摘要
以实验室前期采用cDNA-AFLP技术鉴定的、在低磷胁迫处理小麦叶片中1个呈上调表达的基因转录本(TDF)片段为基础,对该基因的分子特征和转录调控特性进行了较系统研究。主要研究结果如下。
1、BLAST比对结果表明,该TDF与源于蓖麻(Ricinus communis L.)叶绿体磷转运蛋白基因(GenBank登录号为XM_002527885)和拟南芥磷转运蛋白PHT2;1(GenBank登录号为AF515591)具有较高的同源性。在小麦全长cDNA数据库中,获得与该TDF在对应区域序列一致的全长基因(克隆号为SET3_C22,GenBank登录号为AK336079),鉴于有关该基因的分子特征尚未报道,本研究将其命名TaPT2-1。
2、依据TaPT2-1的cDNA序列设计特异引物,扩增了该基因的编码阅读框(open reading frame, ORF)。分析表明,TaPT2-1的cDNA全长为2075 bp,编码568个氨基酸残基。利用生物信息学软件进行TaPT2-1的保守跨膜域预测,发现该蛋白含有13个跨膜区域。
3、系统进化分析发现,TaPT2-1在核苷酸序列水平上与拟南芥(A. thaliana)、马铃薯(S. trberosum)、辣椒(C. frutescens)和茄子(S. melongena)中鉴定的磷转运蛋白基因(PTs)高度同源。表明TaPT2-1与上述PTs在起源上可能具有相同的祖先。
4、采用半定量RT-PCR技术,研究了TaPT2-1在不同供磷水平(正常供磷CK和20μM Pi低磷胁迫)下根系和叶片中的表达水平,结果表明,TaPT2-1的转录本在叶中受到低磷处理的明显诱导;在根系中的表达呈组成型特征,不随介质中的磷素供应状况而发生明显改变。表明TaPT2-1主要参与了叶片中的磷素的转运过程。
5、采用基因组行走技术(genome walking approach),在小麦的基因组中克隆了长度为1744 bp的TaPT2-1启动子区序列。研究发现,TaPT2-1启动子中含有保守的转录调控元件TATA盒和CAAT盒,以及在应答低磷逆境中发挥重要作用的PIBS和Pho-like调控元件。此外,该启动子也含有参与组织优势表达、防御反应应答、生长素和茉莉酸应答以及光照和糖碳代谢相关的重要调控元件。
6、构建了用TaPT2-1启动子驱动报告基因β-葡萄糖苷酸酶基因(GUS)表达的烟草表达系统。研究发现,TaPT2-1启动子驱动GUS在不同磷水平下的表达与半定量RT-PCR的检测结果一致,呈驱动报告基因在叶片中呈明显的低磷诱导特征。此外,该启动子也驱动报告基因在茉莉酸甲酯处理下呈诱导表达。
7、TaPT2-1启动子驱动GUS在低磷处理下叶片中的表达以老叶显著强于新生叶。表明小麦磷转运蛋白基因TaPT2-1主要通过在老叶中诱导增强表达,在改善植株低磷胁迫下的植株磷素转运和利用效率中发挥重要作用。
Previously, a transcript-derived fragment (TDF) in leaves responding to the low-Pi stress was identified in our group based on cDNA-AFLP analysis. The TDF was used for identification of the molecular cheracterization and transcriptional regulation of the corresponding gene in this present study. The main results are as follows:
1. BLAST search analysis suggests that the TDF, a transcript-derived fragment responding to the low-Pi stress, shares high similarities to the phosphate transporter (PT) genes identified in other plant species, including the chloroplast PT gene from Ricinus communis L. (GenBank accession number XM_002527885) and the PT gene from Aarabidopsis thaliana (GenBank accession number AF515591). Based on search of the wheat full-length cDNA database, the full-length gene (clone number SET3_C22, and the GenBank accession number AK336079) coresponding to the TDF was identified. Owing to no report on this gene, it was referred to TaPT2-1.
2. A specific primer pair was designed and the RT-PCR for amlification of the open reading frame (ORF) was successfully performed. It is found that the cDNA full-length of TaPT2-1 is 2075 bp, encoding a 568-aa polypeptide. Based on online analysis with a transmembrane prediction tool, TMprep, thirteen transmembrane domains were identified in TaPT2-1.
3. Phylogenetic analysis displayed that TaPT2-1 shared much higher similarities to other four homologs from Arabidopsis thaliana, Solanum trberosum, Capsicum frutescens, and Solanum melongena. These results have implicated that all of them share a common ansestor along with the evolution process.
