Construction And Analysis Of Mutants From Telomere-Mediated Chromosomal Truncation And Site-Specific Recombination System In Brassica Napus

Rapeseed is an important oil crop. With the development of genetic breeding in Brassica napus, we have realized that the genetic transformation of endogenous and exogenous genes will improve the germ plasm resource of rapeseed. Ulike conventional gene transformation technologies, plant artificial chromosomes provide one solution to the stable expression of multiple transgenes. Plant artificial chromosome is in its beginning stage. This paper focused on cloning and analysis the sequence near the telomeric repeats, construction of mutants from telomere-mediated chromosomal truncation and site-specific recombination system in Brassica napus (Zhongyou 821). The main results are as follows:The cassette-ligation-mediated PCR approach was utilized for cloning the sequence near the telomeric repeats (CCCTAAA) of Brassica napus. Sequences analysis showed that one sequence was highly homologous to the centromeric satellite DNA sequence reported. FISH revealed that this satellite was located primarily at centromeric regions of most chromosomes, and also at some chromosome ends. The sequence was a subtelomeric satellite DNA which possessed variability in the evolution of chromosomes of Brassica species. The same repetitive sequences appeared in both centromere and subtelomere, which accorded with the hypothesis that the centromeres originated from telomeres. The subtelomeric elements were the residues in the centromeric formation. It is significant for studying the function and evolution of chromosomes to analyze the chromosomal function elements and it also improve understanding of the chromosomes in Brassica napus. These will be useful for chromosomal engineering in Brassica napus.With the telomere-mediated chromosomal truncation technology, we have realized chromosomal truncation in Brassica napus and some phenotypic mutations were gotten. We analyzed the different phenotypic mutations including prior florescence, pollen abortion and so on. The chromosomal truncation will result in pollen abortion. We have confirmed the chromosomal truncation in a mutation 1c8-4 and by means of semi-quantitative PCR technology with SSR primes we located the truncation between MR014 and Na14-H12 in linkage groups N5. The model for the telomere-mediated chromosomal truncation were constructed.We utilized the site-specific recombination system such as Cre/loxP and FLP/Frt for constructing the vector to accomplish gene stacking. We got the vector pCre40 including Cre/loxP system, the vector pT18 including FLP/Frt system and the vector pZl including both the systems. With the agrobacterium-mediated genetic transformation, many transgenic plants were gotten. These lay the foundation for gene stacking through site-specific recombination in artificial chromosome.