转冰叶日中花磷转运蛋白McPht转基因水稻对磷素的应答

发布时间：2018-11-14 08:41

【摘要】：磷是植物生命活动过程中所需的大量元素之一,对植物体内的生理以及生化过程具有积极的影响。磷转运蛋白对植物吸收和转运磷元素起重要作用。为了更好地了解植物中磷转运蛋白的功能,从冰叶日中花中分离出一个磷转运蛋白基因McPht,并将其转化到水稻kitaake中。生物信息学分析表明,冰叶日中花磷转运蛋白基因Mc Pht有1071个碱基,编码357个氨基酸,分子量为37.77 kDa,有六个跨膜结构域。为进一步确定Mc Pht蛋白在磷转运蛋白中的分类,进行了亚细胞定位实验,发现其位于线粒体上,属于典型的Pht3亚家族成员。利用qPCR研究了转基因水稻和冰叶日中花在低磷胁迫下磷转运蛋白基因Mc Pht的表达情况,结果表明:在低磷胁迫下,转基因水稻根部和地上部磷转运蛋白基因Mc Pht的表达均高于正常处理;在低磷胁迫下,冰叶日中花地上部磷转运蛋白基因Mc Pht表达高于正常处理。为了进一步探究磷转运蛋白基因Mc Pht在低磷胁迫下的生理功能,我们对转基因和野生型水稻进行了水培和组培实验。结果表明:与野生型相比,低磷胁迫下转基因水稻的生物量,根系活力、叶绿素含量、相对含水量、地上部和根部全氮、全磷含量均增加。对水培和组培的水稻根部进行根系扫描,扫描结果表明:低磷胁迫下转基因水稻的根长,表面积,投影面积和侧根数均高于野生型。为了探究磷转运蛋白基因Mc Pht在低磷胁迫下基因表达特征的变化,对低磷胁迫下转基因和野生型水稻地上部进行了转录组测序。测序结果表明:与野生型相比,转基因水稻共筛选出198个差异表达基因,154个基因上调,44个基因下调;GO功能富集分析显示,上调基因主要集中在细胞质膜,下调基因主要集中在线粒体和细胞质膜;Pathway分析表明:差异基因主要集中在植物次生代谢和植物病原体相互作用过程中,如苯丙素的合成,苯丙氨酸的代谢和次级代谢物的合成等。这些结果为进一步研究低磷胁迫下转基因水稻的分子机制提供了依据。为了进一步验证磷转运蛋白基因Mc Pht的功能,我们利用CRISPR/Cas9技术将水稻中冰叶日中花磷转运蛋白McPht基因同源体Os Pht基因进行敲除,为Mc Pht转运蛋白基因导入水稻Os Pht缺失的突变株系及进一步验证其功能提供可靠的材料支持。结果表明:1)基因编辑后的类型可分为两类:一类为目标编辑片段20个碱基中后6个碱基缺失;另一类为目标编辑片段20个碱基中后8个碱基缺失或突变。2)编辑位点缺失6个碱基的株系占总突变株系的28.6%,编辑位点缺失8个碱基的株系占总突变株系的42.9%,GT碱基突变株系占总突变株系的28.6%。3)突变型水稻的株高、鲜重、根系活力均小于野生型;根系扫描结果显示:突变型水稻的根长、投影面积、表面积和侧根数均大于野生型;Cas-2的叶绿素和可溶性糖含量大于野生型,差异显著,Cas-7的叶绿素和可溶性糖含量小于野生型,但差异不显著。
[Abstract]:Phosphorus is one of the most important elements in plant life, which has a positive effect on physiological and biochemical processes in plants. Phosphorus transporter plays an important role in the absorption and transport of phosphorus in plants. In order to better understand the function of phosphorus transporter in plants, a phosphorous transporter gene McPht, was isolated from the flower of ice leaf and transformed into rice kitaake. Bioinformatics analysis showed that the phosphorous transporter gene Mc Pht contained 1071 bases encoding 357 amino acids with a molecular weight of 37.77 kDa, and six transmembrane domains. In order to further determine the classification of Mc Pht protein in phosphorous transporter, subcellular localization experiments were carried out. It was found that Mc Pht protein was located on mitochondria and belonged to a typical Pht3 subfamily member. QPCR was used to study the expression of phosphorous transporter gene Mc Pht in transgenic rice and ice-leaf flower under low phosphorus stress. The expression of phosphorous transporter gene Mc Pht in root and shoot of transgenic rice was higher than that in normal treatment. Under low phosphorus stress, the Mc Pht expression of P transporter gene was higher than that of normal treatment. In order to further study the physiological function of phosphorous transporter gene Mc Pht under low phosphorus stress, we carried out hydroponic and tissue culture experiments on transgenic and wild type rice. The results showed that compared with wild type, the biomass, root activity, chlorophyll content, relative water content, total nitrogen and total phosphorus content in shoot and root of transgenic rice increased under low phosphorus stress. Root scanning of rice roots in water culture and tissue culture showed that the root length, surface area, projection area and lateral root number of transgenic rice under low phosphorus stress were higher than those of wild type. In order to investigate the changes of gene expression characteristics of phosphorous transporter gene Mc Pht under low phosphorus stress, transcriptome sequencing of transgenic and wild type rice under low phosphorus stress was carried out. Compared with wild type, 198 differentially expressed genes were screened, 154 genes were up-regulated and 44 genes were down-regulated. GO functional enrichment analysis showed that the up-regulated gene was mainly concentrated in the cytoplasmic membrane, and the down-regulated gene was mainly concentrated in the mitochondria and the cytoplasmic membrane. Pathway analysis showed that the differential genes were mainly concentrated in plant secondary metabolism and plant pathogen interaction, such as phenylpropanol synthesis, phenylalanine metabolism and secondary metabolites synthesis. These results provide a basis for further studying the molecular mechanism of transgenic rice under low phosphorus stress. In order to further verify the function of phosphorous transporter gene Mc Pht, we used CRISPR/Cas9 technique to knockout the homologous Os Pht gene of phosphotransport protein McPht gene in rice (Oryza sativa L.). To provide reliable material support for the introduction of Mc Pht transporter gene into mutant lines with Os Pht deletion and further verification of its function. The results showed that: 1) the type of gene editing can be divided into two categories: one is the deletion of the last 6 of the 20 bases of the target editing fragment; In the other category, the lines with 6 bases missing from the 20 bases of the target editing fragment accounted for 28.6g of the total mutation lines. The plant height, fresh weight and root activity of mutant rice with 8 bases missing at editing site were 42.9% and 28.6.3% of total mutant lines, respectively. The root scanning results showed that the root length, projection area, surface area and lateral root number of mutant rice were larger than those of wild type. The content of chlorophyll and soluble sugar in Cas-2 was higher than that in wild type, and the difference was significant. The content of chlorophyll and soluble sugar in Cas-7 was lower than that in wild type, but the difference was not significant.
【学位授予单位】：山西师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511;Q943.2

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