土壤植酸磷高效利用基因GmPAP14生物学功能解析

Phosphorus (P) is one of the least available nutrients that restrict crop production in many ecosystems. Although multiple forms of P exist in soil, only the inorganic form (Pi) dissolved in soil is readily available for uptake by plant root. However, 50%-80% of the total P in agricultural soils exists as organic phosphate, and about 60%-80% of which is myo-inositol hexakisphosphate (phytate). As plant can not directly absorb phytate-P, low availability of P becomes one of the limiting factors to crop growth. Phytase or phosphatase in root and/or root exudation has been demonstrated to be very important for utilizing Pi from phytate in plant rhizosphere. .In our previous study, a novel purple acid phosphatase gene, GmPAP14 (GenBank No. JN967626), was isolated from soybean high P-efficient variety ‘ZH15’. When phytate was added as the sole phosphorus source in culture, the relative expression level of GmPAP14 in ‘ZH15’ was significantly higher than that in ‘NMH’ (P-inefficient genotype), with peak transcription being approximately 35-fold higher in ‘ZH15’ than in ‘NMH’. When GmPAP14 was introduced in Arabidopsis thaliana, the over-expression transgenic plants were more robust under phytate condition compared with the wild-type (WT). .On the basis of the former analysis of GmPAP14 gene, this study proposes to transform different soybean varieties and Arabidopsis thaliana ecotype with GmPAP14 over-expression vector and RNAi vector. And the differences of phenotypic (symptoms of phosphorus deficiency, root architecture, etc.), biology (root dry weight, shoot dry weight, etc.), and physiological characteristics (phytase activity, acid phosphatase activity, phosphorus use efficiency, etc.) between transgenic plants and WT will be studied. Furthermore, we will clone and analyze the promoter sequence of GmPAP14, and will construct a series of promoter deletion fragment::GUS vectors to transform Arabidopsis thaliana for analyzing the regulation mechanisms of GmPAP14 promoter. In addition, we will clone and analyze the DNA sequences of GmPAP14 in different type soybean varieties (P-efficient and P-inefficient genotypes) to study the relationships between sequence differences and GmPAP14 gene function. So the results of this research will be of significance to understanding the molecular mechanisms of soybean purple acid phosphatase and high phosphorus efficiency, as well as to providing functional gene to transgenic breeding in crops.

植酸磷占土壤有机磷含量一半以上，提高其利用效率是解决当前作物低磷问题有效途径。项目组在前期克隆了酸性磷酸酶基因GmPAP14（GenBank No. JN967626），植酸磷处理下，基因在大豆根系诱导表达，磷高效品种中的表达量为低效品种2-35倍，且GmPAP14超表达拟南芥缺磷症状较野生型明显减轻。.在此基础上，本项目以解析GmPAP14生物学功能为目标，利用已有载体继续转化拟南芥与大豆，分析其植酸磷处理下的形态学（缺磷症状、根构型等）、生物学（生物重等）与生理生化（植酸酶活性、磷素利用效率等）性状差异，明确GmPAP14生物学功能，为植酸磷高效利用转基因育种提供功能基因。克隆GmPAP14启动子，利用顺式元件缺失鉴定启动子序列调控元件应答，分析启动子调控机理；克隆GmPAP14的DNA序列，分析磷高效与低效品种间序列差异，研究序列差异与基因功能间的关系，为解析基因作用机理提供依据。

在克隆GmPAP14并初步研究功能基础上，解析其磷高效功能，分析其启动子及调控机理，探讨其在磷高效与低效品种间的序列差异及其与功能间的关系。结果如下：.①分析了启动子对植酸磷胁迫特异应答及组织器官特异表达，探明了启动子调控机制：启动子2568 bp，在种子、子叶、胚根、根尖、顶芽、气孔等部位高效表达，受植酸磷胁迫诱导，在下胚轴、主根、侧根、叶片、根尖表达量高于适磷，且-1635至-2238区段有增强表达关键元件。.②明确了基因有提高植物根际周围植酸磷利用效率的功能，创制了磷高效转基因大豆新种质：基因在磷高效大豆根系植酸磷处理14 d、28 d-70 d表达量高于磷低效大豆，编码蛋白有酸性磷酸酶与植酸酶活性；超表达转基因根系分泌到培养基质的酸性磷酸酶与植酸酶活性显著高于野生型，转基因植株在植酸磷处理下的生长势、地上部、根系重、地上部、根系磷含量较野生型显著增加，花青素、培养基质剩余磷含量显著降低，明确了基因功能，创制了转基因大豆新种质。.③分析了不同磷效率大豆基因序列差异及其与功能间的关系：与磷高效大豆GmPAP14相比，磷低效大豆有1个36 bp插入突变，经转化等位变异载体（磷高效、低效载体pBI121-PAP14z、pBI121-PAP14n），以cDNA载体pBI121-PAP14c为对照，发现植酸磷处理下，转PAP14c、PAP14z株系目的基因转录表达量、酸性磷酸酶与植酸酶活性显著高于转PAP14n株系与野生型；转PAP14z、PAP14c株系植株长势、叶片大小显著优于野生型，转PAP14c、PAP14z与PAP14n株系地上部干重、磷含量显著高于野生型，且以转PAP14c较优，转PAP14n较差，36 bp插入突变抑制了基因磷高效功能。.④开发了功能标记，验证了36 bp等位变异与基因功能间的关系：利用上述等位变异开发功能标记，磷高效、低效大豆分别扩增241 bp、277 bp片段；经扩增大豆磷效率RIL，群体分为2种，PAP14z、PAP14n类型分别有123、130份材料，符合1:1；扩增不同品种发现，17个磷高效大豆有14份扩增241 bp片段，吻合率82.4%，证实该等位变异与基因植酸磷高效利用密切相关，且标记具有应用于分子辅助育种的潜力。.⑤申请专利1项，发表论文4篇，培养研究生5名。

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