钼在草莓中的吸收转运及其对氮代谢调控机制的研究

发布时间：2018-08-05 14:32

【摘要】：钼(molybdenum,Mo)是植物正常生长必不可少的微量元素之一,参与到植物的氮代谢过程中。缺钼的条件下植物容易积累较多的硝态氮从而表现出较低的氮素利用率(nitrogen utilization efficiency,NUE)。提高氮素利用率可以减少化学肥料过多施用带来的负面效应,但是关于钼提高氮素利用率的机理尚不清楚。本研究分别在温室无土栽培和组培条件下进行。以‘章姬’(Fragaria×ananassa Duch.cv.‘Akihime’)草莓为试验材料,在温室无土栽培条件下喷施不同浓度的钼酸钠(sodium molybdate,Na_2MoO_4),试验设置T1(叶面喷施清水)、T2(0.02%Na_2MoO_4)、T3(0.04%Na_2MoO_4)、T4(0.05%Na_2MoO_4)和T5(0.06%Na_2MoO_4)5个处理。研究了其对植株生物量、果实营养元素含量和果实品质(可溶性固形物(total soluble solids,TSS)、抗坏血酸(ascorbic acid,AsA)、可滴定酸(titratable acidity,TA)、芳香物质)、光合作用、钼转运蛋白基因MOT1表达量、植株体内Mo浓度、氮代谢关键酶活性(硝酸还原酶,nitrate reductase,NR;亚硝酸还原酶,NiR;谷氨酰胺合成酶,glutamine synthetase,GS;NADH-谷氨酸合酶,NADH-glutamate synthase,NADH-GOGAT)、氮代谢关键基因(NRT1.1,NRT2.1,NIA,NiR,GS-1,NADH-GOGAT)表达量、15N吸收、分配及利用的影响。在组培条件下,MS固态培养基中加Na_2MoO_4或者钨酸钠(sodium tungstate,Na2WO4),设置CK(-Na_2MoO_4-Na2WO4)、Mo处理(+Na_2MoO_4)、W处理(+Na2WO4)、Mo+W处理(+Na_2MoO_4+Na2WO4)4个处理。研究其对植株表型、根系显微结构及根系活力、叶片显微结构与超显微结构及叶绿素含量、MOT1表达量、钼辅因子合成通路关键基因(CNX2,CNX3,CNX5,CNX6,CNX7,CNX1)表达量、植株体内Mo浓度、氮代谢关键酶活性(NR,NiR,GS,NADH-GOGAT)以及15N利用的影响。主要结果如下:1.适宜浓度的Mo供应能显著提高幼苗的生物量。叶面喷施Mo肥对草莓果实的营养元素(N,Mo,Fe和Cu)含量,TSS,TA和芳香物质含量有一定的提升作用。但是施Mo对果实中的Se和部分芳香物质没有显著影响。0.04%Na_2MoO_4处理在所有处理中表现出最高的果实TSS,N和Fe浓度。0.02%Na_2MoO_4处理的AsA含量显著高于对照。利用顶空固相微萃取法和气质联用技术一共鉴别出97中芳香物质。0.04%Na_2MoO_4处理的6种特征酯类(丁酸甲酯,γ-癸内酯,丁酸乙酯,己酸甲酯,γ-十二内酯,己酸乙酯)含量最高。2.草莓幼苗根部、茎部和叶片的Mo含量随着施Mo量的增加而呈现上升的趋势。0.04%Na_2MoO_4处理的根部的MOT1的表达水平高于其它处理,当Na_2MoO_4的浓度从0.05%增加到0.06%时,根部的MOT1的mRNA水平呈现下降的趋势,培养5天后,叶片的MOT1的表达水平随着喷施Na_2MoO_4浓度的提高而提高;培养10天和15天后,当Na_2MoO_4的浓度从0.00%增加到0.04%时,叶片的MOT1的表达水平呈现升高的趋势,而后随着喷施Na_2MoO_4的提高而呈现下降的趋势。施Mo影响幼苗根部Fe、Cu和Se浓度。0.04%Na_2MoO_4处理的幼苗表现出最高的15N地上部/地下部比例和15N利用率。0.04%Na_2MoO_4处理的氮代谢关键酶活性较高,且硝态氮吸收基因(NRT1.1和NRT2.1)以及硝态氮响应基因的表达水平上调。0.04%Na_2MoO_4处理的根部和叶片的NO3--N浓度低于其它处理,叶片的NH4+-N的浓度较高,Gln/Glu的浓度也较高。而T5处理表现出相反的趋势。因此,叶面喷施0.04%Na_2MoO_4通过调控氮代谢关键酶活性和NO3-吸收同化相关基因的表达提高了根部NO3-向地上部的转运,提高了氮素利用率。3.MS固态培养基中正常加入Na_2MoO_4的草莓幼苗长势较好,叶片为正常绿色,叶肉细胞显微结构及超微结构完整,叶绿体较多,叶绿素含量较高。根系发育正常,根系活力较高,根尖细胞排列整齐紧密,中央髓部细胞体积较大,排列紧密。而缺Mo培养基上生长的幼苗叶片下表皮细胞排列不整齐,叶肉细胞超微结构异常,细胞器及内膜系统受损,叶绿素含量较低,叶片发黄。根系较长,但是长势较弱,根尖细胞微变形,细胞排列疏松,细胞间隙较大。只加Na2WO4和同时加入Na_2MoO_4和Na2WO4的培养基上的幼苗株型变小,长势减弱,细胞器受损,膜结构不清晰,部分叶片表现为黄色,叶绿素含量降低,出现与氮素缺乏类似的症状。施入Na_2MoO_4可以显著诱导草莓地上部和根部FaMOT1的表达,加入抑制剂Na2WO4的FaMOT1表达量则表现出相对下降,且施入Na_2MoO_4幼苗的Mo浓度显著高于其它处理。培养基中加入Na_2MoO_4的组培苗的CNX2和CNX3的表达量较高,培养基加入Na_2MoO_4的组培苗中CNX7和CNX1的表达量高于加入抑制剂的组培苗。加入抑制剂Na2WO4使得NR活性显著降低,加入Na_2MoO_4组培苗的NR活性最高,其它氮代谢关键酶(NiR,GS和NADH-GOGAT)表现出类似的趋势。培养基加入Na_2MoO_4的组培苗的NO3--N浓度低于其它处理,NO2--N和NH4+-N浓度显著高于其它处理。同位素15N-Ca(NO3)2标记进一步表明培养基加入Na_2MoO_4的组培苗吸收利用了较多的15N,转运到地上部的15N也较多,植株的15N利用率最高,而加入抑制剂的组培苗的15N利用率较低。
[Abstract]:Molybdenum (Mo) is one of the essential trace elements essential for the normal growth of plants. It participates in the process of nitrogen metabolism in plants. Under the condition of lack of molybdenum, plants accumulate more nitrate nitrogen and thus show low nitrogen use efficiency (nitrogen utilization efficiency, NUE). Increasing nitrogen use rate can reduce the excessive application of chemical fertilizer. The negative effect is not clear, but the mechanism of increasing nitrogen utilization rate of molybdenum is not clear. This study was carried out under the conditions of soil free cultivation and tissue culture in greenhouse. The experimental materials of "Fragaria x ananassa Duch.cv. 