拟南芥FB1和FB2基因在ABA信号传导中的调控分析

发布时间：2018-04-29 08:57

本文选题：拟南芥 + F-box　；参考：《吉林大学》2017年硕士论文

【摘要】：ABA在植物生命周期中起着关键的作用,例如种子的休眠、萌发、幼苗的生长以及开花等。更重要的是,ABA使植物能够耐受与水相关的胁迫,例如干旱和盐度等。迄今为止,在ABA的信号传导途径中一些基本组分已经被鉴定,比如PROTEIN PHOSPHATASES TYPE 2Cs(PP2Cs),SUCROSENON-FERME-NTING 1-RELATED PROTEIN KINASEs(Sn RK2s)和ABA-RESPONSIVE ELEM-ENTS BINDINGFACTORs(ABFs)等。在2009年,在植物细胞中ABA受体PYRABACTIN RESISTANCE 1/PYR-LIKE/REG-ULATORY COMPONENTS OF ABA RECEPTORs(PYR1/PYLs/RCARs)被证明。当受到外源ABA诱导后,PYR1/PYLs/RCARs受体感知ABA并抑制PP2C的活性使Sn RK2去磷酸化,进而调控下游基因的表达。ABA信号转导通路中基本成分水平的严格控制对于稳态是至关重要的。最近,许多研究已经表明,这些中枢调节因子被泛素26S蛋白酶体系降解。在拟南芥基因组中已经发现了超过1400个编码E3连接酶的基因。这些基因可以分为两组:单亚基型和多亚单位型。F-box蛋白作为SCF复合体最重要的亚基是由快速扩增的真核基因家族编码,在N端包含有40-50个氨基酸组成的保守的F-box结构域,首先在细胞周期蛋白F中被发现并介导了与Skp1的相互作用。F-box蛋白在C端都包含一个或者多个高度可变的蛋白质-蛋白质相互作用结构域。在拟南芥和水稻基因组中分别有692和779个F-box蛋白序列被发现。但大多数F-box家族基因的功能还未知。本研究克隆了两个拟南芥F-box家族基因,对这两个基因在ABA信号传导途径中的功能进行了分析,主要研究结果如下:1.利用Infusion技术,克隆了FB1和FB2基因,成功将其构建到N-egad-LUC植物表达载体上。FB1和FB2基因组全长分别为2283 bp和1337 bp。FB1包含两个外显子和一个内含子,而FB2中不包含内含子。FB1和FB2具有典型的F-box结构域,并与油菜中FB1和FB2亲缘关系最近。2.通过农杆菌介导的蘸花侵染,将重组质粒转化到拟南芥中,利用Basta抗性筛选,Luminometer仪瞬时检测和Western Blot技术,证实得到了FB1和FB2的过表达转基因拟南芥,最终得到T3代纯合转基因植株。另外,从ABRC(Arabidopsis Biological Resource Center)购买到FB1和FB2的T-DNA插入突变体,利用三引物法,鉴定出纯合的突变体。3.利用Gateway技术构建了FB1-YFP融合表达载体,并瞬时转染了拟南芥原生质体进行亚细胞定位分析,结果表明FB1蛋白定位于细胞质和细胞核中。4.分别对FB1和FB2过表达拟南芥株系进行ABA响应表型分析,结果表明FB1和FB2过表达拟南芥种子的萌发率明显高于野生型的,对ABA表现出不敏感,并随着ABA浓度的增加表型更加显著。5.在FB1和FB2过表达以及野生型中,分析ABA相关基因的转录水平,发现FB1和FB2过表达引起ABA信号转导负向调节因子ABI1、ABI2的转录水平升高,正向调节因子ABI3、ABI4的表达量下降;但是如ABF转录因子、Sn RK2和ABI5等ABA信号正向调节因子的表达量出现上调,推测可能存在FB1和FB2降解靶蛋白引起了蛋白水平下降而诱导mRNA转录升高的反馈。
[Abstract]:ABA plays a key role in plant life cycle, such as seed dormancy, germination, seedling growth and flowering. More importantly, ABA enables plants to withstand water-related stresses such as drought and salinity. Up to now, some basic components in the signal transduction pathway of ABA have been identified, such as PROTEIN PHOSPHATASES TYPE 2CsSU PP2CsSU SUCROSENON-FERME-NTING 1-RELATED PROTEIN KINASEs(Sn RK2s and ABA-RESPONSIVE ELEM-ENTS BINDINGFACTORs ABFs. In 2009, ABA receptor PYRABACTIN RESISTANCE 1/PYR-LIKE/REG-ULATORY COMPONENTS OF ABA RECEPTORsPYR1 / PYLs / RCARswere demonstrated in plant cells. When induced by exogenous ABA, the PYR1 / PYLsR / RCARs receptor sensed ABA and inhibited the activity of PP2C so that Sn RK2 was dephosphorylated, and then regulated the expression of downstream genes. Recently, many studies have shown that these central regulatory factors are degraded by the ubiquitin 26 S protease system. More than 1, 400 genes encoding E 3 ligase have been found in Arabidopsis genomes. These genes can be divided into two groups: single subunit type and multiple subunit type. F-box protein, as the most important subunit of SCF complex, is encoded by a rapidly amplified eukaryotic gene family and contains a conserved F-box domain consisting