利用CRISPR/Cas9系统获得水稻ddm1突变体及其表型研究
毕业论文关键词： 水稻； DNA 甲基化； OsDDM1； CRISPR/Cas9；表型研究；表观遗传重组自交系
Acquire ddm1 Mutant by Utilizing CRISPR/Cas9 System andResearch The Phenotype in Rice
Abstract： Plant DNA methylation is a type of classical epigenetic modification, it occurs at 3 sequencecontexts that are CG , CHG and CHH(H=A, C or T), meanwhile, it is capable of repressing repetitivesequences and transposable elements(TEs). Previous study for Arabidopsis ever indicated that,DDM1(Decrease in DNA Methylation 1) is a chromatin remodeler in DNA methylation pathway andencodes the protein in SNF2 family[1]. Except that, it is essential for heterochromatic CHH methylation.Rice(Oryza.Sativa L) is the model plant of monocotyledon, however, the previous study of OsDDM1 inDNA methylation pathway is little in rice. In this research, we treat Nipponbare (Oryza.Sativa L. spp.Japonica, var Nipponbare, AA genome) as a background material, utilize CRISPR/Cas9 which is agenome-editing technique to structure knock-out vector OsDDM1-CRISPR successfully, and transform itinto agrobacterium EHA105 to infect rice callus to acquire Osddm1a/1b homozygous double mutants.Besides that, we observe their phenotype in plant height and panicle development and process hybridizationbetween mutants and wildtype. After this, the prospect of the study is that we can utilize the F1 hybrids selfto generate F2 and screen out the lines whose genotypes were identical to wild-type in OsDDM1a andOsDDM1b, and then acquire epigenetic recombination inbred lines (epiRILs) by continuing to self. We alsocan use epiRILs to locate quantitative trait loci(QTL) and research genes related to growth anddevelopment, disease resistance and stress tolerance and other aspects in rice.
Keywords: rice； DNA methylation； OsDDM1； CRISPR/Cas9； phenotype research； epiRILs
目 录
摘要1
关键词1
Abstract 1
Keywords1
引言 2
1 材料与方法 3
1.1 水稻背景材料 3
1.2 水稻 Osddm1a/1b 纯合双突变体的获得3
1.2.1 OsDDM1 -CRISPR 载体的构建 3
1.2.2 大肠杆菌转化 3
1.2.3 农杆菌电转化与培养 4
1.2.4 水稻愈伤组织诱导与继代培养 4
1.2.5 感菌与共培养 4
1.2.6 愈伤组织的抗生素筛选 5
1.2.7 抗性愈伤组织的诱导分化和生根 5
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