苹果砧木对重金属镉吸收、富集及耐受机制研究

发布时间：2018-03-06 02:10

本文选题：镉　切入点：苹果砧木　出处：《沈阳农业大学》2017年博士论文　论文类型：学位论文

【摘要】：随着人民生活水平的不断提高,果品在人类膳食结构中所占比例越来越大。我国是世界苹果的主产国,苹果在我国居民水果消费中占有重要地位。然而,污水灌溉及化肥农药的大量施用使得果园土壤重金属污染问题日益突出,尤其是Cd污染。嫁接是果树繁殖的重要方式,砧木是果树生产的承接者,对Cd的吸收、转运能力直接影响果树整体Cd耐性及接穗中Cd含量。因此,研究苹果砧木对Cd的吸收、转运以及耐性差异,可为筛选低Cd富集耐性砧木提供理论依据。本试验首先研究了山定子、平邑甜茶、青州林檎和八棱海棠根系Cd2+吸收的时空动态变化规律;随后在生理和分子水平探讨了Cd在苹果砧木各组织转运和对Cd的耐性机理;接着研究褪黑素缓解Cd对苹果砧木的毒害机理;最后探讨了不同苹果嫁接苗对Cd的吸收、转运和耐性差异。主要结果如下:1.通过比较4种苹果砧木根系在50 μM Cd处理下Cd2+吸收的时空动态变化规律和Cd2+在植物体的转运,发现苹果砧木白根根尖600 μm处Cd2+净流量最大,随着距根尖距离的延长,Cd2+净流量逐渐降低。山定子根系和叶柄维管束Cd2+的净流量低于其他3种苹果砧木。代谢抑制剂和通道阻断剂(正钒酸钠、维拉帕米和NEM)处理显著降低了 4种苹果砧木根系Cd2+的净流量。另外,环境Fe3+浓度也影响根系Cd2+的净流量。除山定子外,Cd导致其他3种苹果砧木净光合速率和生物量增量减少。与其他3种苹果砧木相比,Cd胁迫下山定子各组织Cd含量、Cd富集总量,BCF和Tf最小,参与Cd吸收和转运基因如HA4、FRO2-like、NRAMP1、NRAMP3和HMA4转录水平较低,参与Cd解毒相关基因如NAS1和MT2表达较强。由此表明,50 μM Cd处理下山定子对Cd吸收和转运的能力低于其他3种苹果砧木。2.对4种苹果砧木进行较高浓度Cd(300 μM)处理45 d后,所有砧木幼苗的净光合速率、叶绿素含量和生物量均受到了抑制,但对山定子的影响较小,且山定子整株耐性指数(TI)显著高于其他3种苹果砧木。4种苹果砧木中,山定子根、木质部和叶片中Cd含量最低、Cd富集总量和BCF最小。苹果砧木能将Cd固持在细胞壁和液泡中,并将其转化成果胶酸盐或蛋白结合态以及难溶性磷酸盐状态来降低Cd的移动性和毒性。4种苹果砧木生理状态如ROS、糖类和抗氧化系统的变化与对Cd的耐性相一致。与其他3种苹果砧木相比,山定子根和韧皮部中ROS、根和叶片中H202以及根、木质部和韧皮部中MDA含量最低,韧皮部中可溶性糖以及根和叶片淀粉含量最高,抗氧化酶活性和抗氧化物含量较高。说明该试验条件下山定子对Cd的耐受性强于其他3种苹果砧木,这与山定子各组织Cd含量、Cd的亚细胞分布和化学形态,以及抗氧化系统有关。3.通过比较外源褪黑素对30μM Cd处理下山定子和青州林檎幼苗Cd吸收和转运、抗氧化系统及基因表达的影响,发现添加褪黑素后山定子和青州林檎根系Cd2+的净吸收速率、叶片Cd含量以及Tf显著降低,说明褪黑素抑制了苹果砧木对Cd的吸收和转运,且对山定子的抑制作用更明显。与30μMCd处理相比,30μMCd + 0.1μM褪黑素处理减少了苹果砧木根、茎和叶中O2·-、H202和MDA含量,细胞器组分Cd含量和所占比例降低,以及Cd吸收相关基因如NARMAP1表达量降低;抗氧化酶如CAT、GPX、APX和GR活性升高,根和叶片中游离脯氨酸、根中可溶性酚和T-SH、茎和叶片中ASC,根和茎中GSH以及根中褪黑素含量增加,与Cd解毒相关基因如NAS1表达量上调,说明褪黑素能够抑制苹果砧木根系吸收和转运Cd的能力,提高细胞抗氧化水平,有效清除细胞内的ROS,增强苹果砧木对Cd的耐受性。但30μM Cd + 0.1 μM褪黑素处理下青州林檎根和叶片中CAT和GR、根中GPX和APX活性,以及根和叶片中游离脯氨酸、叶片可溶性酚和T-SH、茎和叶片GSH含量低于山定子;参与Cd吸收的基因如HA7和NRAMP1的转录水平高于山定子,与Cd解毒相关基因如NAS1表达量低于山定子。说明褪黑素对缓解山定子Cd胁迫的作用更明显。4.通过比较50μM Cd胁迫下4种苹果嫁接苗对Cd的吸收和转运、抗氧化水平及基因表达的变化,发现Cd导致4种苹果嫁接苗的生物量、光合速率和色素含量降低,但Cd对寒富/山定子的影响最小。寒富/山定子和富士/山定子根系和接穗Cd含量、Cd富集总量和BCF分别小于寒富/青州林檎和富士/青州林檎,说明以山定子为砧木能有效减少Cd在苹果嫁接苗中的富集。Cd胁迫下寒富/山定子木质部、韧皮部和叶片Cd含量及水溶态Cd含量低于其他3种苹果嫁接苗,根和叶片细胞壁组分Cd所占比例最大,细胞器组分Cd含量和比例最小,与Cd吸收和转运相关基因如HA7、FRO2-like、NRAMP1和NRAMP3表达量较低,说明寒富/山定子根系固持Cd的能力较强,能降低Cd在嫁接苗中的移动速率。另外,Cd胁迫下寒富/山定子韧皮部和叶片ROS和MDA含量最低,叶片可溶性酚、ASC和GSH含量最高,与Cd解毒相关的基因如MT2表达量最高,说明寒富/山定子对Cd的忍耐性强于其他3种苹果嫁接苗。
[Abstract]:With the improvement of people's life, the fruit in the human diet in the proportion is increasing. China is the world's leading producers of apple, apple occupies an important position in China's residents consumption of fruit. However, the extensive application of sewage irrigation and fertilizer and pesticide that orchard soil heavy metal pollution problems have become increasingly prominent, especially Cd pollution. Grafting is an important way of fruit breeding, rootstock is to undertake the production of fruit trees, the absorption of Cd, directly affect the content of Cd and Cd in the whole fruit tolerance scion transport capacity. Therefore, the research on absorption of Apple Rootstock Cd, transport and tolerance, which can provide the theoretical basis for screening low Cd enrichment Tolerant Rootstock this test studied first baccata, Pingyi Tiancha, temporal