低磷胁迫下玉米突变体Qi319-96高光效机制的解析及转基因耐盐耐旱棉花新种质的创制

发布时间：2018-01-05 07:12

本文关键词：低磷胁迫下玉米突变体Qi319-96高光效机制的解析及转基因耐盐耐旱棉花新种质的创制　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：一、低磷胁迫下玉米突变体Qi319-96高光效机制的解析作物光合效率的提升与作物产量的提高密切相关,了解植物磷效率与光合作用的关系是农业可持续发展的重要课题。在玉米突变体筛选中发现耐低磷突变体Qi319-96与其来源亲本Qi319相比在低磷胁迫下具有更高的光合效率。本工作在无机磷酸盐充足(+Pi,1000μMKH2P04)和缺乏(-P,5μMKH2P04)条件下培养玉米Qi319-96和Qi319植株,以玉米叶片为材料进行了比较蛋白质组学、基因组学和生理学研究,试图探讨低磷胁迫下Qi319-96高光合效率的分子机制。突变体Qi319-96耐低磷能力的提高与其改善细胞内部Pi的利用效率有关低磷水平限制了植物生长和新陈代谢。在低磷条件下,Qi319和Qi319-96冠部的总磷含量没有显著差异,但Qi319-96的Pi含量比Qi319高52.94%;Qi319-96具有更高的叶绿素含量和光合能力;Qi319和Qi319-96叶片的淀粉和蔗糖含量也不同,Qi319-96叶片比Qi319含有更多的蔗糖。Pi饥饿下,Qi319-96可以更好地调整膜脂质组成,有更高的V-ATPase活性水平,增强了细胞内Pi的循环再利用;叶绿素生物合成得到改善,Qi319-96中几种卡尔文循环和"C02泵"关键酶的活性水平也显著高于Qi319,这导致低磷胁迫下Qi319-96中的光合作用性能优于Qi319。Qi319-96对低磷胁迫耐受性的提高是由于其改善了细胞内部Pi的利用效率,耐低磷玉米新种质可以利用细胞工程手段有效获得。miR395和miR399参与了玉米突变体Qi319-96叶片的Pi水平调控MicroRNA(miRNA)参与了调节植物的生长发育和抗逆反应等多种生理过程。在+P和-P条件下利用测序技术比较了 Qi319和Qi319-96叶片中miRNA的表达水平。在+P下发现有10个已知miRNA家族的23个成员和40个新miRNA呈现差异表达。在-P下有8个玉米已知miRNA家族的34个成员和23个新miRNA在Qi319和Qi319-96间表现出不同表达水平。-P下一些靶基因的表达水平在Qi319和Qi319-96间显著不同,miR395和miR399在玉米突变体Qi319-96叶片的Pi水平调控中扮演着重要角色。Qi319-96在低磷胁迫下有更好的光系统性能和碳固定系统性能磷饥饿降低了玉米叶片的Pn和Gs,但Ci增加,非气孔因素导致了 Pn的下调。低磷下 Fv/Fm、ΦPsⅡ、RC/CS、ABS/CS、ETo/Cs、TRo/CS、Ψo、△Ir/Io、ΦPSⅠ/PSⅡ)和RuBPcase羧化酶羧化活性下调,而DIo/CS、Vj、Vi增加,表明磷饥饿对整个电子传递链造成了伤害,降低了光系统的电子传递能力,吸收光能的热耗散增加,ATP含量降低,碳同化速率下调。不同基因型比较,Qi319-96具有更好的Ψo、△Ir/Io、Φ(PSⅠ/PSⅡ)和RuBPcase羧化酶羧化活性,低磷胁迫下Qi319-96与Qi319相比具有更高的ATP水平和二氧化碳同化能力。叶绿体高丰度差异表达蛋白在Qi319-96低磷耐受性的贡献在于促进光系统的稳定性、协调性和高效性叶绿体作为光合作用的位点,与植物生长和非生物逆境响应密切相关。对Qi319-96和Qi319的叶绿体蛋白质组进行了比较分析,试图了解Qi319-96具有更好碳同化能力的原因。在低磷胁迫下二维凝胶电泳图中,与Qi319相比Qi319-96显著上调的差异表达蛋白为27个,其中24个得到质谱鉴定。这些鉴定的蛋白可分为四类:第一类是光合作用参与蛋白,包括RuBisCO、ATP合酶CF1-β亚基、ATP合酶CF1-α亚基、PSⅡ放氧增强蛋白;第二类是参与光合作用系统稳定的蛋白,包括PSⅡ稳定性/装配因子HCF136、FtsH蛋白、叶绿素a/b结合蛋白;第三类是电子传递链相关蛋白,包括Cytb6/f复合体铁硫亚基、玉米叶铁氧还蛋白NADP+还原酶;第四类为其他蛋白,包括肽-脯酰顺反异构酶、病程相关蛋白等。这些蛋白在Qi319-96的叶绿体中高丰度高累积起到了促进光系统的稳定性、协调性和高效性的作用,在磷饥饿下Qi319-96具有更好的电子传递性能和碳同化能力。以上工作初步揭示了 Qi319-96低磷下高光效的机制,即低磷耐受性的增加是由于细胞内Pi利用效率的提高,低磷胁迫下Qi319-96的光合作用系统具有更好的光系统性能和羧化系统性能。本工作为了解玉米光合作用系统应对低磷胁迫的机制提供了新信息,有望为高光效玉米改良提供新的策略。二、转基因耐盐耐旱棉花新种质的创制干旱、高盐度严重制约棉花的生产和影响纤维质量。生产上迫切需要培育耐盐耐旱棉花品种,以在水分胁迫和/或高盐分条件下维持棉花的生产能力,实现棉花稳产和纤维品质的稳定。在这部分工作中,将来自盐芥的TsVP基因(编码H+-PPase)、来自拟南芥的AtNHX1基因(编码Na+/H+ Antiporter)和来自玉米的ZmPLC1基因(编码PI-PLC)分别导入棉花,采用室内选择结合大田测试的方法,对转TsVP、转TsVP-AtNHX1、转ZmPLC1基因棉花的耐盐性或抗旱性进行了室内和大田测试,选育了耐盐耐旱性提高的转TsVP、转TsVP-AtNHX1、转ZmPLC1基因棉花,培育出一批棉花耐盐抗旱新材料。盐胁迫下TsVP基因的表达提高了转基因棉花的出苗率和在盐碱地中的产量棉花出苗和成苗是盐渍地植棉的关键,盐渍地中棉花的籽棉产量也为人们所关注。对表达TsVP基因和受体(野生型)棉花在盐碱地中的出苗时间、出苗率、成苗率、蕾期叶片碳同化能力、籽棉产量和棉纤维品质等进行测定,以评价选育出的表达TsVP基因的棉花育种材料的应用价值。结果表明在盐碱地中表达TsVP基因棉花的出苗时间与野生型相比可提前2d,且具有更高的出苗率和成苗率。在2013年的大田试验中,表达TsVP的棉花的CO2同化速率显著高于受体LM1138。表达TsVP基因的株系TP1、TP2和TP3的净光合速率分别高于受体30.75%、45.72%和50.61%,PSⅡ实际光化学效率(ΦPSⅡ)也分别高于野生型25.00%、42.50%和45.00%,表达TsVP的棉花具有比受体LM1138更好的碳同化能力和光合电子传递效率。过表达TsPT基因的棉花籽棉产量比野生型平均提高14.81%,与野生型相比棉纤维的品质也有所提高。