Comparative Research On The Morphology And Biochemical Characteristics And Cytoplast Germ Plasm Of Polima CMS And Shaan 2A CMS In Brassica Napus L

CMS is one of the important approaches in heterosis utilization in rapeseed. Polima CMS and Shaan2A CMS are two of the classic and effective CMS lines in Brassica napus L, and these two famous CMS lines have played very important roles in study and utilization of rapeseed heterosis in China even in the world. In early years, study on the two CMS lines was mainly focus on the aspects of planting, genetics as well as utilization of heterosis. Whereas in recent years, study on them was mainly concentrated on the respects of sterility mechanism and molecular biology. The further study on Polima CMS and Shaan2A CMS will have an important significance to probe into molecular mechanism of CMS and use the two CMS lines to service for scientific research and production in rapeseed. This paper summarized the study states of Polima CMS and Shaan2A CMS at home and abroad, introduced the new research progress on the two CMS lines in rapeseed in recent years, systemically researched the two CMS lines on the aspects of morphology and anatomy, physiology and biochemistry, and molecular biology, and afforded the information and basis to further research the two CMS lines in rapeseed. The main results were as follow(for convenience to describe, Polima CMS and Shaan 2A CMS in paper were shortened as PolA and Shaan2A, and their maintainer lines were shortened correspondly as PolB and Shaan 2B):1. Studying on the morphology characteristics such as agronomic traits of plants and modality and structure of flowers in PolA system(PolA, PolB) and Shaan2A system (Shaan2A, Shaan2B), their nuclear-cytoplasm exchanging materials, and their nuclear substitution materials, respectively, the results showed that seedling modality, flower configuration and plant agronomic traits were obviously different between Shaan2A system and PolA system, indicating that the two CMS systems had significantly different genetic backgrounds. In conditions of nuclear-cytoplasm exchanging and nuclear substitution, the two CMS lines with the materials of their same nuclear but different cytoplasm had not significant difference in seedling modality, flower configuration and plant agronomic traits, however the two CMS lines with the materials of their same cytoplasm but different nuclear had obvious difference, indicating that nuclear genome played a decisive role in plant's surface traits.2. The polyacrylamide vertical board gel electrophoresis of Esterase(EST) and Peroxidase(POD) isozymograms were carried through the leaf blade, petiole as well as flower bud organization of 4 materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B in the early flowering season in Brassica napus L. The results showed that between the two CMS lines and their corresponding maintainer lines, EST isozymograms of leaves and petioles had not distinct difference, respectively, but EST isozymograms of flower buds had some. And there were obvious distinctions between EST isozymograms of the two CMS lines, PolA and Shaan2A. The results of POD isozymograms in leaves and petioles,respectively, were not obvious in diversity between the two CMS lines and their corresponding maintainer lines, but the results of POD isozymograms in flower buds had clear difference, and there were obvious distinctions between POD isozymograms of the two CMS lines. Therefore, the two CMS lines had different genetic background.3. Chloroplast DNA(cpDNA) was extracted by seedling leaves from four materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B, and several special gene fragments of cpDNA were amplified by PCR. The result was that the same one target band was found in horizontal submarine agarose gels when 5 pairs of the special gene primers of cpDNA were used respectively to the 4 materials in rapeseed, and amplified products were consistent with expected target fragments. Then cloned and sequenced the RubisCO large subunit(rbcL) gene which was related to photosynthesis. The result showed that the rbcL genes in the four materials were absolutely same. Restriction enzyme digestion and gel electrophoresis of cpDNAs were also performed respectively to the 4 materials using an excess of 4 kinds of enzymes including BamHâ… , EcoRâ… , Hindâ…¢and Pstâ… . The results of restriction enzyme analysis indicated that the bands among 4 materials were same and the enzyme digestion products had no difference, respectively. The above results also reflected that cpDNA sequence was high conservative.4. Mitochondrial DNA(mtDNA) was extracted by tender root and leaf tissues from seedlings of four materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B, and the extracted mtDNA was examined. The results showed that the mean content of mtDNA extracted from tender root tissue was about 3250ng/gFW and about 6.5 times of leaf tissue, significantly higher than that from leaf tissue in the same conditions, and mtDNA extracted from tender root had a higher purity. Results of the agrose gel electrophoresis also showed that mtDNA extracted from tender root had clearer bands than that from leaf. Moreover, the PCR amplifying results of mitochondrial genes showed that the repetition and stability of bands amplified from mtDNA extracted by tender root were much better than that by leaf. So tender root tissue is an ideal material for extracting mtDNA in rapeseed.5. Five sterility-related gene fragments of mtDNA in rapeseed were amplified by PCR to four materials of PolA system and Shaan2A system. The results showed that the same target bands were found by horizontal submarine agarose gels when 3 pairs of the special gene primers of mtDNA were used respectively to the 4 materials, and amplified products were consistent with the expected target fragments. Thereinto, another smaller band was also gotten with primer designed by orf474 and orf159 among 4 materials. The target bands were failed to be found out with primer designed by orf158. One same band was gotten with primer designed by orf224 bewteen PolA and Shaan2A, and amplified products were consistent with expected target fragments. The sequence-mensurating results showed that the sequences of PolA and Shaan2A were also the same at 675bp. In addition, a smaller band was also amplified with primer designed by orf224 bewteen PolB and Shaan2B, which was proved to be DNA fragments consistent with orf224 with 323bp by sequence-mensurating. So guessed that the fragments were relative to the fertility of CMS lines and their maintainer lines.6. With 25 pairs of special primers designed by continuous DNA fragments of the total genome of mtDNA from variety Westar in rapeseed, PCR amplification and Agarose gel electrophoresis were carried on the corresponding DNA section of mtDNA moulds of four materials of PolA system and Shaan2A system. The results showed that total lengths of mtDNAs of four materials were respectively about 200kb, whose framework and arrangement were similar with the known variety Westar. Thereinto, one same expected band was amplified respectively by 11 pairs of specific primers in 4 materials explained that the sections, which covered 44 percent of total genome of mtDNA, might be conservative sequences. Otherwise, one same band was amplified respectively by two pairs of primers in 4 materials, which were different from expected ones. Another pair of primers did not get the amplified products in 4 materials. The above primers respectively got the same band types(or did not get the amplified products) in PCR amplification in 4 materials, therefore forcasted that the homology of the 4 materials were at least 56 percent. However, the other primers had a complicated amplification result in 4 materials explained that these amplification regions, which covered about 44 percent of total genome of mtDNA, might be the variable regions of mtDNA recombination. Some of the primers amplified the different DNA fragments bewteen the CMS lines and their maintainer lines, and some of the primers amplified the different fragments bewteen PolA and Shan2A. Whereas, whether these different fragments have biological function and whether they have any relation with CMS need further study. 7. This paper systemically studied PolA and Shaan2A on three different levels: molecular biology(such as PCR amplification of special gene fragments of cpDNA and mtDNA, sequence-measuring, restriction enzyme analysis of cpDNA), physiology and biochemistry(gel electrophoresis of EST and POD isozymograms), and morphology and anatomy(investigation of plant agronomic trait and observation of flower structure). The total results confirmed that the nuclear genomes of the two CMS lines(PolA and Shaan2A) were obviously different, and their mtDNAs had certain differences thus their cytoplasms also had certain differences, but sequences of the sterility gene(orf224) from PolA and Shaan2A were same. Well then, whether or not were the sterility mechanisms of the two CMS lines completely same? whether or not were there other sterility-related genes on mtDNA of Shaan2A? and so on, these still need further research.