【作者】 汤亚芳；

【导师】 胡承孝；

【作者基本信息】 华中农业大学 ， 植物营养学， 2014， 博士

【摘要】 随着人们对食品安全重视程度的增加,蔬菜作为日常生活中大量消费的食物种类之一,其品质安全也得到广泛关注。蔬菜尤其是叶菜类蔬菜是一种容易积累硝酸盐的作物,加上近几十年来为追求高产大量施用氮肥更加剧了叶菜类蔬菜中硝酸盐的积累。硝酸盐本身对人体无害,但过量摄入的硝酸盐可在细菌作用下反应生成对人体有毒害的物质,增加人体患甲状腺和肠胃系统疾病的可能。小白菜是武汉地区种植密度高,食用量大的叶菜类蔬菜,且品种繁多,从中筛选硝酸盐高、低积累品种,并对品种间硝酸盐吸收、转运、同化差异,硝酸盐吸收代谢的时间动态变化以及氮、钼响应差异进行了研究,现主要研究结果如下：1.武汉地区小白菜硝酸盐积累现状及品种筛选对武汉地区广泛种植的168个小白菜品种硝酸盐含量和生物量进行研究,不同批次种植的小白菜硝酸盐含量和产量存在差异,第二批和第三批的硝酸盐含量和单株产量最高。以我国提出的蔬菜硝酸盐限定标准3100mg/kg为限,三批筛选中分别有24.4%、97.6%和100%的品种超标。结合硝酸盐含量和产量,得到高硝高产品种7个,高硝低产品种9个,低硝高产品种7个,低硝低产品种2个。小白菜不同部位间硝酸盐含量差异较大,叶柄硝酸盐含量是叶片的1.69-2倍。由于品种间叶片硝酸盐含量差异较叶柄大,且叶片硝酸盐含量对外界环境较为敏感,选择叶片为最适宜的采样部位,确定品种96号为叶片硝酸盐高积累品种,品种18号为叶片硝酸盐低积累品种。2.小白菜品种间硝酸盐积累的差异机制以田间筛选获得的叶片硝酸盐高积累品种96号(High nitrate accumulator, H96)和低积累品种18号(Low nitrate accumulator, L18)为试验材料,在水培条件下研究两个品种硝酸盐吸收、转运和同化过程的差异。H96较L18具有较高的硝酸盐吸收能力,根系形态参数—根长、根表面积和根体积分别比L18高18.0%、31.6%和46.5%。根系NRT1.1、NRT2.1相对表达量分别比L18高41.6%和269.6%。硝酸盐转运过程中,两个品种叶片NRT1.1、NRT2.1相对表达量结果与根中相反,表现为L18较H96高279.2%、80.0%。此外,L18叶片硝酸盐同化能力显著高于H96。L18叶片硝酸盐代谢相关酶-NR和GS活性,及其基因-NIA.Glnl、Gln2表达量分别比H96高234.0%、43.9%、105.4%、331.5%和124.8%。同时,L18叶片具有较高的叶绿素含量和较强的光合同化能力,叶片中硝酸盐代谢产物含量,包括氨基酸组分和可溶性蛋白,均显著高于H96。以上结果说明,品种L18叶片硝酸盐含量显著低于H96,受到硝酸盐吸收、转运和同化三个部分共同影响,比较这三个过程对硝酸盐积累影响的贡献,转运和同化过程对硝酸盐含量的影响起决定性作用。3.两个小白菜品种对氮素供应的时间响应差异在营养液栽培条件下对两个小白菜品种L18和H96硝酸盐吸收、转运、同化过程的时间动态变化进行研究,根系对硝酸盐的供应响应最迅速,NRT1.1和NRT2.1表达量急剧增加,5N累积速率最快,含量也显著高于地上部。其中,H96根系表现出对硝酸盐供应的快速响应,NRT1.1和NRT2.1表达量显著高于L18,各处理时间内15N含量也均显著高于L18。同时H96表现出较强的向地上部的转运能力,地上部15N含量显著高于L18。但叶片中L18NRTl.l和NRT2.1表达量分别在12h和24h达到峰值,显著高于H96。在硝酸盐同化过程,两个小白菜品种NR活性及其基因表达受到硝酸盐的强烈诱导,根系和叶片中除12h外,L18NR活性和NIA表达量均显著高于H96,说明L18叶片NR更易受到硝酸盐诱导,具有较强的硝酸盐同化能力。因此,H96具有较强的根系硝酸盐吸收和向地上部转运能力,但L18叶片内硝酸盐再利用以及同化能力相对较强,因而导致了品种间硝酸盐积累的差异。4.两个小白菜品种对氮水平响应差异氮缺乏显著降低两个小白菜品种叶片和叶柄中硝酸盐积累并缩小品种和部位间的差异。高氮处理中,L18和H96叶柄硝酸盐含量是叶片的5.3和2.3倍,H96叶片硝酸盐含量较L18高92.5%,缺氮时部位间和品种间差异均不显著。在不同氮水平下,两个小白菜品种硝酸盐的吸收存在差异。高氮时,H96吸收速率和吸收量显著高于L18,而低氮时,结果相反。同样的结果出现在根系硝酸根转运子表达量上。高氮时,H96根系NRT1.1和NRT2.1表达量较L18高41.7%和264.7%,而低氮时,L18NRT2.1较H96高117.8%,NRT1.1品种间无显著差异。说明品种间硝酸盐吸收差异的反转是由根系转运子,尤其是NRT2.1对氮素的响应来实现的。在高氮水平时,L18叶片具有较强硝酸盐转运能力,NRT1.1和NRT2.1表达量是H96的3.8倍和1.8倍,而在低氮水平时,H96NRT2.1表达量是L18的3倍,因而能将更多的硝酸盐运输进入叶片中进行同化。氮缺乏降低两个品种NR和GS活性,但对品种间差异影响不大。缺氮条件下,尽管L18叶片具有较高的NR活性,但GS活性低于H96,且在硝酸盐代谢产物-可溶性蛋白含量上,H96显著高于L18,说明低氮条件下H96具有较强的硝酸盐同化能力。5.两个小白菜品种对钼水平响应差异钼缺乏显著提高两个小白菜品种根系对硝酸盐的吸收,降低叶片的转运以及同化能力,显著提高硝酸盐含量,但不同品种对于钼水平的响应并不一致。硝酸盐高积累品种H96对钼缺乏较低积累品种L18敏感,表现为硝酸盐吸收、根系NRT1.1、 NRT2.1表达量在缺钼时增加幅度(195.9%、44.2%和52.1%)较L18(146.9%、5.0%和10.3%)高；硝酸盐转运过程,叶片NRT1.1、NRT2.1表达量变化幅度(1252.0%、4181.3%)较L18(702.0%、4024.5%)高；NR和GS活性在钼存在条件下提高47.1倍和64.6%,远高于L18(11.0倍和36.3%)。但L18叶片硝酸盐含量显著低于H96。此外,H96全氮含量也显著高于L18,这与硝酸盐吸收的结果一致。在生物量上,H96受到Mo水平影响较大,缺钼时地上部生物量显著下降,而L18并无明显变化。比较钼缺乏条件下硝酸盐吸收、转运和同化对于两个品种硝酸盐积累的贡献,硝酸盐的同化过程,尤其是硝酸还原酶的差异,决定了硝酸盐积累水平的高低。L18因其具有较强的Mo吸收和利用能力,缺Mo对硝酸盐积累造成的影响较小。综上所述,根系吸收与地上部同化的不一致性是造成小白菜叶片硝酸盐的大量积累的重要原因。其中,H96根系较强的硝酸盐吸收能力是叶片中硝酸盐大量积累的根本原因,L18较强的硝酸盐地上部转运和同化能力是叶片低积累的关键原因。在氮缺乏时,品种间根系吸收、地上部积累和代谢差异缩小,但两个品种硝酸盐吸收出现逆转,这种现象由硝酸根转运子表达量差异造成,由转运子对低氮响应机制不同导致。钼缺乏时,品种的钼利用能力决定硝酸盐吸收、转运、同化过程受到钼缺乏的影响程度,低积累品种因其具有较高的NR活性和较高的钼含量,硝酸盐积累受到影响较小。更多还原

