番茄磷转运蛋白SlPht3基因最小表达框转化小麦对磷素营养的应答

发布时间：2018-11-25 11:15

【摘要】：线粒体磷转运蛋白对冬小麦吸收和转运磷营养有重要的作用。为了更好地了解这种蛋白在植物体中的功能,番茄线粒体磷转运蛋白SlPht3基因被克隆并且通过基因枪技术用线性基因最小表达框转入冬小麦中,在水培和田间试验的不同磷处理条件下验证其在转基因小麦中对磷营养的响应。生物信息学分析表明,番茄线粒体磷转运蛋白SlPht3基因有1074个碱基,编码358个氨基酸,有六个跨膜域,其中有三个是保守跨膜域,定位于线粒体内膜上。半定量RT-PCR表明番茄线粒体磷转运蛋白SlPht3基因在转基因小麦中稳定表达,Southern印迹表明SlPht3基因在小麦中的拷贝数不同,属于低拷贝插入,而且在小麦基因组中的整合形式也不同。水培条件下,磷缺乏时,根据生物量的测定结果知,番茄线粒体磷转运蛋白SlPht3基因的表达促进了转基因小麦地上部的生长和根的发育；根据根系扫描的结果显示,SlPht3基因的表达使转基因小麦总根长、根总的表面积和投影面积明显增加；根据生理生化的测定结果显示,番茄线粒体磷转运蛋白SlPht3基因的表达提高了转基因株系根的活力、根部和地上部酸性磷酸酶的活性,也可能加强了养分从根部向地上部的运输,转基因株系OE-G和OE-T地上部的全氮含量明显高于野生型,而根部的全氮含量与野生型冬小麦相比,差异极显著,转基因株系OE-O和OE-T的地上部全磷含量明显高于野生型,而根部明显低于野生型。而磷充足时,番茄线粒体磷转运蛋白SlPht3基因的表达增加了转基因小麦OE-O和OE-T的根重、根总长、根的总表面积和根的总投影面积。田间试验条件下,磷缺乏时,番茄线粒体磷转运蛋白SlPht3基因的表达增加了小麦在孕穗期的SPAD值和收获期的生物量,尤其是转基因株系OE-O和OE-T的产量明显增加。不同磷处理条件下,转基因株系OE-O和OE-T的秸秆中全磷含量明显增加；转基因株系OE-O籽粒中全磷的含量明显增加。综上所述,磷缺乏时,番茄线粒体磷转运蛋白SlPht3基因的表达可促进转基因小麦根结构发生变化,增加根活力和酸性磷酸酶的活性,提升小麦的光合能力,同时在磷的运输方面起着重要的调节作用。磷充足时,番茄磷转运蛋白SlPht3基因的表达同样可促进转基因小麦OE-O和OE-T的根结构发生变化以及有利于养分在转基因小麦中的运输。
[Abstract]:Mitochondrial phosphorous transporter plays an important role in the absorption and transport of phosphorus nutrition in winter wheat. In order to better understand the function of this protein in plant, the SlPht3 gene of tomato mitochondrial phosphorous transporter was cloned and transferred into winter wheat with linear gene minimal expression frame by gene gun technique. The response to phosphorus nutrition in transgenic wheat was tested under different phosphorus treatments in water culture and field trials. Bioinformatics analysis showed that tomato mitochondrial phosphorous transporter SlPht3 gene had 1074 bases encoding 358 amino acids and had six transmembrane domains three of which were conserved transmembrane domain and located on the mitochondrial intima. Semi-quantitative RT-PCR showed that tomato mitochondrial phosphorous transporter (SlPht3) gene was stably expressed in transgenic wheat. Southern blot showed that SlPht3 gene had different copy number in wheat and belonged to low copy insertion. And the form of integration in the wheat genome is also different. Under the condition of hydroponic culture, under the condition of phosphorus deficiency, according to the results of biomass measurement, the expression of SlPht3 gene of tomato mitochondrial phosphorous transporter promoted the growth of shoot and the development of root of transgenic wheat. The results of root scanning showed that the expression of SlPht3 gene significantly increased the total root length, root surface area and projection area of transgenic wheat. According to the results of physiological and biochemical tests, the expression of SlPht3 gene of tomato mitochondrial phosphorous transporter increased the activity of roots, the activity of acid phosphatase in root and shoot, and the transport of nutrients from root to shoot. The total nitrogen content in shoot of transgenic lines OE-G and OE-T was significantly higher than that of wild type, while the total nitrogen content in root was significantly higher than that in wild type winter wheat. The content of total phosphorus in shoot of transgenic lines OE-O and OE-T was significantly higher than that of wild type, but the content of total phosphorus in root was significantly lower than that in wild type. When phosphorus was sufficient, the expression of SlPht3 gene increased the root weight, total root length, root surface area and total projection area of transgenic wheat OE-O and OE-T. Under the condition of field experiment, the expression of SlPht3 gene increased the SPAD value of wheat at booting stage and the biomass at harvest stage, especially the yield of OE-O and OE-T in transgenic lines. Under different phosphorus treatments, the total phosphorus content in the straw of the transgenic lines OE-O and OE-T was significantly increased, and the total phosphorus content in the grain of the transgenic line OE-O was obviously increased. In conclusion, the expression of SlPht3 gene in tomato mitochondria during phosphorus deficiency could promote the change of root structure, increase the activity of root and acid phosphatase, and enhance the photosynthetic ability of transgenic wheat. At the same time, it plays an important regulatory role in the transport of phosphorus. When phosphorus was sufficient, the expression of SlPht3 gene could also promote the change of root structure of OE-O and OE-T and the transport of nutrients in transgenic wheat.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

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