本文選題：甘藍(lán)型油菜 + 磷高效�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：磷是植物生長發(fā)育不可缺少的礦質(zhì)營養(yǎng)元素,土壤中磷的有效性低已成為作物生長的重要限制因子。油菜是我國重要的油料作物,因需磷較多,對缺磷敏感。探究油菜磷營養(yǎng)高效的生理和分子機(jī)制,為改良油菜的磷效率和挖掘油菜磷高效的基因資源具有重要的理論和實(shí)踐意義。本課題以油菜基因組數(shù)據(jù)庫和課題組前期油菜響應(yīng)缺磷的RNA-seq數(shù)據(jù)為基礎(chǔ),根據(jù)擬南芥控制低磷條件下根構(gòu)型相關(guān)的基因LPR(Low Phosphate Root)序列信息,利用生物信息學(xué)技術(shù)分析甘藍(lán)型油菜LPR家族基因的特征,結(jié)合qRT-PCR、擬南芥遺傳轉(zhuǎn)化等方法,研究BnaLPRs家族基因在低磷脅迫下表達(dá)模式及其BnaA07LPR2基因的功能驗證,揭示BnaLPRs家族基因參與油菜耐低磷脅迫的分子機(jī)理。主要研究結(jié)果如下:1、甘藍(lán)型油菜短期無磷饑餓和長期低磷脅迫下植株的生長和磷的吸收累積以磷高效基因型“鄂油長莢”為材料,采用營養(yǎng)液培養(yǎng)分別設(shè)置短期無磷饑餓和長期低磷脅迫處理,分析植物生長和磷的吸收累積。當(dāng)外界磷源充足時,大部分磷素與光合作用產(chǎn)物用于地上部生長,供應(yīng)根系的相對較少,根冠比較低;而在外界磷源減少時,首先滿足根系的生長發(fā)育,運(yùn)輸?shù)降厣喜康牧自礈p少,導(dǎo)致根冠比增大。在短期無磷饑餓和長期低磷脅迫下,磷利用效率較正常磷處理都有顯著的提高,但根系和地上部的磷利用效率變化有所不同,這表明磷脅迫能促進(jìn)體內(nèi)磷的再利用率,但同一基因型材料的不同部位對磷的利用和分配存在差異。2、甘藍(lán)型油菜LPR家族基因的特征利用擬南芥LPR1和LPR2的基因序列信息和已公布的甘藍(lán)型油菜全基因組序列信息,在甘藍(lán)型油菜中找到4個LPR的同源基因,分別分布在A07、A09、C05、C06四條染色體上。它們氨基酸序列相似性在77-99%之間,核酸序列一致性在36-95%,其中屬BnaC06LPR2和BnaA07LPR2兩個基因之間氨基酸序列和核酸序列的一致性最高。BnaC06LPR2和BnaA07LPR2基因結(jié)構(gòu)相似,只有2個內(nèi)含子,3個外顯子,但不同于AtLPR2,說明在甘藍(lán)型油菜進(jìn)化過程中BnaLPR2s功能發(fā)生分化。同時通過分析保守結(jié)構(gòu)域,發(fā)現(xiàn)LPR基因家族都含有3個多銅氧化酶結(jié)構(gòu)域,通過系統(tǒng)進(jìn)化樹分析進(jìn)一步證實(shí)了基因結(jié)構(gòu)及蛋白保守結(jié)構(gòu)域分析的可靠性。3、甘藍(lán)型油菜LPR基因響應(yīng)低磷脅迫的時空表達(dá)模式分析利用本實(shí)驗室甘藍(lán)型油菜苗期磷饑餓脅迫下的轉(zhuǎn)錄組數(shù)據(jù),共檢測到3個BnaLPR基因的表達(dá),進(jìn)一步的qRT-PCR結(jié)果表明,這種誘導(dǎo)具有短期持續(xù)性,而且在不同的部位對磷饑餓有不同程度的響應(yīng)。在甘藍(lán)型油菜不同組織部位的定量結(jié)果表示,異源四倍體的甘藍(lán)型油菜中BnaLPRs家族基因在長時間的進(jìn)化過程中功能發(fā)生了分化,有不同的表達(dá)模式,在磷信號調(diào)控中發(fā)揮不同的效應(yīng)和生物學(xué)功能。4、甘藍(lán)型油菜BnaA07LPR21基因的功能初步分析利用反向遺傳學(xué)研究BnaLPRs在磷信號調(diào)控中的功能差異,構(gòu)建35S啟動子連接BnaA07LPR2 CDS的超表達(dá)載體,轉(zhuǎn)化野生型擬南芥(Col-0)。通過瓊脂和營養(yǎng)液培養(yǎng)實(shí)驗分析野生型(Col-0)、lpr1突變體和超表達(dá)BnaA07LPR2的擬南芥轉(zhuǎn)基因植株在不同磷、鐵處理下的生長,發(fā)現(xiàn)缺磷會抑制主根生長,缺磷主根長表現(xiàn)為:lpr1OE-BnaA07LPR2Col-0;同時,在轉(zhuǎn)基因植株的根中許多磷饑餓誘導(dǎo)表達(dá)基因均顯著上升,說明BnaA07LPR2可能在油菜磷信號轉(zhuǎn)導(dǎo)通路中起到一定正調(diào)控作用。所以,我們猜測甘藍(lán)型油菜BnaA07LPR2基因與擬南芥中LPR1和LPR2有功能上的分化,超表達(dá)后能緩解部分主根缺磷抑制的現(xiàn)象。
[Abstract]:Phosphorus is an essential mineral nutrient element for plant growth and development. The low availability of phosphorus in the soil has become an important limiting factor for the growth of crops. Rape is an important oil crop in China. It is sensitive to phosphorus deficiency due to high phosphorus requirement. The physiological and molecular mechanism of high phosphorus nutrition in rape is explored to improve the phosphorus efficiency of rapeseed and to tap the high phosphorus of rapeseed. The effective gene resources have important theoretical and practical significance. Based on the rapeseed genome database and the RNA-seq data of the preliminary rapeseed response to the deficiency of phosphorus in the project group, according to the LPR (Low Phosphate Root) sequence information of the root configuration related to the low phosphorus condition in Arabidopsis, the bioinformatics technology is used to analyze the LP of Brassica napus. The characteristics of R family genes, combined with qRT-PCR and Arabidopsis genetic transformation, study the expression pattern of BnaLPRs family gene and the function verification of BnaA07LPR2 gene under low phosphorus stress, and reveal the molecular mechanism of BnaLPRs family genes involved in the low phosphorus stress tolerance of rape. The main results are as follows: 1, short term phosphorus free starvation and long term in Brassica napus The plant growth and phosphorus absorption and accumulation under low phosphorus stress are based on the phosphorus efficient genotype "Hubei oil long pods". The short term phosphorus free starvation and long-term low phosphorus stress treatment are set up in nutrient solution culture to analyze plant growth and phosphorus absorption accumulation. When the external phosphorus source is sufficient, the large part of phosphorus and photosynthesis products are used