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摘要水稻叶片衰老会减少植株干物质的积累，严重影响水稻产量，因此对水稻叶片衰老机制的研究具有重大的理论和实际意义。本实验为了探究叶片衰老调控的分子机理，我们用EMS试剂处理野生型水稻SH，诱变出表现为叶片衰老的突变体，我们将此突变体命名为es2。es2主要表现为叶片枯黄，分蘖较小，穗短小等。我们用突变体es2和野生型NIP进行杂交，得到F2分离群体，发现分离群体中野生型与突变体表型个体分离比约为3:1，说明该突变为隐性突变。我们获得了两千多株突变体表型的F2单株。通过分布水稻的12条染色体的110对在双亲间存在多态的SSR标记，从F2群体中随机挑选20个突变单株，进行DNA混池连锁标记筛选，然后挑选出与候选基因紧密连锁的分子标记，再选取200株突变体进行单株连锁标记，也就是初定位。初定位后扩大模板数量，设计引物，进行精细定位。最终ES2基因被限定在第十二号染色体上P3和P4标记之间。通过测序发现， Os08g16780基因发生135bp的片段缺失，从而造成编码蛋白的大片段缺失，该基因被作为造成es2突变体早衰表型的候选基因。50435
Abstract Rice leaf senescence would cause the reduction of dry matter accumulation, and seriously affect rice yield. So it is important to study the molecular mechanism of rice leaf senescence. In this study, we treated the wild type rice variety SH with a chemical reagent EMS, and obtained a leaf senescence mutant, which was named as es2. The es2 mutant, presented mainly as yellow leaves, small tillers, and short panicle. To clone the ES2 gene, we crossed es2 mutant with wild type NIP, the F2 population were obtained, and the segeregation rate of wild type vs mutant plants was about 3:1, indicating that the es2 was controlled by a recessive gene. To clone the ES2 gene, we obtained 200 mutant plants from the F2 population and 110 pairs of polymorphic SSR markers on 12 chromosomes was applied . A DNA pool with 20 selected mutant plant was used to do the polymerase chain reaction (PCR). Resultly, we found several molecular markers were cosegregated with ES2 gene, which indicated that ES2 gene was linkage with those markers. After applying additional markers. ES2 gene was finally defined on chromosomes 12 between marker P3 and P4. Sequencing analysis of the ORFs located on the region, a 135bp deletion on the gene Os08g16780 was detected, which caused a important catalylic domain deletion. Those data indicated that the deletion on Os08g16780 may led to the leaf senescence phenotype in es2 mutant.
毕业论文关键词：叶片衰老； SSR标记； 基因突变
Keyword: leaf senescence; SSR marker; gene mutation
目录3
引言4
1 材料与方法4
1.1 实验材料.4
1.2 培养方法.4
1.2.1 DNA提取.4
1.2.2 分子标记检测5
1.2.2.1 PCR扩增.5
1.2.2.2 琼脂糖凝胶电泳.5
2 过程与原理5
2.1 实验过程.5
2.1.1 混池连锁标记5
2.1.2 单株连锁标记6
2.1.3 扩大模板数量6
2.1.4 基因克隆6
2.2 实验原理.6
2.2.1 DNA提取原理.6
2.2.1.1 磨样.6
2.2.1.2 水浴.6
2.2.1.3 加氯仿.6
2.2.1.4 加异丙醇.6
2.2.1.5 加乙醇.7
2.2.1.6 加TE溶液或ddH2O7
2.2.2 PCR扩增原理.7
2.2.3 凝胶电泳原理7
3 结果与分析8
3.1水稻叶衰体es2的形态特征.8
3.2水稻叶衰体es2的基因定位10
3.3水稻叶衰体es2基因电泳结果10
3.4野生型与突变体的表达量差异分析,.11
4 讨论与展望12
参考文献：12
致谢13
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