Effects Of Overexpressing Cell Cycle Genes On The Growth And Development Of Rapeseed And Functional Analysis Of The Seed Development Essential Gene ASD In Arabidopsis

Rapeseed (Brassica napus L.) is one of the world’s important oil crops and plays an important role in the future edible oil markets in China. Previously, it is documented that cell cycle genes can regulate the cell growth and development process, and the complexes of CYCD3;1, CYCD2;1and CDKA;1can promote the transit from G1phage to S phage. However, it is unclear how to efficiently improve the quality of rapeseed by overexpressing these genes. In this study, one of the purposes is to investigate the effects on the development and growth of transgenic rapeseed lines through over-expression of three cell cycle genes in rapeseed, and to provide efficient theoretical basis and technologic support for genetic improvement.Screened within different transformation methods, one efficient program was adopted and a lot of transgenic lines of cell cycle genes were obtained through this transformational method. The transcription products were truncated mRNA in AtCYCD2;1(cDNA) overpression lines while the transcription products of genomic CYCD2;1OE lines were normal. The overexpression level of the gene was associated with the copy number of the T-DNA insertion in CYCgD2;1transgenic lines. In CYCgD2;1transgenic plants, the process of G1-S was promoted, but the flowering stage was delayed. In addition, the amount of vegetative growth was also decreased in OE lines. In CYCgD2;1transgenic plants, the polarity of leaf growth was affected, resulting in crinkle and curl leaf. Cells of epidermal layer in leaf became smaller, and did not develop fully. However, in transgenic lines of AtCYCD3;1and AtCDKA;1, no overpression phenotype was observed clearlyIn this study, in order to investigate the genetic mechanism of seed development in dicotyledons, we also carried out a mutant gene (asd) mapping and functional analysis in Arabidopsis thaliana. Genetic analysis showed that it was an embryo-lethal mutant caused by a point mutation, not T-DNA insertion. By mapping-based cloning, the mutant was anchored to the63-KB region of upper arm on Chromosome I. By investigating the seed phenotype in SAKL T-DNA insertion mutants in genes of the63-KB regions, we found that T-DNA insertion mutants in Atlg35680showed the same phenotype as that of asd mutant. It was consistent with the result of sequencing its ORF:there was a piont mutation (A→C) in the982th of Atlg35680, resulting in a change from T (Thr) to P (proline) at the147th aa of ASD protein. Allele test with other two T-DNA insertion lines in Atlg35680and a complementary experiment demonstrated that piont mutation in At1g35680was responsible for the aborted phenotype. Further analyzating the cause of the phenotype, we found that the development of the thylakoids in chloroplasts were inhibited in embryogenesis, resulting in underdevelopment chloroplasts, eventually the development of embryo was blocked in the global stage in siliques. It is the first time indicating that ASD gene is essential to chloroplast biogenesis and embryogenesis.