【作者】 郑泉；

【导师】 郭维明；

【作者基本信息】 南京农业大学 ， 植物学， 2010， 博士

【摘要】 石斛属(Dendrobium)是兰科(Orchidaceae)中最大的属,全球约1500余个野生原种,广泛分布于世界热带、亚热带地区,其中原产我国的有70余种,目前在药用和鲜切花、盆花栽培中广泛应用。春石斛(Spring Dendrobium)在园艺上是指石斛兰中花着生于叶腋,主要自然花期在春季的石斛种或者品种,是目前世界兰花市场最重要的热带兰盆栽花卉之一。为了创制更多有观赏价值的春石斛新品种,在生产和研究上都急需完善相关的育种技术体系。但是采用分子标记技术分析春石斛品种间亲缘关系,和建立稳定高效转化体系并通过转化得到符合目的性状新品种的研究目前均较少本研究根据春石斛主要栽培品种的特点,首先采用AFLP分子标记技术,研究了30个主要栽培品种(系)的亲缘关系,并以其中的一个重要栽培品种(’Sanya’)作为受体材料构建了其类原球茎的再生体系,采用超声波辅助农杆菌诱导法(SAAT)进行CHS和ASACC基因的遗传转化。主要研究结果如下：1.采用改良的CTAB法,在进行细胞核裂解之前先加入核分离缓冲液,成功提取出高品质的春石斛基因组DNA, OD260nm/OD280nm的比率为1.73-1.85。与试剂盒法和普通CTAB法相比,该方法提取的基因组DNA不仅基本排除了春石斛体内多糖及其它次生代谢物质对基因组DNA质量的干扰,得率也较高,完全能满足AFLP等分子生物学试验对DNA质量的要求。2.利用荧光AFLP技术分析了30个春石斛栽培品种(系)的亲缘关系。选择EcoR I/Mse I酶切组合,8对引物组合的选择性扩增共得到1102个条带,其中多态性带占70.6%。利用NTSYS pc Version2.0e软件对扩增数据进行分析,用主坐标分析法(PCOA)和算术平均数的加权配对归类法(UPGMA)等方法对扩增数据进行品种间的亲缘分析,30个品种或品系材料可分为5个类群,类群Ⅰ包含6个品种(系),相似系数在0.70-0.80之间；类群Ⅱ包含3个品种(系),相似系数在0.71-0.76之间；类群Ⅲ包含的品种(系)最多,共13个,其相似系数在0.69-0.84之间；类群Ⅳ包含了7个品种,相似系数在0.68-0.83之间；而类群V仅包含了’Santana Canary’一个品种。3.建立了春石斛品种’Sanya’和’China Doll’类原球茎(PLBs)的再生体系。首先通过盆栽植株的茎尖或腋芽外植体诱导得到无菌苗；再以无菌苗茎基无叶芽茎段为材料,经40KHz超声波预处理10min,接于1/2MS+6-BA0.2mg/L固体培养基上,两个品种分别得到最高10.5%和9%的PLBs诱导率。系列筛选试验表明,采用液体MS+6-BA0.2mg/L两品种的PLBs增殖率均最高,分别为4.57倍／月和4.31倍／月；采用1/2MS+6-BA1.0mg/L+NAA0.2mg/L固体培养基,芽增殖率最高分别为2.90和2.66,这两个春石斛品种的PLBs诱导率与其它品种间存在显著性差异。4.采用超声波辅助农杆菌介导法(SAAT)成功地将CHS和ASACC基因导入春石斛品种’Sanya’的类原球茎中,获得转基因株系。采用农杆菌共侵染60min结合超声波40KHz辅助诱导处理10min,在预培养、共侵染和共培养中均添加100μM AS的处理组合,可使’Sanya’获得最高的转化率。通过Km200mg/L (?)勺筛选,两个基因的转化分别得到63和64个抗性株系,通过GUS、PCR和Southern bolt检测,获得20个转CHS基因株系和10个转ASACC基因株系,最高转化率分别为1.17%和1.04%。经荧光定量RT-PCR检测,其中4个转CHS基因和2个转ASACC基因株系的目的基因相对表达量均显著高于未转化对照,且各株系之间也存在差异,转CHS基因的株系A1和转ASACC基因的B3株系表现出最高的目的基因表达效率。更多还原

