【作者】 罗火林；

【导师】 陈发棣；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2011， 博士

【摘要】 睡莲(Nymphaea spp.)又称水百合,为睡莲科睡莲属多年生宿根性水生植物,其花色艳丽,姿态优美,又能净化水体,具有很高的观赏价值和生态价值。同时,睡莲位于现存被子植物系统树的根部附近位置,被称为ANITA (Amborella- Nymphaeales-Illiciales-Trime-Niaceae-Austrobaileya)类群的成员之一,这引起了植物系统进化领域学者的研究兴趣。而花是植物中包含进化信息最多的器官,因此,对睡莲花器官发育的研究就显得格外重要。但是,睡莲花器官发育的研究刚刚起步,本研究在克隆两个花器官发育关键基因的基础上,通过其在拟南芥中超量表达研究其功能。此外,比较了6个花器官发育基因在两份材料(正常睡莲和一个萼片变成叶子的睡莲)的花器官中表达水平的差异,主要结果如下：1.内参基因的表达稳定性评价以睡莲‘黄公主’Nymphaea’Yellow Prince’)为植物材料,首先通过简并PCR,亚克隆测序以及生物信息学分析,获得5条睡莲内参基因的mRNA序列。然后利用qRT-PCR技术结合生物学软件(geNorm、NormFinder和Lin-RegPCR)评价了8个常用的内参基因(在NCBI数据库中搜到3条睡莲内参基因序列)在睡莲不同组织中以及激素处理和非生物胁迫条件下的表达稳定性。非生物胁迫包括低温、高温、盐、干旱、重金属胁迫等,不同组织包括萼片、花瓣、雄蕊、雌蕊、根、茎和叶,激素处理包括脱落酸(ABA)、赤霉素(GA)和萘乙酸(NAA)处理。我们将得到的荧光定量PCR数据分别用geNorm和NormFinder分析,以便筛选出合适的内参基因。比较geNorm和NormFinder两个软件的分析结果,发现两个软件的分析结果基本相同,只是在不同处理的根中,出现了一些不一致。进一步分析表明,这是由于UBQ11和UBC16存在基因共调节作用而引起的。综合两个软件的分析结果表明,在涉及到多个处理和组织器官时,内参基因AP47和ACT11表达最稳定；在不同组织中,EF1a和ACT11最适合作内参基因；AP47和ACT11在不同处理的根中表达最稳定；而在不同处理下的叶片中,UBC16和ACT11作为内参基因最为可靠。2.AGL6基因的克隆和功能分析根据AGL6同源基因在氨基酸序列上的保守性,设计简并引物,以睡莲‘黄公主’花蕾总RNA为模板,进行RT-PCR扩增,然后亚克隆测序,得到了睡莲AGL6基因的中间片段序列。然后再根据已知序列设计引物进行3’和5’RACE,最后将中间片段、3’末端序列和5’末端序列拼接,得到基因全长。生物信息学分析表明该基因全长927bp,包含一个完整的开放阅读框,编码244个氨基酸,含有保守性很强的MADS结构域和K-结构域,属于AGL6/AGL13亚家族,命名为NsAGL6。qRT-PCR分析表明该基因在所有组织器官中都有表达,在花瓣、萼片和茎尖中表达水平较高,在叶片和根中最低。瞬时表达试验发现,NsAGL6编码的蛋白定位在细胞核上,而酵母单杂交试验表明该基因编码的蛋白单独作用时,不具有转录激活活性。为研究该基因功能,我们将基因的编码框完整地插入到双元载体1301-220中,再通过农杆菌介导,将构建好的表达载体转化拟南芥。经过抗性培养基筛选,GUS染色,RT-PCR检测,鉴定出23个拟南芥转基因株系,转基因T3代用于功能分析。我们发现,无论在长日照还是短日照条件下,转基因植株的花期明显提前,对6个拟南芥开花基因表达的定量分析表明,FT,LFY等多个开花促进因子表达量上升,而负调节因子FLC表达量下降。此外,NsAGL6超表达使拟南芥产生出更多的侧生分枝,对拟南芥分枝相关基因的荧光定量试验发现部分生长素合成与运输相关的基因表达量下降,而且MAX1和MAX4表达量也下降,这暗示着分枝性的提高可能与生长素合成与运输受到影响有关,此外,与MAX调控途径也有关。3.AP2基因的克隆和功能分析利用同样的方法从睡莲中分离出一个AP2同源基因NsAP2,全长1843bp,编码450个氨基酸。序列比对表明该基因含有两个AP2/DREB结构域(AP2/DREB Domain),系统发育分析显示NsAP2属于euAP2亚家族,且与银杏、小买麻藤、云杉等较为原始的植物聚在一个分支,表明睡莲在植物系统演化中的基础地位。在自然生长的条件下,该基因在所有组织器官中都有表达,但是表达量不一,在花瓣、萼片中表达量最高,而在其他器官中,诸如茎尖、根、叶、雄蕊、心皮中表达量相差不是很大。RNA原位杂交试验表明,该基因在花芽分化进程中的所有时期和区域都有表达,但在新分化的器官原基中表达量比较高。瞬时表达试验发现,NsAP2编码的蛋白定位在细胞核上,而酵母单杂交试验表明该基因编码的蛋白单独作用,不具有转录激活活性。同样地,在农杆菌介导下,将该基因转化到拟南芥中,经过抗性培养基筛选,GUS染色,RT-PCR检测,鉴定出15个拟南芥转基因株系,转基因T3代用于功能分析。我们发现,在35S启动子的驱动下,NsAP2在拟南芥中超表达不仅改变拟南芥生殖生长,而且改变拟南芥的营养生长。该基因的过量表达使拟南芥花瓣增多,成了重瓣花,这是AP2基因超表达的典型性状。此外,转基因植株变高。经荧光定量分析发现,赤霉素氧化酶基因GA2ox2和GA2ox7的表达量与野生型拟南芥相比,明显下降,这可能是导致转基因拟南芥植株变高的原因。4.睡莲花器官基因表达模式的研究我们得到一个睡莲变异的材料一睡莲‘蟹爪兰’,其萼片变成叶片状。通过石蜡切片发现‘蟹爪兰’的萼片纵切面具有明显的栅栏组织,该组织结构一般只在叶片中才有。根据6个花器官发育基因AGL6, AP2, AP3, PI,AG,SEP的序列(后4个基因序列来自NCBI)设计引物,利用qRT-PCR和半定量分析这6个基因在正常睡莲和‘蟹爪兰’花中的表达情况。因为睡莲花器官存在过渡类型,如瓣化的雄蕊,我们将睡莲花器官从外到内分为8轮。首先,对比6个基因在两个材料中的表达发现,AP2,AGL6在正常睡莲萼片中表达量很高,但是这两个基因在‘蟹爪兰’的萼片中表达量在所有的花器官中最低,说明这两个基因可能行使A类基因功能。此外,本研究发现,在正常的睡莲材料中,6个基因在所有的样品中都有表达,并且表达量从外轮到内轮呈现梯度变化,AP2和AGL6在外轮花被片中表达量很高,从外到内,表达量逐渐降低；AP3和PI表达量从外轮花瓣到内轮雄蕊,先下降,后上升,在内轮雄蕊中表达量最高,在最外轮(萼片)和最内轮(心皮)的表达量都比较低；AG基因的表达水平从外轮花被片到内轮雄蕊,表达量逐渐上升,而在心皮中表达量偏低；SEP在所有的花器官中都有表达,且表达的差异性不如其他基因显著。这种梯度的变化表明睡莲花器官发育更符合边界衰减模型,在此,我们综合了前人的研究成果,提出了睡莲花器官发育的假定模型。更多还原