'Akihime") strawberry were used to spray different concentrations of sodium molybdate (sodium molybdate, N) under the condition of soil free cultivation in greenhouse. A_2MoO_4), the experiment set up T1 (foliar spraying water), T2 (0.02%Na_2MoO_4), T3 (0.04%Na_2MoO_4), T4 (0.05%Na_2MoO_4) and T5 (0.06%Na_2MoO_4). The plant biomass, fruit nutrition element content and fruit quality (total soluble), ascorbic acid, titratable acid were studied. Table acidity, TA), photosynthesis, MOT1 expression of molybdenum transporter gene, Mo concentration in the plant, activity of key enzymes in nitrogen metabolism (nitrate reductase, nitrate reductase, NR; nitrite reductase, NiR; glutamine synthetase, glutamine synthetase, GS; glutamate synthase, depletion), nitrogen metabolism The expression of key genes (NRT1.1, NRT2.1, NIA, NiR, GS-1, NADH-GOGAT), the effect of 15N absorption, distribution and utilization. Under the condition of tissue culture, Na_2MoO_4 or sodium tungstate (sodium tungstate, Na2WO4) in the solid culture medium of MS (sodium tungstate, Na2WO4), 4 treatments. The plant phenotype, root microstructure and root activity, leaf microstructure and ultrastructure and chlorophyll content, MOT1 expression, the expression of key genes (CNX2, CNX3, CNX5, CNX6, CNX7, CNX1), the concentration of Mo in the plant, the activity of the key enzymes of nitrogen metabolites (NR, NiR, GS, NADH-GOGAT), and the effects of the utilization of the plants. The results are as follows: 1. the suitable concentration of Mo supply can significantly increase the biomass of the seedlings. Spraying Mo fertilizer on the leaf surface can improve the content of nutrient elements (N, Mo, Fe and Cu), TSS, TA and aromatic substance in strawberry fruit. However, the application of Mo on Se and some aromatic substances in the fruit has no significant effect on the treatment of.0.04%Na_2MoO_4 in all treatments. The highest TSS, N and Fe concentrations of.0.02%Na_2MoO_4 were significantly higher than those of the control. A total of 6 kinds of esters (methyl butyrate, gamma decanide, ethyl butyrate, methyl Hexanate, gamma twelve lactone, gamma twelve lactone, ethyl caproate, ethyl hexanoate) were identified by headspace solid phase microextraction and GC-MS. The content of Mo in the roots of the strawberry seedlings with the highest content of.2., the content of the stems and leaves increased with the increase of Mo, and the expression level of MOT1 in the root of the.0.04%Na_2MoO_4 treatment was higher than that of the other treatments. When the concentration of Na_2MoO_4 increased from 0.05% to 0.06%, the mRNA level of MOT1 in the root showed a decline trend, and the leaves was MO after 5 days. The expression level of T1 increased with the increase of the concentration of Na_2MoO_4. When the concentration of Na_2MoO_4 increased from 0% to 0.04% after 10 and 15 days, the expression level of MOT1 in leaves showed a trend of increase, and then decreased with the increase of spraying Na_2MoO_4. Mo influenced Fe, Cu and Se.0.04%Na_2MoO_4 in the roots of seedlings. The seedlings of the treated seedlings showed the highest 15N upper / underground ratio and the 15N utilization rate.0.04%Na_2MoO_4 treated with higher nitrogen metabolism key enzyme activity, and the expression level of nitrate nitrogen