【作者】 陈夕军；

【导师】 徐敬友； 周益军；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2003， 硕士

【摘要】 用抗病基因保守区域合成的38个引物组合成28个引物对，对中国水稻丽江新团黑谷(LTH)近等基因系品种进行扩增。结果表明，不同引物对对同一近等基因系的扩增图谱有很大差异。引物对LM637／LM638扩增谱带最多，共40条；Ptokin1N／Ptokin2N仅扩增1～2条模糊的条带；而Ptokin2-inv1／Ptokin2-inv2扩增后无条带出现。此外，同一引物对(S2／AS1)对不同近等基因系(LTH与Co39)扩增的图谱亦有较大差异。 选择扩增谱带丰富且据不同类型保守区域设计的3对引物XLRRfor／XLRRrev、S1／AS3和Ptokin1／Ptokin2，对江苏27个品种进行扩增。结果指出，3对引物共扩增出清晰可辨的谱带127条，其中多态性带101条，占总数的79.53％。3对引物中扩增谱带和多态性带频率最高的是XLRRfor／XLRRrev，共扩增谱带47条，其中多态性带40条，占85.11％。此外，3对引物扩增的多态性谱带的分子量大小基本无差异。 根据聚类分析，3对引物对或引物组合扩增的RGA图谱可将品种分为多种类型，这些RGA图谱类型与病菌7个小种接种鉴定的抗病表型没有完全的对应关系。其中XLRRfor／XLRRrev引物对其组合的RGA图谱类型似乎与抗病表型有较好的吻合程度，这可能是由于该引物对来自水稻之缘故。 以28对引物对LTH近等基因系品种进行扩增，有11对引物能扩增出特异性条带，共回收特异性片段33个。将回收的片段重新扩增，有11个片段获单一条带。选择其中两个480～490bp片段(HS-1和HS-19)进行探针标记。点杂交试验表明，所有供试品种都有杂交信号，这说明这些片段在各品种中都有相似的序列或部分同源序列。PCR扩增产物电泳后经Southern杂交发现，片段HS-1在含有Pi-ta~2抗性基因的品种F-128-1、NO4中有特异的杂交带出现，这表明此序列可能与抗性基因Pi-ta~2连锁或是抗性基因的一部分。 用引物对XLRRfor／XLRRrev对202个品种进行扩增，在480bp处出现特异带扬州大学硕士学位论文的品种有34个.但与抗性鉴定结果比较发现，即使具有相同特异带的品种其抗病表型也是多样的，这可能与品种抗病表型的遗传复杂性有关。 将特异性片段HS一l克隆测序，全长为478bp.通过在Genbank中进行检索，发现它与Mag。等(1999)在水稻中克隆的一个抗病基因同源序列RGA29有95%的同源性。更多还原

【Abstract】 38 primers synthesized according to resistance gene conserved domains were made up 28 primer pairs. These primer pairs were used to amplify LTH NILs in China. The results showed that the amplified band types in same NILs varied with primer pairs used. The primer pair LM637/LM638 could amplify 40 bands from the varieties, which was the most among 3 primer pairs. Only 1 to 2 bands were amplified by Ptokin1N/Ptokin2N while no band appeared in the amplification of Ptokin2-invl/Ptokin2-inv2. In addition, the bands amplified by the primer pair S1/AS3 were significantly different in two NILs (LTH and Co39).3 primer pairs including XLRRfor/XLRRrev, Ptokin1/Ptokin2 and S1/AS3 were selected to amplified 27 rice cultivars from Jiangsu Province. These primer pairs were designed according to different resistance gene conserved domains and had high ability of the amplification. 127 bands were amplified from 27 rice cultivars by 3 primer pairs, in which 101 bands were polymorphic, with the polymorphic band frequency of 79.53%. The frequency of polymorphic bands amplified by XLRRfor/XLRRrev was the highest among 3 primer pairs, with the sum of 47 bands and the polymorphic band frequency of 85.11%. Furthermore, there were no differences in the molecular weight of polymorphic bands amplified by 3 primer pairs.RGA-PCR maps of rice cultivars amplified by 3 primer pairs or primer pair combinations could be grouped by cluster analysis. There were no completely corresponding relations between these RGA-PCR map groups and resistance phenotypes of rice cultivars. But RGA-PCR maps amplified by XLRRfor/XLRRrev or its combinations had same relations with the resistance phenotypes, which might be because the primer pair came from rice.28 primer pairs were used to amplify LTH NILs varieties. There were 11 primer pairs by which special bands could be amplified. 33 special bands were retrieved and re-amplified, and 11 simple bands were acquired. Two fragments (HS-1 and HS-19) with the size from 480bp to 490bp were selected to sign as probes. There were samesignals in all rice varieties by dot hybridization, which indicated that all rice varieties tested could have same or partial RGA.The results of electrophoresis and southern hybridization showed that HS-1 fragment could detect special signals in rice varieties with resistance gene Pi-ta2, which revealed that this sequence could link with Pi-ta2 gene or be a part of the gene.34 rice varieties had the special band of 480bp among 202 rice varieties amplified with XLRRfor/XLRRrev. By comparison, it was found that although rice varieties had the same special band, their resistance phenotypes could be different, which maybe related to genetic complexity of resistance phenotypes.HS-1 fragment has been cloned and sequenced, and was 478bp. It was 95% identical with RGA29 which was a resistance gene analog cloned from rice by Mago(1999).更多还原