【Abstract】 Nymphaea spp., also known as water lily, is a perennial aquatic flower, and it has high ornamental values due to its colorful flower and good plant architecture. In addition, water lily is economically significant a vegetable and as a source of medicinal compounds, and it is believed to be rather primitive in the evolution of the angiosperms, and thus represents an interesting model for many angiosperm families. The flower is the characteristic feature of the angiosperms, and its evolutionary origin and subsequent diversification are an intriguing topic of botanical research, therefore, it’s important to study the floral organ development in water lily. In this study, two floral organ identify gene (AP2 and AGL6) in water lily was isolated by degenerate PCR and RACE, and its gene function was studied by overexpression in Arabidopsis. Furthermore, to investigate the expression pattern of floral organ identity genes in water lily, six conventional genes was selected. AP3, PI, SEP, AG homologues were available in NCBI database, and expression profile of the six primary floral organ identity genes AP2, AGL6, AP3, PI, AG and SEP in two cultivars showing contrasting floral morphology was determined by quantitative real-time RT-PCR and Semi-quantitative RT-PCR. The main results are as follows:1. Candidate reference genes for gene expression studies in water lilyThe selection of appropriate reference gene(s) is a pre-requisite for the proper interpretation of quantitative real-time polymerase chain reaction data. We report here the evaluation in water lily of eight candidate reference genes, chosen on the basis that they have been employed for this purpose in other plant systems, and Nymphaea’Yellow Prince’ was used throughout. The stability of their expression was tested in various tissues and under various treatments, and was analysed by the three software packages geNorm, NormFinder and BestKeeper. Across all samples, the genes AP47 and ACT11 emerged as the most suitable reference genes. Across tissues, EF1a also exhibited a stable expression pattern in addition to ACT11 and AP47. ACT11 and AP47 were stably expressed in roots subjected to various treatments, but in the leaves of the same plants, the most stably expressed genes were UBC16 and ACT11. IF1 and RPS1 were the least stable genes tested in water lily. The study created a precedent in qPCR analysis dealed with water lily, and the selection of reliable references genes across different tissues and treatments will facilitate qRT-PCR analysis in various research fields on water lily.2. The isolation and function analysis of AGL6 in water lilyThe degenerate primers were design according to the conservative block of the AGL6 homologous proteins, and the AGL6 of water lily was isolated by degenerate PCR and RACE techniques from Nymphaea ’Yellow Prince’. The full length cDNAs of the water lily AGL6, designated NsAGL6 (Accession number:AB495345), were of length 927bp, including a single ORF 732bp, encoding 243 residues. The deduced NsAGL6 polypeptide contained both the conserved MADS- and K-domain, belong to MADS box gene family. MADS-box gene family, involved in floral organ identity, has been divided into 12 subfamilies:AGL6/AGL13, AP1/SQUA, SEPALLATA, AP3/PI/DEF/GLO, AGAMOUS etc., and the phylogenetic analysis placed NsAGL6 within the AGL6/AGL13 lineage.qRT-PCR analysis showed NsAGL6 were expressed throughout all organs at different level, and the transcripts in sepal and petal was the most abundant, and the next was that in shoot apex. NsAGL6 has the typical