The Analyses Of The Relationship Between ABA And WUS And Map-based Cloning Of Three Round Leaf Mutant In Arabidopsis

This thesis is mainly divided into two aspects. Part one mainly analyzes the relationship between ABA and WUS in Arabidopsis. Part two is focused on map-based cloning of three round leaf mutants of Arabidopsis. The first part is mainly focus on the regulation mechanisms of WUS gene and ABA signal transduction.Plant stem cells are the development source of plant tissues and organs, which located in the shoot apical meristem (SAM). However, the formation of the WUS-CLV feedback control loop participates in the development of stem cells in SAM and maintains their homeostasis. As an important plant hormone, Abscisic acid (ABA) not only plays a huge role in plant responses to environmental stress, and also is directly involved in most plant growth and development, but there are few reports on the relationship between ABA and the plant stem cells. Relying on several mutants of WUS gene and mutants of ABA signaling pathway, this research is attempting to resolve the regulation relationship between ABA and WUS,, and further to clarify the effects of ABA on the development of SAM in plants.From the results that the over-expressed WUS mutant and loss-of-function mutant wus-1are all shows different ABA sensitivity compared with WT, the expression of WUS gene is proportional to the amount of ABA signal, that we have verified on the molecular level. We detected the gene expression level in ABA signaling pathway of clv3-2mutant and wus-1mutant in response to exogenous ABA.We searched for the direct target genes of WUS in ABA signal pathway through ChIP, and the results suggested that ABI and HAB1are the direct target genes of WUS. Furthermore, instantaneous transformation of protoplasts provided evidence for that WUS can inhibit the expression of ABI and HAB1. Genetic analyses of sef/abil-1double mutant demonstrated that WUS is on the upstream of ABIl. After the measurement of the size change of SAM before and after applying exogenous ABA to sef and Ws, we found that the SAM of sef mutant is bigger than WT without the applying of exogenous ABA because of the overexpression of WUS. However, with the applying of exogenous ABA, the SAM of sef mutant is significantly smaller than WT. This shows that ABA can suppress the development of SAM by inhibiting the expression of WUS.To sum up, we come to the conclusion that WUS can positively regulate ABA signal transduction through the direct repression of ABI1and HAB1. However, ABA signal can feedback regulate the expression of WUS. ABA regulate the growth and development of plants through this mechanism.The second part is mainly focus on the map-based cloning of several round-leaf mutant in Arabidopsis. Leaf is an important vegetative organ and an important aspect of plant morphogenesis, it has been preliminarily elucidated about the molecular regulation network in recent years. But more clear mechanism between each control factor is still not very clear. This study was used map-based cloning combining with the high-throughput sequencing technologies, several round leaf mutants in Arabidopsis as materials, trying to find out new genes controlling morphogenesis, and further to clarify the molecular network during the leaf development.We got three round leaf mutants lry、lcbs and spr through EMS mutagenesis, and cloned genes control leaf morphogenesis based on the three mutants. The main results are as following:(1) The phenotype of the mutants:The three mutants became round at different levels, while lry showed the yellow leaves, plant dwarf and retarded growth; lcbs and spr have the same origin, but lcbs showed curl leaf and spr showed flat blade with shorter petiole.(2) Genetic analysis was carried out on the mutant by hybridism. F1were all showed the same phenotype as the wild type, while character separation happened in F2. This demonstrated that the mutant was recessive.(3) Using the map-based cloning combined with high-throughput sequencing technology, the mutant gene have been successfully cloned in lry, and the gene encodes β subunits of an tryptophan synthase. For that this gene has been studied in detail, we had to abandon it and turn to other two genes. The mutated gene in Icbs was mapped between250Kb on chromosome4and the mutated gene in spr was mapped between1Mb on chromosome1. Both mutants needs more fine mapping to find the mutant gene.