【Abstract】 Vegetable, as large consumed food in daily life, its qulity and safty were paid attention by Chinese peple. Leaf vegetables are classified as high nitrate accumulation crops and this phenomenon is aggravated by high nitrogen fertilizer application in vegetable production. Nitrate itself is not harmful to human, while its reductant may increase risks of thyroid and gastric illnesses. Widly planted of Chinese cabbage [Brassica campestris L. ssp. Chinensis (L.)] in Wuhan and high consumption force us to mininise any adverse effects on human health. Based on various cultivars in Chinese cabbge, we have conducted high and low nitrate accumulate cultivars screening and studied nitrate uptake, translocation and assimilation differences between cultivars. Differences of nitrate uptake and assimilation between cultivars in response to nitrate and molybdenum were also examined. The main results are as follow:1. Nitrate accumulation in Chinese cabbage in Wuhan and nitrate accumulators screening experimentsThe nitrate content and biomass of168Chinese cabbage cultivars in Wuhan have been studied. The experimental results show that there were differences in nitrate concentration and biomass were observed in cultivars. The highest nitrate concentration and biomass were observed in the third and second screening experiments respectively. Nitrate concentration exceeding3100mg/kg were observed in24.4%,97.6%and100%of the cultivars in the three screening experiments respectively. On the basis of nitrate content and biomass, we choosed7cultivars defined as high nitrate high biomass cultivars,9cultivars defined as high nitrate low biomass cultivars,7cultivars defined as low nitrate high biomass cultivars and2cultivars defined as low nitrate low biomass cultivars. Nitrate content in different plant tissues were also determined. Petiole nitrate content in the third and fourth experiments were1.69and2times higher than in leaves. Due to high sensitivity, leaves were considered to be the best tissues for evaluating nitrate accumulation in plant. L18was defined as a low nitrate leaf accumulatior and H96was defined as a high leaf nitrate accumulator.2. Differences in the mechanism of nitrate accumulation between cultivarsThe high nitrate accumulator-H96and the low nitrate accumulator-L18, from the field screening experiments, were used in a hydroponic culture to investigate genotypic differences in nitrate uptake, translocation and assimilation between the two Chinese cabbage cultivars. H96could uptake more nitrate than L18in the root but had lower transport into leaf cells and assimilation in the leaf. It was show that root morphology parameters (length, surface area and volume) of H96were18.0%,31.6%and46.5%higher than for L18respectively. Nitrate transporters NRT1.l and NRT2.1transcription levels in roots were41.6%and269.6%higher than those of L18respectively. In process of nitrate translocation, NRT1.1and NRT2.1expressions in the leaf blades of the two cultivars were opposite to these in the roots, L18NRT1.1and NRT2.1expressions were279.2%and80.0%higher than H96. In addition, nitrate