摘要
植物根系对磷的吸收和磷在细胞和组织间的传输,是通过位于细胞原生质膜上的磷转运蛋白介导的需能主动运输过程。本项研究针对迄今有关小麦磷转运蛋白基因研究和报道较少的现状,利用现代生物信息学技术和分子生物学技术,对部分小麦磷转运蛋白的分子特征、表达特性进行较全面的研究。主要研究结果如下:
1、通过在国际生物信息学网站进行小麦磷转运蛋白基因查找,获得4个尚未进行特征分析、表达和功能研究的小麦磷转运蛋白基因,GenBank登录号分别为AF488415、AJ830009、AY293827和AY293828。本研究依次命名为TaPT1、TaPT2、TaPT3和TaPT4。
2、TaPT1~TaPT4的编码氨基酸数量变化在467~555之间,分子量变化在57.44~60.86之间,含有在11至14个跨膜域,以及蛋白激酶C位点和酪蛋白激酶磷酸化位点。表明TaPT1~TaPT4具有植物种属磷转运蛋白基因共有的结构特征。各供试小麦磷转运蛋白基因的表达特征存在较大差异。TaPT1在正常供磷水平下,根叶中的均有表达,低磷胁迫条件下,根系中检测不到该基因的转录本,而叶片中表达表现为随低磷胁迫时间延长不断增加;TaPT2和TaPT3的表达表现为根系特异表达的特征,随着低磷胁迫时间延长,表达水平也不断增强。TaPT4在根叶及不同供磷水平下呈组成型表达特征。
3、采用染色体步移技术克隆TaPT2的启动子,克隆的启动子全长1237 bp。分析表明,在TaPT2启动子的翻译起始位点(ATG)上游附近,含有转录调控的保守元件TATA盒和CAAT盒。此外,含有应答低磷胁迫逆境能力的PHO-like元件(GACGTGG, -18 bp)。表明该PHO-like元件可能介导了TaPT2对低磷胁迫的应答。
4、利用DNA重组技术,构建了融合TaPT2启动子的双元表达载体pCAMBIA3301-TaPT2-Gus,以及用TaPT2不同缺失片段长度驱动报告基因Gus表达的双元表达载体。为进一步揭示TaPT2的转录调控机制奠定了基础。
5、以通过GenBank查找获得的1个栽培一粒小麦磷转运蛋白基因(TabPT1, GenBank登录号:AF488415)为基础,对该基因的分子特征、表达特性进行了研究。研究表明,TabPT1是普通小麦TaPT1的祖先,在分子特征和表达特性上与TaPT1一致。构建了融合TabPT1启动子及其不同缺失片段的双元表达载体,为进一步揭示该基因的转录调控机制和功能奠定了基础。
The uptake by the roots and the transportation in cells and tissues of inorganic phosphorus (Pi) were mediated by the phosphate transporters which are located at the cytoplasm membrane. The process of uptake and transportation of Pi is necessary of the energy supply. So far, few studies about of wheat (Triticum aestivum L.) phosphate transporters have been reported. In this study, the molecular characterization, expression patterns of several phosphate transporter genes derived from wheat and its ancestor Triticum boeoticum were analyzed based on modern bioinformatic approach and molecular biological techniques.
1. Based on serches of wheat phosphate transporter genes in international bioinformatics website (NCBI), four wheat phosphate transporter genes with uncharacterized, unknown expression patterns and functions were obtained. The GenBank accession numbers were AF488415, AJ830009, AY293827, and AY293828. In this study, they were designated as TaPT1, TaPT2, TaPT3, and TaPT4, respectively.
2. The translated amino acids of TaPT1~TaPT4 changed from 467~555, with the morlecular weights of 57.44~60.86 and 11 to 14 conserved transmembrane domains, and protein kinase C activation sites, and casein phosphorylation sites. Therefore, TaPT1~TaPT4 own the conserved structural properties of phosphate transporters in plant species. There were dramatic differences on the expression patterns of the tested phosphate transporter gens. Under normal Pi-supply condition, there were similar transcripts of TaPT1 in roots and leaves, with a pattern that no transcripts detected in roots under low-Pi condition and elevated expression levels with the extension of low-Pi treatment. The expressions of TaPT2 and TaPT3 were shown root specific, also increased in transcripts with the extension of low-Pi treatment. The expression patter of TaPT4 in roots and leaves under various-Pi conditions was shown to be constitutive.
3. Using genome walking approach, the promoter region of TaPT2 with 1237 bp length was isolated. The conserved regulatory elements functional on transcriptional regulation, such as TATA box and CAAT box were identified. The Pi-starvation response element PHO-like (GACGTGG, -18 bp) was also figured out in TaPT2 promoter, indicating that this element possibly involved in the low-Pi cue responding of TaPT2.
4. Using DNA recombinant technology, the binary cassette of TaPT2-Gus, and series of cassettes in which the reporter gene Gus being driven by the deleted promoter fragments have been constructed. This work would provide the basis for further exploration of the transcriptional regulation of TaPT2 in the future.
5. Based on a DNA clone (GenBank accession number: AF488415), a phosphate transporter gene TabPT1 was identified. The molecular characterization, expression pattern were evaluated. It is found that the TabPT1 was the ancestor of TaPT1, the phosphate transporter gene which was former characterized. There were same molecular characterization and expression patterns between TabPT1 and TaPT1. The binary cassettes fused the open reading frame (ORF), the TabPT1 promoter and its series of deleted promoter fragments have been constructed, aiming at further understanding of the transcriptional regulation and function in the future.
引文
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