Location And Cloning Identification Of Arabidopsis Mutant TRANSPARENT TESTA GLABRA3

As a model plant,Arabidopsis thaliana has been extensively used in studying various mechanisms of plant functions.The research team in which the author has been working obtained a mutant TRANSPARENT TESTA GLABRA3(ttg3)in studying the transgenic Arabidopsis Col-0(Annat2Pro-GUS)and subsequently its homozygote.The mutant ttg3 exhibited some unique traits like late flowering,drought tolerance and longer pods,etc.,of which some are important in researches involving flower industry and crop production.The mutant ttg3 plants differ from the wild-type Col-0 plants with the following features: less or absence of epidermal hairs in leaves,more rosette leaves,delayed flowering time,larger flowers,colorless seed coats,and longer pods.In addition,ttg3 mutants also exhibited higher resistance to salt and drought stresses.A phenotypic analysis showed that the epidermal hairs,flower size,flowering duration and colour of seed coats were all controlled by recessive genes,and their ratio of phenotypic segregation well matched the Mendel's law of inheritance.The ttg3 mutation sites were initially positioned on the chromosome 5 of Arabidopsis thaliana,a region about 5M between molecular markers A21(2,476,150)and NGA139(8,428,133)with the screening of molecular markers by using the map-based cloning method.Following the preliminary positioning results,the the mutant ttg3 genes were sent to a company for sequencing and bioinformatics(Sangon Biotech)for sequencing,and the sequence comparison revealed that the mutant genes were missing in a region of 41 kb between8,358,391-8,400,311 on the chromosome 5,confirmed by designing primers at both ends of the predicted region.Analysis to the positioning interval according to the positioning results and by using the TAIR website(https://www.arabidopsis.org/)published data showed that there were 9candidate genes in the mutation region: AT5G24470,AT5G24480,AT5G24490,AT5G24500,AT5G24510,AT5G24520,AT5G24530,AT5G24540,AT5G24550,among which AT5G24520 is the TTG1 gene that was previously reported,while AT5G24540 and AT5G24550 are homologous genes.Primers were designed according to the sequences of the candidate genes,transformation vectors were then constructed,and genetic transformation was conducted with the Agrobacterium infiltration method.When the transformed line was purified and homologously confirmed,the phenotype of the transformed line was identified,indicating that there were fewer rosette leaves and shorter flowering duration in the transgenic plants,which were complementary to the to ttg3 mutants.Our study may have laid the foundation in illustrating the key role of ttg3 genes in regulating the growth and development of plants.