水稻OsCYP2及其互作蛋白OsZFP调控侧根生长的功能研究

发布时间：2018-01-12 00:19

本文关键词：水稻OsCYP2及其互作蛋白OsZFP调控侧根生长的功能研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：侧根是水稻胚后发育的重要器官,在水稻根的构型建立中起着举足轻重的作用。尽管前人的研究报道中已对水稻侧根的形态和生理作用有了较详尽的描述,但是对于控制水稻侧根形成的相关基因所知仍然不多,因此对水稻侧根发生机制的研究及关键基因的克隆具有重要意义。本研究以水稻品种'爱知旭'野生型(WT)、OsCYP2基因过表达和干扰三种基因型植株为材料,通过对其表型、表达谱验证以及互作蛋白OsZFP的基因克隆和功能分析,研究了 OsCZP2和OsZFP两个基因在生长素信号通路上对侧根发生的影响,取得创新性结果如下:1、OsCYP2是一个在水稻各组织器官中广泛表达的基因,其干扰之后导致侧根形成受阻,侧根数量显著减少。GUS染色结果显示,颖壳、叶、茎和根等组织部位均呈现明显的蓝色信号,其中颖壳颜色最深,表明OsCYP2的启动子为组成型表达启动子。外源生长素IAA处理前后,OsIAA1、OsIAA8和OsIAA31基因的表达量在OsCYP2干扰植株中无显著性差异,表明其依赖于OsCYP2信号途径。并且,除OsIAA1和OsIAA20,OsCZP2-RNAi植株中其余4个生长素响应基因(OsIAA8、11、23和31)的诱导表达量与WT相比均显著下降,表明OsCZP2干扰植株对生长素处理存在一定的不敏感性。2、RNA-seq结果显示,与WT相比,OsCYP2-RNAi的31515个差异表达基因中有3158个表现显著性差异,其中1267个基因呈上调趋势,1891个呈下调趋势。11个显著差异表达基因与侧根形成有关,4个差异基因与生长素有关。RT-PCR验证结果表明以上相关基因的上/下调趋势与RNA-seq结果一致,由KEGG数据库分析得到差异表达基因的前20个富集pathway中核糖体富集最显著,其次是次生代谢生物合成和类黄酮合成。3、构建了水稻cDNA文库,通过酵母双杂交筛选,获得3个阳性互作蛋白,之后经 GST-pulldown 体外互作验证,OsCYP2 与 OsZFP(Os01g0252900)存在互作。经同源性比对,OsZFP属CCHC型锌指结构蛋白;聚类分析表明,在拟南芥、大麦、高粱和玉米中高度保守,而在人类、酵母和小麦没有此结构域。4、利用反向遗传学方法,对水稻OsZFP基因调控侧根生长发育的生物学功能进行研究。以水稻品种'爱知旭'野生型为试验材料,构建了 PCAMBIA1300-OsZFP-RNAi表达载体并获得多个稳定遗传转化株系。结果表明,T2代OsZFP-RNAi植株同样出现水稻侧根形成受阻,侧根缺失的表型。之后构建OsZFP基因与'GFP融合的表达载体并进行水稻原生质体转化,亚细胞定位结果表明OsZFP蛋白定位于细胞核。外源生长素IAA处理下,RT-PCR结果显示OsZFP-RNAi植株中六个Aux/IAA基因(OsIAA1、2、6、20、23和24)与无激素处理相比没有显著差异,表明OszFP基因被干扰之后,植株的生长素信号途径受到影响。
[Abstract]:The lateral root is an important organ development after rice embryo, in rice root configuration plays an important role in the establishment. Although previous research reports have been on rice lateral root morphological and physiological function of a more detailed description, but the genes controlling rice lateral root formation is still not known, so cloning research and the key gene on pathogenesis of rice root has important significance. In this study, rice cultivar 'Aichi Asahi' wild type (WT), OsCYP2 gene expression and interference of three genotypes as materials, based on their phenotype, expression and verification of interaction protein OsZFP gene cloning and functional analysis of OsCZP2 and OsZFP two genes in auxin signaling pathway on lateral root formation, the innovative results are as follows: 1, OsCYP2 is a widely expressed in various tissues and organs of rice genes, after its interference Lead to lateral root formation is blocked, the number of lateral roots was significantly reduced..GUS staining showed that glume, leaf, stem and root tissues showed a clear signal to the blue part, wherein the glume color deep, showed that the OsCYP2 promoter for constitutive expression promoter. The exogenous auxin IAA before and after the treatment, OsIAA1, expression of OsIAA8 and the OsIAA31 gene had no significant difference in plant OsCYP2 interference, showed that the OsCYP2 dependent signaling pathway. And, in addition to OsIAA1 and OsIAA20, the remaining 4 auxin response genes in OsCZP2-RNAi mutants (OsIAA8,11,23 and 31) induced expression of WT decreased significantly, OsCZP2 interference showed that plants on auxin.2 sensitivity the RNA-seq results showed that compared with WT, the 31515 difference expression of OsCYP2-RNAi 3158 showed significant differences in gene, of which 1267 genes were up-regulated and 1891 were downregulated.11 The formation of a gene with the roots significantly differentially expressed genes and auxin, 4 different.RT-PCR validation results showed that the above genes / downward trend was consistent with RNA-seq, KEGG database by analysis of gene expression difference obtained before the 20 enrichment of pathway ribosomes in the enrichment was most significant, followed by secondary metabolism and biosynthesis of flavonoid biosynthesis.3 construction of rice cDNA library by yeast two hybrid screening, 3 positive interaction protein after GST-pulldown in vitro interaction test, OsCYP2 and OsZFP (Os01g0252900) interaction. By homology, OsZFP belongs to CCHC type zinc finger protein; cluster analysis showed that in Arabidopsis, barley, sorghum and maize highly conserved in human, yeast and wheat without the.4 domain, using the method of reverse genetics, biological function of rice OsZFP gene regulation of lateral root growth and development To study. With the rice variety 'Aichi Asahi' wild type as the experimental materials, the expression vector PCAMBIA1300-OsZFP-RNAi was constructed and a stable genetic transformation strain. The results showed that the T2 generation of OsZFP-RNAi plants also appear rice lateral root formation is blocked, root missing. After constructing the phenotypic expression vector of OsZFP gene and'GFP fusion and rice protoplast transformation, subcellular localization results showed that OsZFP protein was localized in the nucleus. The exogenous auxin IAA, RT-PCR results showed that six Aux/IAA genes in OsZFP-RNAi mutants (OsIAA1,2,6,20,23 and 24) and non hormone treatment had no significant difference compared with that after OszFP gene was interfered, the auxin signaling pathway was affected.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511

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