in the upper part of the earth. The root is relatively low and the root cap is relatively low, and when the external phosphorus source is reduced, the growth and development of the root system are first met, the phosphorus source in the upper part of the ground is reduced and the root crown ratio is increased. The phosphorus utilization efficiency is significantly higher than that of normal phosphorus treatment under the short-term phosphorus free hunger and the long-term low phosphorus stress, but the phosphorus benefit of the root and the upper part of the ground is significantly improved. The changes in efficiency vary, which indicates that phosphorus stress can promote the reutilization of phosphorus in the body, but there is a difference in the utilization and distribution of phosphorus in different parts of the same genotypic material.2. The characteristics of LPR family genes in Brassica napus are the sequence information of LPR1 and LPR2 in Arabidopsis thaliana and the whole genome sequence information of Brassica napus. 4 homologous genes of LPR were found in Brassica napus, which were distributed on the four chromosomes of A07, A09, C05 and C06 respectively. Their amino acid sequence similarity was between 77-99% and nucleic acid sequence consistency in 36-95%, which was the highest consistency of the amino acid sequence and nucleic acid sequence between the two genes of BnaC06LPR2 and BnaA07LPR2, and the highest.BnaC06LPR2 and BnaA07LPR2. The gene structure is similar, with only 2 introns and 3 exons, but different from AtLPR2, it shows that BnaLPR2s function differentiation in the process of Brassica napus evolution. At the same time, through the analysis of the conservative domain, it is found that the LPR gene family contains 3 multi copper oxidase domains, and the gene structure and protein are further confirmed by phylogenetic tree analysis. The reliability of the conservative domain analysis (.3), the spatio-temporal expression pattern of LPR gene in Brassica napus was analyzed by the time and space expression pattern of low phosphorus stress. The expression of 3 BnaLPR genes was detected by the transcriptional data of phosphorus starvation under the seedling stage of Brassica napus in the laboratory. The further qRT-PCR results showed that the induction had short duration and was not at the same time. The same parts respond to phosphorus starvation in varying degrees. Quantitative results in different tissue parts of Brassica napus indicate that the BnaLPRs family genes in the allotetraploid Brassica napus are differentiated in a long period of evolution, with different expression patterns and different effects and biological functions in the regulation of phosphorus letter numbers. .4, preliminary analysis of the function of BnaA07LPR21 gene in Brassica napus (Brassica napus) using reverse genetics to study the function difference of BnaLPRs in the regulation of phosphorus signal, construct the overexpression vector of 35S promoter to connect BnaA07LPR2 CDS, transform wild type Arabidopsis (Col-0). Analysis of wild type (Col-0), lpr1 mutant and overwatch through agar and nutrient solution culture experiment. The growth of BnaA07LPR2 transgenic plants under different phosphorus and iron treatments showed that phosphorus deficiency could inhibit the growth of the main root, and the main root length of phosphorus deficiency was lpr1OE-BnaA07LPR2Col-0. At the same time, many phosphorous starvation induced genes increased significantly in the roots of the transgenic plants, indicating that BnaA07LPR2 may play a role in the phosphorous signal transduction pathway of rapeseed. Therefore, we speculate that the BnaA07LPR2 gene of Brassica napus has functional differentiation with LPR1 and LPR2 in Arabidopsis thaliana. After overexpression, it can alleviate the inhibition of phosphorus deficiency in some main roots.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S565.4

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