因表达分析准确定量所需的最适内参基因数目。对组织器官和光周期(长日/短日)样品来讲,分别需要用3个内参基因对结果进行标准化,而蓝光/红光和不同品种样品仅需要2个内参基因就能保证定量结果的准确性。发育时期样品需要4个内参基因同时使用以保证结果的准确性。3.从大豆中克隆到19个PEBP基因,包括9个FT同源基因,4个TFL1同源基因,4个CEN同源基因和1个MFT同源基因。这些基因位于10条不同染色体上,FT类基因具有成对排列于同一条染色体的短的区段的倾向,TFL1和CEN的4个不同拷贝均位于不同的染色体上。7对基因之间同源性高于90%,而且多位于不同染色体上,可能与大豆古四倍体的特性有关。4.除CEN4外,大豆PEBP基因均具有4个外显子和3个内含子的保守基因结构。第二个和第三个外显子的长度最为保守,在所有基因间多为62bp和41bp。多数大豆的FT/TFL1基因具备该类基因的典型特征,9个配基结合位点非常保守。5.在洋葱表皮、拟南芥原生质体和大豆幼叶中瞬时表达表明大豆9个FT均定位于细胞质和细胞核中。6.长日条件下,不同纬度来源的大豆品种FTL1/2和FTL3 mRNA表达量与开花时间成正相关,短日条件下的FT的表达量没有显著差别,相对应的开花时间也没有差异。大豆FT类(FTL和TSF)和TFL1类(TFL1和CEN)基因在不同发育时期的表达模式基本相反,大多数FT同源基因在开花期表达量较高,而TFL1同源基因在营养生长的早期表达水平较高;多数大豆FT基因在营养生长阶段的表达呈逐渐升高的趋势,并在开花期达到峰值,表明其可能在大豆的开花调控中起重要的作用。GmFTL1,GmFTL2和GmFTL3过表达拟南芥均导致极端早花表型。PEBP家族基因在不同物种间具有高度的保守性。7.多数FT mRNA在开花期叶柄中高水平表达,比在叶片中表达量高30～50倍,这种表达的组织特异性是在其他植物中尚未见报道,初步推测叶柄在大豆开花调控中具有重要功能。更多还原

【Abstract】 Phosphatidylethanolamine-binding protein(PEBP) gene family members are of important roles in controlling flowering time in plants.As a key member of PEBP gene family,FT(Flowering Locus T) protein or/and mRNA are one of the principal components of florigen.An analysis of soybean PEBP gene family aimed to clarify their definite roles and positions in soybean flowering network may be helpful to elucidate the molecular mechanism underlying flowering control in soybean(Glycine max),a typical short day(SD) crop and a model plant in the early study of photoperiodic flowering.In the present study,19 members of soybean PEBP gene family were cloned and bioinfomatically analyzed. Their spatio-temporal expression profiles across different developmental stages and in various tissues/organs were performed by quantitative real-time reverse transcript PCR(qRT-PCR). Furthermore,functional analysis of three FT-like genes was performed in transgenic Arabidopsis.As the accuracy of qRT-PCR mainly relies on the selection of proper internal reference controls.We perform the gene stability analysis of 14 candidate reference genes in soybean among 116 samples including different developmental stages,various tissues/organs and different photoperiod treatments.The main results were as follows:ⅰACT11,UKN1 and UKN2 were the most stably expressed reference genes among different tissues/organs,whereas SKIP16,UKN1 and MTP showed the most expression stabilities in samples from different developmental stages.In LD and SD photoperiodic treatment,ACT11,TUA5 and TIP41 were the most stably expressed.TIP41,UKN1 and UKN2 showed the most expression stabilities in blue and red light photoperiodic treatment.ACT11,UKN2 and TUB4 performed the best in different cultivars. Generally,SKIP16,UKN1 and UKN2 showed excellent expression stabilities across all the experimental samples.ⅱTo determine the optimal number of genes required for accurate normalization in soybean gene expression studies,a default cut-off value of 0.15 as proposed by geNorm was adopted.For the samples of different cultivars and blue and red photoperiodic treatment,two reference genes are necessary for optimal normalization.For samples of LD and SD photoperiodic treatments,three genes would be necessary and sufficient to achieve accurate normalization.In contrast,for developmental stages and tissue/organ samples,a combination of four genes is recommended for the accurate normalization.ⅲNineteen PEBP genes in soybean were cloned,including 9 FT homologs,4 TFL1 homologs,4 CEN homologs and 1 MFT homolog.They were located on 10 different chromosomes,and FT-like genes seemed proning to parallelly locate in a short section on the same chromosome.In contrast,the different copies of TFL1 and CEN genes were located on the four different chromosomes,respectively. There were 7 pairs of genes which shared high identities of more than 90 percent,and locate on the different chromosomes.This may be due to the characteristics of soybean genome,an ancient tetraploid.ⅳAll the soybean PEBP genes except for CEN4 possessed a conserved exon-intron structure with 4 exons and 3 introns.The second and third exons were most conserved among all the PEBP genes, which is 62bp and 41bp in length,respectively.Most of soybean FT/TFL1 proteins shared almost identical ligand-binding sites with their counterparts in other plants reported.ⅴThe transient expression using particle bombardment in three different systems including onion epidermal cells,Arabidopsis proplast and soybean leaves was employed to investigate the subcellular location of 9 soybean FT homologs.All the analysis reached to the same patterns that all soybean FT homologs were localized in both nuclear and cytoplasm as Arabidopsis FT did.ⅵThe mRNA expression level of GmFTL1/2 and GmFTL3 was positively correlated with the flowering time of different cultivars from different latitudes under long day(LD) conditions.There is no significant differences in FT mRNA expression and flowering time among the different cultivars in short day(SD) conditions.Soybean FT-like genes and TFL1-like genes showed generally opposite expression patterns across different developmental stages.Most of the FT-like genes showed high expression at the anthesis,while TFL1 genes showed high expression at the earlier vegetative stages.The mRNA expression of most of FT genes increased gradually along with the vegetative growth and peaked at the flowering.Overexpression of GmFTL1,GmFTL2 and GmFTL3 in Arabidopsis all resulted in extremely early flowering.These results demonstrated that FT genes may be important in soybean flowering time control and the function of FT was highly conserved in soybean.ⅶThe mRNA expression of a majority of FT homologs was detected with a relatively high abundance in petioles,which was 30～50 folds higher than that in leaves.This indicated that the petiole may function(at least in part) on the floral induction in soybean.更多还原