摘要：水稻是世界主要粮食作物之一，水稻病害是阻碍水稻高产、稳产的重要因素。染色质重构蛋白是染色质重塑的主要调控者，可利用水解ATP 释放的能量改变染色质构型，进而调控基因的表达。水稻中有40个SNF2类染色质重构蛋白，它们中大部分蛋白的功能尚待研究。ISWI属于SNF2染色质重构蛋白中的Iswi亚类，被证明在拟南芥发育和核小体调控中起重要作用。本实验通过CRISPR/Cas9系统构建了ISWI基因OsCHR11的功能缺失突变体，观察到突变体出现了生长发育缺陷表型。通过对突变体植株接种水稻白叶枯菌PXO99之后的抗病性分析，初步证明OsCHR11基因是水稻抗病性的负调控因子，参与水稻抗病性调控的基因网络。

关键词：水稻；染色质重构蛋白；抗病性；CRISPR技术

The Mutant Generation and Disease Resistance Phenotype Identification Study of OsCHR11 in Rice

Abstract：Rice is one of the main grain crops in the world and rice disease is an important factor that hinders the high yield and stable yield of rice.As a major player of chromatin remodeling, chromatin remodeling ATPases regulate global gene expression by utilizing energy derived from ATP hydrolysis to alter histone-DNA contacts within the nucleosome. There are 40 putative Sucrose Non-Fermenting 2 (SNF2) ATPases in rice, most of which have unknown functions. The Imitation Switch (ISWI) gene belongs to the Iswi subgroup of the SNF2 ATPases, and ISWI in Arabidopsis is involved in development and nucleosome regulation.In this study, loss-of-function mutants of ISWI gene OsCHR11 was constructed by CRISPR/Cas9 system. It was observed that the mutant showed a growth and development defect phenotype.The resistance analysis of mutants after inoculation of biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99 showed that the OsCHR11 gene was a negative regulator of rice disease resistance and it was involved in the gene network of rice resistance regulation.

Key words: Rice；Chromatin remodeling protein；disease resistance; CRISPR technology

目  录

摘要1

关键词1

Abstract1

Key words1

1  引言1

1.1  染色质重塑概述1

1.2  SNF2类染色质重塑蛋白概述2

1.3  CRISPR技术概述2

2  材料与方法3

2.1  材料3

2.1.1  转基因材料3

2.1.2  菌株与质粒3

2.1.3  非生物材料4

2.2  试验方法4

2.2.1  水稻愈伤组织培养4

2.2.2  载体构建5

2.2.2.1  筛选靶点5

2.2.2.2  引物设计5

2.2.2.3  PCR扩增5

2.2.2.4  建立酶连接体系5

2.2.2.5  转化产物筛选6

2.2.3  农杆菌转化6

2.2.4  纯和突变体的筛选鉴定6

2.2.5  抗病性状的鉴定6

3  结果与分析7

3.1  突变位点分析7

3.2  野生型与突变体生长表型比较7

3.3  OsCHR11以及抗病相关基因的表达分析8

3.4  Oschr11抗病表型分析 8

4  讨论8

致谢9

参考文献9

OsCHR11突变体的产生及其抗病表型鉴定

1  引言

水稻(Oryza sativa L.)是世界上最主要的三大粮食作物之一，播种面积占世界粮食总面积的1/5，年产量占世界粮食总产量的1/4。目前我国约60% 的人口以水稻为主食[1]。水稻稳产及增产对保障我国粮食安全及国民经济的发展具有重要意义。然而，水稻在生长的过程中常会遭到白叶枯病、稻瘟病和纹枯病等病害的危害，给水稻产量造成严重损失。长期的生产实践证明，水稻抗病品种的选育和利用是防治水稻病害最经济有效的措施[2]。因此，挖掘在水稻抗病调控过程中起重要作用的基因，研究基因的作用机理，对培育水稻抗病新品种具有重要意义。

1.1  染色质重塑概述