【作者】 佟友丽；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 露地菊(Dendranthema×gandiflorum)是重要的园林绿化花卉之一,具有植株低矮,开花繁密,耐粗放管理等特性,有一定的社会经济价值。但是其盛花期通常处于深秋季节,往往因温度骤降导致观赏价值降低。通过遗传转化途径引入重要的抗逆基因,有望提高菊花抵御低温的能力。本研究以11个北方主要栽培露地菊品种(‘金不换’、‘矮红’、‘蜂窝粉’、‘雪涛’、‘秋艳’、‘粉玫瑰’、‘紫秋裳’、‘富丽’、‘金凤凰’、‘东林瑞雪’、‘袖珍红’)和以这些品系为母本,在杂交子代中选育的16个综合性状优良的露地菊品系(‘东林1号’、‘东林2号’、‘东林3号’、‘东林4号’、‘东林5号’、‘东林6号’、‘东林7号’、‘东林9号’‘东林9号’、‘东林9号’、‘东林11号’‘东林12号’‘东林13号’、‘东林14号’、‘东林15号’、‘东林16号’)为试材,在对这27个露地菊品种和品系耐低温能力筛选的基础上,选择其中耐寒能力较强的4个露地菊品系和品种,进行了以叶片为外植体直接诱导不定芽的研究。同时从拟南芥中克隆了AtproDH和P5CS基因,并应用Gateway克隆技术构建了植物表达载体,进行P5CS基因转化露地菊的研究。主要研究结果如下:1、露地菊抗冻品种和品系的筛选利用受伤率和伤害指数两项指标对27个露地菊品系进行耐低温筛选,筛选出‘金不换’、‘雪涛’、‘富丽’、‘东林4号’、‘东林6号’、‘东林9号’、‘东林13号’等7个耐低温品系,同时发现‘粉玫瑰’、‘东林1号’、‘东林2号’三个品系的耐低温能力最差;通过对不同自然低温处理时期,7个品系的电导率、丙二醛含量、脯氨酸含量、过氧化氢酶、果糖、葡萄糖和可溶性糖等指标的测定发现,‘雪涛’和‘东林4号’的耐低温能力要强于其它品系,尤其是‘东林4号’对低温的调节能力较强,在低温胁迫下,能够调节渗透途径减轻低温的伤害。2、低温胁迫下,露地菊蛋白质表达谱分析利用二维液相色谱技术,分离了‘东林4号’露地菊品系的低温处理和对照样品的总蛋白,通过ProteoVue软件分析发现:处理样品与对照相比共有蛋白差异点36个,其中显著增强表达的蛋白差异点有26个。选取10个差异色谱峰,经过蛋白质质谱鉴定了3个植物耐低温相关蛋白质,分别是:假定的钙离子运输ATP酶,铁氧还-硫氧还蛋白还原酶,假定的细胞色素C氧化酶。3、抗冻基因的克隆及植物表达载体的构建通过RT-PCR方法从拟南芥中克隆了P5CS和AtproDH的cDNA全长基因。利用Gateway克隆技术分别构建了AtproDH和P5CS两个基因的植物表达载体。4、露地菊遗传转化体系的建立建立‘东林4号’、‘东林6号’、‘东林9号’叶片外植体再生体系,发现不同基因型叶片外植体的分化率差异明显。‘东林9号’的叶片外植体分化率最高。‘东林13号’未获得以叶片为外植体的不定芽。‘东林4号’最适分化培养基为:MS+3 mg/L BA+0.1mg/L2,4-D,叶片分化率为10%;‘东林6号’最适分化培养基为:MS+2 mg/L BA+1 mg/LNAA,叶片分化率为82%;‘东林9号’最适分化培养基为:MS+0.5 mg/L BA+1.5 mg/LNAA,该品系植株上层幼嫩叶片分化率为95%,中下层叶片分化率均小于50%;潮霉素对以叶片为外植体的分化起到很强的抑制作用,在潮霉素含量为5 mg/L的培养基中叶片即可失去分化能力。‘东林9号’品系组培再生芽在含有30 mg/L潮霉素的培养基中全部死亡。通过农杆菌LBA4404介导,初步建立了P5CS基因转入露地菊的遗传转化体系,经过PCR鉴定得到了一个阳性株系。更多还原

【Abstract】 Chrysanthemum(Dendranthema×gandiflorum) used for outdoor planting is one of the most important flowers in the landscape greening.It has specific characteristics such as short height with a heavy bloom,planting need not careful management and possessing social and economic values.The blooming stage of chrysanthemum grown outdoors is usually at late autumn and its ornamental values deteriorate when sudden declining of temperature.In these cases,tolerance to low temperature of chrysanthemum for outdoor planting would be improved through transfering related genes to cold tolerance by genetic transformation.In this study, materials used were 11 major cultivars in northern parts of China(Jinbuhuan","Aihong", ’Fengwofen’,’Xuetao’,’Qiuyan’,’Fenmeigui’,’Ziqiushang’,’Fuli’,’Jinfenghuang’, ’Donglinruixue’,and ’Xiuzhenhong’) and 16 lines selected in comprehensive evaluation from filial generations of maternd parents(’Donglin 1’,’Donglin 2’,’Donglin 3’,’Donglin 4’, ’Donglin 5’,’Donglin 6’,’Donglin 7’,’Donglin 8’,’Donglin 9’,’Donglin 10’,’Donglin 11’, ’Donglin 12’,’Donglin 13’,’Donglin 14’,’Donglin 15’,’Donglin 16’).After screening 27 lines of chrysanthemums under freezing treatments,four lines were selected to establish regeneration system by inducing direct production of adventitious buds from leaf explants.The plant expression vectors of AtproDH and P5CS genes,which were cloned from seedling of Arabidopsis thaliana,were