of 40-50 amino acids at the N-terminal. First of all, it was found and mediated the interaction with Skp1 in cyclin F. F-box protein contains one or more highly variable protein-protein interaction domains at the C-terminal. In Arabidopsis thaliana (Arabidopsis thaliana) and rice (Oryza sativa) genomes, 692 and 779 F-box protein sequences were found, respectively. But the function of most F-box family genes remains unknown. In this study, two Arabidopsis F-box family genes were cloned and their functions in ABA signaling pathway were analyzed. The main results are as follows: 1. Using Infusion technique, FB1 and FB2 genes were cloned and successfully constructed into the N-egad-LUC plant expression vector. The genome length of. FB1 and FB2 were 2283 BP and 1337 bp.FB1, respectively, containing two exons and one intron, respectively. However, FB2 does not contain introns. FB1 and FB2 have typical F-box domain, and have the closest relationship with FB1 and FB2 in rapeseed. The recombinant plasmid was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens mediated by dipping flower. The transient detection of Basta resistance sieve and Western Blot technique confirmed the overexpression of FB1 and FB2 in Arabidopsis thaliana. Finally, the T 3 fusion transgenic plants were obtained. In addition, the T-DNA insertion mutants of FB1 and FB2 were purchased from ABRC(Arabidopsis Biological Resource Center, and the homozygous mutants .3were identified by three-primer method. The fusion expression vector of FB1-YFP was constructed by Gateway technique, and the subcellular localization analysis of Arabidopsis protoplasts was carried out. The results showed that FB1 protein was located in cytoplasm and nucleus. The ABA response phenotype of Arabidopsis thaliana lines with FB1 and FB2 overexpression was analyzed. The results showed that the germination rate of FB1 and FB2 overexpression Arabidopsis seeds was significantly higher than that of wild-type Arabidopsis plants, and was insensitive to ABA, and the phenotype was more significant with the increase of ABA concentration. In the overexpression of FB1 and FB2 and wild type, the transcriptional level of ABA related genes was analyzed. It was found that the overexpression of FB1 and FB2 resulted in an increase in the transcription level of ABA signal transduction negative regulatory factor ABI1 and ABI2, and a decrease in the expression of positive regulatory factor ABI3, ABI4. However, the expression of ABA signal positive regulatory factors such as ABF transcription factors, such as Sn RK2 and ABI5, was up-regulated, which suggested that there might be feedback that the degradation of FB1 and FB2 could induce the decrease of protein level and induce the increase of mRNA transcription.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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