The Mechanisms Of Fertility Changeover In Thermo-Sensitive TsCMS7311 Line Of Chinese Cabbage

The thermo-sensitive male sterile of Chinese cabbage (Brassica campestris L. ssp. Pekinensis) play an extremely important role in heterosis utilization, but the mechanism of temperature regulating fertility is still not clear. The technologies of cDNA-AFLP and the two-dimensional GEL electrophoresis of protein were used to research the mechanism of temperature regulating fertility transform at two levels of transcription and expression respectively. The differences of gene transcript in little buds (little bud≤1.0㎜, including apical meristem) and gene express in little buds (little bud≤1.5㎜) were continuously analyzed. The main results are as follows:1. A cDNA-AFLP (cDNA Amplified Fragment Length Polymorphism) technology was established, which was suitable for analyzing the gene differentially-expression of Chinese cabbage. The RNA extracted from small buds (≤1.0㎜, including apical meristem ) was used to synthesize 1st Strand cDNA with PrimeScriptTM 1st Strand cDNA Synthesis Kit. The 2st Strand cDNA was synthesized with the suitable proportion of DNA polymeraseⅠand RNase H and the appropriate reaction time, and then to carry on the AFLP analysis. In a word, the technology had lots merits. Such as stable zymograms, high quality 2st Strand cDNA, low false positive, and few RNA template.2. The plants of TsCMS7311 with flower buds were treated under low temperature first, then the RNA was extracted from small buds (≤1.0㎜, including apical meristem ) of them in 10 continues days , and were analyzed with cDNA-AFLP. The results showed that 212 differentially-expressed fragments were obtained with 256 primer pairs, and which was divided into two species and four zymogram types as the day going. In which, one mainly species was inducting produce zymogram types, including type"absent→present→absent"and type"absent→present", totalled 175 fragments. The other species was inducting silence zymogram types, including type"present→absent→present"and type"present→absent", totalled 175 fragments.3. 100 ESTs with 100-700bp length were obtained by recovered, cloned and sequenced. 34 ESTs of these 100 ESTs were highly homologous (>62%) with the known proteins of GeneBank. By Blastx search to NCBI dbEST, it was found that the low temperature inducing fertility-changeover was related to many proteins, such as metabolism, energy, defense, cell construction, protein synthesis and transport, transcription and translation, Cellular communication, signal transduction mechanism correlative protein and so on, which maybe form a network. 49 ESTs of these 100 ESTs were highly homologous (>90%) with the known ESTs of GeneBank, in which 47 ESTs comes from Brassica campestris. The other 17 ESTs had not homologous sequences of GeneBank. As implied that they were new gene fragment. Three ESTs with length longer 150bp were submitted to GeneBank. They were: EST80: GenBank_Accn FE905220, dbEST_Id 54764252; EST182: GenBank_Accn FE905221, dbEST_Id 54764253; EST162: GenBank_Accn FE905222, dbEST_Id 54764254.4. The EST-58, EST-114, EST-188 were amplified by In silico cloning and the RT-PCR was used to confirm the authenticity. Two new genes of Chinese cabbage were obtained, which are xyloglucan endotransglycosylase (XET) gene (GenBank_Accn is EU579461) and PAS (PASTICCINO) gene (GenBank_Accn is EU650783). And one new sequence (GenBank_Accn is FE905223; dbEST_Id is 54764255) full length is 877bp, come from EST-188. The new sequence come from EST-58, whose full length is 1196bp, contained a complete ORF, encoding 292 animo acids. The confirm result of RT-PCR is identity to it with 98%. Homology analyse showed that it was the Chinese cabbage gene of XET. The new sequence come from EST-114, whose full length is 1036bp, contained a complete ORF, encoding 229 animo acids. The confirm result of RT-PCR is identity to it with 98%. Homology analyse showed that it was the Chinese cabbage gene of PAS. The new sequence comes from EST-188, whose full length is 877bp. The confirm result of RT-PCR is identity to it with 96%. Whose ORF was not complete. So it was submitted to GeneBank as EST.5. Established an effective method ofβ-EST recycling and purification from Chinese cabbage. Namely, riched the goalβ-EST with one-dimensional active GEL electrophoresis and cutting goal bands technology, and further purified with self-made two-dimensional GEL electrophoresis. The one-dimensional active GEL was only dyed two margins in order to denote the goal band area middle to reclaim. The reclaimed bands were made up to rude powder by electroelution and freeze-dry respectively, and then were further separated by two-dimensional GEL electrophoresis. In the two-dimensional GEL electrophoresis, the first dimensional IEF was optimized using vertical slab made in China, which is economy and effective. After IEF,the GEL was dyed and cut the goal bands. These goal bands were separated by the second dimensional SDS GEL further. This method reduced the non-goal bands disturbance, and made the analysis results more reliable.6. Obtained the partial amino acid sequences of the specialβ-EST poly-peptides. By the separation and purification of two-dimensional GEL electrophoresis, 6 kinds poly-peptides related to fertility changeover were obtained. The poly-peptides with molecular weight 57.1KD and 62KD were analyzed by Q-TOF mass spectrum. We obtained three sequences of short peptides from 57.1KD poly-peptide and two sequences of short peptides from 62KD poly-peptide, and the two short peptides sequences of 62KD poly-peptide are same as that from the 57.1KD poly-peptide. The result indicates that the sequences of the two polypeptides might have high similarity.7. By comparing the sequence with protein database of GeneBank, it was found that the three short peptides obtained were highly homologous (60-100%) with protein p27SJ.