【作者】 谌谋华；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2003， 硕士

【摘要】 柑橘是我国重要的经济作物，其产量位居世界第3位。病害一直是作物生产的主要问题。在现有的柑橘栽培品种中抗病资源有限，如何从近缘属中克隆到相应的抗病基因是未来进行转基因抗病育种的关键问题。 提高植物抗病性是植物育种的重要目标之一。随着现代生物技术的不断发展，利用植物基因工程进行植物抗病育种已经成为一种重要手段。到目前，人们已经通过图位克隆和转座子标签等方法分离克隆到一些植物抗病基因。 本研究是根据已克隆的植物抗病基因保守序列设计简并引物，用PCR及RT—PCR的方法从柑橘抗病材料中扩增出抗病基因类似物(resistance gene analog，RGA)，并对其中部分RGA的碱基序列及其编码的氨基酸序列进行了分析。同时随机挑选了一个RGA作为探针，进行基因组RFLP分析，所取得的主要结果如下： 1．建立了提取总RNA的体系。经凝胶电泳和紫外分光光度计检测，其总RNA的质量基本达到分子生物学实验技术要求，能够用于RT—PCR分析及Northern杂交分析。 2．根据已知抗病基因保守结构域设计了多对简并引物。采用PCR方法，分别从枳壳、九里香、黄皮、国庆1号基因组及枳壳、国庆1号的cDNA中扩增得到以500bp为主的电泳带(特征带)。分析全部的PCR结果发现，所有的引物对均能在感病材料或抗病材料基因组DNA及它们cDNA中扩增到以500bp为主带的电泳图。因此，用现有的简并引物无法直接对感病和抗病材料进行抗病基因的筛选，可能需要进一步设计特异性更高的引物来进行筛选。 3．分别对枳壳基因组DNA PCR产物及RT—PCR产物克隆成功的重组子进行测序。将得到的25个RGA采用美国国立生物技术信息中心BLAST程序及DNAsis软件进行分析发现：所有的RGA与已克隆的部分植物抗病基因(水稻Xa1、拟南芥RPS2、烟草N、亚麻L6)在碱基序列和氨基酸序列上均有一定程度的同源性(5.7％-25.3％)；RGAs之间也存在很高的同源性(27.2％-100％)。如何进一步对这些RGAs进行筛选还有待研究。 4．选取RGAn-17-2作为探针，进行柑橘基因组DNA的RFLP分析。从杂交结果可以看出：RGAn-17-2在不同的柑橘材料(枳壳、九里香、黄皮、国庆1号)中都有若干同源片段。更多还原

【Abstract】 Citrus is an important economic crop of our country . Its yield resides the 3rd in the world .The diseases are key problems that limit crop productions. There are limited disease resistance materials in the current cultivars. Cloning the resistance gene from citrus relatives is the base for transformation breeding for citrus.To improve the resistance ability is a main target in plant breeding. With development of modern bio - technique, the plant genetic engineering shows good potential for disease resistance breeding. Up to now, some plant resistance genes have been cloned. These R genes are mainly cloned by means of positional cloning or map - based cloning, transposon tagging etc..In this study, the degenerate primers were designed according to the conservative domain of plant disease resistance genes. The resistance gene or resistance gene analogy (RGA) fragments were amplified from DNA or cDNA of resistance disease plant materials of citrus by PCR. Some of these RGA are cloned and sequenced. The main results are as follows:1. We established a protocol to extract and purify total RNA from Citrus. RNA was detected by gel electrophoresis and ultraviolet spectrophotometer. Its quality was fine for subsequent molecular biology experiment.2. The domain of amino acid identity in R genes were used to design degenerate primers. The fragments of 500bp were amplified by PCR and RT-PCR from Poncirus trifoliata, Clausena lansium, Murraya paniculata, Guoqing NO.1 satsuma mandarin(C. unshiu), but there are no difference among materials. So, it’ s difficult to separate R gene by PCR directly.3. Twenty-five recombinants of PCR and RT-PCR were sequenced. These RGAs have been analyzed by BLAST on internet and aligned by DNAsis. All of them got some homology score at amino acid level (5. 7%-25. 3%)with the resistance genes (N gene from tobacco , RPS4 gene from Arabidopsis , L6 from flax and Xa1 from rice). The results also show high homology (27. 2%-100%) among RGAs. To screen the real resistance gene needs further research.4. RFLP analysis indicates homologous fragments are encompassed among citrus genome DNA blotted by RGAn-17-2, which shows different copy numbers from P.trifoliata , C. lansium , M.paniculata, Guoqing NO. 1 satsuma mandarin(C. unshiu).更多还原