The Screening And Identification Of Telomere-mediated Artifical Minichromosoms In Brassica Napus

This study was based on the privious study on mini-chromosomes of Brassica napus var. Westar in Hu Zanmin’s research group, IGDB, CAS. The plants with the artificial mini-chromosomes were identified by FISH and PCR. The plants with only one pair artificial mini-chromesomes was screened and obtained by selfing and/or hybridization. It was also investigated if the foreign genes could be inserted into the minichromosome of Brassica napus. This study could provide a new technical approach and a new material for the research on molecular breeding, and the strcture and function of chromosomes in Brassica napus. The main results were listed below.1. The expanding of the library of the transgenic plants through selfing and/or hybridization between pWY86-5-73 and pWY86-5-75 transgenic lines which both have telomere-mediated chromosomal truncation. These two transgenic lines was screened and stably inherited plants which have artificial mini-chromosomes was got in this process.;2. All pWY86-5 each generation transgenic plants were screened by spraying of herbicides and PCR methods according to the characteristics pWY86-5 plasmid constructs. The positive plants were bred and with repeat this process we got the transgenic lines which have a specific number of mini-chromosomes that was expected..3. The mini-chromosomes in pWY86-5 transgenic lines’ each generations which was identified as positive plants by PCR method was observed through FISH. The probe which is made by pWY96 structure was hybridized with the bud which was picking from the pWY86-5 transgenic lines;4. The karyotyping for transgenic lines which have artificial mini-chromosomes was initially completed by using BAC clone L17 from the BAC library of Brassica rapa as a probe, hybridized with pWY86-5-73 transgenic lines and pWY86-5-75 transgenic lines which both have chromosome truncation together with the probe 45 S which is the probe of telomeric repeats and the probe 5S which is the probe of centromere repeats;5. Transgenic lines of cre1301 was got by the transformation of vectors with cre-lox site-specific recombination box to Westar and the continue cultivate, provided support to verify the integration of exogenous genes to the material of B. napus mini-chromosome.