温室内育种盘的种子出苗试验结果表明,当NaCl浓度大于100mM时,转基因株系50%种子出苗所需的时间显著少于受体LM1138。因此,我们的工作表明转TsVP基因棉花可用于提高盐渍地中棉花的经济产量和改善棉纤维质量。转AtNHX1-TsVP双基因提高了棉花的耐盐性和盐碱地中的籽棉产量为了提高棉花的高盐耐受能力,在春棉GK35中共表达了AtNHX1和TsVP基因。无论在温室盐胁迫下还是在东营盐碱地中,棉花叶片的相对含水量下降,但表达AtNHX1-TsVP的株系AT3比WT具有更高的相对水含量,表达TsVP-AtNHX1基因增强了棉花细胞的保水能力。在温室盐胁迫下或者在盐碱地中转基因株系AT3叶片中阳离子总摩尔数分别比WT提高35.79%和23.87%,叶片饱和渗透势也比WT分别降低23.88%和22.01%,这些细胞具有较低的饱和渗透势,有助于转基因棉花在盐胁迫下维持更好的水分吸收和持水能力,保持较高的相对水含量,促使表达AtNHX1-TsVP的棉花维持更高的光合作用能力。光合参数测定表明,表达TsVP-AtHNX1的棉花比WT具有更高的Pn和Gs。在盐碱地中表达AtNHX1-TsVP的棉花的出苗时间与受体GK35相比可提前2d。转AtNHX1-TsVP基因棉花具有比野生型(29.53%)更高的出苗率(53.22%)和比野生型(25.299%)更高的成苗率(49.45%),在萌发期和苗期呈现出更高的耐盐能力。转AtNHX1-TsVP基因棉花在盐碱地中表现出较高的籽棉产量,与野生型相比,平均增产22.46%。转AtNHX1-TsVP基因棉花耐盐性的增强可能与叶中更多的Na+、K+和Ca2+累积相关,这些阳离子的大量积累可能是AtNHX1-TsVP基因共表达导致的结果。阳离子在细胞中的适度积累有利于在盐胁迫下植物细胞维持离子稳态和细胞渗透势,从而赋予叶片细胞更高的相对水含量和维持较高的碳同化能力。转AtNHX1-TsVP基因棉花具有提高盐碱地中棉花产量的潜力,可应用于提高我国盐渍田的籽棉产量。转ZmPLC1基因提高了棉花的耐旱性和干旱胁迫下的籽棉产量PI-PLC在植物耐旱机制中起着重要作用。利用农杆菌介导法将ZmPLC1基因导入受体鲁棉研19(Lu19)中获得了表达ZmPLC1的棉花,Southern印迹分析、qRT-PCR和PI-PLC活性测定结果表明ZmPLC1基因整合到棉花基因组中并在细胞中有效表达。在苗期、现蕾期及花期三个阶段研究了表达ZmPLC1的棉花对干旱逆境的耐受性。结果表明,在干旱生境下,表达ZmPLC1的株系PC1、PC2、PC3具有比受体Lu19更高的相对含水量、更好的渗透调节、改善的光合作用速率、更低的离子泄漏、更小的脂质膜过氧化和具有更高的籽棉产量。在新疆大田自然干旱条件下,转基因株系PC1、PC2、PC3与野生型(WT)相比,蕾花期叶片具有更好的二氧化碳同化速率,更值得关注的是,在大田干旱条件下表达ZmPLC1的株系具有更高的经济产量。表达ZmPLC1的株系PC1、PC2、PC3与WT相比显著增强了对干旱逆境的耐受性,且这种耐受性的提高与干旱胁迫下细胞积累更多溶质和ABA含量变化有关。依据温室和大田的测试结果,转TsVP、TsVP-AtHNX1和ZmPLC1基因的棉花提高了抗逆性,在干旱或高盐分条件下具有更高的籽棉产量。TsVP、TsVP-AtNHX1、ZmPLC1基因是可用于棉花抗逆改良的优良候选基因。
[Abstract]:One, the photosynthetic efficiency of crops and improve crop maize mutant Qi319-96 analysis of high efficiency mechanism of low phosphorus stress is closely related to the improvement of yield, understand the relationship between plant phosphorus efficiency and photosynthesis is an important issue in the sustainable development of agriculture. In maize mutant found in low phosphorus tolerant mutant Qi319-96 and its source is higher than the parent Qi319 photosynthetic efficiency under low phosphorus stress. The work in inorganic phosphate sufficient (+Pi, 1000 MKH2P04) and short (-P, 5 MKH2P04) of Qi319-96 and Qi319 plants of maize cultivation conditions in maize leaves as materials for comparative proteomics research, genomics and physiology, attempts to explore low phosphorus under the stress of Qi319-96 high photosynthetic efficiency. The molecular mechanism of low phosphorus tolerance mutant Qi319-96 increased with the improvement of the utilization efficiency of Pi cells on low phosphorus levels on plant growth and new Metabolism. Under low phosphorus conditions, no significant differences in total phosphorus content of the crown of the Qi319 and Qi319-96 Qi319-96, but the content of Pi is 52.94% higher than that of Qi319; Qi319-96 has higher chlorophyll content and photosynthetic capacity; the content of starch and sucrose in leaves of Qi319-96 