黄土高原丛枝菌根真菌（AMF）提高刺槐抗旱性机制

发布时间：2018-03-11 07:42

  本文选题：丛枝菌根真菌　切入点：刺槐　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：本文通过黄土高原半干旱区5种主要林木刺槐(Robinia pseudoacacia)、杜松(Juniperus communis)、青杨(Populus cathayana)、沙棘(Hippophae rhamnoides)和旱柳(Salix matsudana)根际丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)资源调查,分析了AMF与土壤因子的关系,研究了干旱胁迫下AMF对刺槐幼苗生长、光合作用、抗氧化酶活性等生理生化指标的影响,AMF对刺槐幼苗抗氧化酶基因和水孔蛋白基因表达的调控,初步揭示了AMF提高刺槐的抗旱机制。得出以下主要结果:1.黄土高原半干旱区AMF资源及其与土壤和气候因子的相关性从刺槐、杜松、青杨、沙棘和旱柳5种林木根际土壤分离到8属21种AMF:管柄囊霉属(Funneliformis)7种,为优势属,幼套近明囊霉(Claroideoglomus etunicatum)和网状球囊霉(Glomus reticulatum)是优势种。土壤脲酶、过氧化氢酶和全磷是影响AMF总侵染率和孢子密度最直接的因子;转化酶是影响AMF物种丰富度、Shannon-Wiener多样性指数和Shannon均匀度指数最直接的因子。从6个不同半干旱区刺槐根际土壤分离得到9属23种AMF:管柄囊霉属6种,为优势属,根内根生囊霉(Rhizophagus intraradices)、两型管柄囊霉(Fun.dimorphicum)、聚丛根生囊霉(R.aggregatum)、单孢管柄囊霉(Fun.monosporum)、凹坑管柄囊霉(Fun.multiforum)、地管柄囊霉(Fun.geosporum)和幼套近明囊霉是优势种。通径分析表明降雨量是影响AMF状况和多样性指数最直接的因子。2.干旱胁迫条件下,AMF对刺槐光合作用的影响采用人工模拟干旱法研究根内根生囊霉(R.irregularis)对刺槐生长、叶绿素含量、气体交换参数、叶绿素荧光参数等的影响。结果发现,接种R.irregularis能够提高刺槐叶绿素a、类胡萝卜素、Fm、qP、NPQ和ΦPSII值,正常水分条件下,分别提高120.8%、44.7%、28.6%、3.4%、4.2%和7.0%;干旱胁迫条件下,分别提高69.5%、265.0%、12.8%、4.3%、3.8%和7.5%,与对照相比差异均达显著水平。菌根化刺槐叶片净光合速率和气孔导度也显著高于对照,正常水分条件下,分别增加16.4%和12.8%;干旱胁迫条件下,分别增加14.3%和8.1%。说明干旱胁迫条件下AMF与刺槐共生提高了宿主叶片PSII光化学活性与光合电子传递能力,保持高的光能利用效率与光合作用潜力,干旱胁迫条件下菌根化刺槐能更好地进行光合作用。3.干旱胁迫条件下,AMF对刺槐抗氧化能力的影响采用人工模拟干旱法研究根内根生囊霉(R.irregularis)对刺槐抗氧化能力的影响。结果发现,接种R.irregularis后,刺槐根系O2、H_2O2和MDA含量降低,分别较对照显著降低20.5%、33.1%和26.8%,说明接种AMF缓解了干旱胁迫对刺槐造成的氧化损伤。正常水分条件下,菌根化刺槐叶片和根系超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)活性分别增加53.6%和33.3%、20.4%和16.3%、20.9%和10.3%、67.4%和36.4%、2.7%和11.0%;干旱胁迫条件下,分别增加1.1%和27.2%、25.7%和23.5%、13.5%和9.8%、78.3%和58.2%、22.7%和14.4%,其中干旱胁迫条件下接种与对照叶片SOD、POD、CAT、APX和GR活性差异均达显著水平。说明菌根化刺槐可以通过增强自身的抗氧化酶活性来清除干旱胁迫产生的活性氧。4.干旱胁迫条件下,AMF对刺槐抗氧化酶基因表达的影响采用实时荧光定量PCR(qRT-PCR)技术研究了R.irregularis对刺槐根、茎和叶片铜锌超氧化物歧化酶基因(Cu/Zn-SOD)、抗坏血酸过氧化物酶基因(APX)和谷胱甘肽还原酶(GR)基因表达的影响。结果发现,接种AMF上调了刺槐根、茎和叶片Cu/Zn-SOD、APX和GR基因的表达。正常土壤水分条件下,接种AMF上调了刺槐根、茎和叶片APX基因的表达,分别为对照的3.31、3.17和3.06倍;接种AMF也上调了刺槐叶片GR基因的表达,为对照的3.92倍。干旱胁迫条件下,接种AMF的刺槐根、茎和叶片Cu/Zn-SOD基因表达均表现为上调,分别为对照的1.47、1.62和1.49倍;接种AMF也上调了刺槐根系GR基因的表达,为对照的1.96倍。说明菌根化刺槐通过提高其根、茎和叶片Cu/Zn-SOD、APX和GR基因的表达来调控活性氧代谢,使其抗旱性增强。5.干旱胁迫条件下,AMF对刺槐水孔蛋白基因表达的影响采用qRT-PCR技术研究了接种R.irregularis对刺槐根、茎和叶片水孔蛋白(Aquaporin,AQP)基因表达的影响。从刺槐根部克隆得到RpTIP1;1、RpTIP1;3、RpTIP2;1、RpPIP1;1、RpPIP1;3和RpPIP2;1 6个水孔蛋白基因,6个RpAQPs cDNA全长为825~1201 bp,含有750~870 bp开放阅读框,编码249~289个氨基酸,分子量为25.37~31.06 kDa,等电点为5.09~8.96,RpAQPs编码的氨基酸序列与蒺藜苜蓿的同源性达89%~97%。组织表达分析表明,刺槐RpPIP基因在根部高表达,而RpTIP基因(RpTIP1;3除外)在叶片高表达。干旱胁迫下,接种AMF的刺槐RpTIP2;1和RpPIP2;1基因在根中的表达表现出上调趋势,分别较对照提高54.7%和79.4%;在茎部,菌根化刺槐RpTIP1;1、RpTIP2;1和RpPIP2;1基因的相对表达量分别为对照的1.17、1.83和1.15倍;成熟叶片中,菌根化刺槐RpTIP2;1和RpPIP2;1基因相对表达量分别为对照的1.44和1.61倍。水孔蛋白家族基因在控制水分运输过程中发挥的作用不尽相同,接种AMF能通过促进刺槐水孔蛋白基因的表达来增强植物对干旱胁迫的适应性。
[Abstract]:The 5 main forest of Robinia pseudoacacia by semi arid area of Loess Plateau (Robinia pseudoacacia), Du Song (Juniperus communis), poplar (Populus cathayana), sea buckthorn (Hippophae rhamnoides) and willow (Salix matsudana) in rhizosphere of arbuscular mycorrhizal fungi (Arbuscular mycorrhizal, fungi, AMF) resource survey, analyzes the relationship between AMF and soil factors study on photosynthesis under drought stress, the growth of AMF, the effects of physiological and biochemical indexes of Robinia pseudoacacia seedlings, antioxidant enzyme activity, regulation of