Studies on Extracting Mitochondrial DNA and Gene Expressions in Brassica Napus L
|School||Nanjing Agricultural College|
|Keywords||Brassica napus mitochondrial DNA extraction and purification gene expression|
Plenty of researches had indicated that CMS (Cytoplasmic Male Sterility, CMS) is related to mitochondrial DNA (mtDNA). In this study, technology system for extracting and purifying mitochondrial DNA is established, and expressions of 16 mitochondrial genes and 1 gene frgment orf141 were detected for illustrating molecular mechanism of the CMS in Brassica napus. The results are as follows:(1) Establishment of a technical system for extracting highly purified mitochondrial DNA:A higher efficiency and better technology system for extracting mtDNA was established and optimized by comparing with yield and quality of mtDNA from different extracting programmes. Optimized experimental procdure is described as:Fresh etiolated seedlings from 7 days-old were cut into small pieces, rapidly broken up. The extract was filtered through one layer of cheesecloth and four layers of 100 mesh nylon gauze and centrifuged at 3,000 rpm for 15min. The supernatant was centrifuged at 17,000g for 15 min. Pellet was collected and then resuspended in a suspension buffer, centrifuged at 3,000 rpm for 10min. Supernatant was took and DNase I was added in. Restriction enzyme digestion incubated at room temperature for 30 min, then keep in 4℃for 1h, it was terminate by the adding of EDTA. The suspension was centrifuged at 18.000g for 20min, crude mitochondrial pellet was accumulated. The pellet was resuspended and layered on a discontinuity step gradient consisting of 1.45M and 1.2M sucrose solutions. The gradient was centrifuged at 72.000g for 90min. Purified mitochondria were removed from the 1.45M-1.2M interphase, diluted, mixed and then centrifuged at 20.000g for 20 min. The pellet was highly purified mitochondria. Lysed with improved SDS-Proteinase K alkaline method, high purified mtDNA was separated. Determination of mitochondrial DNA concentration and purity showed the mtDNA was highly purified and was of good-quality.(2) Gene expressions of mtDNA from Brassica napus cytoplasmic male sterile lines:The CMS line H526A used in this research were developed from protoplast fusion hybrid between Brassic napus L. and Descurainia Sophia. To research the CMS mechanism, total RNAs were extracted from flowers and buds of the CMS line H526A and the corresponding fertile hybridized combination F1, subsequently were transcribed into cDNA. The results of realtime PCR are as follow:In buds, the expressions of orf222 and orfl41 were obviously higher in CMS line than that in its fertile F1:the expression of cox2-1 was slightly higher than that in fertile F1; while all other genes were lower. Orf222 was predicted as a CMS candidate gene, and cox2-1 may play an important role in CMS. There were five genes orf222. cox2-1, cob, rps7. atpl and orfl41 whose expressions in CMS were distinctive differences in buds and flowers, while other genes were found with no significant expressions differences.