and Qi319 is different, Qi319-96 leaves contain more sugar than hunger.Pi Qi319, Qi319-96 can better adjust the membrane lipid composition, have a higher level of V-ATPase activity, enhanced intracellular Pi recycling; chlorophyll biosynthesis improved, activity level of Qi319-96 in several Calvin cycle and "C02 pump" key enzyme is significantly higher than that of Qi319, which leads to low phosphorus stress photosynthesis performance under Qi319-96 the superior Qi319.Qi319-96 tolerance to low phosphorus stress increase is due to the improvement of the utilization efficiency of Pi inside cells, tolerance to low phosphorus maize germplasm can by cell engineering means Effect of.MiR395 and miR399 in the Qi319-96 mutant of maize leaf Pi level control MicroRNA (miRNA) involved in the regulation of plant growth and development and stress responses and other physiological processes. In comparison with the miRNA Qi319 and Qi319-96 in the leaves of the expression level of +P and -P by sequencing conditions. +P found 10 known as the miRNA family of 23 members and 40 new miRNA DifferentiallyExpressed in -P. There are 8 known maize miRNA family of 34 members and 23 new miRNA in Qi319 and Qi319-96 showed the expression level of some target genes of different expression levels of.-P in Qi319 and Qi319-96 between miR395 and miR399 was significantly different. In the regulation of maize mutant Qi319-96 leaf Pi levels play an important role in the performance of optical system performance and phosphorus carbon fixation system has an important role in hunger.Qi319-96 has better under low phosphorus stress reduced maize leaf Pn and Gs, but Ci Increase of non stomatal factors leads to the downregulation of Pn. Under low phosphorus Fv/Fm, Phi Ps II, RC/CS, ABS/CS, ETo/Cs, TRo/CS, O, Ir/Io, Phi PS I /PS II) down, and RuBPcase carboxylase carboxylation activity and DIo/CS, Vj, Vi increased, showed that phosphate starvation caused damage the electron transport chain, reduced the electron optical system transmission capacity, heat dissipation of absorbed light energy increased, ATP content decreased, carbon assimilation rate decreased. Comparing different genotypes, Qi319-96 has better o, Delta Ir/Io, phi (PS I and /PS II) RuBPcase carboxylase carboxylation activity, low phosphorus under duress Qi319-96 compared with Qi319 has higher level of ATP and carbon dioxide assimilation. The protein is to promote the stability of optical system in Qi319-96 low phosphorus tolerance with high abundance of chloroplast expression differences, coordination and efficiency of photosynthesis and chloroplast as sites, plant growth and abiotic stress response. Cut. Chloroplast proteome of Qi319-96 and Qi319 were analyzed. The reason of trying to understand Qi319-96 has better carbon assimilation capacity under low