absorption gene (NRT1.1 and NRT2.1) and nitrate nitrogen response gene up regulated the NO3--N concentration of the root and leaf of.0.04%Na_2MoO_4 treatment was lower than that of other treatments. The concentration of NH4+-N was higher and the concentration of Gln/Glu was higher, while T5 treatment showed the opposite trend. Therefore, the foliar spraying 0.04%Na_2MoO_4 enhanced the transport of NO3- to the top of the root by regulating the activity of key enzyme activities of nitrogen metabolism and the expression of NO3- assimilation related genes, and increased the normal addition of Na_ in the solid-state medium of nitrogen utilization ratio.3.MS. The growth trend of strawberry seedlings in 2MoO_4 is better, the leaf is normal green, the microstructure and ultrastructure of the mesophyll cells are complete, the chloroplasts are more, the chlorophyll content is higher, the root development is normal, the root activity is high, the root tip cells are arranged neatly and closely, the central marrow cells are large and arranged closely. The leaves are short under the leaves of the seedlings grown on the Mo medium. The epidermal cells were not arranged regularly, the ultrastructure of the mesophyll cells was abnormal, the organelles and the intima system were damaged, the chlorophyll content was low, the leaves were yellow and the roots were long, but the growth potential was weak, the root tip cells were slightly deformed, the cells were loosely arranged and the cell gaps were large. The plant types on the medium with Na2WO4 and Na_2MoO_4 and Na2WO4 were smaller. The cell organ is impaired, the organelle is damaged, the membrane structure is not clear, some leaves are yellow, the content of chlorophyll is reduced, and there is a similar symptom with nitrogen deficiency. Na_2MoO_4 can significantly induce the expression of FaMOT1 in the upper and root parts of the strawberry, and the amount of the FaMOT1 surface of the inhibitor Na2WO4 shows a relative decline and is applied to the Na_2MoO_4 seedlings. The concentration of Mo was significantly higher than that of other treatments. The expression of CNX2 and CNX3 in tissue culture seedlings with Na_2MoO_4 was higher. The expression of CNX7 and CNX1 in the tissue culture medium with Na_2MoO_4 was higher than that of the tissue culture seedlings which were added to the culture medium. The addition of inhibitor Na2WO4 made the NR activity significantly lower, and the NR activity of the group of Na_2MoO_4 tissue cultured seedlings was the highest. The key enzyme of nitrogen metabolism (NiR, GS and NADH-GOGAT) showed a similar trend. The concentration of NO3--N in the culture medium with Na_2MoO_4 was lower than that of other treatments, and the concentration of NO2--N and NH4+-N was significantly higher than that of other treatments. The isotopic 15N-Ca (NO3) 2 marker further indicated that the tissue culture seedlings of the culture medium added to Na_2MoO_4 were absorbed and utilized more 15N and transported to the ground. There were more 15N in the upper part, and the highest utilization rate of 15N in plants. The 15N utilization rate of tissue culture seedlings with inhibitors was lower.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S668.4

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