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以巴西橡胶“热研7-33-97”古铜期嫩叶为材料,采用酶解去壁低渗法来制备中期染色体标本,及利用显微分离方法随机分离橡胶树单条染色体,分离后的单染色体分别用接头引物介导PCR(LA-PCR)和单细胞全基因组扩增方法进行体外扩增。结果表明:经LA-PCR扩增获得了200~2 500 bp之间的DNA片段,单细胞全基因组扩增后产物大小为300~2 500 bp。对橡胶基因组DNA酶切后制备探针,经Southern杂交证实了扩增产物均来自橡胶树基因组。单细胞全基因组扩增法相比较于LA-PCR,具有操作简单、用时短、扩增片段长、产物适用多个平台等优点,将更适用于单条染色体高通量测序等相关方面的研究。
In this study, medium-term chromosomal specimens were prepared by enzymolysis wall-less osmotic method using the bronze-aged young leaves of Brassica chinensis 7-39-97 , and single chromosomes of Hevea brasiliensis were randomly separated by microscopical separation. Of single chromosomes were amplified in vitro using adapter primer-mediated PCR (LA-PCR) and single cell whole genome amplification. The results showed that a DNA fragment of 200-2 500 bp was amplified by LA-PCR. The size of single-cell whole genome amplified product was 300-2 500 bp. After digesting the genomic DNA of the rubber, the probe was prepared and confirmed by Southern blotting that the amplified product was from the rubber tree genome. Compared with LA-PCR, single-cell genome-wide amplification method is more suitable for single-chromosome high-throughput sequencing and other related researches because of its advantages of simple operation, short time, long amplified fragments and multiple platforms for products.