小黑杨PsnOBP1在转基因烟草次生生长中的功能研究

发布时间：2018-12-15 16:09

【摘要】：OBP1 (OBF binding protein)为Dof (DNA binding with one finger)类转录因子,属于锌指蛋白超家族(zinc finger super-family)的成员,是一类植物特有的转录因子。OBP1转录因子能够通过调控细胞周期蛋白基因参与植物的生长发育过程。本项研究从小黑杨(Populus simonii × P. nigra)中克隆了OBP1的全长cDNA序列,因其与拟南芥OBP1相似性达99%,因此命名为PsnOBP1。通过对PsnOBP1基本特性与过表达烟草主要生长与次生性状分析,得到以下结论。(1) PsnOBP1基因编码248个氨基酸,其分子量为25.5 kDa,等电点为8.57,属于锌指蛋白超家族的Dof转录因子。(2) PsnOBP1组织表达特异性研究表明,PsnOBP1基因在小黑杨的各个组织中均有表达,但在木质部表达量很高,其次为根部及叶部组织。洋葱表皮细胞瞬时表达研究发现,PsnOBP1编码蛋白定位在细胞核中。利用酵母单杂交对PsnOBP1转录结合能力进行分析,结果表明PsnOBP1能与次生细胞壁形成基因(如MYB61, MYB63, MYB43, MYB92)启动子区域高频率元件GA-motif、GARE-motif、TCA和HSE结合。(3)过表达PsnOBP1烟草主要生长性状与次生性状分析表明,相比野生型,转基因株系的茎秆强度、叶绿素含量、纤维长度和宽度和纤维素含量均显著增加,次生细胞壁厚的明显增加,而木质素含量显著下降；转基因株系次生细胞壁形成相关功能基因的表达均发生了不同程度的变化,其中纤维素(CesA4, CesA7, CesA8)、半纤维素形成基因(CesA4, CesA7, CesA8)表达量显著上调,而木质素形成基因(IRX8,IRX9, FRA8)表达量下调；转基因株系生长发育正常,能正常开花结实，，且株高、生物量与野生型相比,无显著差异。(4)拟南芥AtOBP1突变体的次生性状分析：与正常的野生型拟南芥相比株高与叶片大小无明显变化,茎部染色观察,拟南芥AtOBP1突变体与野生型相比较,纤维素染色区域变暗,木质素染色区域较深。
[Abstract]:OBP1 (OBF binding protein) is a member of Dof (DNA binding with one finger) transcription factor, which belongs to the zinc finger protein superfamily (zinc finger super-family. OBP1 transcription factors can participate in the growth and development of plants by regulating cyclin genes. In this study, the full-length cDNA sequence of OBP1 was cloned from (Populus simonii 脳 P. nigra). Because of its similarity with OBP1 of Arabidopsis thaliana, it was named PsnOBP1.. By analyzing the basic characteristics of PsnOBP1 and the main growth and secondary characters of over-expressed tobacco, the following conclusions are obtained: (1) the PsnOBP1 gene encodes 248 amino acids with a molecular weight of 25.5 kDa, isoelectric point of 8.57; Dof transcription factors belonging to the zinc finger protein superfamily. (2) the specific expression of PsnOBP1 in tissues showed that PsnOBP1 gene was expressed in all tissues of Populus nigra, but it was highly expressed in xylem, followed by root and leaf tissues. Transient expression of onion epidermis cells revealed that PsnOBP1 encoded proteins were located in the nucleus. The transcriptional binding ability of PsnOBP1 was analyzed by yeast single hybridization. The results showed that PsnOBP1 could interact with the high frequency element GA-motif,GARE-motif, in the promoter region of secondary cell wall forming gene (such as MYB61, MYB63, MYB43, MYB92). (3) Analysis of the main growth and secondary characters of over-expressed PsnOBP1 tobacco showed that the stem strength, chlorophyll content, fiber length and width and cellulose content of transgenic lines increased significantly compared with wild type. The thickness of secondary cell wall increased and lignin content decreased significantly. The expression of functional genes related to secondary cell wall formation in transgenic lines changed in varying degrees, in which the expression of cellulose (CesA4, CesA7, CesA8) and hemicellulose forming gene (CesA4, CesA7, CesA8) were significantly up-regulated. However, the expression of lignin forming gene (IRX8,IRX9, FRA8) was down-regulated. The transgenic lines have normal growth, normal flowering and fruiting, plant height and biomass compared with wild type. (4) Analysis of secondary characters of Arabidopsis AtOBP1 mutants: the plant height and leaf size of Arabidopsis thaliana had no significant change compared with the normal wild type Arabidopsis thaliana. The AtOBP1 mutants of Arabidopsis thaliana were compared with wild type by stem staining. Cellulose staining area darkened, lignin staining area was deeper.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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