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作 者 ：郭丽 郭程瑾 路文静 李小娟 肖凯

期 刊 ：植物营养与肥料学报 2014年 20卷 4期 页码：877-884

关键词：小麦(Triticum aestivum L.)；磷转运蛋白基因；低磷胁迫；磷素吸收；干物质生产；

Keywords:Wheat (Triticum aestivum L.), Phosphate transporter gene, Pi deprivation, phosphate acquisition, dry mass production,

摘 要 ：【目的】植株对介质中磷素的吸收及磷素在体内器官组织间的转运，是通过位于细胞质膜上的磷转运蛋白（PT）介导完成的。高亲和PT在介导植物对低磷逆境下的磷素吸收中发挥重要作用。本研究以小麦中国春遗传背景的整套B染色体双端体为材料，对小麦高亲和PT基因TaPht1; 4的染色体定位特征及其与低磷下小麦品种磷效率的联系进行系统研究，旨在为今后小麦品种磷效率分子鉴定和磷高效遗传改良提供依据。【方法】采用水培法培养中国春（CS）及其遗传背景B染色体组双端体幼苗。三叶期时收获各供试材料根系，提取各材料基因组DNA，通过PCR特异扩增TaPht1; 4，鉴定TaPht1; 4在染色体上定位。通过对各供试材料三叶期幼苗进行24 h低磷胁迫获取丰缺磷处理根叶样本，采用半定量RT-PCR及实时定量PCR分析TaPht1; 4在丰缺磷下的表达。采用上述幼苗培养、 丰缺磷处理和基因表达分析技术，研究不同磷吸收效率小麦品种磷效率参数和TaPht1; 4表达特征。【结果】 1）与CS及其他双端体材料能特异扩增目标基因不同，在3BS中未扩增到目标基因TaPht1; 4； 采用半定量RT-PCR和qPCR对丰、 缺磷下CS和各双端体根、 叶中TaPht1; 4的表达研究表明，丰磷下各供试材料根、 叶中均检测不到TaPht1; 4 表达，缺磷下各供试材料叶片中也均未检测到TaPht1; 4表达，但在根中除3BS未检测到TaPht1; 4 表达外，CS和其他双端体均具有较高的TaPht1; 4表达水平。表明TaPht1; 4定位在3B染色体长臂，呈低磷诱导和根系特异表达特征。2）丰磷下，3BS单株干重与CS没有差异； 缺磷下，与CS相比，3BS单株干重显著降低。表明缺少TaPht1; 4及所在3B染色体长臂后，植株干物质生产能力受到较大影响，这可能与因缺乏该染色体臂丧失TaPht1; 4造成低磷下植株的磷素吸收能力降低密切相关。3）对丰、 缺磷下不同磷吸收效率6个小麦品种TaPht1; 4 的表达水平以及单株干重、 全磷含量、 磷累积量和磷效率研究表明，缺磷下各小麦品种表现为随品种磷吸收效率提高，TaPht1; 4表达水平也随之增高。表明TaPht1; 4 表达水平与低磷下小麦品种磷素吸收能力和干物质积累具有紧密联系。【结论】小麦高亲和PT基因TaPht1; 4 定位在3B长臂。低磷条件下，3BS的单株干重和磷累积量较CS显著降低。丰、 缺磷下，不同磷吸收效率小麦品种TaPht1; 4 表达水平与植株干重和单株磷累积量密切相关。TaPht1; 4 能显著增强小麦在低磷下磷素吸收能力，可作为小麦品种耐低磷能力的参考分子评价指标。

Abstract:【Objectives】The acquisition of inorganic phosphate (Pi) and the Pi translocation across the organs and tissues in plants is mediated by phosphate transporters (PTs) located at the cytoplasmic membranes. The PTs with high-affinity property play critical roles in mediating the Pi absorption by plants under the Pi-limited condition. Currently, the molecular characterization and biological functions of PTs in wheat were few reported. In this study, using Chinese spring (CS) and its ditelosimic lines of B chromosome as materials, the localization on chromosome as well as expression patterns of TaPht1; 4, a highaffinity PT gene in wheat, was systematically studied under sufficient- and deficient-Pi  conditions. In addition, the relationship between the expression levels of TaPht1; 4 and the plant phosphorus use efficiencies across various wheat cultivars under supply of lower phosphorous was determined to provide molecular basis for evaluation of phosphorus use efficiency across wheat cultivars and guidance on genetic improvement for high phosphorus usage in wheat.【Methods】The hydroponic culture method was used to cultivate seedlings of Chinese spring (CS) and its ditelosimic lines of B chromosome. The roots and leaves of all tested materials were separately harvested at three-leaf growth stage and subjected to extraction of genome DNA. PCR analysis was performed using the gene specific primers of TaPht1; 4  to identify the chromosome location of TaPht1; 4. The roots and leaves of all tested materials under conditions of sufficient- and deficient-Pi by treated with 24 h low-Pi stress were sampled and subjected to expression analysis of TaPht1; 4   gene by semiquantitative RT-PCR and real-time PCR. Using the similar methods of seedling culture, treatments of sufficient- and deficient-Pi, and gene expression analysis approaches, the parameters of phosphorus use efficiency and the expression patterns of TaPht1; 4 in wheat cultivars were investigated.【Results】 1) In contrast to CS and other its ditelosimic lines of B chromosome, TaPht1; 4  was not amplified by PCR in 3BS. Moreover, the expression analysis of TaPht1; 4 by semiquantitative RT-PCR and real-time PCR revealed that CS and all ditelosimic lines of B chromosome both in the roots and leaves under the condition of sufficient- Pi did not exhibit the expression of TaPht1; 4,  and similar result was shown in the leaves under the condition of deficient-Pi. However, the transcripts of TaPht1; 4 were detected in the roots of CS as well as in the ditelosimic lines, and was not detected in that of 3BS. Therefore, TaPht1; 4 is located at the long arm of 3B and exhibits expression patterns of induction by low-Pi stress, and has root-specific expression. 2)Under the condition of sufficient-Pi, no variations on plant dry mass was observed between the 3BS and CS. By contrast, the plant dry mass of 3BS was significantly lower than that of CS under the condition of deficient-Pi. These results indicate that the plant dry matter production is largely affected by TaPht1; 4 and the long arm of 3B that situates in the TaPht1; 4. Deletion of TaPht1; 4 by missing this chromosome arm can result in significant reduction in plant dry mass under deprivation of Pi. 3)Investigations on the expression levels of TaPht1; 4，the plant dry mass, total phosphorus contents, accumulative phosphorus amount per plant, and phosphorus use efficiencies in six wheat cultivars with contrasting Pi acquisition capacity indicated that the expression levels of TaPht1; 4 was positively correlated with the Pi acquisition capacities and phosphorus use efficiencies under the condition of deficient-Pi.【Conclusions】The wheat high affinity PT gene, TaPht1; 4 is located at the long arm of 3B. Under the condition of deficient-Pi, the plant dry mass and accumulative amounts of phosphorus per plant of 3BS were significantly lower than those of CS. The expression levels of TaPht1; 4 across the wheat cultivars with contrasting Pi acquisition capacity were closely associated with their plant dry masses and accumulative amounts of phosphorus per plant under conditions of sufficient- and deficient-Pi. Taken together, TaPht1; 4 plays critical roles in regulating plant Pi acquisition in wheat under low-Pi stress, and can act as one of valuable molecular criteria in evaluating the capacity to tolerate low-Pi stress in wheat.

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