constructed by Gateway clone technique.P5CS gene had been transformed to chrysanthemums.The main results are shown as follows:1.Screening freezing tolerant cultivars and lines of chrysanthemumTwenty seven lines of chrysanthemum grown outdoors were screened using ’injury rate’ and ’injury index’.There were 7 cultivars and lines,which were relatively high tolerant to freezing treatment,i.e.,’Jinbuhuan’,’Xuetao’,’Fuli’,’Donglin 4’,’Donglin 6’,’Donglin 9’, and ’Donglin 13’.There were 3 cultivars and lines,which were less tolerant,i.e.,’Fenmeigui’, ’Donglin 1’,and ’Donglin 2’;The determination of electrolyte leakage,and contents of free proline,MDA,CAT,fructose,glucose and soluble sugars on 7 lines of chrysanthemums at different stages freezing showed that ’Xuetao’ and ’Donglin 4’ were more tolerant than others, and that especially ’Donglin 4’ was able to regulate its osmotic pathway to alleviate the ’injury degree’ caused by freezing conditions.2.Protein expression profile of chrysanthemum under low temperature stressTwo-dimensional liquid chromatography was applied to fractionate and analyze total protein taken from ’Donglin 4’ leaves grown at low temperature and at normal condition. Chromatography peaks of fraction were transformed to gel graph by ProteoVue software.The 36 different protein points were found,and expression in 26 of them were enhanced.Three proteins correlation with low-temperature tolerance were identification by MS:Putative calcium-transporting ATPase 10,Ferredoxin-thioredoxin reductase and Putative cytochrome c oxidase subunitⅡPS17.3.Cloning freezing tolerant genes and constructing plant expression vectorsP5CS and AtproDH were cloned from cDNA of Arabidopsis thaliana by RT-PCR.The expression vectors of AtproDH and P5CS were constructed by Gateway clone technique.4.Constructing transformation system of chrysanthemumThe obvious difference in differentiation rate of leaf explants was revealed among gene types of ’Donglin 4’,’Donglin6’ and ’Donglin9’.The highest differentiation rate was obtained in leaf explants of ’Donglin 9’.No adventitious bud produced from leaf explants of ’Donglin 13’.For ’Donglin 4’,the most suitable culture medium(MSCM) was MS+3 mg/L BA+0.1 mg/L 2,4-D but the bud differentiation rate was only 10%.For ’Donglin 6’,the bud differentiation rate was 82%,and MSCM was MS+2 mg/L BA+1 mg/L NAA.For ’Donglin 9’, MSCM was MS+0.5 mg/L BA+1.5 mg/L NAA.The bud differentiation rate in upper young leaves of’Donglin 9’ was as high as 95%,but the differentiation rate being lower than 50%for leaves at middle and lower parts of stems.Hygromycin reduced bud differentiation of leaf explants.’Donglin 9’ lost ability to differentiation in the culture medium containing 5 mg/L hygromycin and differentiated buds died when soaked in solution at 30 mg/L hygromycin.Transformation system of P5CS in chrysanthemums for outdoor planting was established by A.tumefaciens LBA4404.Positive transgenic plants proved by PCR identification was obtained.更多还原