Analysis Of The Phenotype And Molecular Mechanism Of Abnormal Flower Development Line(AFDL) In Arabidopsis Thaliana

Based on the studies of homeotic flower mutants in Arabidopsis, Antirrhinum and Petunia, we realized the flower development is under the control of a complex genetic network. The molecular mechanisms of the flower induction and the flower development become the core and hotspot in plant development biology. A-class gene APETALA1(AP1) is not only a floral identy gene but also a floral meristem identity gene, playing a vital role in the regulation of flower organ identity, development and the morphology of the inflorescence. In this study, we use crossing, RT-PCR, complementary experiment, histochemical GUS assay and microarray to study the regulation mechanisms of the AP1 expression in the ABNORMAL FLOWER DEVELOPMENT LINE(AFDL) of Arabidopsis thaliana. The major findings are described as followings:1. The genetic analysis of AFDL showed that AFDL is absent of petals and with a stacked inflorescence meristems, but only in rare cases showed a conjoined or caespitose stems. The crossing between AFDL and wild-type, ap1-5, ap1-8 or ap2-1 was carried, and the phenotype of th F1 population and the segregation in the F2 plants suggested AFDL is affected by a recessive gene. We analysis the genes expression that are related to AP1 on the flower pathway, the result showed the expression of the genes downstream of AP1 are correspond to the declined AP1, the decreased expression of AP1 in AFDL is resulted from an unknown pathway.2. We cloned the-3579 bp promoter and the code region of the AP1 with the genomic DNA and cDNA of AFDL and wild-type, the result of sequence analysis showed there are no different occurs at the level of nucleotide sequence. Thus we consider that the declined AP1 expression was due to the transcriptional regulation. The analysis of the promoter indicated some cis-elements responed to the stress and stimulation from environment, for instance, there are three CArG boxes located in-2999 bp,-1752 bp and-18 bp upstream the transcriptional sites that are the binding sites for MADS domain proteins.3. The AFDL was used as the material to analysis the function of CArG box in the regulation of AP1. We construct various forms of AP1 promoter to drive the expression of AP1 in AFDL, which were constructed in pCAMBIA1304 with 35S::AP1,-1752 CArG1+2::AP1,-1752 CArG1::AP1,-1379 CArG2::AP1 and-1379 △CArG::AP1. Based on the distribution of the extent of the recovered phenotypes of transgenic plants, we consided that the CArG box1 and CArG box2 are the binding sites of the repressor and they play a negative role in the regulation. In addition, CArG box1 and CArG box2 are not essential for the expression of AP1. Five constructs of pCAMBIA1304-pAP1::GUS were obtained to analysis the effect of CAr G box on the expression pattern. Histochemical GUS activity assay showed that the CArG box 1 and CArG box 2 are non-functional on the AP1 expression pattern, but there are unidentifired elements that play a rloe on the expression specificity outside the-1752 bp upstream of the transcriptioanl start site. Therefore, while the GUS staining showed in carples, silique and pod in the absence of these elements.4. Microarray analysis of AFDL vs widetype showed that the expression of some genes in the abnormal flower development line are changed during the vegetative growth. These genes mostly involved in the process of detect and responed to the light and circadian rhythm, temperature, water and salt or the stimulation of endogenous hormones, and the metabolism of carbohydrate as glucan and starch; The level of other genes expressed in the inflorescence was changed during the reproductive growth, being involved in the biological process of flower development, meristem maintenance and differentiation, etc.. AFDL is a valuable material to analyze the molecular regulatory network of floral induction and development of flower.5. A few of genes in AFDL seedling was more than fold-change≥2(P<0.01). We chosen 18 genes to analyze their functions on the flower development. Among these genes we found the transgenic plants with AT1G56300 and AT1G65490 showed a phenotype of multi-stems stacked or caespitose stems. In addition, the development of the euphylla and the root are postponed, the number of lateral root is increased, the length of root and the amount of root hair are reduced. Genes expression in these transgenic plants are measured by RT-qPCR, the level of genes in different line is coincidenced with their phenotype. We presume the two genes are relative to the development and differentiation of the meristem in embryo stage. This also suggests there may be a relationship between the flower development and the vascular development.