水稻雄性不育基因OsPL1在花药发育中的机制解析

论文总字数：29821字

摘 要

我国水稻育种至今经历了三次重大革命，水稻雄性不育系的应用和研究始终具有广阔前景，该方面理论的丰富与发展更是对分子育种具有重要意义。在本论文中，水稻雄性不育基因OsPL1材料是通过⁶⁰Co_γ射线辐射诱变籼稻品种3037得到，利用免疫荧光染色分析、qRT-PCR扩增、互补载体构建等技术，经过表型鉴定、遗传分析、基因定位和结构分析、花药半薄树脂切片分析、花粉母细胞减数分裂进程分析、蛋白表达鉴定等过程，最终得出结论：OsPL1基因为一隐性单核基因突变，定位在水稻10号染色体上，突变后导致该基因的第6外显子内基因序列由TTCCGC突变成GTCCCTC，且突变体中出现AvaII酶酶切位点，可用于后期基因型酶切鉴定。其基因在穗中的花药组织中表达量最高。此外，OsPL1基因的突变并不影响雄配子减数分裂的进程，但延迟了绒毡层的凋亡进程，使得花粉发育异常无花粉释放，由此造成水稻雄性不育。

关键词：水稻（Oryza sativa），雄性不育，OsPL1基因，花药发育，绒毡层

Abstract：After three major revolutions in rice breeding, the application and research of rice male sterile still has a broad prospect, especially the rich and the development in its theory is of great significance for the molecular breeding. In this study, the OsPL1 mutant material is obtained by ⁶⁰Co_γ irradiation induced the mutation of an indica rice variety 3037. Using the immunofluorescence staining analysis, qRT-PCR amplification and complementary alalysis, , together with the phenotypic identification, genetic analysis, gene localization and structural analysis, anther semi-thin resin section analysis, pollen mother cell meiosis process analysis, protein expression identification and other processes, we concluded that the OsPL1 mutation, as a recessive mononuclear gene mutations, locates on the rice chromosome 10, and its mutation occurs on the 6^th exon of the gene, thus lead to the original gene sequence TTCCGC into the GTCCCTC. Moreover, this mutation can be cut with the restriction endonuclease enzyme Avall , which is used to identify the genetype of OsPL1. The expression of OsPL1 gene are mainly distributed in the plasma membrane, and had the highest expression in anther tissue; The mutation of OsPL1 gene did not affect the process of the meiosis of male gametes, but delayed the transfer of the tapetum, causing the abnormal pollen development , and finally resulting in the rice male sterility.

Keywords：rice (Oryza sativa), male sterility, OsPL1, anther development, tapetum

目 录

1 引言 3

1.1 水稻雄性生殖的发育过程 3

1.1.1 水稻雄蕊及花药的发育 3

1.1.2 花药壁绒毡层的发育过程 5

1.2 水稻雄性不育研究进展 6

1.2.1 水稻雄性不育系的分类 6

1.2.2 水稻雄性不育的不育机制 6

1.2.3 研究思路和技术手段 8

1.3 本研究的目的和意义 9

2 材料与方法 9

2.1 实验材料 9

2.1.1 水稻材料 9

2.1.2 实验试剂及仪器 9

2.2 实验方法 10

2.2.1 材料种植与实验群体构建 10

2.2.2 统计学分析确定遗传类型 10

2.2.3 KI-I₂染色观察花粉 10

2.2.4 水稻总DNA的提取与基因结构分析 11

2.2.5 水稻组织RNA的提取与表达量分析 11

2.2.6 载体构建及水稻植株转化 13

2.2.7 水稻花药半薄树脂切片 13

2.2.8 特异蛋白的免疫荧光染色分析 14

2.2.9 测序及数据来源 14

3 结果与分析 14

3.1 OsPL1突变体的表型鉴定 14

3.2 OsPL1突变体遗传分析 15

3.3 OsPL1基因结构与突变位点鉴定分析 15

3.4 OsPL1突变体的互补验证 16

3.5 花药半薄树脂切片分析 17

3.6 花粉母细胞染色体表型分析 17

3.7 花粉母细胞染色体免疫荧光染色分析 18

3.8 OsPL1基因功能缺陷对绒毡层发育相关基因的表达量影响 18

结 论 20

参考文献 21

在校期间已获成果 26

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