Isolation, Expression And Function Analysis Of Floral Organ Identify Genes In Water Lily

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.