| The open reading frame (ORF) orf224 and orf138 in mitochondrial genome are important reasons of Pol CMS and Ogu CMS infertility, using the specific expression of these ORFs can control plant fertility and create new male sterile lines. Using constitutive promoter ED35S and specific promoter BcA9 of cabbage anther, we constructed four different expression plasmids which carried cytoplasmic male sterility gene Pol CMS orf224 and Ogu CMS orf138, in addition, on the basis of our research about regeneration and genetic transformation of Chinese cabbage, we acquired transgenic plants which carried gene orf224 via agrobactrium mediated transformation method. This study can be a foundation of new germplasm resources of male sterile lines and the research about the mechanism of cytoplasmic male sterility.The details were as follows:Firstly, CMS specific sequence was amplified from radish CMS B1-5# by PCR amplifying, then cloned and sequenced, Sequence analysis revealed that we had acquired the full length of gene orf138, and its homology with orf138 published in NCBI was 99%.Secondly, the recombinant plasmid pMD18-T-CMS7311-orf224 and expression vector pWR306 were digested by Xbaâ… a nd Sacâ… and then linked directionally, thus we constructed the plant expression vector pWR-CMS7311-orf224 which was verified by PCR amplification and restriction enzyme digestion. On the basis of pWR-CMS7311-orf224, pWR-CMS7311- orf224 and pMD18-T-BcA9 were digested by Hindâ…¢and BamHâ… then linked, so pWR- BcA9-orf224 plant expression plasmid was acquired which carried cabbage anther promoter BcA9 and gene orf224. The results of PCR amplification and restriction enzyme digestion showed that the fragment of BcA9 was introduced into pWR-CMS7311-orf224 plasmid. The two plant expression vector of orf224 was transformed into Agrobacterium strain EHA105 by means of rapid frozen thaw method.Thirdly, the recombinant plasmid pMD18-T-orf138 and expression vector pWR306 were digested by BamHâ… a nd Salâ… and then linked directionally, therefore the plant expression vector pWR- orf138 was constructed, the results of PCR amplification and restriction enzyme digestion showed that the fragment of gene orf138 was introduced into pWR306 plasmid and the expression vector successfully. On the basis of pWR-orf138, pWR-orf138 and pMD18-T-BcA9 were digested by Hindâ…¢and BamHâ… and then linked, so constitutive promoter ED35S was replaced by cabbage anther promoter BcA9, PCR amplification and restriction digestion enzyme showed that we got the plant expression plasmid pWR-BcA9-orf138 successfully. The two plant expression plasmids of orf138 were transformed into Agrobacterium strain EHA105 by means of rapid frozen thaw method.Fourthly, the cotyledonary segments from six genotype were used to study the effects of the combinations of 6-BA and NAA, state of seedling, cotyledonary segments cutting and inoculation methods on regeneration, The results showed that the best stage for culture in vitro (5-6 days after sowing) was when seedling 06J28 was growing upright with two green and lat cotyledons, and no true leaf were obvious. The seedling was cut at the cross of hypocotyls and cotyledon firstly, then the apical bud and the 1/3 top of cotyledon was cut off, lastly inserted into culture medium vertically. Cotyledon segments were shifted to the medium of (MS+7.0mg.L-16-BA+0.5mg.L-1) for 2-3 days, then transferred to the medium of (MS+5.5 mg.L-16-BA+0.5mg.L-1NAA) inducing adventitious shoots. The explants of orange Chinese cabbage 06J28 got the highest regeneration ratio, which was 90.4%, and its maximum number of regeneration buds was 1.14 per explants,Fifthly, according to the research about the sensitivity of Chinese cabbage cotyledonary segments 's to Hyg and Cef, the days of preculture, concentrations of Agrobacterium tumefaciens, the days of common culture and the time of being infected on the optimum culture medium, the high frequency transgenic transformation system of Chinese cabbage was established: regenerated buds were precultured for 2-3 days on bud differentiation culture medium (MS+7.0mg.L-16-BA+0.5mg.L-1NAA), and inoculated with EHA-105 Agrobacterium tumefacious for 5 minutes, cocultured for 2-3 days on (MS+5.5mg.L-16-BA+ 0.5mg.L-1NAA), then transferred to screening-culture medium (MS+5.5mg.L-16-BA+0.5 mg.L-1NAA+10mg.L-1AS+5mg.L-1Hyg+500mg.L-1Cef) to screen resistant buds. Medium were changed every two weeks, then nine Hyg -resistant plants were obtained.Lastly, PCR analysis of 9 06J28 transgenic plants, 5 of them showed the same positive band as 690 bp; through examination of report gene GUS, four of the 5 transgenic plants showed blue, Non-transgenic plants didn't show blue through GUS inoculation. By RT-PCR preliminary testing, two 06J28 transgenic lines were obtained with band of 690bp, so the integration of the pWR-CMS7311-orf224gene into Chinese cabbage genome DNA was confirmed in some degree. Southern Blotting is developing. |
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