characters of MADS genes, and it was localized to the nucleus by transient expression assay in onion epidermis cells. Yeast one-hybrid analysis indicated the NsAGL6 did not possess transcriptional activation activity. In order to examine NsAGL6 functions, we produced transgenic Arabidopsis ecotype Columbia plants carrying the coding region of NsAGL6 driven by the CaMV 35S promoter. All transgenic lines showed an early flowering phenotype by sublimating the flowering genes. Furthermore, we found that branching of axillary shoots was more pronounced in the overexpression lines, and qRT-PCR analysis showed the shoot-branching regulated genes CYP79B2, DFL1, PIN1, MAX1 and MAX4 were down-regulated.3. The isolation and function analysis of AP2 in water lilyThe AP2 homologous was isolated by degenerate PCR and RACE techniques from Nymphaea ’Yellow Prince’. The full length cDNAs of the water lily AP2, designated NsAP2 (Accession number:AB495343), were of length 1843bp, which contain a single 1350bp ORF, encoding polypeptides consisting of 449 residues. The deduced NsAP2 polypeptide contained two characteristic AP2 domains, and the phylogenetic analysis placed NsAP2 within the euAP2 lineage.It was showed NsAP2 were expressed throughout all organs at different level, and the mRNA level in sepal and petal was the most abundant by qRT-PCR. In addition, the domain of NsAP2 gene transcripts spreads from the floral meristem to include the sepal primordial at early stage, and then expanded to include the petal, stamen, and carpel primordial by in situ RNA hybridization analysis. NsAP2 was a typical AP2/DREB transcription factor, and it was localized to the nucleus. Yeast one-hybrid analysis indicated the NsAP2 did not possess transcriptional activation activity. In the overexpression lines, the plants produced an excess of petal, and the shape of petal was a litter different from that of wild type. In addition, the plant height of transgenic lines was higher than that of WT, mainly correlated with the inhibition of GA2ox2 and GA2ox7.4. The expression of floral organ identity genes in contrasting water lilyThe floral organs of typical eudicots such as Arabidopsis thaliana are arranged in four characteristic whorls, namely the sepal, petal, stamen and carpel, and the "ABC" floral organ identity model has been based on this arrangement. However, the floral organs in most basal angiosperms are spirally arranged with a gradual transition from the inside to outside, and an alternative model referred to as "fading borders" was developed to take account of this. The flower morphology of the water lily was tested against the "fading borders" model by determining the expression profile of the six primary floral organ identity genes AP2, AGL6, AP3, PI, AG and SEP in two cultivars with contrasting floral morphology(Nymphaea ’Tina’ and Nymphaea ’Nang Kwag’). In addition, to get accurate floatation of the genes expression level from outer to inner, we divided the floral organs into eight whorls according to morphological features. All these genes were expressed throughout the flower, but their expression level changed gradually from the outside of the flower to its inside. This pattern was consistent with the "fading borders" model.更多还原