A Study On The Structure, Expression And Evolution Of Floral Organ Identity Genes CsAP1, CsAP3 And CsSEP3 From Chloranthus Spicatus (Chloranthaceae)

Flowers are reproductive structures that characterize angiosperms. The formation and development of flowers are determined by a complex regulatory system of numerous genes. The evolution of flower can thus be regarded as the evolution of this regulatory system. Consequently, to fully understand the origin and diversification of flowers and angiosperms, one must carefully study the function and evolution of genes in floral development. Chloranthaceae is a representative taxon in basal angiosperms. Compared with the well-studied model plants, such as the eudicot Arabidopsis thaliana and Antirrhinum majus, and the monocot Oryza sativa and Zea mays, plants in the Chloranthaceae have much reduced flowers. Loss of perianth as well as androecium or gyneocium makes some plants (such as species in genera Sarcandra and Chloranthus) produce the simplest bisexual flowers (consisting of only one stamen and one carpel) in angiosperms while others (such as species in genera Ascarina and Hedyosmum) generate the simplest unisexual flowers (only one stamen in male flower and one carpel in female flower) in angiosperms. For this reason, a deep study on the floral genes of Chloranthaceae will not only improve our understanding of the mechanism by which the flowers and angiosperms had originated and diversified, but also help gain insight into the mechanism by which reduced flowers had evolved. In this thesis, six MADS-box genes that are potentially important for the initiation and development of perianth were isolated from the Chloranthus spicatus inflorescences and flowers. Investigated also for these genes are the sequence structure, expression pattern, phylogenetic relationship and evolutionary constraints. Based on these results, mechanisms by which Chloranthus-like flowers were produced and perianths were lost are discussed. The main results are shown as follows: 1. A cDNA library was constructed for the flower of Chloranthus spicatus. This library construction employs SMART kit manufactured by Clontech using LD PCR method and package protein by Stratagene. The liter of the primary library is about 5 × 106 pfu, with recombinant ratio of at least 90%, and the size of most inserts are larger than 0.5 kb. Thus, the library is of good quality and provides a solid basis for future studies. 2. CsAP1, CsAP1a, CsAP1b, CsAP1c, CsAP3 and CsSEP3 genes were isolated from Chloranthus spicatus. Analysis of amino acid sequences reveals that they are all MIKCc-type MADS-box genes. Phylogenetic analysis indicates that CsAP1, CsAP1a, CsAP1b and CsAP1c are clustered with AP1/SQUA-like genes, and CsAP3 and CsSEP3 are clustered with AP3/DEF-like genes and SEP1/2/3/4-like genes, respectively. CsAP1a, CsAP1b and CsAP1c have arisen via gene duplication; however, possibly only CsAP1a is functional since abnormality occurs to the coding sequences of CsAP1b and CsAP1c. Sequence analysis suggested that CsAP1 and CsSEP3 are functional. In CsAP3, however, a point mutation from Guanine (G) to Thymine (T) in the C-terminus may compromise the function of this gene because the protein sequence encoded by this gene contains an incomplete paleoAP3 motif. 3. CsAP1 is expressed in the spike meristems, young inflorescences, young bracts, floral primordia, androecial primordia, and developing stamens. CsAP3 is exclusively expressed in male floral organs. The gene is transcribed in the stamen primordia, developing stamens and thecae. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates in the floral primordia, young bracts, stamen and carpel primordia, thecae, and ovules. The expression pattern of CsAP1 reflects the ancestral role of class A function genes in determining floral meristems; the expression pattern of CsAP3 reveals the innate expression of class B function genes in male organs and reflects their ancestral role in the differentiation between male and female organs; the expression pattern of CsSEP3 reflects the role of class E function genes in providing a "floral context".4. Evolutionary force on known genes in Chloranthus spicatus is also analyzed. With comparison to most close homologues, CsAP1, CsAP3, CsPI and CsAG1 have been undergoing purifying selection with comparable intensities; CsAG2 and CsSEP3 are affected by stronger purifying selection, and CsAP1a may have been under relaxed purifying selection. This result suggests that all genes but CsAP1a may not change function during evolution. 5. Conclusively, genes relevant to perianth development still exist in the perianthless flowers of Chloranthus spicatus, and they have not changed their functions. This suggests that the "ABC" and "quartet" models proposed for eudicots and monocots can also be applied to a basal angiosperm group such as Chloranthaceae. The lack of perianth may be due to more complex mechanisms. The non-sense mutation in CsAP3 may be the consequence of rather than the cause for the loss of perianth.