Identification And Genetic Positioning Related To Embryo And Endosperm Development In Arabidopsis Thaliana

As a kind of typical model organism,Arabidopsis plays an important role in investigating embryo and endosperm development mechanisms.The zygote develops into the embryo and the suspensor,while the fertilized central cell contributes to the endosperm which would be eventually degraded.We cloned target genes of mutants related to embryo and endosperm in Arabidopsis thaliana by Genome Walking and Map-based cloning techniques.Genome Walking,also known as Chromosome Walking,is an effective method for cloning target genes of T-DNA insert mutants in Arabidopsis,However,we could not obtained the target genes of Arabidopsis point to mutation by Genome Walking technique.Thus,the map-based cloning was used to clone the mutable sites of genes in Arabidopsis mutants related to embryo and endosperm development.The map-based cloning mainly relies on the linkage of mutant phenotype and gene or chromosomal molecular marker position.The basic principle of map-based cloning is that the functional gene in the genome has a relatively stable locus.Then we constantly narrowed the candidate region with the molecular markers closely linked to the mutable site.Eventually we could obtain the target genes and validated the function of the target genes through genetic transformation and functional complementarity.There is a small genome,high density molecular markers and the whole genome sequences in Arabidopsis,so we could effectively obtain the target genes of Arabidopsis mutants by map-based cloning.In this paper,we sought for the target genes in Arabidopsis mutants which have defects during embryos andendosperm development by using Genome Walking and Map-based cloning techniques.Main results of this work are described as follows:1.The phenotype observation of the T-DNA random insert mutants in Arabidopsis showed that the 097-1 mutant embryos stopped at early torpedo and endosperm nuclei were swollen and scarce.The 241-1 and 275-3 mutants had abnormal embryos at the global stage,but their endosperms were normal.The 241-4 and 578-1 mutants displayed the abnormal embryos from the 2-cell stage and endosperm was swollen and scarce.The 350 and 535-1 mutants displayed the abnormal embryos at the global stage,and endosperm was swollen and scarce.And then,we obtained the target genes of these mutants by Genome Walking,and verified the target genes by genetics,cell biology,and molecular biology methods.The results showed that the phenotypes and genotypes were inconsistent in the 097-1,241-1,241-4,275-3,350,535-1,and 578-1 mutants.Therefore,we eliminated the unnecessary genetic background of these mutants by hybridization with wild type(Ler).Then,we identified the T-DNA insertions and phenotypes of hybrid progeny.The results showed that the plants without T-DNA insertion still appeared the defective embryo and endosperm phenotype.Therefore,we could conclude that the phenotypes of these mutants were not caused by T-DNA insertion,while might be not T-DNA insertion mutants.2.We analyzed the phenotypes and genetic stability of the 241-1,241-4,and 578-1 mutants by the experimental methods such as genetics,cell biology,and molecular biology.The results showed that the phenotypes of the 241-1,241-4,and 578-1 mutants were stable in the progenies of hybridization and backcrossing.The abortion rates were close to 25%and the separation ratios were close to 2:1 in the 241-1,241-4,and 578-1 mutants,which was identified that the 241-1,241-4,and 578-1 mutants were implicit mutations controlled by single genes and were death of homozygous embryos.Thus,we chose BC2F1 generation as genetic mapping group.In the process of hybridization,abnormal male gametophyte plant from the 241-2 mutant was isolated,and the separation ratios were close to 3:1,concluding that the phenotype of the 241-2 mutant was not caused by T-DNA insertion and not T-DNA insertion mutant.Therefore,we could obtain the target genes by map-based cloning.Preliminary positioning analysis indicated that the mutable site of 241-1 mutant was localized between molecular makers T22H22 and R18I in the chromosome 1,the mutable site of 241-2 mutant between molecular makers F7J7 and F17L22 in the chromosome 4,the mutable site of 241-4 mutant between molecular makers MDE13 and MZA15 in the chromosome 5,and the mutable site of 578-1 mutant between molecular makers F13H10 and T1024 in the chromosome 2.Similarly,we analyzed the phenotypes and genetic stability of the 097-1,350 and 535-1 mutants.The results showed that the phenotypes of the 097-1,350 and 535-1 mutants were stable in the processes of hybridization and backcrossing,but the abortion rates and the separation ratios were not stable.Thus we were not sure wheather the phenotypes of 241-4,578-1 and 241-1 mutants are controlled by single gene,and it remains to be further research.