轉(zhuǎn)冰葉日中花磷轉(zhuǎn)運蛋白McPht轉(zhuǎn)基因水稻對磷素的應(yīng)答
發(fā)布時間：2018-11-14 08:41
【摘要】：磷是植物生命活動過程中所需的大量元素之一,對植物體內(nèi)的生理以及生化過程具有積極的影響。磷轉(zhuǎn)運蛋白對植物吸收和轉(zhuǎn)運磷元素起重要作用。為了更好地了解植物中磷轉(zhuǎn)運蛋白的功能,從冰葉日中花中分離出一個磷轉(zhuǎn)運蛋白基因McPht,并將其轉(zhuǎn)化到水稻kitaake中。生物信息學分析表明,冰葉日中花磷轉(zhuǎn)運蛋白基因Mc Pht有1071個堿基,編碼357個氨基酸,分子量為37.77 kDa,有六個跨膜結(jié)構(gòu)域。為進一步確定Mc Pht蛋白在磷轉(zhuǎn)運蛋白中的分類,進行了亞細胞定位實驗,發(fā)現(xiàn)其位于線粒體上,屬于典型的Pht3亞家族成員。利用qPCR研究了轉(zhuǎn)基因水稻和冰葉日中花在低磷脅迫下磷轉(zhuǎn)運蛋白基因Mc Pht的表達情況,結(jié)果表明:在低磷脅迫下,轉(zhuǎn)基因水稻根部和地上部磷轉(zhuǎn)運蛋白基因Mc Pht的表達均高于正常處理;在低磷脅迫下,冰葉日中花地上部磷轉(zhuǎn)運蛋白基因Mc Pht表達高于正常處理。為了進一步探究磷轉(zhuǎn)運蛋白基因Mc Pht在低磷脅迫下的生理功能,我們對轉(zhuǎn)基因和野生型水稻進行了水培和組培實驗。結(jié)果表明:與野生型相比,低磷脅迫下轉(zhuǎn)基因水稻的生物量,根系活力、葉綠素含量、相對含水量、地上部和根部全氮、全磷含量均增加。對水培和組培的水稻根部進行根系掃描,掃描結(jié)果表明:低磷脅迫下轉(zhuǎn)基因水稻的根長,表面積,投影面積和側(cè)根數(shù)均高于野生型。為了探究磷轉(zhuǎn)運蛋白基因Mc Pht在低磷脅迫下基因表達特征的變化,對低磷脅迫下轉(zhuǎn)基因和野生型水稻地上部進行了轉(zhuǎn)錄組測序。測序結(jié)果表明:與野生型相比,轉(zhuǎn)基因水稻共篩選出198個差異表達基因,154個基因上調(diào),44個基因下調(diào);GO功能富集分析顯示,上調(diào)基因主要集中在細胞質(zhì)膜,下調(diào)基因主要集中在線粒體和細胞質(zhì)膜;Pathway分析表明:差異基因主要集中在植物次生代謝和植物病原體相互作用過程中,如苯丙素的合成,苯丙氨酸的代謝和次級代謝物的合成等。這些結(jié)果為進一步研究低磷脅迫下轉(zhuǎn)基因水稻的分子機制提供了依據(jù)。為了進一步驗證磷轉(zhuǎn)運蛋白基因Mc Pht的功能,我們利用CRISPR/Cas9技術(shù)將水稻中冰葉日中花磷轉(zhuǎn)運蛋白McPht基因同源體Os Pht基因進行敲除,為Mc Pht轉(zhuǎn)運蛋白基因?qū)胨綩s Pht缺失的突變株系及進一步驗證其功能提供可靠的材料支持。結(jié)果表明:1)基因編輯后的類型可分為兩類:一類為目標編輯片段20個堿基中后6個堿基缺失;另一類為目標編輯片段20個堿基中后8個堿基缺失或突變。2)編輯位點缺失6個堿基的株系占總突變株系的28.6%,編輯位點缺失8個堿基的株系占總突變株系的42.9%,GT堿基突變株系占總突變株系的28.6%。3)突變型水稻的株高、鮮重、根系活力均小于野生型;根系掃描結(jié)果顯示:突變型水稻的根長、投影面積、表面積和側(cè)根數(shù)均大于野生型;Cas-2的葉綠素和可溶性糖含量大于野生型,差異顯著,Cas-7的葉綠素和可溶性糖含量小于野生型,但差異不顯著。
[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.
【學位授予單位】：山西師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S511;Q943.2
本文編號：2330685
[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.
【學位授予單位】：山西師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S511;Q943.2
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