assimilation capacity of L18was significantly higher than H96in leaves. It was shown that nitrate assimilation enzymes-NR, GS and those gene-NIA、Gln1、Gln2relative expressions of L18were234.0%,43.9%,105.4%,331.5%and124.8%higher than those of H96respectively. Both chlorophyll content and photosynthesis of L18were higher than those of H96. Nitrate assimilation products-Glu, total amino acid, soluble protein content in the leaf of L18were all significantly higher than those of H96. The results above suggested that nitrate accumulation differences were due to differential capacities for uptake, mechanisms for nitrate transport in leaves and assimilation of nitrate. Comparing the contribution of three aspects in nitrate accumulation, the latter two aspects contributed more of low nitrate concentration in the leaf blade.3. Genotypic differences of nitrate uptake, translocation and assimilation in response to nitrate provisionA hydroponic culture experiment was conducted to investigate genotypic difference of nitrate uptake, translocation and assimilation in response to nitrate provision. The results showed that NRT1.1and NRT2.1expressions in roots of the two cultivars were sharply increased in response to nitrate supply.15N accumulate rate and contents in roots was higher than in leaves. Meanwhile NRT1.1and NRT2.1expressions and5N contents in tissueses of H96were significantly higher than for L18. However, the results in leaves were reversed, NRT1.1and NRT2.1expressions in leaves of L18were peaked at12h and24h and significantly higher than those of H96. In process of nitrate reduction, NR activity and NIA expression of two cultivars were induced by nitrate supply, and NR activity and NIA expression of L18were significantly higher than those of H96, except for12h in leaves. It was suggested that nitrate assimilation capacity of L18was stronger than H96.4. Genotypic difference of two Chinese cabbage cultivars in response to N levelsDifferences of nitrate concentration between cultivars and in tissues were decreased in response to N deficiency. Nitrate content in tissues decreased under a-N treatment. Nitrate contents in petioles of L18and H96were5.3times and2.3times higher than in leaves. The leaf nitrate content of H96was higher than of L18significantly under the+N treatment, while no significant difference was observed in tissues or between cultivars under the-N treatment. Nitrate uptake was different between the two cultivars under N treatments. Nitrate uptake rate and amount of H96were higher than for L18under+N, while the results were reversed under-N. Similar results were observed in root nitrate transporters expressions. NRT1.1and NRT2.1expressions in roots of H96were41.7%and264.7%higher than those of L18under the+N treatment, while NRT2.1expression of L18was117.8%higher than H96, no significant difference was observed in NRT1.1. It was suggested that differences of nitrate uptake between cultivars was based on NRT2.1expression differences. In the process of nitrate translocation, NRT1.1and NRT2.1expression in leaves of