variation. Roots Cd2+ absorption of Qingzhou LinQin and eight ribs; then in physiological and molecular level of Cd in Guo Zhenmu. The organization of transport and Cd tolerance mechanism; then study melatonin alleviates the poisoning mechanism of Cd on Apple Rootstock; finally discusses the absorption of different apple grafted on Cd, translocation and tolerance differences. The main results are as follows: 1. by comparing the 4 kinds of Apple Rootstock Roots in 50 M Cd treatment Cd2+ absorption dynamic changes of Cd2+ in plants and transport, found the net Cd2+ 600 m flow of Apple Rootstock root increased with white maximum distance from root tip, Cd2+ net flow decreased gradually. The net flow of Cd2+ root and petiole vascular bundle is lower than other 3 kinds of crab apple rootstocks. Metabolic inhibitors and channel blockers (orthovanadate Na, Vera Pammy and NEM) significantly decreased the net flow of 4 kinds of Apple Rootstock Roots of Cd2+. In addition, Fe3+ concentration also affects the net flow of root Cd2+. Except Cd baccata, lead to other 3 kinds of net photosynthetic rate of Apple Rootstocks The rate and biomass increment decreased. Compared with the other 3 kinds of apple rootstocks, Cd stress down tissue Cd content stator, Cd enrichment amount, BCF and Tf was the smallest, involved in Cd uptake and transport genes such as HA4, FRO2-like, NRAMP1, NRAMP3 and HMA4 transcription level is low, involved in Cd detoxification related genes such as NAS1 and MT2 expression strong. It showed that 50 M Cd processing ability of Cd stator down the absorption and transport of lower than the other 3 types of Apple Rootstock.2. high concentration Cd on 4 rootstocks (300 M) after 45 D, the net photosynthetic rate of all rootstock seedlings, chlorophyll content and biomass was inhibited however, the impact of Yama Joko and Yama Joko is small, the whole plant tolerance index (TI) Yama Joko root was significantly higher than the other 3 kinds of Apple Rootstock.4 apple rootstocks, and Cd content of the xylem and leaves the lowest, Cd and BCF. The minimum amount of Apple Rootstock Cd can be fixed in the cell wall and Vacuole, and convert the results of gel acid salt or protein bound and soluble state of Cd to reduce the mobility and toxicity of.4 Apple Rootstocks with physiological state changes such as ROS, sugar and antioxidant system consistent with the Cd tolerance. Compared with other 3 kinds of Apple Rootstock Roots and phloem cells in ROS, the root and leaf H202 and root MDA content in xylem and phloem of the lowest in the phloem of root and leaf soluble sugar and starch content is the highest, higher antioxidant enzyme activity and antioxidant content. The test under the condition of strong tolerance to the stator Cd than the other 3 apple rootstocks, and the the Cd content of the tissue cells, subcellular distribution and chemical forms of Cd, and the.3. antioxidant system by comparing the effects of exogenous melatonin on the 30 M Cd down the stator and Qingzhou LinQin seedling Cd uptake and translocation, antioxidant system and medium Because of the