Cloning And Differential Expression Of Genes Related To Cytoplasmic Male Sterility In Chinese Cabbage

Cytoplasmic male sterility (CMS) has been an ideal system to produce hybrid seed of Chinese cabbage (Brassica campestris syn. rapa L. ssp. pekinensis). In order to elucidate the molecular basis of CMS in Chinese cabbage, some molecular biology protocols were developed, and the genes related to CMS were cloned and its different expression were investigated. The main results were as follows:1.Established a proper method of mitochondrial DNA (mtDNA)extraction from Chinese cabbage. The mitochondria was extracted from the inner leaves of CMS lines and their maintainer lines by using differential centrifugation and sucrose shelf method, then the mtDNA was extracted by SDS-Proteinase K method. Compared with other methods, this protocol spent less time and money, and the extracted mtDNA had good quality for further research.2.Established an effective and quick protocol for total RNA isolation from flower bud of Chinese cabbage. Which is that based on the acidic phenol - guanidine thiocyanate - chloroform RNA isolation method, the secondary extraction was omitted and one more step of ultracenentrifuging was added. Compared with other methods, the modified protocol had higher efficiency and spent less time. The extracted RNA had good quality and was used successfully in reverse transcription and cDNA-RAPD.3.Based on the nucleotide sequence of specific fragment orf224 associated with"Polima"CMS in Brassica napus, two primers were synthesized and applied to polymerase chain reaction (PCR) with the templates of mitochondrial DNA of new CMS line CMS7311 and its maintainer line 7311 of Chinese cabbage. A special amplified fragment obtained only in CMS7311 and was cloned into pMD18-T vector. Sequencing analysis revealed that this fragment had no variation compared with Polima-orf224 reported, which means CMS7311 and Polima CMS probably have a similar sterile mechanism and the variation of CMS in Brssica napus is conservative.