AMF on gene expression of antioxidant enzymes of Robinia pseudoacacia seedlings and aquaporin genes, revealed that the AMF mechanism to improve the drought resistance of Robinia pseudoacacia. The main results are as follows: correlation of AMF resources in semi arid region of the Loess Plateau and its 1. with soil and climate factors from locust, Du Song, poplar, willow and seabuckthorn 5 kinds of tree rhizosphere soil from 8 genera and 21 species of Trichoderma AMF: capsule tube handle (Funneliformis) for 7. The dominant genera, young set of near Ming (Claroideoglomus etunicatum) and fungus Glomus reticulate (Glomus reticulatum) is the dominant species. Soil urease, catalase and total phosphorus are the factors influencing AMF colonization and spore density directly; invertase was influenced by AMF factor Shannon-Wiener species richness, diversity index and Shannon evenness the index directly. 9 genera and 23 species of AMF: tube handle pouch mildew genera and 6 species isolated from the rhizosphere soil of Robinia pseudoacacia in 6 different semi arid region, was the dominant genus in the roots, the root fungus (Rhizophagus intraradices), type two (Fun.dimorphicum) tube handle pouch mildew, leaf clumping fungus (R.aggregatum) the tube handle, single spore fungus (Fun.monosporum), pit tube handle pouch mildew (Fun.multiforum), pipe handle pouch mildew (Fun.geosporum) and the set of near Ming capsule mold is dominant. The path analysis showed that rainfall is the impact of AMF status and diversity index of the most direct factor.2. stem Under drought stress, artificial drought of root root fungus with AMF on Photosynthesis of Robinia pseudoacacia (R.irregularis) on the growth of Robinia pseudoacacia, chlorophyll content, gas exchange parameters, the effect of chlorophyll fluorescence parameters. The results showed that R.irregularis inoculation can improve the locust chlorophyll a, carotenoid, Fm, qP, NPQ and with PSII, under normal water condition, increased by 120.8%, respectively, 44.7%, 28.6%, 3.4%, 4.2% and 7%; under drought stress, increased by 69.5%, respectively, 265%, 12.8%, 4.3%, 3.8% and 7.5%, compared with the control difference reached significant level. The mycorrhizal locust leaf net photosynthetic rate and stomatal conductance was significantly higher than that in control, under normal water condition, an increase of 16.4% and 12.8% respectively; under drought stress, respectively increased 14.3% and 8.1%. AMF and Robinia pseudoacacia under drought stress increased leaf PSII symbiotic host photochemical activity and photosynthetic power Sub transmission capacity, maintain the high light energy utilization efficiency and photosynthetic potential of mycorrhizal fungi of Robinia pseudoacacia under drought stress to.3. photosynthesis under drought stress, artificial drought of root root fungus with AMF on antioxidant capacity of black locust (R.irregularis) effects on antioxidant capacity of Robinia pseudoacacia. The results showed that R.irregularis after inoculation, Robinia pseudoacacia roots decreased H_2O2 and O2, MDA content, were significantly lower than the CK 20.5%, 33.1% and 26.8%, AMF inoculation alleviated the drought stress on oxidative damage caused by normal locust. Water conditions, mycorrhizal locust leaves and roots of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) activity