OsPT2基因过表达对低磷胁迫下菜用大豆结瘤固氮及氮代谢的影响

发布时间：2018-01-02 13:33

本文关键词：OsPT2基因过表达对低磷胁迫下菜用大豆结瘤固氮及氮代谢的影响　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：菜用大豆,又称毛豆,指在鼓粒末期籽粒饱满而尚未老熟时采青食用的大豆[Glycinemax(L.)Merrill],是大豆的专用型品种。豆科植物根瘤中的固氮过程是一个高耗能过程,有较高的磷素需求。然而由于磷素在土壤中易被固定、难以移动导致土壤中能被植物吸收利用的有效磷含量较低,土壤缺磷已成为共生固氮豆科植物生产的重要制约因素。因此,培育低磷环境下高效固氮的豆科品种对减少氮肥、磷肥的施用以及增强农业的可持续性有重要意义。水稻磷转运蛋白基因OsPT2受低磷诱导显著增强表达,主要起到转运植物体内磷素的作用。在前期研究中我们已获得转OsPT2基因磷高效利用的T3代菜用大豆种子,本研究以此为试验材料,将分离纯化的日本慢生根瘤菌(Bradyrhizobium japonicum)接种于转基因植株,采用蛭石盆栽的方法进行低磷处理试验,探究OsPT2基因的过表达能否提高转基因植株的结瘤固氮及氨同化能力,为培育氮磷协同高效的菜用大豆新品种提供理论依据和新种质。主要研究结果如下:1、从大豆新鲜根瘤中分离纯化得到3个分离物,分别命名为NAU1301、NAU1302、NAU1303,通过菌落形态观察以及采用16S-23SrDNA基因间隔序列分析技术进行菌种鉴定,确认了分离的3个菌株均为Bradyrhizobium japonicum(日本慢生根瘤菌)。结瘤实验进一步确认了分离物为根瘤菌,均能与菜用大豆('新辽鲜'品种)共生结瘤并具备固氮能力,对植株生长具有促进作用。2、采用砂培方法,设置5个磷水平5、10、20、40、1000 μM (正常磷水平),研究低磷胁迫对菜用大豆结瘤和固氮能力的影响。低磷条件下植株各器官有效磷和总磷含量显著低于正常磷水平;低磷胁迫显著降低了菜用大豆根瘤的生长发育和固氮能力,表现为株高、叶面积、地上部生物量、根瘤干重和根瘤体积显著降低,植株总氮含量呈下降趋势。低磷处理下根瘤干重降低的幅度比地上部干重、根系干重降低的幅度大,表明和植株生长相比,根瘤生长对缺磷更敏感。荧光定量PCR分析显示,和正常磷水平相比,在根瘤发育过程中(0-11 d),结瘤关键基因GmENOD40-1、GmENOD40-2、GmNIN1a、GmNIN1b、GmRIC1 和 GmRIC2 的表达量在低磷处理的不同时间点显著降低;早期结瘤信号识别的受体激酶基因GmNFR1a/β、GmNFR5a/β、GmSymRKα/β的表达量在低磷和正常磷处理之间并无显著差异。表明低磷胁迫通过抑制若干结瘤关键基因的表达而抑制根瘤后期的发育、成熟,但对早期根瘤菌结瘤因子信号的识别过程没有影响。3、以三个T3代纯合株系转OsPT2基因菜用大豆(编号12PT2-1-1-1、12PT2-2-1-3和12PT2-4-3-1)为试验材料,采用蛭石盆栽的方法进行低磷实验,探究低磷条件下OsPT2过表达对菜用大豆结瘤固氮和氨同化能力的影响。结果显示,低磷条件下T3代转基因植株的生长表现、种子重量和根瘤发育均显著优于野生型。T3代转基因植株的磷含量、总氮含量及酰脲浓度显著高于野生型植株,说明过表达OsPT2基因促进了低磷条件下转基因植株体内磷、氮、酰脲的积累。荧光定量PCR分析显示,低磷处理24d和32d,早期结瘤素基因(GmENOD40-1、GmENOD40-2),豆血红蛋白基因(GmLba)和谷氨酰胺合成酶基因(GmGS1β1、GmGS1β2)在T3代转基因植株的表达量显著高于野生型;GmGS1β1和GmGS1β2基因表达量的增加和转基因植株中GS酶活性的增加一致。GmLba基因表达量的增加和转基因植株中豆血红蛋白含量的增加相一致。以上结果表明,低磷胁迫下OsPT2基因过表达显著促进了菜用大豆体内磷素的积累,增强结瘤固氮相关基因的表达,进而提高了转基因菜用大豆的固氮和氨同化能力。
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【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S643.7

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