phosphorus stress. Under the two-dimensional gel electrophoresis, compared with that of the Qi319 significantly up-regulated the expression of Qi319-96 protein was 27, 24 of them were identified. These proteins can be identified divided into four categories: the first category is a protein involved in photosynthesis, including RuBisCO, ATP, CF1- synthase beta subunit of ATP synthase CF1- subunit, PS II oxygen enhanced protein; the second is involved in protein stability of photosynthesis system, including the PS II / assembly stability factor HCF136, FtsH protein, chlorophyll a/b binding protein; third is the electron transport chain related proteins, including Cytb6/f complex iron sulfur subunit, maize leaf ferredoxin reductase NADP+; fourth kinds of other proteins, including peptide prolyl CIS trans isomerase, pathogenesis related protein, this. Some of the Qi319-96 protein in chloroplast in high abundance high accumulation to promote the stability of optical system, function of coordination and efficiency, in under phosphorus starvation Qi319-96 has better electron transport properties and carbon assimilation. The above work reveals the mechanism of high efficiency of Qi319-96 is increased under low phosphorus, low phosphorus tolerance is due to the intracellular Pi utilization efficiency, performance of optical system performance and carboxylation system under low phosphorus stress photosynthesis system Qi319-96 has better. This work as understanding the mechanism of maize photosynthesis system in response to low phosphorus stress and provided new information, is expected to provide a new strategy for high photosynthetic efficiency of maize improved. Two, the creation of drought salt and drought tolerance of transgenic cotton germplasm, high salinity severely restricts the production and quality of cotton fiber. The effects of production on the urgent need to cultivate salt tolerant drought tolerant cotton varieties in water stress and Maintain / cotton production capacity or high salt conditions, yield and fiber quality of cotton to achieve stability. In this part, from thellungiellahalophila TsVP gene (encoding H+-PPase), AtNHX1 gene from Arabidopsis thaliana (encoding Na+/H+ and Antiporter) from maize ZmPLC1 gene (encoding PI-PLC) were introduced into cotton by indoor selection method combined with test field, on TsVP, TsVP-AtNHX1, ZmPLC1 transgenic cotton salt resistance and drought resistance of indoor and field tests, the breeding of salt tolerance improved drought tolerance to TsVP, TsVP-AtNHX1, ZmPLC1 transgenic cotton, cultivate a group of Cotton Drought Tolerance expression of new materials. Under salt stress TsVP gene enhances the transgenic cotton germination rate and yield of cotton in saline alkali soil in germination and seedling is the key of salinized