were increased by 53.6% and 33.3%, 20.4% and 16.3%, 20.9% and 10.3%, 67.4% and 36.4%, 2.7% and 11% ; under drought stress condition, increased by 1.1% and 27.2% respectively, 25.7% and 23.5%, 13.5% and 9.8%, 78.3% and 58.2%, 22.7% and 14.4%, which under drought stress inoculation and control leaves SOD, POD, CAT, APX and GR activity significantly. The mycorrhizal Robinia pseudoacacia through its antioxidant activity to remove the drought stress by reactive oxygen.4. drought stress condition, the effect of AMF on locust antioxidant enzyme gene expression by real-time fluorescent quantitative PCR (qRT-PCR) of R.irregularis on locust roots, stems and leaves of copper zinc superoxide dismutase gene (Cu/Zn-SOD), ascorbate peroxidase gene (APX) and glutathione reductase (GR) gene expression in vitro. The results showed that AMF inoculation increased locust root, stems and leaves of Cu/Zn-SOD, the expression of APX and GR genes. The normal soil moisture conditions, inoculation of AMF raised locust roots, stems and The expression of APX gene in leaves, respectively 3.31,3.17 and 3.06 times of inoculation; AMF also upregulated the expression of GR gene of Robinia pseudoacacia leaves, was 3.92 times of the control. Under drought stress, Robinia pseudoacacia roots inoculated with AMF, the expression of stem and leaf of Cu/Zn-SOD gene were up-regulated, respectively, according to 1.47,1.62 and 1.49 times AMF; inoculation increased the expression of GR gene of Robinia pseudoacacia roots, was 1.96 times of the control. The mycorrhizal Robinia pseudoacacia by improving its roots, stems and leaves of Cu/Zn-SOD, the expression of APX and GR genes to regulate the metabolism of active oxygen, which enhanced.5. drought resistance under drought stress, effect of AMF on the expression of aquaporin gene of Robinia pseudoacacia the study of locust root inoculation of R.irregularis using qRT-PCR technology, stems and leaves of aquaporins (Aquaporin, AQP) gene expression of RpTIP1 from locust Root Clone; 1, RpTIP1; 3, RpTIP2; 1, RpPIP1; 1, RpPIP1; 3 and RpPIP2; 16 Aquaporin 6, RpAQPs cDNA was 825~1201 BP, containing a 750~870 open reading frame of BP encoding 249~289 amino acids, molecular weight of 25.37~31.06 kDa, the isoelectric point was 5.09~8.96, RpAQPs encoding the amino acid sequence of Medicago truncatula homology of 89%~97%. tissue expression analysis showed that RpPIP gene is highly expressed in the roots of black locust, and RpTIP gene (RpTIP1; except 3) is highly expressed in leaves. Under drought stress, Robinia pseudoacacia inoculated with AMF RpTIP2; 1 and RpPIP2; 1 gene expression in root showed upward trend, respectively increased 54.7% and 79.4%; in the stems, mycorrhizal locust RpTIP1; 1, RpTIP2; 1 and RpPIP2; the relative expression of 1 genes were controlled 1.17,1.83 and 1.15 times; in mature leaves of mycorrhizal locust RpTIP2; 1 and RpPIP2; 1 gene expression levels were decreased to 1.44 and 1.61 times. The aquaporin family genes in the control of water transport process The role of AMF is different, and inoculation can enhance the adaptability of plants to drought stress by promoting the expression of the Robinia Robinia water pore protein gene.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S792.27
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本文编号：1597211