L18were3.8times and1.8times higher than those of H96under the+N treatment, while NRT2.1expression in leaves of H96was3times higher than L18under the-N treatment. Thus, more nitrate were transported into leaves for reduction than for L18. NR and GS activities were decreased by the-N treatment, with no difference between cultivars. NR activity in leaves of L18was significantly higher, but its GS activity and nitrate assimilation production-soluble protein contents were lower than for H96under the-N treatment. These above results suggest that nitrate assimilation capacity of H96was higher than L18under the-N treatment.5. Genotypic difference of two Chinese cabbage cultivars in response to Mo levelsMo deficiency significantly increased nitrate uptake and nitrate transporters-NRT1.1and NRT2.1expressions in roots, decreased nitrate transport into leaves and enzyme (NR, GS) activities which were involved in nitrate assimilation, while genotypic difference of two cultivars were shown in response to Mo treatments. High nitrate accumulator-H96was more sensitive to Mo deficiency. It was shown that increase rate of nitrate uptake, NRT1.1and NRT2.1expressions in root of H96were (195.9%,44.2%and52.1%) higher than for L18(146.9%,5.0%and10.3%) response to Mo deficiency. In the process of nitrate translocation, decreased rates of NRT1.1and NRT2.1expressions in leaves of H96under Mo deficiency (1252.0%,4181.3%) were higher than those of L18(702.0%,4024.5%). NR and GS activities of H96were increased47.1times and64.6%which higher than L18(11.0times and36.3%). Nitrate assimilation, especially NR activity was most impacted by Mo deficiency. In addition, the nitrate content in leaves of L18was lower than for H96. Related to the nitrate uptake result, total N of H96was significantly higher than for L18. Comparing contributions of uptake, translocation and assimilation to nitrate accumulation under conditions of Mo deficiency, uptake and assimilation capacities were determined in nitrate accumulation in leaf. Based on high Mo utilization, the nitrate assimilation capacity of L18, especially NRA, contributed to the low nitrate content in response to Mo deficiency.In conclusion, inconsistency between root uptake and shoot assimilation capacity is main reason cause nitrate accumulation in leaf of Chinese cabbage. H96had a great capacity of nitrate uptake in roots, which was a basic reason for nitrate high accumulation in leaves. Nitrate high translocation and assimilation capacities in leaves were a key reason for nitrate low accumulation in leaves. Differences in root nitrate uptake, shoot accumulation and assimilation between the two cultivars were minimized under-N treatment, while the results of root nitrate uptake were reversed. This was caused by nitrate transporter expression changes in the roots. Changes of nitrate uptake, translocation and assimilation in response to Mo deficiency were based on Mo efficiency of two cultivars. Due to high NR activity and Mo efficiency, nitrate concentration in leaves of L18was less impacted by Mo deficiency.更多还原