4. Based on semi-quantitative RT-PCR analysis, the expression patterns of TaPT2-1 in leaves and roots under various Pi-supply condition were detected. It was ovserved that the transcripts of TaPT2-1 in leaves were significantly induced by the low-Pi stress. Whereas the expression of TaPHY2;1 in roots was shown to be constitutive, no obivious alteration detected with the changes of Pi-supply. Therefore, TaPT2-1 is possibly involved in the Pi recycling process in leaves under the Pi-stress condition.
5. Using the genome walking approach, the promoter region of TaPT2-1 with a 1744 bp length was cloned from cv. Shixin828. The putative TaPT2-1 promoter contains the conserved boxes, TATA and CAAT, two playing important roles on gene transcriptional regulation. In addition, TaPT2-1 promoter also contains two regulatory elements, PIBS and Pho-like, being invoved in the responding to low-Pi stress signaling. Furthermore, some potential key regualtory elements, such as those involved in tissue-specific, defense response, auxin and salicylic acid responding, and signaling of light, sugar, and carbon, were also identified in TaPT2-1 promoter.
6. The transgenic tobacco plants with the integrated TaPT2-1 promoter: GUS were generated. GUS histochemical staining analysis in the roots and leaves of the transgenic plants was performed. The results of GUS staining in roots and leaves under various Pi-supply conditions were in accordance with the TaPT2-1 transcripts detected based on RT-PCR analysis, showing a pattern to be induced expression in leaves. Also, the expression level of GUS was also induced by external treatment of salicylic acid.
7. The expression level of reporter gene GUS under the control of TaPT2-1 promoter was much more induced in old leaves compared with those of novels. The distinct expression of low-Pi induced and leaf-predominantly expression of TaPT2-1 especially in old leaves suggest that this wheat phosphate transporter gene plays an critical role on cellelur transportation of Pi and improvement of Pi use efficiency in plants under low-Pi stress condition.
引文
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[49]谷俊涛,鲍金香,王效颖等.利用cDNA-AFLP技术分析小麦应答低磷胁迫的特异表达
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[35] Daram P, Brunner S, Persson B L, et al. Functional analysis and cellspecific expression of a phosphate transporter from tomato [J]. Planta, 1998, 206:225-233.
[36] Leggewie G, Wilmitzer L, Riesmeier J W. Two cDNAs from potatoare able to complement a phosphate uptake-deficient yeast mutant: i-dentification of phosphate transporters from higher plants [J]. Plant Cell, 1997, 9: 381-392.
[37] Liu H, Trieu A T, Blaylock L A, et al. Cloning and characterization of two phosphate transporter genes are differentially regulated inplant tissues by phosphorus [J]. Plant Physiol, 1998, 116: 91-99.
[38] Ai P, Sun S, Zhao J, et al. Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation [J]. Plant J, 2009, 57: 798-809.
[39] Guo B, Jin Y, Wussler C, et al. Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters [J]. New Phytol, 2008, 177: 889-898.
[40] Seo H M, Jung Y, Song S, et al. Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice [J]. Biotechnol Lett, 2008, 30: 1833-1838.
[41] Mitsukawa N, Okumura S, Shirano Y, et al. Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions [J]. Proc Natl Acad Sci USA, 1997, 94:7098-102.
[42] Daram P, Brunner S, Rausch C, et al. Pht2;1 encodes a low affinity phosphate transporter from Arabidopsis [J]. Plant Cell, 1999, 11:2153-2166.
[43] Tittarelli A, Milla L, Vargas F, et al. Isolation and comparative analysis of the wheat TaPT2 promoter: identification in silico of new putative regulatory motifs conserved between monocots and dicots [J]. J Exp Bot, 2007, 58: 2573-2582.
[44] Smith F W, Ealing P M, Dong B, Delhaize E. The cloning of two Arabidopsis genes belonging to a phosphate transporter family [J]. Plant Journal, 1997, 11: 83-92.
[45] Okumura S, Mitsukawa N, Shirano Y, Shibata D. Phosphate transporter gene family of Arabidopsis thaliana [J]. DNA Research, 1998, 5: 261-269.
[46] Karthikeyan A S, Varadarajan D K, Mukatira U T, et al. Regulated expression of Arabidopsis phosphate transporters [J]. Plant Physiology, 2002, 130: 221-233.
[47] Rae A L, Cybinski D H, Jarmey J M, Smith F W. Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family [J]. Plant Molecular Biology, 2003, 53: 27-36.
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[49]谷俊涛,鲍金香,王效颖等.利用cDNA-AFLP技术分析小麦应答低磷胁迫的特异表达