【作者】 范爱丽；

【导师】 张鲁刚；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2009， 博士

【摘要】 线粒体基因组中的开放阅读框orf224和orf138分别是造成PolCMS、OguCMS雄性不育的主要原因,利用这些开放阅读框的特异性表达有可能控制植物育性表达、创制新的雄性不育材料。本研究分别构建了含有组成型启动子ED35S和白菜花药特异启动子（BcA9）的两类不同来源的大白菜PolCMS orf224细胞质雄性不育基因和OguCMS orf138细胞质雄性不育基因的植物表达载体。进一步优化了大白菜离体再生体系,并以携带pWR-CMS7311-orf224表达载体质粒的农杆菌进行了大白菜遗传转化体系研究,获得了转基因植株,为大白菜不育系种质资源创新及细胞质雄性不育机理研究奠定了基础。本试验的主要研究结果如下:1利用PCR技术从萝卜细胞质雄性不育材料B1-5#线粒体基因组中扩增出特异片断orf138,对其进行克隆、测序及序列分析。序列分析表明:获得orf138基因的全序列,开放阅读框完整,该基因与NCBI上报道的orf138序列同源性为99%。2用XbaⅠ和SacⅠ双酶切重组质粒pMD18-T-CMS7311-orf224和表达载体pWR306后,将CMS7311-orf224基因与线性表达载体pWR306进行定向连接,构建了细胞质雄性不育线粒体基因CMS7311-orf224的植物表达载体pWR-CMS7311-orf224,通过酶切和PCR验证pWR-CMS7311-orf224载体构建正确。在构建pWR-CMS7311-orf224基础上,再用BamHⅠ和HindⅢ双酶切pWR-CMS7311-orf224质粒和pMD18-T-BcA9质粒,将pWR-CMS731-orf224表达载体与BcA9启动子定向连接,把ED35S组成型启动子置换成BcA9白菜花药启动子,得到pWR-BcA9-orf224植物表达载体,酶切和PCR验证pWR-BcA9-orf224载体构建正确。采用快速冻融法,将orf224两个表达载体质粒分别导入农杆菌EHA105。3用BamHⅠ和SalⅠ双酶切重组质粒pMD18-T-orf138和表达载体pWR306后,将orf138基因与线性表达载体pWR306进行定向连接,构建了细胞质雄性不育线粒体基因orf138的植物表达载体pWR-orf138,通过酶切和PCR验证pWR-orf138载体构建正确。在构建pWR-orf138基础上,再利用BamHⅠ和HindⅢ双酶切pWR-orf138质粒和pMD18-T-BcA9质粒,将pWR-orf138表达载体与BcA9启动子定向连接,ED35S组成型启动子被置换成BcA9白菜花药启动子,得到pWR-BcA9-orf138植物表达载体,通过酶切和PCR验证pWR-BcA9-orf138载体构建正确。采用快速冻融法,将orf138两个表达载体质粒分别导入农杆菌EHA105。4建立了的大白菜子叶段离体再生体系:最优苗态为两片子叶绿色将展平、第一片真叶未露出、小苗直立生长时（播种5-6 d）。最佳切割和接种方式是保留单片子叶,在生长点处（即子叶柄基部和下胚轴交接处）,先横切,然后纵切,去除顶芽,切除子叶顶端的1/3,竖直插入培养基。优化的培养程序为外植体先在MS+7.0 mg.L^-16-BA+0.5 mg.L^-1NAA的培养基上启动培养2-3 d;再转入MS+5.5 mg.L^-16-BA+0.5 mg.L^-1NAA的芽诱导培养基培养,再生频率最高。在供试的6种基因型材料中,橙色大白菜06J28子叶段再生频率最高,为90.4%,每块外植体再生芽数最高达1.14个。5建立了大白菜遗传转化体系:技术要点是先进行大白菜子叶段切割,子叶段在芽启动培养基(MS+7.0 mg.L^-16-BA+0.5 mg.L^-1NAA)上预培养2-3 d;投入到活化的农杆菌菌液中浸染5 min,然后在芽诱导培养基(MS+5.5 mg.L^-16-BA+0.5 mg.L^-1NAA)上共培2-3 d,再转入筛选培养基(MS+5.5 mg.L^-16-BA+0.5 mg.L^-1NAA+10 mg.L^-1AS+5 mg.L^-1Hyg+ 500 mg.L^-1Cef)中筛选抗性芽。每两周更换一次培养基,最后获得9株06J28大白菜抗性植株。6用携带pWR-CMS7311-orf224质粒的EHA105农杆菌转化06J28大白菜,在获得9株抗性苗中,其中5个植株PCR检测扩增出约690 bp条带,这5个PCR阳性转化植株中有4株GUS染色呈现蓝色。进一步RT-PCR检测有2株在690 bp左右处有明显的条带,初步判断此基因已转入大白菜基因组中,并已经转录。Southern Blotting正在进行中。更多还原

【Abstract】 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 XbaⅠa nd SacⅠand 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 HindⅢand BamHⅠthen 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 BamHⅠa nd SalⅠand 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 HindⅢand BamHⅠand 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.更多还原