【Abstract】 Dendrobium is the biggest genus within Orchidaceae. There are more than1500wild ancestors in the world which distribute mainly in the tropical&subtropical areas in which more than70species of Dendrobium originated from China. Spring Dendrobium is a group of Dendrobium representing blooming in spring, and is also one of the top grade pot flowers which are very popular in the international orchid market. In order to create more valuable new cultivars of Spring Dendrobium, the related breeding technilogy needed to be solved both in production and research. However, the research on genetic relationship between species or cultivars by molecular markers, constracting the highly efficient and stable transformation system, and getting the new cultivars consistent with the purpose gene by transgenic breeding technology, are still limited.For the first time, this study was intended to determine genetic relatedness of popular pot ornamental Sping Dendrobium cultivars and varieties materials using AFLP markers with near-infrared fluorescence-labeled primers, providing a reference for the parent selection in the breeding of Spring Dendrobium. Then,’Sanya’ as an important commercial cultivar was selected for the transgenic explant. The in vitro regeneration system of it by PLBs was studied. An efficient method of sonication-assisted-Agrobacterium-mediated transformation (SAAT) was developed for it. Agrobacterium tumefaciens (LBA4404) harboring ASACC gene or CHS gene was used to transform PLBs explants of’Sanya’. Our main research results as follows:1. Using modified CTAB method, before carrying out nuclear lysis buffer into the nuclear separation, can be successfully extracted high-quality gDNA. The ratios of OD260nm/OD280n of the genomic DNA extracted by the modified CTAB methods were respectively1.85and1.73. The comprehensive results from electrophoresis, ratios of OD260nm and OD280nm and Fluorescent-AFLP amplification suggested that the modified CTAB can produce high quality genomic DNA while the conventional CTAB and Genomic DNA Kit methods are not suitable for the DNA extraction from Spring Dendrobium;2. The genetic relationship of30Spring Dendrobium cultivars (or varieties) were analyzed using amplified fragment length polymorphism (AFLP) markers with near-infrared fluorescence-labeled primers. Eight EcoRI+3bases/MseI+3bases primer set combinations were used in this investigation. Each selected primer set generated113-158scorable fragments. A total of1102AFLP fragments were detected, of which778were polymorphic (70.6%). An un-weighted pair-group method of the arithmetic averages (UPGMA), principal coordinate analysis (PCOA), and bootstrap analysis were used to analyze the genetic relationships. The30cultivars or varieties were separated into five clusters. Cluster Ⅰ contains6varieties materials that are either from Senlan No.l to Senlan No.6with Jaccard’s similarity coefficients ranging from0.70to0.80. All of these6varieties materials came from Taiwan, and were derived from somaclonal variants or sports; Just3cultivars are positioned in cluster Ⅱ ranging from0.71to0.77, and also origined from Taiwan; Cluster Ⅲ has13cultivars(or varieties), Jaccard’s similarity coefficients varied from0.69to0.84. Seven cultivars(or varieties) from Senlan No.15to’Snowboy Romance’were situated in cluster IV with Jaccard’s similarity ranging from0.68to0.83; Only’Santana Canary’was positioned in cluster V.3. The regeneration and proliferation system in vitro of’Sanya’and’China Doll’by PLBs was studied. At first, the plantlet in vitro were obtained from explants, then, PLBs were induced by basal stem without bud of the plantlet as the explants, which were pretreated with40KHz Ultrosonic Wave for10mines, then were respectively cultured in1/2MS+6-BA0.2mg/L solid culture medium. The induction rates of two cultivars were10.5%and9%, while the control did not induce the formation of PLBs. And then were respectively cultured in MS+6-BA0.2mg/L liquid medium for PLBs proliferation. At last, proliferation rates of PLBs of’Sanya’and’China Doll’as4.57and4.31times per month in liquid medium. Cultured by1/2MS+6-BA1.0mg/L+NAA0.2mg/L solid medium for buds culture could get the greatest propagation rate as2.90and2.66. There are distinct different to induced rate of PLBs among different Spring Dendrobium cultivars.4. An efficient and reproducible transformation method of sonication-assisted-Agrobacterium-mediated transformation (SAAT) was developed for’Sanya’. assisted with Ultrasonic Wave40KMz for10min and inculture for60min. By using a series of co-cultivation, filteated on the1/2MS medium supplemented with200mg/L Km, shoot initiation and root inducing media,63positive lines of trans-CHS gene and64lines of trans-ASACC gene were recovered. Analysis of positive materals by GUS、PCR and Southern hybridization confirmed that,20of trans-CHS gene and10of trans-ASA CC gene positive plants were transgenic lines. The highest transformation efficiency respectively was1.17%and1.04%. cDNA analysis of the trans-CHS and trans-ASACC lines by Real-time RT-PCR show that their comparative quantification of NPTⅡ gene were high distinctly compare with control. B3of the trans-ASACC gene lines and A1of the trans-CHS gene lines had highest comparative quantification among these transgenic lines.更多还原