cotton, cotton seed cotton yield in saline soil has attracted more attention. The expression of TsVP gene and receptor (wild type) of cotton in saline alkali soil in the germination time, germination rate, seedling rate, leaf carbon assimilation ability of bud stage, seed cotton yield and fiber quality were determined. The application value of cotton breeding materials selected by the evaluation of TsVP gene expression. The results show that compared the expression of TsVP gene cotton in saline soil in emergence time and wild type 2D in advance, and has a higher germination rate and seedling rate of field experiment. In 2013, CO2 assimilation rate expression of TsVP cotton was significantly higher than that of LM1138. receptor TsVP gene expression strains TP1, TP2 and TP3 optical net photosynthetic rate were higher than that of receptor 30.75%, 45.72% and 50.61%, the actual photochemical efficiency (phi PS II PS II) were also higher than that of the wild type 25%, 42.50% and 45%, the expression of TsVP in cotton compared with carbon assimilation receptor LM1138 better and photosynthetic electron transfer efficiency. Over expression of TsP T gene cotton yield is 14.81% higher than the wild type on average, compared with the wild type cotton fiber quality is also improved. The greenhouse breeding disc of seed germination test results showed that when the concentration of NaCl is greater than 100mM, 50% transgenic seed germination time required significantly less than the receptor LM1138. therefore, our work suggests that TsVP cotton can be used to improve the yield and quality of cotton fiber cotton economy in saline soil. AtNHX1-TsVP transgenic cotton improves salt tolerance and saline alkali soil in seed cotton yield in order to improve the high salt tolerance of cotton, cotton in spring GK35 AtNHX1 and TsVP gene were expressed both in greenhouse under salt stress. Or in Dongying saline soil, cotton leaf relative water content decreased, but the expression of AtNHX1-TsVP strain AT3 has higher relative water content than WT, enhanced the expression of TsVP-AtNHX1 gene in cotton Cell water retention capacity. In the greenhouse under salt stress or in saline alkali cations in transgenic lines AT3 in leaves of the total number of moles were higher than WT 35.79% and 23.87%, leaf saturated osmotic potential than WT were decreased by 23.88% and 22.01%, these cells have low saturation osmotic potential, contribute to transgenic cotton in salt under the stress of maintaining better moisture absorption and water holding capacity, higher relative water content, the expression of AtNHX1-TsVP in cotton maintained higher photosynthetic capacity. The photosynthetic parameters indicated that the expression of TsVP-AtHNX1 cotton had higher Pn and Gs. expression of AtNHX1-TsVP cotton in saline alkali