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利用CRISPR/Cas9系统获得水稻ddm1突变体及其表型研究

时间:2019-06-30 08:14来源:毕业论文
植物DNA甲基化是一种经典的表观遗传学修饰,发生在3种序列环境中:CG,CHG和CHH(H=A,C和T),它的功能是抑制植物基因组中重复序列和转座子的表达

摘要: 植物 DNA 甲基化是一种经典的表观遗传学修饰, 发生在 3 种序列环境中: CG, CHG 和 CHH(H=A, C 和 T),它的功能是抑制植物基因组中重复序列和转座子的表达。此前有研究表明, 拟南芥(Arabidopsis thaliana L.) DDM1(Decrease in DNA Methylation 1) 是其 DNA 甲基化途径中一个重要的染色质重塑因子, 它能够编码 SNF2 家族的蛋白质,进而引发染色质重塑[1]; 同时, 它也被异染色质序列的 CHH 甲基化所需要。水稻(Oryza.Sativa L.)是单子叶模式植物, 而此前对 OsDDM1 在 DNA 甲基化途径中作用的研究很少。 在本研究中,我们以粳稻日本晴(Oryza.Sativa L. spp. Japonica, varNipponbare, AA genome)为背景, 利用 CRISPR/Cas9 基因组编辑技术, 成功构建了 OsDDM1-CRISPR的基因敲除载体,转化入农杆菌 EHA105 并侵染水稻愈伤组织,从而获得了 4 种具有不同突变类型的 Osddm1a/1b 纯合双突变体; 我们还观察到突变体与野生型相比具有株高变矮和穗高度不育的表型,并利用突变体与野生型日本晴进行杂交。在此之后对本研究的展望是, 从杂交种 F1 代自交产生的 F2 代中, 筛选出野生型基因型的株系,并不断自交,从而获得表观遗传重组自交系 (epigeneticrecombination inbred lines, epiRILs),利用该群体定位数量性状位点(quantitative trait loci, QTL), 并研究与水稻生长发育、抗病性和抗逆境胁迫等方面相关的基因。36757
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. 源`自!六^维"论^文;网www.lwfree.cn
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 利用CRISPR/Cas9系统获得水稻ddm1突变体及其表型研究:http://www.lwfree.cn/shengwu/20190630/35328.html
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