Gene Cloning And Functional Analysis Of Mutants With Aberrantly Branched Trichomes In Arabidopsis Thaliana

The trichomes in plants have been widely used as a research model for the regulation of plant cell development.In order to explore new components that regulate the development of plant trichomes,we performed large-scale screening in Arabidopsis.In this thesis,we reported the finding of four mutants with aberrantly branched trichomes,in which two of the mutants were single branching.Through map-based cloning and genetic complementation experiments,we proved that the two mutants are new allelic mutants of the STI gene,and both of them are recessive mutations.The STI gene has been reported to be a key factor in the regulation of trichome branching.The expression product of this gene is composed of three functional domains,the largest of which is located in the middle and similar with the sequence of the eukaryotic DNA polymerase III?-subunit.Two PEST domains are located at the N-terminus,and each has a nuclear localization signal(NLS)at the N-terminus and C-terminus of the protein.Our biochemical and molecular assays had demonstrated that the first PEST domain located at the N-terminus plays an important role in regulating the trichome branches of plants.It can interact with BRACHLESS TRICHOME(BLT),a key factor that regulates cell morphology and internal replication.Previous studies have shown that the function of STI in regulating trichome branches is not related to DNA replication,actin,or the polymerization of microtubule cytoskeleton.Our study shows that there are direct-acting domains between STI and BLT.This result reveals that STI has played a potential role in cell morphogenesis.The other mutant we screened had almost no trichome in its rosette leaves.We named it F08-01.Position cloning and genetic complementation experiments confirmed that the mutant gene is a novel allele of GLABROUS1(GL1).The 228-amino acid protein encoded by GL1 is known to belong to the R2R3 MYB transcription factor.Real-time quantitative RT-PCR revealed that the expression of GL1 m RNA was significantly reduced in F08-01,and overexpression of the GLI gene in the F08-01 mutant restored the trichome development defects of F08-01,indicating that F08-01 is a new mutant allelic of GL1.GL2 positively regulates the initiation of trichomes,and serve as the last control elementto trigger trichome initiation.We isolated a mutant that could reverse the gl2-3 trichome developmental defects,and was named M25-01.The trichomes on M25-01 rosette leaves were similar to the wild type.Genetic analysis showed that there may be a semi-dominant mutation site linked to GL2 in M25-01.Molecular analysis of the M25-01 GL2 locus revealed that the T-DNA insertion that caused the gl2-3 mutant phenotype was still exist in M25-01.But the slight expression of GL2 mRNA suggests that the reduced expression of GL2 mRNA makes M25-01 possible reversal of the phenotype of gl2-3 trichomes loss.Our results lay the foundation for the further cloning of the M25-01 gene and the discovery of new factors that may interact with GL2.