soil than WT emergence time ahead of 2D. AtNHX1-TsVP transgenic cotton is compared with the wild type receptor GK35 (29.53%) was higher than the rate (53.22%) and wild type (25.299%) higher survival rate (49.45%), at the stage of germination and seedling showing Salt tolerance higher. AtNHX1-TsVP transgenic cotton in saline alkali soil showed higher seed cotton yield, compared with the wild type, the average yield of AtNHX1-TsVP transgenic cotton 22.46%. enhanced salt tolerance and leaves more Na+, K+ and Ca2+ accumulation, a large amount of these cationic accumulation may be the result of co expression of AtNHX1-TsVP gene in the cell. The cationic moderate accumulation in favor of ion homeostasis and cellular osmotic potential of plant cells under salt stress, thus giving leaf cell relative water content higher and maintain higher carbon assimilation capacity. AtNHX1-TsVP transgenic cotton has increased the yield of cotton in saline land in the potential, can be applied to improve our salted field seed cotton yield. ZmPLC1 transgenic cotton improves drought tolerance under drought stress and the yield of PI-PLC plays an important role in plant drought tolerance mechanism in use. Agrobacterium tumefaciens mediated transformation of ZmPLC1 gene into Lumianyan 19 receptor (Lu19) in the expression of ZmPLC1 cotton, Southern blot analysis, the test results of the qRT-PCR and PI-PLC activity showed that ZmPLC1 gene was integrated into the cotton genome and in cells express effectively. At the seedling stage, bud stage and florescence three stages of tolerance the expression of ZmPLC1 on Cotton Drought stress. The results show that in the arid habitat. The expression of ZmPLC1 strains PC1, PC2, PC3 with relative water content is higher than Lu19 receptor, better osmotic adjustment, improved photosynthetic rate, lower ion leakage, lipid peroxidation and membrane less is more high yield. In the field of Xinjiang under natural drought conditions, transgenic lines PC1, PC2, PC3 (WT) compared with the wild type, CO2 assimilation rate of leaf bud and flowering period is more and more concern is that in the field under drought condition ZmPLC1 strain has higher economic yield expression. The expression of ZmPLC1 strains PC1, PC2, PC3 compared with WT significantly enhanced tolerance to drought stress, and the increased resistance to drought stress and cell accumulation more solute and ABA content. According to the test results, the greenhouse and in field TsVP, TsVP-AtHNX1 and ZmPLC1 genes in cotton increased resistance, higher seed yield in.TsVP, drought or high salt conditions TsVP-AtNHX1, ZmPLC1 gene can be used for good candidate gene to improve the resistance of cotton.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S562;S513

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