【作者】 庞洪泉；

【导师】 樊龙江；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2004， 硕士

【摘要】 作物的抗性基因研究一直是各国科学家关注的重点。本文主要研究了(1)甜菜(Beta vulgaris L．)基因组中NBS-LRR类抗性基因序列特征；(2)甜菜再生体系的构建及其影响因子；(3)农杆菌介导转化甜菜体系的构建及优化。主要研究结果如下： 1．甜菜基因组TIR-Type类抗性基因序列分析 大部分植物抗性基因蛋白质是由核苷酸绑定位点和亮氨酸富集区(NBS-LRR)构成的。根据保守NBS-LRR残基设计的简并引物，扩增了双子叶植物甜菜TIR-Type类抗性基因序列，共获得12个表达的NBS-LRR类基因序列。序列分析说明，这些序列中的点突变，如核苷酸替代、插入、缺失很可能是甜菜NBS-LRR类基因多样性的主要原因。序列联配和系统发育树结果显示，甜菜NBS-LRR类基因属于无TIR型抗性基因亚族，且克隆获得的其他所有序列均属于该亚族。用TIR型特异性引物获得的PCR产物在超过16，000条甜菜EST序列的搜索中，均未发现典型的TIR型NBS-LRR类基因。该结果表明双子叶植物甜菜基因组中TIR型BNS-LRR类基因可能缺失，这是首次在双子叶植物中发现该现象，此前只在单子叶植物(如水稻)中发现该抗性基因亚族缺失。 2．甜菜再生体系的构建及其影响因子 为了建立甜菜的高频再生体系，确定影响再生的主要因子，采用了正交设计方法，通过不同浓度的激素配比、TIBA浓度、蔗糖浓度、预培养时间、外植体的筛选，提出了双八甜菜品种离体培养再生频率较高的培养基配方(MS+NAA0.4mg／L+BAP1.0mg／L+TIBA0.4mg／L+3％蔗糖+0.8％琼脂)、最适宜的外植体(叶柄)和最佳的预培养时间(3周)。本研究建立的高频再生体系再生频率达到76.7％。利用MS+KT0.1mg／L+NAA0.8mg／L和MS+NAA1.0mg／L为生根培养基，生根率达到100％，再生植株移栽的成活率也达到了100％。 3．甜菜遗传转化体系构建 为了能有效地杀除共培养后残余的农杆菌和筛选转化体，测定了双八甜菜品种抗生素的敏感性。经实验证明，离体培养叶柄时，Kan的浓度为150mg／L为宜。种抗生素的敏感性。经实验证明，离体培养叶柄时，Kan的浓度为15Omg/L为宜。在所用的梭节青霉素、氨节青霉素和头抱霉素三种抑菌抗生素中，根据它们对芽再生的影响，应首选狡节青霉素。为建立农杆菌介导甜菜转化的稳定体系，以gus基因在叶片中的表达频率为指标，对影响转化频率的几个重要因子进行了优化。通过对菌液浓度、预培养时间、感染时间、共培养时间以及诱导分化温度的梯度实验得出，菌液浓度0D60(，二1.0、预培养2天、感染15分钟、共培养3天、诱导分化温度为25一30℃时，gus的表达频率最高。更多还原

【Abstract】 Disease resistance of crops is an important topic in scientific field. In this paper, we (1) analyzed NBS-LRR-type resistance gene analogues in the Sugarbeet (Beta vulgarisL.) Genome; (2) established a tissue culture or regeneration system and (3) an effective transformation system by Agrobacterium for sugar beet. The main results follow as:1. The analysis of TIR-type resistance gene analogues in the sugar beet genomeThe majority of known plant resistance genes encode proteins with conserved nucleotide binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic and sequence alignment analysis revealed that NBS-LRR type resistance genes of sugar beet belong to non-TIR-type NBS-LRR and all other clones are this subfamily. PCR amplifications based on the primers specific for TIR were researched in EST database of sugar beet that contains 16,000 sequences. The results showed that we could not find the representative TIR NBS-LRR-containing resistance genes. This indicated that TIR NBS-LRR-containing resistance genes might miss in sugar beet genome. This instance was the fist reported in dicotyledon and only covered in monocotyledon (such as Oryza sative L.).2. Establishment of regeneration system of sugar beetIn order to establish an effective regeneration system of sugarbeet and research key factors, we investigated the concentration of NAA, BAP,TIBA and sucrose, the time of pre-culture and the type of explant forregeneration of sugar beet. The results showed that the regenerationfrequency reached 76.7% by medium with MS+MS+NAA0. 4mg/L+BAPl. Omg/L+TIBA0. 4mg/L+3%Scurose+0. 8%agar, the bestexplant is petioles, and the most condign time is three weeks. Therhizogenesis mediums are MS+KT0.1mg/L+NAAO. 8mg/L and MS+NAA1. Omg/L, andthe rhizogenesis frequency reached 100% in those mediums. The livabilityof regeneration plantlet is 100%. This formula is fit for other cultivarsin Beta vulgar is L.3 Establishment of an effective transformation system by Agrobacteriumof sugarbeet varietyTo effective eliminate Agrobactiu/nand screen of transformated cells after co-cultivation, sensitivity of cultivar Shuang-eight of sugar beet cultured in vitro to four antibiotics was tested. Results showed that petiole was sensitive to knamycin and the suitable concentration is 150mg/L. Among carbenicillin, ampicillin and cefotaxime used for elimination of hgrobacterium, carbenicillin should be preferable based on their effects on shoot regeneration.In order to establish an effective transformation system of sugarbeet, several key factors (the concentration of Agrobacterium, pre-culture time, infective time, co-culture time and polarization temperature) on in the process of Agrobacten’urn-mediated transformation were optimized based on the frequency of transient expression of gus gene. The highest frequency was observed when explants that had been pre-cultivated two days were immersed in bacterium suspension with OD600 value about 1. 0 for 15 minutes followed by co-cultivated for about three days, and then they were cultured in 25-30T.更多还原