influence on the expression of melatonin, found that adding Malus baccata and Qingzhou Cd2+ net of mallus doumeri root absorption rate, leaf Cd content and Tf decreased significantly, indicating that melatonin inhibits the absorption and transport of Cd Apple Rootstock, and the inhibition of m.baccata was more obvious. Compared with 30 MCd, 30 MCd + 0.1 M melatonin treatment reduces the Apple Rootstock Roots, stems and leaves of O2, H202 and MDA content, Cd content and cell component proportion decreased, and the absorption of Cd related genes such as NARMAP1 expression decreased; antioxidant enzymes such as CAT, GPX, APX increased and GR activity, free proline in roots and leaves. Soluble phenol and T-SH in root, stem and leaf ASC, increase in root and stem and root GSH in melatonin content, and Cd toxin genes such as NAS1 up-regulated, indicating that melatonin can inhibit the ability of Apple Rootstock root uptake and translocation of Cd cells, improve the antioxidant The level of effective removal of intracellular ROS, enhance the tolerance of Apple Rootstocks on Cd. But 30 M Cd + 0.1 M melatonin treatment CAT and GR in roots and leaves of Malus in Qingzhou, GPX and APX activity in roots, and the roots and leaves of free proline, soluble phenol and T-SH, and stem leaf GSH content is lower than in Malus baccata; Cd absorption such as gene transcription of HA7 and NRAMP1 in the high mountain stator, and Cd detoxification related genes such as NAS1 low expression in the mountains. The stator of melatonin on Cd stress relieving m.baccata more obvious by comparing the.4. 50 M Cd under the stress of the absorption and transport of Cd 4 kinds of Apple grafted seedlings, the expression level and antioxidant genes, found that Cd causes 4 kinds of apple seedlings biomass, photosynthetic rate and pigment content decreased, but the influence of Cd on Hanfu Hanfu. The minimum / m.baccata / baccata and Fuji / Cd content and scion root cells, C D and BCF were less than the amount of Hanfu / Qingzhou / Qingzhou Fuji mallus doumeri and mallus doumeri, which can effectively reduce the Cd baccata as rootstock grafted seedlings in apple.Cd enrichment under the stress of Hanfu expressed in xylem, phloem and leaf Cd content and water soluble Cd content is lower than the other 3 kinds of apple seedlings, root and leaf cell wall components Cd the largest proportion of organelles component content of Cd and the ratio of the minimum, associated with the absorption and transportation of Cd genes such as HA7, FRO2-like, NRAMP1 and NRAMP3 low expression, indicating Hanfu / root Cd strong ability of holding cells, can reduce the rate of the mobile Cd in grafted seedlings in addition, under the stress of Cd / m.baccata Hanfu phloem and leaf ROS and MDA content was the lowest, soluble phenol, ASC and GSH were the highest, and Cd toxin genes such as the expression of MT2 was highest, that of Hanfu / strong tolerance to Cd in which he expressed 3 kinds of apple Grafted seedlings.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S661.1

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